Structural and Biochemical Studies of TIGAR (TP53-induced Glycolysis and Apoptosis Regulator)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, H.; Jogl, G

2009-01-01

Activation of the p53 tumor suppressor by cellular stress leads to variable responses ranging from growth inhibition to apoptosis. TIGAR is a novel p53-inducible gene that inhibits glycolysis by reducing cellular levels of fructose-2,6-bisphosphate, an activator of glycolysis and inhibitor of gluconeogenesis. Here we describe structural and biochemical studies of TIGAR from Danio rerio. The overall structure forms a histidine phosphatase fold with a phosphate molecule coordinated to the catalytic histidine residue and a second phosphate molecule in a position not observed in other phosphatases. The recombinant human and zebra fish enzymes hydrolyze fructose-2,6-bisphosphate as well as fructose-1,6-bisphosphate but notmore » fructose 6-phosphate in vitro. The TIGAR active site is open and positively charged, consistent with its enzymatic function as bisphosphatase. The closest related structures are the bacterial broad specificity phosphatase PhoE and the fructose-2,6-bisphosphatase domain of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The structural comparison shows that TIGAR combines an accessible active site as observed in PhoE with a charged substrate-binding pocket as seen in the fructose-2,6-bisphosphatase domain of the bifunctional enzyme.« less

The characteristics of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferases from Sansevieria trifasciata leaves and Phaseolus coccineus stems.

PubMed

Kowalczyk, S

1987-01-01

Three different molecular forms of pyrophosphate-dependent phosphofructokinase have been isolated: one from Sansevieria trifasciata leaves and two from Phaseolus coccineus stems. The form isolated from S. trifasciata has the molecular weight of about 115,000. The apparent molecular weights for the two forms from mung bean were approximately 220,000 and 450,000. All three forms have the same pH optima, an absolute requirement for Mg2+ ions both in the forward and reverse reaction, but differ in their sensitivity toward fructose 2,6-bisphosphate. Kinetic properties of the partially purified enzymes have been investigated in the presence and absence of fructose 2,6-bisphosphate. Pyrophosphate-dependent phosphofructokinase from S. trifasciata exhibited hyperbolic kinetics with all substrates tested. The saturation curves of the enzyme (form A) from mung bean for pyrophosphate, fructose 6-phosphate and fructose 1,6-bisphosphate were sigmoidal in the absence of fructose 2,6-bisphosphate. In the presence of fructose 2,6-bisphosphate these kinetics became hyperbolic.

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, G.; Singer, A.; Lunin, V. V.

2009-02-06

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed amore » compact, globular shape with two {alpha}/{beta}-sandwich domains. The core fold of GlpX is structurally similar to that of Li{sup +}-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr{sup 90} is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.« less

Effect of low shear modeled microgravity on phenotypic and central chitin metabolism in the filamentous fungi Aspergillus niger and Penicillium chrysogenum.

PubMed

Sathishkumar, Yesupatham; Velmurugan, Natarajan; Lee, Hyun Mi; Rajagopal, Kalyanaraman; Im, Chan Ki; Lee, Yang Soo

2014-08-01

Phenotypic and genotypic changes in Aspergillus niger and Penicillium chrysogenum, spore forming filamentous fungi, with respect to central chitin metabolism were studied under low shear modeled microgravity, normal gravity and static conditions. Low shear modeled microgravity (LSMMG) response showed a similar spore germination rate with normal gravity and static conditions. Interestingly, high ratio of multiple germ tube formation of A. niger in LSMMG condition was observed. Confocal laser scanning microscopy images of calcofluor flurophore stained A. niger and P. chrysogenum showed no significant variations between different conditions tested. Transmission electron microscopy images revealed number of mitochondria increased in P. chrysogenum in low shear modeled microgravity condition but no stress related-woronin bodies in fungal hyphae were observed. To gain additional insight into the cell wall integrity under different conditions, transcription level of a key gene involved in cell wall integrity gfaA, encoding the glutamine: fructose-6-phosphate amidotransferase enzyme, was evaluated using qRT-PCR. The transcription level showed no variation among different conditions. Overall, the results collectively indicate that the LSMMG has shown no significant stress on spore germination, mycelial growth, cell wall integrity of potentially pathogenic fungi, A. niger and P. chrysogenum.

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, Leads to Altered Carbohydrate Metabolism in Cancer Cells*

PubMed Central

Tan, Bo; Dong, Sucai; Shepard, Robert L.; Kays, Lisa; Roth, Kenneth D.; Geeganage, Sandaruwan; Kuo, Ming-Shang; Zhao, Genshi

2015-01-01

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD+ biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD+ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500–3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed. PMID:25944913

Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.

2009-05-14

ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. Themore » A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.« less

Human fructose-1,6-bisphosphatase gene (FBP1): Exon-intron organization, localization to chromosome bands 9q22.2-q22.3, and mutation screening in subjects with fructose-1,6-bisphosphatase deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

El-Maghrabi, M.R.; Jiang, W.

1995-06-10

Fructose-1,6-bisphosphatase (EC 3.1.3.11) is a key regulatory enzyme of gluconeogenesis that catalyzes the hydrolysis of fructose-1,6-bisphosphate to generate fructose-6-phosphate and inorganic phosphate. Deficiency of fructose-1,6-bisphosphatase is associated with fasting hypoglycemia and metabolic acidosis because of impaired gluconeogenesis. We have cloned and characterized the human liver fructose-1,6-bisphosphatase gene (FBP1). FBP1, localized to chromosome bands 9q22.2-q22.3 by fluorescence in situ hybridization, consists of seven exons that span > 31 kb, and the six introns are in the same position as in the rat gene. FBP1 was screened for mutations in two subjects with fructose-1,6-bisphosphatase deficiency. Four nucleotide substitutions were identified, two ofmore » which were silent mutations in the codons for Ala-216 (GCT {yields} GCC) and Gly-319 (GGG {yields} GGA). The other substitutions were in intron 3, a C {yields} T substitution 7 nucleotides downstream from the splice donor site, and in the promoter region, an A {yields} T substitution 188 nucleotides upstream from the start of transcription. These nucleotide substitutions were also found in normal unaffected subjects and thus are not the cause of fructose-1,6-bisphosphatase deficiency in the two subjects studied. The molecular basis of hepatic fructose-1,6-bisphosphatase deficiency in these subjects remains undetermined but could result from unidentified mutations in the promoter that decrease expression or from mutations in another gene that indirectly lead to decreased fructose-1,6-bisphosphatase activity. 18 refs., 3 figs., 3 tabs.« less

Laser Raman spectra of mono-, oligo- and polysaccharides in solution

NASA Astrophysics Data System (ADS)

Barrett, T. W.

We examined the Raman spectra of thirteen sugars—seven monosaccharides, two disaccharides, one trisaccharide and three polysaccharides—in the wavelength range 200—1700 cm -1 and (i) varied the phosphate buffered solution from pH 6.0 to 8.5 at constant ionic strength of 0.1 and (ii) varied HCl solutions from pH 0.8 to 5.0. As is to be expected with molecules containing COH groupings, all the molecular spectra are distinct. Of the thirteen sugars examined, only D-fructose 1,6-diphosphate (FDP) demonstrated spectral changes for the pH range 6.0—8.5 in phosphate buffer; but all exhibited band intensity enhancement in HCl at the lower pHs, but not band wavenumber changes. The results indicate that: (i) changes in the pH of the major intracellular buffer, phosphate, toward acidity, are able to hydrolyze the 1-phosphate group of FDP and the relative concentration of fructose 1-phosphate to fructose 6-phosphate is indicated by the intensity ratio of the 982 and 1080 cm -1 bands; (ii) it appears that all phosphate groups of FDP are hydrolyzed at pH 0.8 in HCl; and (iii) although conditions of extreme acidity are able to hydrolyze other sugars examined, there is no major degradation.

Formation of a Soluble Amylopectin-Like Polysaccharide in Potato Tubers 1

PubMed Central

Frydman, Rosalia B.; Cardini, Carlos E.

1967-01-01

When potato sprouts or potato tuber slices were incubated with 0.1 m glucose 1-phosphate, a soluble amylopectin-like polysaccharide was excreted to the medium. This polysaccharide was found to be a very good primer for phosphorylase and a poor one for starch synthetase. Beside the formation of this extracellular polysaccharide, a more branched intracellular polysaccharide could be isolated. This polysaccharide was an excellent primer for starch synthetase. Fructose 6-phosphate, glucose 6-phosphate, fructose 1,6-diphosphate, glucose or sucrose could not substitute for glucose 1-phosphate. 2,4-Dinitrophenol or nitrogen did not affect the excretion of the polysaccharide. Some properties of these 2 polysaccharides are described. PMID:16656546

Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice.

PubMed

Messner, Christoph B; Driscoll, Paul C; Piedrafita, Gabriel; De Volder, Michael F L; Ralser, Markus

2017-07-11

The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

Fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase during liver regeneration.

PubMed Central

Rosa, J L; Ventura, F; Carreras, J; Bartrons, R

1990-01-01

Glycogen and fructose 2,6-bisphosphate levels in rat liver decreased quickly after partial hepatectomy. After 7 days the glycogen level was normalized and fructose 2,6-bisphosphate concentration still remained low. The 'active' (non-phosphorylated) form of 6-phosphofructo-2-kinase varied in parallel with fructose 2,6-bisphosphate levels, whereas the 'total' activity of the enzyme decreased only after 24 h, similarly to glucokinase. The response of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from hepatectomized rats (96 h) to sn-glycerol 3-phosphate and to cyclic AMP-dependent protein kinase was different from that of the enzyme from control animals and similar to that of the foetal isoenzyme. PMID:2173548

Fructose metabolism in the cerebellum.

PubMed

Funari, Vincent A; Crandall, James E; Tolan, Dean R

2007-01-01

Under normal physiological conditions, the brain utilizes only a small number of carbon sources for energy. Recently, there is growing molecular and biochemical evidence that other carbon sources, including fructose, may play a role in neuro-energetics. Fructose is the number one commercial sweetener in Western civilization with large amounts of fructose being toxic, yet fructose metabolism remains relatively poorly characterized. Fructose is purportedly metabolized via either of two pathways, the fructose-1-phosphate pathway and/or the fructose-6-phosphate pathway. Many early metabolic studies could not clearly discriminate which of these two pathways predominates, nor could they distinguish which cell types in various tissues are capable of fructose metabolism. In addition, the lack of good physiological models, the diet-induced changes in gene expression in many tissues, the involvement of multiple genes in multiple pathways involved in fructose metabolism, and the lack of characterization of some genes involved in fructose metabolism have complicated our understanding of the physiological role of fructose in neuro-energetics. A recent neuro-metabolism study of the cerebellum demonstrated fructose metabolism and co-expression of the genes specific for the fructose 1-phosphate pathway, GLUT5 (glut5) and ketohexokinase (khk), in Purkinje cells suggesting this as an active pathway in specific neurons? Meanwhile, concern over the rapid increase in dietary fructose, particularly among children, has increased awareness about how fructose is metabolized in vivo and what effects a high fructose diet might have. In this regard, establishment of cellular and molecular studies and physiological characterization of the important and/or deleterious roles fructose plays in the brain is critical. This review will discuss the status of fructose metabolism in the brain with special reference to the cerebellum and the physiological roles of the different pathways.

Determination of fructose metabolic pathways in normal and fructose-intolerant children: A sup 13 C NMR study using (U- sup 13 C)fructose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gopher, A.; Lapidot, A.; Vaisman, N.

1990-07-01

An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-(U-{sup 13}C)fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of {sup 13}C NMR spectra of plasma glucose. Significantly lower values ({approx}3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitativemore » determination of the metabolic pathways of fructose conversion to glucose was derived from {sup 13}C NMR measurement of plasma ({sup 13}C)glucose isotopomer populations. The finding of isotopomer populations of three adjacent {sup 13}C atoms at glucose C-4 ({sup 13}C{sub 3}-{sup 13}C{sub 4}-{sup 13}C{sub 5}) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only {approx}50% of the total amount of hepatic fructose conversion to glucose. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of ({sup 13}C)glucose formation from a trace amount of (U-{sup 13}C)fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism.« less

Ion-exchange chromatography separates activities synthesizing and degrading fructose 2,6-bisphosphate from C3 and C4 leaves but not from rat liver

NASA Technical Reports Server (NTRS)

Macdonald, F. D.; Chou, Q.; Buchanan, B. B.

1987-01-01

Fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were separated on the basis of charge from leaves of C3 (spinach, lettuce, and pea) and C4 (sorghum and amaranthus) plants but not from rat liver--a tissue known to contain a bifunctional enzyme with both activities. [2-32P]Fructose 2,6-bisphosphate binding experiments also suggest that the major forms of these activities reside on different proteins in leaves.

Synthesis of glycogen from fructose in the presence of elevated levels of glycogen phosphorylase a in rat hepatocytes.

PubMed

Ciudad, C J; Massagué, J; Salavert, A; Guinovart, J J

1980-03-20

Incubation of hepatocytes with glucose promoted the increase in the glycogen synthase (-glucose 6-phosphate/+glucose 6-phosphate) activity ratio, the decrease in the levels of phosphorylase a and a marked increase in the intracellular glycogen level. Incubation with fructose alone promoted the simultaneous activation of glycogen synthase and increase in the levels of phosphorylase a. Strikingly, glycogen deposition occurred in spite of the elevated levels of phosphorylase a. When glucose and fructose were added to the media the activation of glycogen synthase was always higher than when the hexoses were added separately. On the other hand the effects on glycogen phosphorylase were a function of the relative concentrations of both sugars. Inactivation of glycogen phosphorylase occurred when the fructose to glucose ratio was low while activation took place when the ratio was high. The simultaneous presence of glucose and fructose resulted, in all cases, in an enhancement in the deposition of glycogen. The effects described were not limited to fructose as D-glyceraldehyde, dihydroxyacetone, L-sorbose, D-tagatose and sorbitol, compounds metabolically related to fructose, provoked the same behaviour.

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

PubMed

Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

2016-04-01

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

Differences in the roles of a glutamine amidotransferase subunit of pyridoxal 5'-phosphate synthase between Bacillus circulans and Bacillus subtilis.

PubMed

Itagaki, Shiori; Haga, Minami; Oikawa, Yuji; Sakoda, Ayaka; Ohke, Yoshie; Sawada, Hiroshi; Eguchi, Tadashi; Tamegai, Hideyuki

2013-01-01

BtrC2 of the butirosin producer Bacillus circulans is a non-catalytic subunit of 2-deoxy-scyllo-inosose (DOI) synthase that is involved in butirosin biosynthesis, and also a homolog of glutamine amidotransferase subunit (PdxT) of pyridoxal 5'-phosphate (PLP) synthase of Bacillus subtilis. BtrC2 has been found to have functions in B. circulans both in primary and secondary metabolism. In this study, we investigated the properties of PdxT of B. subtilis in order to determine whether the property of enzyme stabilization is universal among PdxT homologs. Complementation with PdxT in the btrC2 disruptant of B. circulans restored the growth and short-term production of antibiotics, but long-term production of antibiotics cannot be restored. Additionally, PdxT did not bind physically with or stabilize BtrC. Our results indicate that the function of BtrC2 in secondary metabolism is specific properties, not universal among PdxT homologs.

Pyrophosphate-Dependent Fructose-6-Phosphate 1-Phosphotransferase Induction and Attenuation of Hsp Gene Expression during Endosperm Modification in Quality Protein Maize1[C][W][OA

PubMed Central

Guo, Xiaomei; Ronhovde, Kyla; Yuan, Lingling; Yao, Bo; Soundararajan, Madhavan P.; Elthon, Thomas; Zhang, Chi; Holding, David R.

2012-01-01

Quality Protein Maize (QPM) is a hard-endosperm version of the high-lysine opaque2 (o2) maize (Zea mays) mutant, but the genes involved in modification of the soft o2 endosperm are largely unknown. Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the ATP-independent conversion of fructose-6-phosphate to fructose-1,6-bisphosphate in glycolysis. We found a large increase in transcript and protein levels of the α-regulatory subunit of PFP (PFPα) in QPM endosperm. In vitro enzyme assays showed a significant increase in forward PFP activity in developing endosperm extracts of QPM relative to the wild type and o2. An expressed retrogene version of PFPα of unknown function that was not up-regulated in QPM was also identified. The elevated expression levels of a number of ATP-requiring heat shock proteins (Hsps) in o2 endosperm are ameliorated in QPM. PFPα is also coinduced with Hsps in maize roots in response to heat, cold, and the unfolded protein response stresses. We propose that reduced ATP availability resulting from the generalized Hsp response in addition to the reduction of pyruvate, orthophosphate dikinase activity in o2 endosperm is compensated in part by increased PFP activity in QPM. PMID:22158678

Chloroplast Phosphofructokinase

PubMed Central

Kelly, Grahame J.; Latzko, Erwin

1977-01-01

Chloroplast phosphofructokinase from spinach (Spinacia oleracea L.) was purified approximately 40-fold by a combination of fractionations with ammonium sulfate and acetone followed by chromatography on DEAE-Sephadex A-50. Positive cooperative kinetics was observed for the interaction between the enzyme and the substrate fructose 6-phosphate. The optimum pH shifted from 7.7 toward 7.0 as the fructose 6-phosphate concentration was taken below 0.5 mm. The second substrate was MgATP2− (Michaelis constant 30 μm). Free ATP inhibited the enzyme. Chloroplast phosphofructokinase was sensitive to inhibition by low concentration of phosphoenolpyruvate and glycolate 2-phosphate (especially at higher pH); these compounds inhibited in a positively cooperative fashion. Inhibitions by glycerate 2-phosphate (and probably glycerate 3-phosphate), citrate, and inorganic phosphate were also recorded; however, inorganic phosphate effectively relieved the inhibitions by phosphoenolpyruvate and glycolate 2-phosphate. These regulatory properties are considered to complement those of ADP-glucose pyrophosphorylase and fructosebisphosphatase in the regulation of chloroplast starch metabolism. PMID:16660079

Regulations and roles for alternative pathways of hexose metabolism in plants

Treesearch

Clanton C. Black; Laszlo Mustardy; S.S. Sung; P.P. Kormanik; D.-P. Xu; Nachman Paz

1987-01-01

Plants have two cytoplasmic pathways of glycolysis and gluconeogenesis for the reversible interconversion of fructose 6-phosphate (F-6-P) and fructose 1,6-bisphosphate (F-1,6,-P2). One pathway is described as a maintenance pathway that is catalyzed by a nucleotide triphosphate-dependent phosphofructokinase (EC 2.7.1.11; ATP-PFK) glycolytically and a F-1,6...

Crystal Structure of Heart 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase (PFKFB2) and the Inhibitory Influence of Citrate on Substrate Binding

PubMed Central

Crochet, Robert B.; Kim, Jeong-Do; Lee, Herie; Yim, Young-Sun; Kim, Song-Gun; Neau, David; Lee, Yong-Hwan

2016-01-01

The heart-specific isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8Å, respectively. Citrate, a TCA cycle intermediate and well-known inhibitor of PFKFB2, co-crystallized in the 2-kinase domains of both orthologues, occupying the fructose-6-phosphate binding-site and extending into the γ-phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ-phosphate site by citrate proved highly consequential to the binding of co-complexed ATP analogues. The bovine structure, which co-crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co-complexed with AMPPNP, which, unlike ADP, contains a γ-phosphate. The presence of this γ-phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding-pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate’s inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate’s negative feed-back loop of the glycolytic pathway via PFKFB2. PMID:27802586

Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

PubMed Central

Glenn, Katie

2015-01-01

ABSTRACT Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and Pi) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13:657–663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCE Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains. PMID:25605308

Allosteric regulation of Lactobacillus plantarum xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp).

PubMed

Glenn, Katie; Smith, Kerry S

2015-04-01

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and Pi) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657-663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Proteomic and biochemical basis for enhanced growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium.

PubMed

Kumar, Arvind; Rai, Lal Chand

2015-01-01

Proteomics and biochemical analyses were used to unravel the basis for higher growth yield of Enterobacter sp. LCR1 on insoluble phosphate medium compared to soluble. Proteomic analysis using 2-DE, MALDI-TOF/MS and LC-MS revealed the involvement of nine proteins. Down-regulation of fructose bisphosphate aldolase with decreased concentrations of glucose-6-phosphate and fructose-6-phosphate indicated diminished glycolysis. However, up-regulation of phosphoglycerate mutase, increase in the activities of 6-phosphogluconate dehydratase, 2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrogenase suggested induction of Entner-Doudoroff and pentose phosphate pathways. These pathways generate sufficient energy from gluconic acid, which is also used for biosynthesis as indicated by up-regulation of elongation factor Tu, elongation factor G and protein disulfide isomerase. Increased reactive oxygen species (ROS) formation resulting from organic acid oxidation leads to overexpressed manganese superoxide dismutase and increased activities of catalase and ascorbate peroxidase. Thus the organism uses gluconate instead of glucose for energy, while alleviating extra ROS formation by oxidative defense enzymes. Copyright © 2014 Elsevier GmbH. All rights reserved.

Structure of fructose bisphosphate aldolase from Bartonella henselae bound to fructose 1,6-bisphosphate.

PubMed

Gardberg, Anna; Abendroth, Jan; Bhandari, Janhavi; Sankaran, Banumathi; Staker, Bart

2011-09-01

Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=72.39, b=127.71, c=157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site.

ChREBP regulates fructose-induced glucose production independently of insulin signaling

PubMed Central

Kim, Mi-Sung; Krawczyk, Sarah A.; Doridot, Ludivine; Fowler, Alan J.; Wang, Jennifer X.; Trauger, Sunia A.; Noh, Hye-Lim; Kang, Hee Joon; Meissen, John K.; Blatnik, Matthew; Kim, Jason K.; Lai, Michelle; Herman, Mark A.

2016-01-01

Obese, insulin-resistant states are characterized by a paradoxical pathogenic condition in which the liver appears to be selectively insulin resistant. Specifically, insulin fails to suppress glucose production, yet successfully stimulates de novo lipogenesis. The mechanisms underlying this dysregulation remain controversial. Here, we hypothesized that carbohydrate-responsive element-binding protein (ChREBP), a transcriptional activator of glycolytic and lipogenic genes, plays a central role in this paradox. Administration of fructose increased hepatic hexose-phosphate levels, activated ChREBP, and caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis in mice. Activation of ChREBP was required for the increased expression of glycolytic and lipogenic genes as well as glucose-6-phosphatase (G6pc) that was associated with the effects of fructose administration. We found that fructose-induced G6PC activity is a major determinant of hepatic glucose production and reduces hepatic glucose-6-phosphate levels to complete a homeostatic loop. Moreover, fructose activated ChREBP and induced G6pc in the absence of Foxo1a, indicating that carbohydrate-induced activation of ChREBP and G6PC dominates over the suppressive effects of insulin to enhance glucose production. This ChREBP/G6PC signaling axis is conserved in humans. Together, these findings support a carbohydrate-mediated, ChREBP-driven mechanism that contributes to hepatic insulin resistance. PMID:27669460

Breaking the Dogma of Aldolase Specificity: Simple Aliphatic Ketones and Aldehydes are Nucleophiles for Fructose-6-phosphate Aldolase.

PubMed

Roldán, Raquel; Sanchez-Moreno, Israel; Scheidt, Thomas; Hélaine, Virgil; Lemaire, Marielle; Parella, Teodor; Clapés, Pere; Fessner, Wolf-Dieter; Guérard-Hélaine, Christine

2017-04-11

d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis and Physicochemical Characterization of D-Tagatose-1-phosphate: The Substrate of the Tagatose-1-Phosphate Kinase TagK in the PTS-mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis

PubMed Central

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I.; Thompson, John; Joris, Bernard; Battistel, Marcos D.

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multi-component PEP-dependent:tag-PTS present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by 31P and 1H NMR spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-Tagatose catabolic Pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TFHis6) of Escherichia coli. The active fusion enzyme was named TagK-TFHis6. Tag-1P and D-fructose-1-phosphate (Fru-1P) are substrates for the TagK-TFHis6 enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate (Tag-6P) and D-fructose-6-phosphate (Fru-6P) are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific enzyme II (EIITag) in E.coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and EI, to restore the phosphate transfer is demonstrated. PMID:26159072

Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

PubMed

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I; Thompson, John; Joris, Bernard; Battistel, Marcos D

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated. © 2015 S. Karger AG, Basel.

High-Level Production of the Low-Calorie Sugar Sorbitol by Lactobacillus plantarum through Metabolic Engineering▿

PubMed Central

Ladero, Victor; Ramos, Ana; Wiersma, Anne; Goffin, Philippe; Schanck, André; Kleerebezem, Michiel; Hugenholtz, Jeroen; Smid, Eddy J.; Hols, Pascal

2007-01-01

Sorbitol is a low-calorie sugar alcohol that is largely used as an ingredient in the food industry, based on its sweetness and its high solubility. Here, we investigated the capacity of Lactobacillus plantarum, a lactic acid bacterium found in many fermented food products and in the gastrointestinal tract of mammals, to produce sorbitol from fructose-6-phosphate by reverting the sorbitol catabolic pathway in a mutant strain deficient for both l- and d-lactate dehydrogenase activities. The two sorbitol-6-phosphate dehydrogenase (Stl6PDH) genes (srlD1 and srlD2) identified in the genome sequence were constitutively expressed at a high level in this mutant strain. Both Stl6PDH enzymes were shown to be active, and high specific activity could be detected in the overexpressing strains. Using resting cells under pH control with glucose as a substrate, both Stl6PDHs were capable of rerouting the glycolytic flux from fructose-6-phosphate toward sorbitol production with a remarkably high efficiency (61 to 65% glucose conversion), which is close to the maximal theoretical value of 67%. Mannitol production was also detected, albeit at a lower level than the control strain (9 to 13% glucose conversion), indicating competition for fructose-6-phosphate rerouting by natively expressed mannitol-1-phosphate dehydrogenase. By analogy, low levels of this enzyme were detected in both the wild-type and the lactate dehydrogenase-deficient strain backgrounds. After optimization, 25% of sugar conversion into sorbitol was achieved with cells grown under pH control. The role of intracellular NADH pools in the determination of the maximal sorbitol production is discussed. PMID:17261519

Genetics Home Reference: hereditary fructose intolerance

MedlinePlus

... is responsible for the second step in the metabolism of fructose, which breaks down the molecule fructose-1-phosphate into other molecules called glyceraldehyde and dihydroxyacetone phosphate. ALDOB gene mutations reduce the function of the enzyme, impairing its ability ...

Structure of fructose bisphosphate aldolase from Encephalitozoon cuniculi

PubMed Central

Gardberg, Anna; Sankaran, Banumathi; Davies, Doug; Bhandari, Janhavi; Staker, Bart; Stewart, Lance

2011-01-01

Fructose bisphosphate aldolose (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxy­acetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources. Bioinformatic analysis of the genome of the eukaryotic microsporidian parasite Encephalitozoon cuniculi revealed an FBPA homolog. The structures of this enzyme in the presence of the native substrate FBP and also with the partial substrate analog phosphate are reported. The purified enzyme crystallized in 90 mM Bis-Tris propane pH 6.5, 18% PEG 3350, 18 mM NaKHPO4, 10 mM urea for the phosphate-bound form and 100 mM Bis-Tris propane pH 6.5, 20% PEG 3350, 20 mM fructose 1,6-­bisphosphate for the FBP-bound form. In both cases protein was present at 25 mg ml−1 and the sitting-drop vapour-diffusion method was used. For the FBP-bound form, a data set to 2.37 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group C2221, with unit-cell parameters a = 121.46, b = 135.82, c = 61.54 Å. The structure was refined to a final free R factor of 20.8%. For the phosphate-bound form, a data set was collected to 2.00 Å resolution. The space group was also C2221 and the unit-cell parameters were a = 121.96, b = 137.61, c = 62.23 Å. The structure shares the typical barrel tertiary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. The quaternary structure is dimeric. This work provides a direct experimental result for the substrate-binding conformation of the product state of E. cuniculi FBPA. PMID:21904050

Structure of fructose bisphosphate aldolase from Encephalitozoon cuniculi.

PubMed

Gardberg, Anna; Sankaran, Banumathi; Davies, Doug; Bhandari, Janhavi; Staker, Bart; Stewart, Lance

2011-09-01

Fructose bisphosphate aldolose (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources. Bioinformatic analysis of the genome of the eukaryotic microsporidian parasite Encephalitozoon cuniculi revealed an FBPA homolog. The structures of this enzyme in the presence of the native substrate FBP and also with the partial substrate analog phosphate are reported. The purified enzyme crystallized in 90 mM Bis-Tris propane pH 6.5, 18% PEG 3350, 18 mM NaKHPO(4), 10 mM urea for the phosphate-bound form and 100 mM Bis-Tris propane pH 6.5, 20% PEG 3350, 20 mM fructose 1,6-bisphosphate for the FBP-bound form. In both cases protein was present at 25 mg ml(-1) and the sitting-drop vapour-diffusion method was used. For the FBP-bound form, a data set to 2.37 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group C222(1), with unit-cell parameters a=121.46, b=135.82, c=61.54 Å. The structure was refined to a final free R factor of 20.8%. For the phosphate-bound form, a data set was collected to 2.00 Å resolution. The space group was also C222(1) and the unit-cell parameters were a=121.96, b=137.61, c=62.23 Å. The structure shares the typical barrel tertiary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. The quaternary structure is dimeric. This work provides a direct experimental result for the substrate-binding conformation of the product state of E. cuniculi FBPA.

Structure of fructose bisphosphate aldolase from Bartonella henselae bound to fructose 1,6-bisphosphate

PubMed Central

Gardberg, Anna; Abendroth, Jan; Bhandari, Janhavi; Sankaran, Banumathi; Staker, Bart

2011-01-01

Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxy­acetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 72.39, b = 127.71, c = 157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site. PMID:21904049

Investigation on the mechanism by which fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes.

PubMed Central

Niculescu, L; Veiga-da-Cunha, M; Van Schaftingen, E

1997-01-01

In isolated hepatocytes in suspension, the effect of sorbitol but not that of fructose to increase the concentration of fructose 1-phosphate and to stimulate glucokinase was abolished by 2-hydroxymethyl-4-(4-N,N-dimethylamino-1-piperazino)-pyrimidine (SDI 158), an inhibitor of sorbitol dehydrogenase. In hepatocytes in primary culture, fructose was metabolized at approximately one-quarter of the rate of sorbitol, and was therefore much less potent than the polyol in increasing the concentration of fructose 1-phosphate and the translocation of glucokinase. In cultures, sorbitol, commercial mannitol, fructose, D-glyceraldehyde or high concentrations of glucose caused fructose 1-phosphate formation and glucokinase translocation in parallel. Commercial mannitol was contaminated by approx. 1% sorbitol, which accounted for its effects. The effects of sorbitol, fructose and elevated concentrations of glucose were partly inhibited by ethanol, glycerol and glucosamine. Mannoheptulose increased translocation without affecting fructose 1-phosphate concentration. Kinetic studies performed with recombinant human beta-cell glucokinase indicated that this sugar, in contrast with N-acetylglucosamine, binds to glucokinase competitively with the regulatory protein. All these observations indicate that translocation is promoted by agents that favour the dissociation of the glucokinase-regulatory-protein complex either by binding to the regulatory protein (fructose I-phosphate) or to glucokinase (glucose, mannoheptulose). They support the hypothesis that the regulatory protein of glucokinase acts as an anchor for this enzyme that slows down its release from digitonin-permeabilized cells. PMID:9003425

Investigation on the mechanism by which fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes.

PubMed

Niculescu, L; Veiga-da-Cunha, M; Van Schaftingen, E

1997-01-01

In isolated hepatocytes in suspension, the effect of sorbitol but not that of fructose to increase the concentration of fructose 1-phosphate and to stimulate glucokinase was abolished by 2-hydroxymethyl-4-(4-N,N-dimethylamino-1-piperazino)-pyrimidine (SDI 158), an inhibitor of sorbitol dehydrogenase. In hepatocytes in primary culture, fructose was metabolized at approximately one-quarter of the rate of sorbitol, and was therefore much less potent than the polyol in increasing the concentration of fructose 1-phosphate and the translocation of glucokinase. In cultures, sorbitol, commercial mannitol, fructose, D-glyceraldehyde or high concentrations of glucose caused fructose 1-phosphate formation and glucokinase translocation in parallel. Commercial mannitol was contaminated by approx. 1% sorbitol, which accounted for its effects. The effects of sorbitol, fructose and elevated concentrations of glucose were partly inhibited by ethanol, glycerol and glucosamine. Mannoheptulose increased translocation without affecting fructose 1-phosphate concentration. Kinetic studies performed with recombinant human beta-cell glucokinase indicated that this sugar, in contrast with N-acetylglucosamine, binds to glucokinase competitively with the regulatory protein. All these observations indicate that translocation is promoted by agents that favour the dissociation of the glucokinase-regulatory-protein complex either by binding to the regulatory protein (fructose I-phosphate) or to glucokinase (glucose, mannoheptulose). They support the hypothesis that the regulatory protein of glucokinase acts as an anchor for this enzyme that slows down its release from digitonin-permeabilized cells.

Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains.

PubMed

Wang, Yipeng; San, Ka-Yiu; Bennett, George N

2013-08-01

NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.

D-tagatose, a stereoisomer of D-fructose, increases blood uric acid concentration.

PubMed

Buemann, B; Toubro, S; Holst, J J; Rehfeld, J F; Bibby, B M; Astrup, A

2000-08-01

D-Fructose has been found to increase uric acid production by accelerating the degradation of purine nucleotides, probably due to hepatocellular depletion of inorganic phosphate (Pi) by an accumulation of ketohexose-1-phosphate. The hyperuricemic effect of D-tagatose, a stereoisomer of D-fructose, may be greater than that of D-fructose, as the subsequent degradation of D-tagatose-1-phosphate is slower than the degradation of D-fructose-1-phosphate. We tested the effect of 30 g oral D-tagatose versus D-fructose on plasma uric acid and other metabolic parameters in 8 male subjects by a double-blind crossover design. Both the peak concentration and 4-hour area under the curve (AUC) of serum uric acid were significantly higher after D-tagatose compared with either 30 g D-fructose or plain water. The decline in serum Pi concentration was greater at 50 minutes after D-tagatose versus D-fructose. The thermogenic and lactacidemic responses to D-tagatose were blunted compared with D-fructose. D-Tagatose attenuated the glycemic and insulinemic responses to a meal that was consumed 255 minutes after its administration. Moreover, both fructose and D-tagatose increased plasma concentrations of cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1). The metabolic effects of D-tagatose occurred despite its putative poor absorption.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of fructose-1,6-bisphosphate aldolase from Escherichia coli.

PubMed

Zhang, Li; Guo, Zheng; Huang, Jing; Liu, Meiruo; Wang, Yuandong; Ji, Chaoneng

2014-10-01

Fructose-1,6-bisphosphate aldolase is one of the most important enzymes in the glycolytic pathway and catalyzes the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The full-length fbaB gene encoding fructose-1,6-bisphosphate aldolase class I (FBPA I) was cloned from Escherichia coli strain BL21. FBPA I was overexpressed in E. coli and purified. Biochemical analysis found that the optimum reaction temperature of FBPA I is 330.5 K and that the enzyme has a high temperature tolerance. Crystals of recombinant FBPA I were obtained by the sitting-drop vapour-diffusion technique in a condition consisting of 19 mg ml(-1) FBPA I in 0.1 M Tris pH 9.0, 10%(w/v) polyethylene glycol 8000 and diffracted to 2.0 Å resolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 217.7, b = 114.9, c = 183.9 Å, β = 124.6°. The asymmetric unit of these crystals may contain ten molecules, giving a Matthews coefficient of 2.48 Å(3) Da(-1) and a solvent content of 50.5%.

Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.

PubMed

Zgiby, S M; Thomson, G J; Qamar, S; Berry, A

2000-03-01

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.

Production of 2-deoxyribose 5-phosphate from fructose to demonstrate a potential of artificial bio-synthetic pathway using thermophilic enzymes.

PubMed

Honda, Kohsuke; Maya, Shohei; Omasa, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Ohtake, Hisao

2010-08-02

Six thermophilic enzymes from Thermus thermophilus were used to construct an 'artificial bio-synthetic pathway' for the production of 2-deoxyribose 5-phosphate from fructose. By a simple operation using six recombinant Escherichia coli strains producing the thermophilic enzymes, respectively, fructose was converted to 2-deoxyribose 5-phosphate with a molar yield of 55%. Copyright 2010 Elsevier B.V. All rights reserved.

Regulatory insights into the production of UDP-N-acetylglucosamine by Lactobacillus casei

PubMed Central

Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio; Yebra, María J.

2012-01-01

UDP-N-acetylglucosamine (UDP-GlcNAc) is an important sugar nucleotide used as a precursor of cell wall components in bacteria, and as a substrate in the synthesis of oligosaccharides in eukaryotes. In bacteria UDP-GlcNAc is synthesized from the glycolytic intermediate D-fructose-6-phosphate (fructose-6P) by four successive reactions catalyzed by three enzymes: glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM) and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). We have previously reported a metabolic engineering strategy in Lactobacillus casei directed to increase the intracellular levels of UDP-GlcNAc by homologous overexpression of the genes glmS, glmM and glmU. One of the most remarkable features regarding the production of UDP-GlcNAc in L. casei was to find multiple regulation points on its biosynthetic pathway: (1) regulation by the NagB enzyme, (2) glmS RNA specific degradation through the possible participation of a glmS riboswitch mechanism, (3) regulation of the GlmU activity probably by end product inhibition and (4) transcription of glmU. PMID:22825354

Isolation and properties of a Bacillus subtilis mutant unable to produce fructose-bisphosphatase.

PubMed Central

Fujita, Y; Freese, E

1981-01-01

A Bacillus subtilis mutation (gene symbol fdpA1), producing a deficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified. An fdpA1-containing mutant did not produce cross-reacting material. It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate. Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B. subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase. In other words, during gluconeogenesis B. subtilis must be able to bypass this reaction. Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis. The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes. Images PMID:6257649

Effects of Cadmium and Mercury on the Upper Part of Skeletal Muscle Glycolysis in Mice

PubMed Central

Ortega, Fernando; Westerhoff, Hans V.; Gelpí, Josep Lluis; Centelles, Josep J.; Cascante, Marta

2014-01-01

The effects of pre-incubation with mercury (Hg2+) and cadmium (Cd2+) on the activities of individual glycolytic enzymes, on the flux and on internal metabolite concentrations of the upper part of glycolysis were investigated in mouse muscle extracts. In the range of metal concentrations analysed we found that only hexokinase and phosphofructokinase, the enzymes that shared the control of the flux, were inhibited by Hg2+ and Cd2+. The concentrations of the internal metabolites glucose-6-phosphate and fructose-6-phosphate did not change significantly when Hg2+ and Cd2+ were added. A mathematical model was constructed to explore the mechanisms of inhibition of Hg2+ and Cd2+ on hexokinase and phosphofructokinase. Equations derived from detailed mechanistic models for each inhibition were fitted to the experimental data. In a concentration-dependent manner these equations describe the observed inhibition of enzyme activity. Under the conditions analysed, the integral model showed that the simultaneous inhibition of hexokinase and phosphofructokinase explains the observation that the concentrations of glucose-6-phosphate and fructose-6-phosphate did not change as the heavy metals decreased the glycolytic flux. PMID:24489641

Expedient synthesis of C-aryl carbohydrates by consecutive biocatalytic benzoin and aldol reactions.

PubMed

Hernández, Karel; Parella, Teodor; Joglar, Jesús; Bujons, Jordi; Pohl, Martina; Clapés, Pere

2015-02-16

The introduction of aromatic residues connected by a C-C bond into the non-reducing end of carbohydrates is highly significant for the development of innovative structures with improved binding affinity and selectivity (e.g., C-aril-sLex). In this work, an expedient asymmetric "de novo" synthetic route to new aryl carbohydrate derivatives based on two sequential stereoselectively biocatalytic carboligation reactions is presented. First, the benzoin reaction of aromatic aldehydes to dimethoxyacetaldehyde is conducted, catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovar I. Then, the α-hydroxyketones formed are reduced by using NaBH4 yielding the anti diol. After acetal hydrolysis, the aldol addition of dihydroxyacetone, hydroxyacetone, or glycolaldehyde catalyzed by the stereocomplementary D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate aldolase is performed. Both aldolases accept unphosphorylated donor substrates, avoiding the need of handling the phosphate group that the dihydroxyacetone phosphate-dependent aldolases require. In this way, 6-C-aryl-L-sorbose, 6-C-aryl-L-fructose, 6-C-aryl-L-tagatose, and 5-C-aryl-L-xylose derivatives are prepared by using this methodology. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The first case of a complete deficiency of diphosphoglycerate mutase in human erythrocytes.

PubMed

Rosa, R; Prehu, M O; Beuzard, Y; Rosa, J

1978-11-01

An inherited and complete deficiency of diphosphoglycerate mutase was discovered in the erythrocytes of a 42-yr-old man of French origin whose blood hemoglobin concentration was 19.0 g/dl. Upon physical examination he was normal with the exception of a ruddy cyanosis. The morphology of his erythrocytes was also normal and there was no evidence of hemolysis. The erythrocyte 2,3-diphosphoglycerate level was below 3% of normal values and, as a consequence, the affinity of the cells for oxygen was increased. Diphosphoglycerate mutase activity was undetectable in erythrocytes as was that of diphosphoglycerate phosphatase. The activities of all the other erythrocyte enzymes that were tested were normal except for nomophosphoglycerate mutase which was diminished to 50% of the normal value. The levels of reduced glutathione, ATP, fructose 1,6-diphosphate, and of triose phosphates were elevated, whereas those of glucose 6-phosphate and fructose 6-phosphate were decreased. This report sheds new light on the role of diphosphoglycerate mutase in the metabolism of erythrocytes.

The first case of a complete deficiency of diphosphoglycerate mutase in human erythrocytes.

PubMed Central

Rosa, R; Prehu, M O; Beuzard, Y; Rosa, J

1978-01-01

An inherited and complete deficiency of diphosphoglycerate mutase was discovered in the erythrocytes of a 42-yr-old man of French origin whose blood hemoglobin concentration was 19.0 g/dl. Upon physical examination he was normal with the exception of a ruddy cyanosis. The morphology of his erythrocytes was also normal and there was no evidence of hemolysis. The erythrocyte 2,3-diphosphoglycerate level was below 3% of normal values and, as a consequence, the affinity of the cells for oxygen was increased. Diphosphoglycerate mutase activity was undetectable in erythrocytes as was that of diphosphoglycerate phosphatase. The activities of all the other erythrocyte enzymes that were tested were normal except for nomophosphoglycerate mutase which was diminished to 50% of the normal value. The levels of reduced glutathione, ATP, fructose 1,6-diphosphate, and of triose phosphates were elevated, whereas those of glucose 6-phosphate and fructose 6-phosphate were decreased. This report sheds new light on the role of diphosphoglycerate mutase in the metabolism of erythrocytes. Images PMID:152321

Fructose-bisphosphate aldolase a is a potential metastasis-associated marker of lung squamous cell carcinoma and promotes lung cell tumorigenesis and migration.

PubMed

Du, Sha; Guan, Zhuzhu; Hao, Lihong; Song, Yang; Wang, Lan; Gong, Linlin; Liu, Lu; Qi, Xiaoyu; Hou, Zhaoyuan; Shao, Shujuan

2014-01-01

Fructose-bisphosphate aldolase A (ALDOA) is a key enzyme in glycolysis and is responsible for catalyzing the reversible conversion of fructose-1,6-bisphosphate to glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. ALDOA contributes to various cellular functions such as muscle maintenance, regulation of cell shape and mobility, striated muscle contraction, actin filament organization and ATP biosynthetic process. Here, we reported that ALDOA is a highly expressed in lung squamous cell carcinoma (LSCC) and its expression level is correlated with LSCC metastasis, grades, differentiation status and poor prognosis. Depletion of ALDOA expression in the lung squamous carcinoma NCI-H520 cells reduces the capabilities of cell motility and tumorigenesis. These data suggest that ALDOA could be a potential marker for LSCC metastasis and a therapeutic target for drug development.

A new level of regulation in gluconeogenesis: metabolic state modulates the intracellular localization of aldolase B and its interaction with liver fructose-1,6-bisphosphatase.

PubMed

Droppelmann, Cristian A; Sáez, Doris E; Asenjo, Joel L; Yáñez, Alejandro J; García-Rocha, Mar; Concha, Ilona I; Grez, Manuel; Guinovart, Joan J; Slebe, Juan C

2015-12-01

Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein-protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein-protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K(+). We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the co-expression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes. © 2015 Authors; published by Portland Press Limited.

Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060.

PubMed

Rozova, O N; Khmelenina, V N; Trotsenko, Y A

2012-03-01

The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His(6)-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.

Glucose Induces Protein Targeting to Glycogen in Hepatocytes by Fructose 2,6-Bisphosphate-Mediated Recruitment of MondoA to the Promoter

PubMed Central

Petrie, John L.; Al-Oanzi, Ziad H.; Arden, Catherine; Tudhope, Susan J.; Mann, Jelena; Kieswich, Julius; Yaqoob, Muhammad M.; Towle, Howard C.

2013-01-01

In the liver, a high glucose concentration activates transcription of genes encoding glucose 6-phosphatase and enzymes for glycolysis and lipogenesis by elevation in phosphorylated intermediates and recruitment of the transcription factor ChREBP (carbohydrate response element binding protein) and its partner, Mlx, to gene promoters. A proposed function for this mechanism is intracellular phosphate homeostasis. In extrahepatic tissues, MondoA, the paralog of ChREBP, partners with Mlx in transcriptional induction by glucose. We tested for glucose induction of regulatory proteins of the glycogenic pathway in hepatocytes and identified the glycogen-targeting proteins, GL and PTG (protein targeting to glycogen), as being encoded by Mlx-dependent glucose-inducible genes. PTG induction by glucose was MondoA dependent but ChREBP independent and was enhanced by forced elevation of fructose 2,6-bisphosphate and by additional xylitol-derived metabolites. It was counteracted by selective depletion of fructose 2,6-bisphosphate with a bisphosphatase-active kinase-deficient variant of phosphofructokinase 2/fructosebisphosphatase 2, which prevented translocation of MondoA to the nucleus and recruitment to the PTG promoter. We identify a novel role for MondoA in the liver and demonstrate that elevated fructose 2,6-bisphosphate is essential for recruitment of MondoA to the PTG promoter. Phosphometabolite activation of MondoA and ChREBP and their recruitment to target genes is consistent with a mechanism for gene regulation to maintain intracellular phosphate homeostasis. PMID:23207906

Dietary phenolic acids reverse insulin resistance, hyperglycaemia, dyslipidaemia, inflammation and oxidative stress in high-fructose diet-induced metabolic syndrome rats.

PubMed

Ibitoye, Oluwayemisi B; Ajiboye, Taofeek O

2017-12-20

This study investigated the influence of caffeic, ferulic, gallic and protocatechuic acids on high-fructose diet-induced metabolic syndrome in rats. Oral administration of the phenolic acids significantly reversed high-fructose diet-mediated increase in body mass index and blood glucose. Furthermore, phenolic acids restored high-fructose diet-mediated alterations in metabolic hormones (insulin, leptin and adiponectin). Similarly, elevated tumour necrosis factor-α, interleukin-6 and -8 were significantly lowered. Administration of phenolic acids restored High-fructose diet-mediated increase in the levels of lipid parameters and indices of atherosclerosis, cardiac and cardiovascular diseases. High-fructose diet-mediated decrease in activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase) and increase in oxidative stress biomarkers (reduced glutathione, lipid peroxidation products, protein oxidation and fragmented DNA) were significantly restored by the phenolic acids. The result of this study shows protective influence of caffeic acid, ferulic acid, gallic acid and protocatechuic acid in high-fructose diet-induced metabolic syndrome.

Fructose-Bisphosphate Aldolase A Is a Potential Metastasis-Associated Marker of Lung Squamous Cell Carcinoma and Promotes Lung Cell Tumorigenesis and Migration

PubMed Central

Hao, Lihong; Song, Yang; Wang, Lan; Gong, Linlin; Liu, Lu; Qi, Xiaoyu; Hou, Zhaoyuan; Shao, Shujuan

2014-01-01

Fructose-bisphosphate aldolase A (ALDOA) is a key enzyme in glycolysis and is responsible for catalyzing the reversible conversion of fructose-1,6-bisphosphate to glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. ALDOA contributes to various cellular functions such as muscle maintenance, regulation of cell shape and mobility, striated muscle contraction, actin filament organization and ATP biosynthetic process. Here, we reported that ALDOA is a highly expressed in lung squamous cell carcinoma (LSCC) and its expression level is correlated with LSCC metastasis, grades, differentiation status and poor prognosis. Depletion of ALDOA expression in the lung squamous carcinoma NCI-H520 cells reduces the capabilities of cell motility and tumorigenesis. These data suggest that ALDOA could be a potential marker for LSCC metastasis and a therapeutic target for drug development. PMID:24465716

Metabolism of Mannose in Cultured Primary Rat Neurons.

PubMed

Rastedt, Wiebke; Blumrich, Eva-Maria; Dringen, Ralf

2017-08-01

Glucose is the main peripheral substrate for energy production in the brain. However, as other hexoses are present in blood and cerebrospinal fluid, we have investigated whether neurons have the potential to metabolize, in addition to glucose, also the hexoses mannose, fructose or galactose. Incubation of primary cerebellar granule neurons in the absence of glucose caused severe cell toxicity within 24 h, which could not be prevented by application of galactose or fructose, while the cells remained viable during incubation in the presence of either mannose or glucose. In addition, cultured neurons produced substantial and almost identical amounts of lactate after exposure to either glucose or mannose, while lactate production was low in the presence of fructose and hardly detectable during incubations without hexoses or with galactose as carbon source. Determination of the K M values of hexokinase in lysates of cultured neurons for the hexoses revealed values in the micromolar range for mannose (32 ± 2 µM) and glucose (59 ± 10 µM) and in the millimolar range for fructose (4.4 ± 2.3 mM), demonstrating that mannose is efficiently phosphorylated by neuronal hexokinase. Finally, cultured neurons contained reasonable specific activity of the enzyme phosphomannose isomerase, which is required for isomerization of the hexokinase product mannose-6-phosphate into the glycolysis intermediate fructose-6-phosphate. These data demonstrate that cultured cerebellar granule neurons have the potential and express the required enzymes to efficiently metabolize mannose, while galactose and fructose serve at best poorly as extracellular carbon sources for neurons.

Trivalent chromium inhibits TSP-1 expression, proliferation, and O-GlcNAc signaling in vascular smooth muscle cells in response to high glucose in vitro.

PubMed

Ganguly, Rituparna; Sahu, Soumyadip; Chavez, Ronaldo J; Raman, Priya

2015-01-15

Trivalent chromium (Cr(3+)) is a mineral nutrient reported to have beneficial effects in glycemic and cardiovascular health. In vitro and in vivo studies suggest that Cr(3+) supplementation reduces the atherogenic potential and lowers the risk of vascular inflammation in diabetes. However, effects of Cr(3+) in vascular cells under conditions of hyperglycemia, characteristic of diabetes, remain unknown. In the present study we show that a therapeutically relevant concentration of Cr(3+) (100 nM) significantly downregulates a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in human aortic smooth muscle cells (HASMC) stimulated with high glucose in vitro. Promoter-reporter assays reveal that this downregulation of TSP-1 expression by Cr(3+) occurs at the level of transcription. The inhibitory effects of Cr(3+) on TSP-1 were accompanied by significant reductions in O-glycosylation of cytoplasmic and nuclear proteins. Using Western blotting and immunofluorescence studies, we demonstrate that reduced protein O-glycosylation by Cr(3+) is mediated via inhibition of glutamine: fructose 6-phosphate amidotransferase, a rate-limiting enzyme of the hexosamine pathway, and O-linked N-acetylglucosamine (O-GlcNAc) transferase, a distal enzyme in the pathway that controls intracellular protein O-glycosylation. Additionally, we found that Cr(3+) attenuates reactive oxygen species formation in glucose-stimulated HASMC, suggesting an antioxidant effect. Finally, we report an antiproliferative effect of Cr(3+) that is specific for high glucose and conditions triggering elevated protein O-glycosylation. Taken together, these findings provide the first cellular evidence for a novel role of Cr(3+) to modulate aberrant vascular smooth muscle cell function associated with hyperglycemia-induced vascular complications. Copyright © 2015 the American Physiological Society.

Glycogen synthase activation by sugars in isolated hepatocytes.

PubMed

Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

1988-07-01

We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

Elucidation of new condition-dependent roles for fructose-1,6-bisphosphatase linked to cofactor balances

PubMed Central

Kilian, Stephanus G.; du Preez, James C.

2017-01-01

The cofactor balances in metabolism is of paramount importance in the design of a metabolic engineering strategy and understanding the regulation of metabolism in general. ATP, NAD+ and NADP+ balances are central players linking the various fluxes in central metabolism as well as biomass formation. NADP+ is especially important in the metabolic engineering of yeasts for xylose fermentation, since NADPH is required by most yeasts in the initial step of xylose utilisation, including the fast-growing Kluyveromyces marxianus. In this simulation study of yeast metabolism, the complex interplay between these cofactors was investigated; in particular, how they may affect the possible roles of fructose-1,6-bisphosphatase, the pentose phosphate pathway, glycerol production and the pyruvate dehydrogenase bypass. Using flux balance analysis, it was found that the potential role of fructose-1,6-bisphosphatase was highly dependent on the cofactor specificity of the oxidative pentose phosphate pathway and on the carbon source. Additionally, the excessive production of ATP under certain conditions might be involved in some of the phenomena observed, which may have been overlooked to date. Based on these findings, a strategy is proposed for the metabolic engineering of a future xylose-fermenting yeast for biofuel production. PMID:28542187

Mechanisms of aldehyde-induced adenosinetriphosphatase activities of kinases.

PubMed

Rendina, A R; Cleland, W W

1984-10-23

Aldehyde analogues of the normal alcohol substrates induce ATPase activities by glycerokinase (D-glyceraldehyde), fructose-6-phosphate kinase (2,5-anhydromannose 6-phosphate), fructokinase (2,5-anhydromannose or 2,5-anhydrotalose), hexokinase (D-gluco-hexodialdose), choline kinase (betaine aldehyde), and pyruvate kinase (glyoxylate). Since purified deuterated aldehydes give V and V/K isotope effects near 1.0 for glycerokinase, fructokinase with 2,5-anhydro[1-2H]talose, hexokinase, choline kinase, and pyruvate kinase, the hydrates of these almost fully hydrated aldehydes are the activators of the ATPase reactions. Fructose-6-phosphate kinase and fructokinase with 2,5-anhydro[1-2H]mannose show V/K deuterium isotope effects of 1.10 and 1.22, respectively, suggesting either that both hydrate and free aldehyde may be activators (predicted values are 1.37 if only the free aldehyde activates the ATPase) or, more likely, that the phosphorylated hydrate breaks down in a rate-limiting step on the enzyme while MgADP is still present and the back-reaction to yield free hydrate in solution is still possible. 18O was transferred from the aldehyde hydrate to phosphate during the ATPase reactions of glycerokinase, fructose-6-phosphate kinase, fructokinase, and hexokinase but not with choline kinase or pyruvate kinase. Thus, direct phosphorylation of the hydrates by the first four enzymes gives the phosphate adduct of the aldehyde, which decomposes nonenzymatically, while with choline kinase and pyruvate kinase the hydrates induce transfer to water (metal-bound hydroxide or water with pyruvate kinase on the basis of pH profiles). Observation of a lag in the release of phosphate from the glycerokinase ATPase reaction at 15 degrees C supports the existence of a phosphorylated hydrate intermediate with a rate constant for breakdown of 0.035-0.043 s-1 at this temperature. Kinases that phosphorylate creatine, 3-phosphoglycerate, and acetate did not exhibit ATPase activities in the presence of keto or aldehyde analogues (N-methylhydantoic acid, D-glyceraldehyde 3-phosphate, and acetaldehyde, respectively), possibly because of the absence of an acid-base catalytic group in the latter two cases. These analogues were competitive inhibitors vs. the normal substrates, and in the latter case, the hydrate of acetaldehyde was shown to be the inhibitory species on the basis of the deuterium isotope effect on the inhibition constant.

13C nuclear magnetic resonance and gas chromatography-mass spectrometry studies of carbon metabolism in the actinomycin D producer Streptomyces parvulus by use of 13C-labeled precursors.

PubMed Central

Inbar, L; Lapidot, A

1991-01-01

Fructose and glutamate metabolism was monitored in cell suspensions of streptomyces parvulus by 13C nuclear magnetic resonance. The experiments were performed for cells grown with various 13C sources in a growth medium containing D-[U-13C]fructose, L-[13C]glutamate, or L-[U-13C]aspartate and with nonlabeled precursors to compare intracellular pools in S. parvulus cells at different periods of the cell life cycle. The transport of fructose into the cells was biphasic in nature; during rapid transport, mannitol, fructose, and glucose 6-phosphate were accumulated intracellularly, whereas during the passive diffusion of fructose, the intracellular carbohydrate pool comprised mainly trehalose (1,1'-alpha-alpha-D-glucose). The regulation of fructokinase activity by the intracellular intermediates may play an important role in fructose catabolism in S. parvulus. Transaldolase activity in S. parvulus was determined from the 13C nuclear magnetic resonance labeling pattern of trehalose carbons obtained from cells grown in medium containing either L-[U-13C]aspartate or L-[U-13C]glutamate. Only carbons 4, 5, and 6 of the disaccharide were labeled. Isotopomer analysis of the trehalose carbons led us to conclude that the flux through the reverse glycolytic pathway, condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate, makes at best a minor contribution to the 13C-labeled glucose units observed in trehalose. The pentose pathway and transaldolase activity can explain the labeling pattern of 4,5,6-13C3 of trehalose. Moreover, the transfer of the 13C label of L-[U-13C]aspartate into the different isotopomers of trehalose C4, C5, and C6 by the transaldolase activity allowed us to calculate the relative fluxes from oxaloacetate via gluconeogenesis and through the tricarboxylic acid cycle. The ratio of the two fluxes is approximately 1. However, the main carbon source for trehalose synthesis in S. parvulus is fructose and not glutamate or aspartate. The 13C enrichment and isotopomer population, measured by nuclear magnetic resonance and gas chromatography-mass spectrometry, of the actinomycin D peptide ring enabled us to specify the origins of the five amino acids of actinomycin D. Threonine and proline exhibited isotopomer populations similar to that of the extracellular L-[13C]glutamate, indicating that protein catabolism is the origin of their 13C label, whereas the isotopomer populations of sarcosine and N-methylvaline were similar to those of the new intracellular pool of S. parvulus that originated from D-[U-13C]fructose during the production of actinomycin D. PMID:1744035

Dinuclear copper(II) complexes with {Cu2(mu-hydroxo)bis(mu-carboxylato)}+ cores and their reactions with sugar phosphate esters: A substrate binding model of fructose-1,6-bisphosphatase.

PubMed

Kato, Merii; Tanase, Tomoaki; Mikuriya, Masahiro

2006-04-03

Reactions of CuX2.nH2O with the biscarboxylate ligand XDK (H2XDK = m-xylenediamine bis(Kemp's triacid imide)) in the presence of N-donor auxiliary ligands yielded a series of dicopper(II) complexes, [Cu2(mu-OH)(XDK)(L)2]X (L = N,N,N',N'-tetramethylethylenediamine (tetmen), X = NO3 (1a), Cl (1b); L = N,N,N'-trimethylethylenediamine (tmen), X = NO3 (2a), Cl (2b); L =2,2'-bipyridine (bpy), X = NO3 (3); L = 1,10-phenanthroline (phen), X = NO3 (4); L = 4,4'-dimethyl-2,2'-bipyridine (Me2bpy), X = NO3 (5); L = 4-methyl-1,10-phenanthroline (Mephen), X = NO3 (6)). Complexes 1-6 were characterized by X-ray crystallography (Cu...Cu = 3.1624(6)-3.2910(4) A), and the electrochemical and magnetic properties were also examined. Complexes 3 and 4 readily reacted with diphenyl phosphoric acid (HDPP) or bis(4-nitrophenyl) phosphoric acid (HBNPP) to give [Cu2(mu-phosphate)(XDK)(L)2]NO3 (L = bpy, phosphate = DPP (11); L = phen, phosphate = DPP (12), BNPP (13)), where the phsophate diester bridges the two copper ions in a mu-1,3-O,O' bidentate fashion (Cu...Cu = 4.268(3)-4.315(1) A). Complexes 4 and 6 with phen and Mephen have proven to be good precursors to accommodate a series of sugar monophosphate esters (Sugar-P) onto the biscarboxylate-bridged dicopper centers, yielding [Cu2(mu-Sugar-P)(XDK)(L)2] (Sugar-P = alpha-D-Glc-1-P (23a and b), D-Glc-6-P (24a and b), D-Man-6-P (25a), D-Fru-6-P (26a and b); L = phen (a), Mephen (b)) and [Cu2(mu-Gly-n-P)(XDK)(Mephen)2] (Gly-n-P = glycerol n-phosphate; n = 2 (21), 3 (22)), where Glc, Man, and Fru are glucose, mannose, and fructose, respectively. The structure of [Cu2(mu-MNPP)(XDK)(phen)2(CH3OH)] (20) was characterized as a reference compound (H2MNPP = 4-nitrophenyl phosphoric acid). Complexes 4 and 6 also reacted with d-fructose 1,6-bisphosphate (D-Fru-1,6-P2) to afford the tetranuclear copper(II) complexes formulated as [Cu4(mu-D-Fru-1,6-P2)(XDK)2(L)4] (L = phen (27a), Mephen (27b)). The detailed structure of 27a was determined by X-ray crystallography to involve two different tetranuclear complexes with alpha- and beta-anomers of D-Fru-1,6-P2, [Cu4(mu-alpha-D-Fru-1,6-P2)(XDK)2(phen)4] and [Cu4(mu-beta-D-Fru-1,6-P2)(XDK)2(phen)4], in which the D-Fru-1,6-P2 tetravalent anion bridges the two [Cu2(XDK)(phen)2]2+ units through the C1 and C6 phosphate groups in a mu-1,3-O,O' bidentate fashion (Cu...Cu = 4.042(2)-4.100(2) A). Notably, the structure with alpha-D-Fru-1,6-P2 demonstrated the presence of a strong hydrogen bond between the C2 hydroxyl group and the C1 phosphate oxygen atom, which may support the previously proposed catalytic mechanism in the active site of fructose-1,6-bisphosphatase.

Effects of oral D-tagatose, a stereoisomer of D-fructose, on liver metabolism in man as examined by 31P-magnetic resonance spectroscopy.

PubMed

Buemann, B; Gesmar, H; Astrup, A; Quistorff, B

2000-10-01

D-tagatose, which is a stereoisomer of D-fructose, is phosphorylated to D-tagatose-1-phosphate by fructokinase in the liver. Because of a slow degradation rate of D-tagatose-1-phosphate, this substance may accumulate, and ingested D-tagatose may therefore cause a longer lasting reduction in inorganic phosphate (Pi) and adenosine triphosphate (ATP) levels in the liver compared with D-fructose. Similar to what is seen in patients with hereditary fructose intolerance, this may increase purine nucleotide degradation and thereby increase uric acid production. The effect of 30 g D-tagatose or D-fructose administered orally on ketohexose-1-phosphates, ATP, and Pi levels in the liver was studied by 31P-magnetic resonance spectroscopy (PMRS) in 5 young male volunteers. Blood and urine were collected to detect a possible increased uric acid production. A peak at 5.2 ppm assigned as D-tagatose-1-phosphate equivalent to about 1 mmol/L was found in the spectrum within 30 minutes after D-tagatose was administered in all subjects. Concomitantly, ATP was reduced by about 12% (P < .05). Both effects had vanished after 150 minutes. Serum uric acid concentration was increased by 17% 50 minutes after D-tagatose (P < .05) and did not reach baseline level when the experiment was terminated 230 minutes after the load. Although renal fractional extraction of uric acid decreased by approximately 12%, this could not explain the acute hyperuricemic effect of D-tagatose. No changes in 31PMRS spectra or serum uric acid concentration were found after D-fructose. These results suggest that a moderate intake of D-tagatose may affect liver metabolism by phosphate trapping despite the fact that the sugar may only be incompletely absorbed in the gut.

Rational Design, Synthesis and Evaluation of First Generation Inhibitors of the Giardia lamblia Fructose-1,6-biphosphate Aldolase

PubMed Central

Li, Zhimin; Liu, Zhengang; Cho, Dae Won; Zou, Jiwen; Gong, Maozhen; Breece, Robert M.; Galkin, Andrey; Li, Ling; Zhao, Hong; Maestas, Gabriel D.; Tierney, David L.; Herzberg, Osnat; Dunaway-Mariano, Debra; Mariano, Patrick S.

2011-01-01

Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn2+ binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (Ki = 14 μM) that is comparable to that of FBP (Km = 2 μM) or its inert analog TBP (Ki = 1 μM). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3 Å) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn2+. The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn2+ are not consistent with a strong interaction. To determine if Zn2+coordination occurs in the GlFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn2+ coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn2+ coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn2+ coordination to binding of 8 to GlFBPA. PMID:21333622

Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

PubMed

Gordon, G L; Doelle, H W

1976-08-16

The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

Ketose induced respiratory inhibition in isolated hepatocytes.

PubMed

Martínez, P; Carrascosa, J M; Núñez de Castro, I

1987-06-01

The addition of 10 mM fructose or 10 mM tagatose to a suspension of hepatocytes caused respiratory inhibition, whereas no change in oxygen uptake was observed following the addition of glucose. However, incubations in the presence of fructose showed a high, aerobic glycolytic activity. Tagatose is phosphorylated to tagatose 1-phosphate but is not further metabolized by cell free liver extract. Moreover, the addition of fructose to glucagon treated cells also caused the Crabtree-like effect. The concentration of adenine nucleotides and inorganic phosphate (Pi) in the mitochondrial and cytosolic compartments during incubation (time 30 min) was determined by the digitonin fractionation procedure. In the presence of 10 mM fructose or tagatose, the total adenine nucleotide pools decreased by 40%; however, glucose produced no change. The addition of ketoses diminished the asymmetric distribution of extramitochondrial (ATP/ADP)e ratio and intramitochondrial (ATP/ADP)i ratio. At the same time the total mitochondrial Pi fell from 17 mM to 6-7 mM. The mitochondrial membrane potential (-161 mV) in the presence of fructose showed no changes during the 30 min experimental period. An increase in the NADH/NAD+ ratio was observed. These results suggest that in hepatocytes the inhibition of respiration is not necessarily linked with the enhanced aerobic glycolysis, by competition for common substrates.

Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs.

PubMed

Bolado-Martínez, E; Acedo-Félix, E; Peregrino-Uriarte, A B; Yepiz-Plascencia, G

2012-01-01

Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri.

Characterization of a Salmonella sugar kinase essential for the utilization of fructose-asparagine.

PubMed

Biswas, Pradip K; Behrman, Edward J; Gopalan, Venkat

2017-04-01

Salmonella can utilize fructose-asparagine (F-Asn), a naturally occurring Amadori product, as its sole carbon and nitrogen source. Conversion of F-Asn to the common intermediates glucose-6-phosphate, aspartate, and ammonia was predicted to involve the sequential action of an asparaginase, a kinase, and a deglycase. Mutants lacking the deglycase are highly attenuated in mouse models of intestinal inflammation owing to the toxic build-up of the deglycase substrate. The limited distribution of this metabolic pathway in the animal gut microbiome raises the prospects for antibacterial discovery. We report the biochemical characterization of the kinase that was expected to transform fructose-aspartate to 6-phosphofructose-aspartate during F-Asn utilization. In addition to confirming its anticipated function, we determined through studies of fructose-aspartate analogues that this kinase exhibits a substrate-specificity with greater tolerance to changes to the amino acid (including the d-isomer of aspartate) than to the sugar.

Regulation of 2-carboxyarabinitol 1-phosphatase.

PubMed

Holbrook, G P; Galasinski, S C; Salvucci, M E

1991-11-01

The regulation of 2-carboxyarabinitol 1-phosphatase (CA 1-Pase) by phosphorylated effectors was studied with enzyme purified from tobacco (Nicotiana tabacum) leaves. CA 1-Pase activity was most stimulated by fructose 1,6-bisphosphate, exhibiting an A(0.5) value of 1.9 millimolar and a 10-fold enhancement of catalysis. With ribulose-1,5-bisphosphate, the A(0.5) was 0.6 millimolar, and maximal stimulation of activity was 5.3-fold. Among the monophosphates, 3-phosphoglycerate and phosphoglycolate were more potent positive effectors than glyceraldehyde 3-phosphate, glucose 1-phosphate, glucose 6-phosphate, and dihydroxyacetone phosphate. Stimulation of CA 1-Pase by ribulose-1,5-bisphosphate and fructose 1,6-bisphosphate increased V(max) but did not appreciably alter K(m) (2-carboxyarabinitol 1-phosphate) values. Inorganic phosphate appeared to inhibit CA 1-Pase noncompetitively with respect to 2-carboxyarabinitol 1-phosphate, exhibiting a K(i) of 0.3 millimolar. The results suggest that these positive and negative effectors bind to a regulatory site on CA 1-Pase and may have a physiologial role in the light regulation of this enzyme. Related experiments with CA 1-Pase inactivated by dialysis in the absence of dithiothreitol show that partial reactivation can be achieved in the presence of a range of reducing reagents, including dithiothreitol, cysteine, and reduced glutathione. This could imply an ancillary involvement of sulfhydryl reduction during light activation of CA 1-Pase in vivo. The enzyme was thermally stable up to 35 degrees C, in contrast to ribulose-1,5-bisphosphate carboxylase/oxygenase activase which lost activity above 30 degrees C. The activation energy for CA 1-Pase was calculated to be 56.14 kilojoules per mole.

Regulation of 2-Carboxyarabinitol 1-Phosphatase 1

PubMed Central

Holbrook, Gabriel P.; Galasinski, Scott C.; Salvucci, Michael E.

1991-01-01

The regulation of 2-carboxyarabinitol 1-phosphatase (CA 1-Pase) by phosphorylated effectors was studied with enzyme purified from tobacco (Nicotiana tabacum) leaves. CA 1-Pase activity was most stimulated by fructose 1,6-bisphosphate, exhibiting an A0.5 value of 1.9 millimolar and a 10-fold enhancement of catalysis. With ribulose-1,5-bisphosphate, the A0.5 was 0.6 millimolar, and maximal stimulation of activity was 5.3-fold. Among the monophosphates, 3-phosphoglycerate and phosphoglycolate were more potent positive effectors than glyceraldehyde 3-phosphate, glucose 1-phosphate, glucose 6-phosphate, and dihydroxyacetone phosphate. Stimulation of CA 1-Pase by ribulose-1,5-bisphosphate and fructose 1,6-bisphosphate increased Vmax but did not appreciably alter Km (2-carboxyarabinitol 1-phosphate) values. Inorganic phosphate appeared to inhibit CA 1-Pase noncompetitively with respect to 2-carboxyarabinitol 1-phosphate, exhibiting a Ki of 0.3 millimolar. The results suggest that these positive and negative effectors bind to a regulatory site on CA 1-Pase and may have a physiologial role in the light regulation of this enzyme. Related experiments with CA 1-Pase inactivated by dialysis in the absence of dithiothreitol show that partial reactivation can be achieved in the presence of a range of reducing reagents, including dithiothreitol, cysteine, and reduced glutathione. This could imply an ancillary involvement of sulfhydryl reduction during light activation of CA 1-Pase in vivo. The enzyme was thermally stable up to 35°C, in contrast to ribulose-1,5-bisphosphate carboxylase/oxygenase activase which lost activity above 30°C. The activation energy for CA 1-Pase was calculated to be 56.14 kilojoules per mole. PMID:16668528

Proteomic Prediction of Breast Cancer Risk: A Cohort Study

DTIC Science & Technology

2008-03-01

under denaturing conditions and its subsequent concentration on a C4 column (complete removal of guanidium hydrochloride was difficult and adversely... Glucosamine --fructose-6-phosphate aminotransferase [isomerizing] 2 (EC 2.6.1.16) (Hexoseph 216 (Q13415) Origin recognition complex subunit 1 (Replication

Glycolytic intermediates and adenosine phosphates in rat liver at high altitude /3,800 m/.

NASA Technical Reports Server (NTRS)

Cipriano, L. F.; Pace, N.

1973-01-01

Liver tissue obtained from adult rats exposed to 3800 m altitude for intervals ranging from 1.5 hr to 63 days was examined by enzymatic analysis. During the first 3 hr of exposure, an immediate decrease in rephosphorylation of high-energy phosphates led to reduced glycogenesis and eventual pileup of AMP, pyruvate, fructose 1,6-diphosphate, glucose 6-phosphate, and glucose. This was accompanied by a reduction of pentose phosphate pathway activity. After 3 to 6 hr, a secondary adjustment of substrate concentrations occurred along with the apparent facilitation of phosphofructokinase. This secondary adjustment appears to increase anaerobic production of ATP and represents a significant intracellular contribution to the acclimatization process at high altitude.

Hibiscus sabdariffa calyx palliates insulin resistance, hyperglycemia, dyslipidemia and oxidative rout in fructose-induced metabolic syndrome rats.

PubMed

Ajiboye, Taofeek O; Raji, Hikmat O; Adeleye, Abdulwasiu O; Adigun, Nurudeen S; Giwa, Oluwayemisi B; Ojewuyi, Oluwayemisi B; Oladiji, Adenike T

2016-03-30

The effect of Hibiscus sabdariffa calyx extract was evaluated in high-fructose-induced metabolic syndrome rats. Insulin resistance, hyperglycemia, dyslipidemia and oxidative rout were induced in rats using high-fructose diet. High-fructose diet-fed rats were administered 100 and 200 mg kg(-1) body weight of H. sabdariffa extract for 3 weeks, starting from week 7 of high-fructose diet treatment. High-fructose diet significantly (P < 0.05) increased the serum levels of blood glucose, insulin, total cholesterol (TC), triacylglycerol (TAG), low-density lipoprotein cholesterol (LDLc) and very-low-density lipoprotein cholesterol (VLDLc), with a concomitant reduction in high-density lipoprotein cholesterol (HDLc). These alterations were significantly ameliorated by the extract. High-fructose diet-mediated decreases in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-red) and glucose 6-phosphate dehydrogenase (Glc 6-PD) were significantly (P < 0.05) attenuated. Altered levels of reduced glutathione (GSH) and glutathione disulfide (GSSG) were significantly (P < 0.05) restored to normal. High-fructose diet-mediated increases in the concentrations of malondialdehyde, conjugated dienes, lipid hydroperoxides, protein carbonyl and percentage fragmented DNA were significantly (P < 0.05) lowered by the Hibiscus extract. Overall, aqueous extract of H. sabdariffa palliates insulin resistance, hyperglycemia, dyslipidemia and oxidative rout in high-fructose-induced metabolic syndrome rats. © 2015 Society of Chemical Industry.

Carbohydrate metabolism in erythrocytes of copper deficient rats.

PubMed

Brooks, S P J; Cockell, K A; Dawson, B A; Ratnayake, W M N; Lampi, B J; Belonje, B; Black, D B; Plouffe, L J

2003-11-01

Dietary copper deficiency is known to adversely affect the circulatory system of fructose-fed rats. Part of the problem may lie in the effect of copper deficiency on intermediary metabolism. To test this, weanling male Long-Evans rats were fed for 4 or 8 weeks on sucrose-based diets containing low or adequate copper content. Copper deficient rats had significantly lower plasma and tissue copper as well as lower plasma copper, zinc-superoxide dismutase activity. Copper deficient rats also had a significantly higher heart:body weight ratio when compared to pair-fed controls. Direct measurement of glycolysis and pentose phosphate pathway flux in erythrocytes using (13)C NMR showed no differences in carbon flux from glucose or fructose to pyruvate but a significantly higher flux through the lactate dehydrogenase locus in copper deficient rats (approximately 1.3 times, average of glucose and glucose + fructose measurements). Copper-deficient animals had significantly higher erythrocyte concentrations of glucose, fructose, glyceraldehyde 3-phosphate and NAD(+). Liver metabolite levels were also affected by copper deficiency being elevated in glycogen and fructose 1-phosphate content. The results show small changes in carbohydrate metabolism of copper deficient rats.

Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens.

PubMed

Kaplan, J B; Merkel, W K; Nichols, B P

1985-06-05

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.

REGULATION OF METABOLISM, IN VIVO,

DTIC Science & Technology

The concentrations of 35 critical metabolites, including glycogen, trehalose , components of the glycolytic pathway and citric acid cycle, amino acids...sites of regulation: the phosphorylation of fructose-6-phosphate; the cleavage of trehalose ; and the phosphorolysis of glycogen were established in

Interaction of ethacrynic acid with control sites of renal glucose metabolism.

PubMed

Fúlgraff, G; Dingler-Núnemann, H

1975-01-01

Ethacrynic acid stimulates in vitro concentration dependent renal gluconeogenesis from substrates which enter the gluconeogenic pathway at the level of the triosephosphates like glycerol or fructose or from substrates which have to pass the oxaloacetate shuttle like pyruvate or from intermediary products of fatty acid oxydation or citrate cycle. Our results suggest that a site of action of ethacrynic acid in this metabolic aspect is the enzyme system fructose diphosphatase/frutose-6-phosphate kinase and eventually additionally pyruvate carboxylase.

Influence of some mononucleotides and their corresponding nucleosides on the metabolism of carbohydrates in the isolated rat diaphragm muscle

PubMed Central

Beloff-Chain, Anne; Betto, P.; Bleszynski, W.; Catanzaro, Raffaella; Chain, E. B.; Dmitrovskii, A. A.; Longinotti, L.; Pocchiari, F.

1965-01-01

1. The influence of ATP on glucose metabolism was studied in the isolated rat diaphragm; it was shown that ATP increases the oxidation of glucose and the aerobic conversion of glucose into lactate, whereas it decreases glycogen synthesis. There was no influence of ATP on the anaerobic formation of lactate from glucose. 2. A maximum effect of ATP on the oxidation of glucose (about 160% increase) was obtained in the presence of 10mm-ATP; in the presence of 2mm-ATP the effect was about 65%, and was approximately constant from 10 to 90min. incubation period. 3. In a phosphate-free tris-buffered medium the oxidation of glucose was considerably decreased, but the percentage stimulation by ATP was about the same as in a phosphate-buffered medium. 4. ATP was shown to increase the oxidation of fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 1,6-diphosphate and, to a much smaller extent, pyruvate. 5. ADP stimulated the oxidation of glucose to the same extent as ATP at a concentration of 2mm and the effect with AMP was only slightly less; IMP and adenosine had only a small stimulatory effect at this concentration, whereas inosine had no effect. PMID:16749165

Effects of elevated CO sub 2 concentrations on glycolysis in intact Bartlett pear fruit. [Pyrus communis L

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kerbel, E.L.; Kader, A.A.; Romani, R.J.

1988-04-01

Mature intact Bartlett pear fruit (Pyrus communis L.) were stored under a continuous flow of air or air + 10% CO{sub 2} for 4 days at 20{degree}C. Fruit kept under elevated CO{sub 2} concentrations exhibited reduced respiration (O{sub 2} consumption) and ethylene evolution rates, and remained firmer and greener than fruit stored in air. Protein content, fructose 1,6-bisphosphate levels, and ATP:phosphofructokinase and PPi:phosphofructokinase activities declined, while levels of fructose 6-phosphate and fructose 2,6-bisphosphate increased in fruit exposed to air + 10% CO{sub 2}. These results are discussed in light of a possible inhibitory effect of CO{sub 2} at the sitemore » of action of both phosphofructokinases in the glycolytic pathway, which could account, at least in part, for the observed reduction in respiration.« less

Rational Design Synthesis and Evaluation of New Selective Inhibitors of Microbial Class II (Zinc Dependent) Fructose Bis-phosphate Aldolases

DOE Office of Scientific and Technical Information (OSTI.GOV)

R Daher; M Coincon; M Fonvielle

2011-12-31

We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows Ki against class II Fbas from various pathogens in the nM range, with very highmore » selectivity (up to 105). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.« less

Metabolic changes induced during adaptation of Saccharomyces cerevisiae to a water stress.

PubMed

Singh, K K; Norton, R S

1991-01-01

When exponentially growing Saccharomyces cerevisiae was transferred from a normal high water activity growth medium (aw 0.997) to a medium containing 8% NaCl low water activity growth medium (aw 0.955), glycerol accumulation during the first eight hours of the adaptation was both retarded and greatly diminished in magnitude. Investigation of the underlying reasons for the slow onset of glycerol accumulation revealed that not only was overall glycerol production reduced by salt transfer, but also the rates of ethanol production and glucose consumption were reduced. Measurement of glycolytic intermediates revealed an accumulation of glucose-6-phosphate, fructose-6-phosphate, fructose 1,6 bisphosphate and phosphoenolpyruvate in S. cerevisiae 3 to 4 h after transfer to salt, suggesting that one or more glycolytic enzymes were inhibited. Potassium ions accumulated in S. cerevisiae after salt transfer and reached a maximum about 6 h after transfer, whereas the sodium ion content increased progressively during the adaptation period. The trehalose content also increased in adapting cells. It is suggested that inhibition of glycerol production during the initial period of adaptation could be due to either the inhibition of glycerol-3-phosphate dehydrogenase by increased cation content or the inhibition of glycolysis, glycerol being produced glycolytically in S. cerevisiae. The increased accumulation of glycerol towards the end of the 8-h period suggests that the osmoregulatory response of S. cerevisiae involves complex sets of adjustments in which inhibition of glycerol-3-phosphate dehydrogenase must be relieved before glycerol functions as a major osmoregulator.

Two enzymes with redundant fructose bisphosphatase activity sustain gluconeogenesis and virulence in Mycobacterium tuberculosis.

PubMed

Ganapathy, Uday; Marrero, Joeli; Calhoun, Susannah; Eoh, Hyungjin; de Carvalho, Luiz Pedro Sorio; Rhee, Kyu; Ehrt, Sabine

2015-08-10

The human pathogen Mycobacterium tuberculosis (Mtb) likely utilizes host fatty acids as a carbon source during infection. Gluconeogenesis is essential for the conversion of fatty acids into biomass. A rate-limiting step in gluconeogenesis is the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate by a fructose bisphosphatase (FBPase). The Mtb genome contains only one annotated FBPase gene, glpX. Here we show that, unexpectedly, an Mtb mutant lacking GLPX grows on gluconeogenic carbon sources and has detectable FBPase activity. We demonstrate that the Mtb genome encodes an alternative FBPase (GPM2, Rv3214) that can maintain gluconeogenesis in the absence of GLPX. Consequently, deletion of both GLPX and GPM2 is required for disruption of gluconeogenesis and attenuation of Mtb in a mouse model of infection. Our work affirms a role for gluconeogenesis in Mtb virulence and reveals previously unidentified metabolic redundancy at the FBPase-catalysed reaction step of the pathway.

Two enzymes with redundant fructose bisphosphatase activity sustain gluconeogenesis and virulence in Mycobacterium tuberculosis

PubMed Central

Ganapathy, Uday; Marrero, Joeli; Calhoun, Susannah; Eoh, Hyungjin; de Carvalho, Luiz Pedro Sorio; Rhee, Kyu; Ehrt, Sabine

2015-01-01

The human pathogen Mycobacterium tuberculosis (Mtb) likely utilizes host fatty acids as a carbon source during infection. Gluconeogenesis is essential for the conversion of fatty acids into biomass. A rate-limiting step in gluconeogenesis is the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate by a fructose bisphosphatase (FBPase). The Mtb genome contains only one annotated FBPase gene, glpX. Here we show that, unexpectedly, an Mtb mutant lacking GLPX grows on gluconeogenic carbon sources and has detectable FBPase activity. We demonstrate that the Mtb genome encodes an alternative FBPase (GPM2, Rv3214) that can maintain gluconeogenesis in the absence of GLPX. Consequently, deletion of both GLPX and GPM2 is required for disruption of gluconeogenesis and attenuation of Mtb in a mouse model of infection. Our work affirms a role for gluconeogenesis in Mtb virulence and reveals previously unidentified metabolic redundancy at the FBPase-catalysed reaction step of the pathway. PMID:26258286

Long range molecular dynamics study of interactions of the eukaryotic glucosamine-6-phosphate synthase with fructose-6-phosphate and UDP-GlcNAc.

PubMed

Miszkiel, Aleksandra; Wojciechowski, Marek

2017-11-01

Glucosamine-6-phosphate synthase (EC 2.6.1.16) is responsible for catalysis of the first and practically irreversible step in hexosamine metabolism. The final product of this pathway, uridine 5' diphospho N-acetyl-d-glucosamine (UDP-GlcNAc), is an essential substrate for assembly of bacterial and fungal cell walls. Moreover, the enzyme is involved in phenomenon of hexosamine induced insulin resistance in type II diabetes, which makes of it a potential target for anti-fungal, anti-bacterial and anti-diabetic therapy. The crystal structure of isomerase domain from human pathogenic fungus Candida albicans has been solved recently but it doesn't reveal the molecular mechanism details of inhibition taking place under UDP-GlcNAc influence, the unique feature of eukaryotic enzyme. The following study is a continuation of the previous research based on comparative molecular dynamics simulations of the structures with and without the enzyme's physiological inhibitor (UDP-GlcNAc) bound. The models used for this study included fructose-6-phosphate, one of the enzyme's substrates in its binding pocket. The simulation results studies demonstrated differences in mobility of the compared structures. Some amino acid residues were determined, for which flexibility is evidently different between the models. Importantly, it has been confirmed that the most fixed residues are related to the inhibitor binding process and to the catalysis reaction. The obtained results constitute an important step towards understanding of the inhibition that GlcN-6-P synthase is subjected by UDP-GlcNAc molecule. Copyright © 2017 Elsevier Inc. All rights reserved.

The effect on some enzymes of rat tissue of diets low in fat content.

PubMed

Bartley, W; Dean, B; Taylor, C B; Bailey, E

1967-05-01

1. Rats of two strains were kept on three different diets; one was a commercial diet of rat pellets, one contained about 80% of sucrose and 20% of casein and was supplemented with corn oil, and the third was a similar diet without the corn oil. 2. On the commercial diet, the specific activities of pyruvate kinase, glucose 6-phosphate dehydrogenase and fructose 1,6-diphosphatase in the livers of one strain of rats (strain A) were 1.5-3 times those in the other strain (strain B). When the diet high in sucrose and supplemented with corn oil was given, there were large increases in the specific activity of pyruvate kinase, glucose 6-phosphate dehydrogenase and fructose 1,6-diphosphatase in the livers of strain A rats. With strain B rats the increases were much smaller. Omission of corn oil from the diet caused a threefold increase in the specific activity of glucose 6-phosphate dehydrogenase in strain B rats, but had little effect on other enzymes. 3. The enzymes of the kidneys and hearts of strain A rats were also more active than those of strain B rats. In strain A rats, the specific activities of pyruvate kinase and fructose 1,6-diphosphatase in the kidney increased when the sucrose content of the diet was high, but in the kidneys of strain B rats there was little change. 4. In strain A rats, the specific activity of pyruvate kinase in the heart more than doubled with the high-sucrose-corn oil diet and increased threefold when corn oil was omitted. No changes were seen in strain B rats. 5. In strain A rats, omission of corn oil from the diet increased the ability of the kidneys to synthesize glucose from lactate. 6. In strain B rats, addition of corn oil to the diet resulted in a decrease in the liver in the specific activity of ATP citrate lyase and in the ability to incorporate acetate into lipid.

Rational Design Synthesis and Evaluation of First Generation Inhibitors of the Giardia Lamblia Fructose-1 6-biphosphate Aldolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Z Li; Z Liu; D Cho

2011-12-31

Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn{sup 2+} binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (K{sub i} = 14 {micro}M) that is comparable tomore » that of FBP (K{sub m} = 2 {micro}M) or its inert analog TBP (K{sub i} = 1 {micro}M). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3 {angstrom}) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn{sup 2+}. The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn{sup 2+} are not consistent with a strong interaction. To determine if Zn{sup 2+} coordination occurs in the GlFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn{sup 2+} coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn{sup 2+} coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn{sup 2+} coordination to binding of 8 to GlFBPA.« less

Rational design, synthesis and evaluation of first generation inhibitors of the Giardia lamblia fructose-1,6-biphosphate aldolase.

PubMed

Li, Zhimin; Liu, Zhengang; Cho, Dae Won; Zou, Jiwen; Gong, Maozhen; Breece, Robert M; Galkin, Andrey; Li, Ling; Zhao, Hong; Maestas, Gabriel D; Tierney, David L; Herzberg, Osnat; Dunaway-Mariano, Debra; Mariano, Patrick S

2011-04-01

Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn(2+) binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (K(i)=14μM) that is comparable to that of FBP (K(m)=2μM) or its inert analog TBP (K(i)=1μM). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3Å) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn(2+). The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn(2+) are not consistent with a strong interaction. To determine if Zn(2+)coordination occurs in the GlFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn(2+) coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn(2+) coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn(2+) coordination to binding of 8 to GlFBPA. Copyright © 2010 Elsevier Inc. All rights reserved.

[Fructose and fructose intolerance].

PubMed

Buzás, György Miklós

2016-10-01

Although fructose was discovered in 1794, it was realised in recent decades only that its malabsorption can lead to intestinal symptoms while its excessive consumption induces metabolic disturbances. Fructose is a monosaccharide found naturally in most fruits and vegetables. Dietary intake of fructose has gradually increased in the past decades, especially because of the consumption of high fructose corn syrup. With its 16.4 kg/year consumption, Hungary ranks secondly after the United States. Fructose is absorbed in the small intestine by facilitated transport mediated by glucose transporter proteins-2 and -5, and arrives in the liver cells. Here it is transformed enzymatically into fructose-1-phosphate and then, fructose-1,5-diphosphate, which splits further into glyceraldehyde and dihydroxyacetone-phosphate, entering the process of glycolysis, triglyceride and uric acid production. The prevalence of fructose intolerance varies strongly, depending on the method used. The leading symptoms of fructose intolerance are similar, but less severe than those of lactose intolerance. Multiple secondary symptoms can also occur. A symptom-based diagnosis of fructose intolerance is possible, but the gold standard is the H 2 breath test, though this is less accurate than in lactose testing. Measuring fructosaemia is costly, cumbersome and not widely used. Fructose intolerance increases intestinal motility and sensitivity, promotes biofilm formation and contributes to the development of gastrooesophageal reflux. Long-term use of fructose fosters the development of dental caries and non-alcoholic steatohepatitis. Its role in carcinogenesis is presently investigated. The cornerstone of dietary management for fructose intolerance is the individual reduction of fructose intake and the FODMAP diet, led by a trained dietetician. The newly introduced xylose-isomerase is efficient in reducing the symptoms of fructose intolerance. Orv. Hetil., 2016, 157(43), 1708-1716.

Structural Basis for Catalysis of a Tetrameric Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pegan, Scott D.; Ruskseree, Kamolchanok; Franzblau, Scott G.

2009-03-04

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one-third of the world's population in its latent form. The emergence of multidrug-resistant and extensive drug-resistant strains has highlighted the need for new pharmacological targets within M. tuberculosis. The class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since it is upregulated in latent TB. Since the structure of MtFBA has not been determined and there is little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering themore » pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and fructose 1,6-bisphosphate at 1.5, 2.1, and 1.3 {angstrom}, respectively. Through these structures, it was discovered that MtFBA belongs to a novel tetrameric class of type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for better predictability of the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Substrates and products were observed in geometries poised for catalysis; in addition, unexpectedly, the hydroxyl-enolate intermediate of dihydroxyacetone phosphate was also captured and resolved structurally. These concise new details offer a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes.« less

PPi-dependent phosphofructotransferase (phosphofructokinase) activity in the mollicutes (mycoplasma) Acholeplasma laidlawii.

PubMed Central

Pollack, J D; Williams, M V

1986-01-01

A PPi-dependent phosphofructotransferase (PPi-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1, 6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430-fold). PPi was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or Pi could not substitute for PPi. The PPi-dependent reaction (2.0 mM PPi) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (less than or equal to 300 microM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1, 6-P2 to F-6-P, required Pi; arsenate could substitute for Pi, but not PPi or any other nucleotide tested. The computer-derived kinetic constants (+/- standard deviation) for the reaction in the PPi-driven direction of F-1, 6-P2 were as follows: v, 38.9 +/- 0.48 mM min-1; Ka(PPi), 0.11 +/- 0.04 mM; Kb(F-6-P), 0.65 +/- 0.15 mM; and Kia(PPi), 0.39 +/- 0.11 mM. A. laidlawii B-PG9 required PPi not only for the PPi-phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity. a dependency unknown in any other organism. In A. laidlawii B-PG9, the PPi requirement may be met by reactions in this organism already known to synthesize PPi (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PPi instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process. PMID:3001032

Effects of glucose-6-phosphate dehydrogenase deficiency on the metabolic and cardiac responses to obesogenic or high-fructose diets.

PubMed

Hecker, Peter A; Mapanga, Rudo F; Kimar, Charlene P; Ribeiro, Rogerio F; Brown, Bethany H; O'Connell, Kelly A; Cox, James W; Shekar, Kadambari C; Asemu, Girma; Essop, M Faadiel; Stanley, William C

2012-10-15

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzymopathy that affects cellular redox status and may lower flux into nonoxidative pathways of glucose metabolism. Oxidative stress may worsen systemic glucose tolerance and cardiometabolic syndrome. We hypothesized that G6PD deficiency exacerbates diet-induced systemic metabolic dysfunction by increasing oxidative stress but in myocardium prevents diet-induced oxidative stress and pathology. WT and G6PD-deficient (G6PDX) mice received a standard high-starch diet, a high-fat/high-sucrose diet to induce obesity (DIO), or a high-fructose diet. After 31 wk, DIO increased adipose and body mass compared with the high-starch diet but to a greater extent in G6PDX than WT mice (24 and 20% lower, respectively). Serum free fatty acids were increased by 77% and triglycerides by 90% in G6PDX mice, but not in WT mice, by DIO and high-fructose intake. G6PD deficiency did not affect glucose tolerance or the increased insulin levels seen in WT mice. There was no diet-induced hypertension or cardiac dysfunction in either mouse strain. However, G6PD deficiency increased aconitase activity by 42% and blunted markers of nonoxidative glucose pathway activation in myocardium, including the hexosamine biosynthetic pathway activation and advanced glycation end product formation. These results reveal a complex interplay between diet-induced metabolic effects and G6PD deficiency, where G6PD deficiency decreases weight gain and hyperinsulinemia with DIO, but elevates serum free fatty acids, without affecting glucose tolerance. On the other hand, it modestly suppressed indexes of glucose flux into nonoxidative pathways in myocardium, suggesting potential protective effects.

Effects of glucose-6-phosphate dehydrogenase deficiency on the metabolic and cardiac responses to obesogenic or high-fructose diets

PubMed Central

Hecker, Peter A.; Mapanga, Rudo F.; Kimar, Charlene P.; Ribeiro, Rogerio F.; Brown, Bethany H.; O'Connell, Kelly A.; Cox, James W.; Shekar, Kadambari C.; Asemu, Girma; Essop, M. Faadiel

2012-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzymopathy that affects cellular redox status and may lower flux into nonoxidative pathways of glucose metabolism. Oxidative stress may worsen systemic glucose tolerance and cardiometabolic syndrome. We hypothesized that G6PD deficiency exacerbates diet-induced systemic metabolic dysfunction by increasing oxidative stress but in myocardium prevents diet-induced oxidative stress and pathology. WT and G6PD-deficient (G6PDX) mice received a standard high-starch diet, a high-fat/high-sucrose diet to induce obesity (DIO), or a high-fructose diet. After 31 wk, DIO increased adipose and body mass compared with the high-starch diet but to a greater extent in G6PDX than WT mice (24 and 20% lower, respectively). Serum free fatty acids were increased by 77% and triglycerides by 90% in G6PDX mice, but not in WT mice, by DIO and high-fructose intake. G6PD deficiency did not affect glucose tolerance or the increased insulin levels seen in WT mice. There was no diet-induced hypertension or cardiac dysfunction in either mouse strain. However, G6PD deficiency increased aconitase activity by 42% and blunted markers of nonoxidative glucose pathway activation in myocardium, including the hexosamine biosynthetic pathway activation and advanced glycation end product formation. These results reveal a complex interplay between diet-induced metabolic effects and G6PD deficiency, where G6PD deficiency decreases weight gain and hyperinsulinemia with DIO, but elevates serum free fatty acids, without affecting glucose tolerance. On the other hand, it modestly suppressed indexes of glucose flux into nonoxidative pathways in myocardium, suggesting potential protective effects. PMID:22829586

Genomic and fluxomic analysis of carbohydrate metabolism in Bifidobacterium spp: human symbiotic bacteria

USDA-ARS?s Scientific Manuscript database

Bifidobacteria are gram-positive microorganisms widely applied in fermented dairy products due to their health-promoting effects. Bifidobacterium ssp. may also represent up to 91% of microbial gut population in the infant colon, but considerably less in adults. Fructose-6 phosphate phosphoketolase...

Starch Biosynthesis in Developing Wheat Grain 1

PubMed Central

Keeling, Peter L.; Wood, John R.; Tyson, R. Huw; Bridges, Ian G.

1988-01-01

We have used 13C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of 13C between carbons atoms 1 and 6 of [1-13C] or [6-13C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the 13C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the 13C sugars were supplied in vivo to the intact plant. The 13C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of 13C in all glucosyl carbons was quantified by 13C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background 13C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues. PMID:16666140

Mannitol metabolism during pathogenic fungal–host interactions under stressed conditions

PubMed Central

Meena, Mukesh; Prasad, Vishal; Zehra, Andleeb; Gupta, Vijai K.; Upadhyay, Ram S.

2015-01-01

Numerous plants and fungi produce mannitol, which may serve as an osmolyte or metabolic store; furthermore, mannitol also acts as a powerful quencher of reactive oxygen species (ROS). Some phytopathogenic fungi use mannitol to stifle ROS-mediated plant resistance. Mannitol is essential in pathogenesis to balance cell reinforcements produced by both plants and animals. Mannitol likewise serves as a source of reducing power, managing coenzymes, and controlling cytoplasmic pH by going about as a sink or hotspot for protons. The metabolic pathways for mannitol biosynthesis and catabolism have been characterized in filamentous fungi by direct diminishment of fructose-6-phosphate into mannitol-1-phosphate including a mannitol-1-phosphate phosphatase catalyst. In plants mannitol is integrated from mannose-6-phosphate to mannitol-1-phosphate, which then dephosphorylates to mannitol. The enzyme mannitol dehydrogenase plays a key role in host–pathogen interactions and must be co-localized with pathogen-secreted mannitol to resist the infection. PMID:26441941

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

PubMed

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway.

PubMed Central

Rosey, E L; Oskouian, B; Stewart, G C

1991-01-01

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus. PMID:1655695

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum1

PubMed Central

Vanlerberghe, Greg C.; Feil, Regina; Turpin, David H.

1990-01-01

The onset of anaerobiosis in darkened, N-limited cells of the green alga Selenastrum minutum (Naeg.) Collins elicited the following metabolic responses. There was a rapid decrease in energy charge from 0.85 to a stable lower value of 0.6 accompanied by rapid increases in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also a large increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase, can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase activity. These changes resulted in an approximately twofold increase in the rate of starch breakdown indicating a Pasteur effect. The Pasteur effect was accompanied by accumulation of d-lactate, ethanol and succinate as fermentation end-products, but not malate. Accumulation of succinate was facilitated by reductive carbon metabolism by a partial TCA cycle (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). An initial stoichiometric decline in aspartate and increases in succinate and alanine suggests that aspartate catabolism provides an initial source of carbon for reduction to succinate under anoxic conditions. These observations allow us to develop a model for the regulation of anaerobic carbon metabolism and a model for short-term and long-term strategies for succinate accumulation in a green alga. PMID:16667805

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: I. Regulation of Carbon Metabolism and Succinate as a Fermentation Product.

PubMed

Vanlerberghe, G C; Feil, R; Turpin, D H

1990-11-01

The onset of anaerobiosis in darkened, N-limited cells of the green alga Selenastrum minutum (Naeg.) Collins elicited the following metabolic responses. There was a rapid decrease in energy charge from 0.85 to a stable lower value of 0.6 accompanied by rapid increases in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also a large increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase, can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase activity. These changes resulted in an approximately twofold increase in the rate of starch breakdown indicating a Pasteur effect. The Pasteur effect was accompanied by accumulation of d-lactate, ethanol and succinate as fermentation end-products, but not malate. Accumulation of succinate was facilitated by reductive carbon metabolism by a partial TCA cycle (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). An initial stoichiometric decline in aspartate and increases in succinate and alanine suggests that aspartate catabolism provides an initial source of carbon for reduction to succinate under anoxic conditions. These observations allow us to develop a model for the regulation of anaerobic carbon metabolism and a model for short-term and long-term strategies for succinate accumulation in a green alga.

Charge stabilization and entropy reduction of central lysine residues in fructose-bisphosphate aldolase.

PubMed

St-Jean, Miguel; Blonski, Casimir; Sygusch, Jurgen

2009-06-02

Fructose-1,6-bisphosphate muscle aldolase is an essential glycolytic enzyme that catalyzes reversible carbon-carbon bond formation by cleaving fructose 1,6-bisphosphate to yield dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde phosphate. To elucidate the mechanistic role of conserved amino acid Asp-33, Asn-33 and Ser-33 mutants were examined by kinetic and structural analyses. The mutations significantly compromised enzymatic activity and carbanion oxidation in presence of DHAP. Detailed structural analysis demonstrated that, like native crystals, Asp-33 mutant crystals, soaked in DHAP solutions, trapped Schiff base-derived intermediates covalently attached to Lys-229. The mutant structures, however, exhibited an abridged conformational change with the helical region (34-65) flanking the active site as well as pK(a) reductions and increased side chain disorder by central lysine residues, Lys-107 and Lys-146. These changes directly affect their interaction with the C-terminal Tyr-363, consistent with the absence of active site binding by the C-terminal region in the presence of phosphate. Lys-146 pK(a) reduction and side chain disorder would further compromise charge stabilization during C-C bond cleavage and proton transfer during enamine formation. These mechanistic impediments explain diminished catalytic activity and a reduced level of carbanion oxidation and are consistent with rate-determining proton transfer observed in the Asn-33 mutant. Asp-33 reduces the entropic cost and augments the enthalpic gain during catalysis by rigidifying Lys-107 and Lys-146, stabilizing their protonated forms, and promoting a conformational change triggered by substrate or obligate product binding, which lower kinetic barriers in C-C bond cleavage and Schiff base-enamine interconversion.

Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues.

PubMed

Larrabee, M G

1990-11-15

A classic equation that has frequently been used to estimate the fraction of glucose metabolized by the pentose phosphate pathway, using 14CO2 data, is more simply re-derived with careful consideration of the assumptions involved and the conditions under which it is applicable. The equation is shown to be unreliable for non-homogeneous tissues, depending on the fraction of triose phosphate converted to CO2. The formula in question is as follows: ([1]CO2/G-[6]CO2/G)/(1-[6]CO2/G) = 3Fmet./(1 + 2Fmet.) where [1]CO2 and [6]CO2 are output rates of carbons 1 and 6 of glucose respectively to CO2, G is the rate of glucose uptake and Fmet. is the fraction of the glucose that is metabolized to CO2 and triose phosphate by the pentose phosphate pathway, allowing for recycling of an appropriate fraction of the fructose-6-phosphate produced by the pathway. This analysis illustrates the importance of suitably testing any equation that assumes homogeneity before application to non-homogeneous tissues.

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

2014-08-01

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional Analysis of PDX2 from Arabidopsis, a Glutaminase Involved in Vitamin B6 Biosynthesis1[W][OA

PubMed Central

Tambasco-Studart, Marina; Tews, Ivo; Amrhein, Nikolaus; Fitzpatrick, Teresa B.

2007-01-01

Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5′-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1. PMID:17468224

Key glycolytic branch influences mesocarp oil content in oil palm.

PubMed

Ruzlan, Nurliyana; Low, Yoke Sum Jaime; Win, Wilonita; Azizah Musa, Noor; Ong, Ai-Ling; Chew, Fook-Tim; Appleton, David; Mohd Yusof, Hirzun; Kulaveerasingam, Harikrishna

2017-08-29

The fructose-1,6-bisphosphate aldolase catalyzed glycolysis branch that forms dihydroxyacetone phosphate and glyceraldehyde-3-phosphate was identified as a key driver of increased oil synthesis in oil palm and was validated in Saccharomyces cerevisiae. Reduction in triose phosphate isomerase (TPI) activity in a yeast knockdown mutant resulted in 19% increase in lipid content, while yeast strains overexpressing oil palm fructose-1,6-bisphosphate aldolase (EgFBA) and glycerol-3-phosphate dehydrogenase (EgG3PDH) showed increased lipid content by 16% and 21%, respectively. Genetic association analysis on oil palm SNPs of EgTPI SD_SNP_000035801 and EgGAPDH SD_SNP_000041011 showed that palms harboring homozygous GG in EgTPI and heterozygous AG in EgGAPDH exhibited higher mesocarp oil content based on dry weight. In addition, AG genotype of the SNP of EgG3PDH SD_SNP_000008411 was associated with higher mean mesocarp oil content, whereas GG genotype of the EgFBA SNP SD_SNP_000007765 was favourable. Additive effects were observed with a combination of favourable alleles in TPI and FBA in Nigerian x AVROS population (family F7) with highest allele frequency GG.GG being associated with a mean increase of 3.77% (p value = 2.3E -16 ) oil content over the Family 1. An analogous effect was observed in yeast, where overexpressed EgFBA in TPI - resulted in a 30% oil increment. These results provide insights into flux balances in glycolysis leading to higher yield in mesocarp oil-producing fruit.

Engineering the donor selectivity of D-fructose-6-phosphate aldolase for biocatalytic asymmetric cross-aldol additions of glycolaldehyde.

PubMed

Szekrenyi, Anna; Soler, Anna; Garrabou, Xavier; Guérard-Hélaine, Christine; Parella, Teodor; Joglar, Jesús; Lemaire, Marielle; Bujons, Jordi; Clapés, Pere

2014-09-22

D-Fructose-6-phosphate aldolase (FSA) is a unique catalyst for asymmetric cross-aldol additions of glycolaldehyde. A combination of a structure-guided approach of saturation mutagenesis, site-directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800-fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose-like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross-aldol additions of glycolaldehyde to N-carbobenzyloxyaminoaldehydes to furnish between 80-98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum.

PubMed

Wang, Zhihao; Chan, Siu Hung Joshua; Sudarsan, Suresh; Blank, Lars M; Jensen, Peter Ruhdal; Solem, Christian

2016-11-01

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

PubMed Central

2014-01-01

Background Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt C ) and one located on the natural occurring plasmid pBM19 (tkt P ). Results Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn2+ and Mg2+. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni2+, ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKTC: kcat/KM: 264 s-1 mM-1; TKTP: kcat/KM: 231 s-1 mM) and ribulose 5-phosphate (TKTC: kcat/KM: 109 s-1 mM; TKTP: kcat/KM: 84 s-1 mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKTC: kcat/KM: 108 s-1 mM; TKTP: kcat/KM: 71 s-1 mM) and fructose 6-phosphate (TKTC kcat/KM: 115 s-1 mM; TKTP: kcat/KM: 448 s-1 mM). Conclusions Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt P , but not of tkt C during growth with methanol [J Bacteriol 188:3063–3072, 2006] argues for TKTP being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed. PMID:24405865

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus.

PubMed

Markert, Benno; Stolzenberger, Jessica; Brautaset, Trygve; Wendisch, Volker F

2014-01-09

Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt(C)) and one located on the natural occurring plasmid pBM19 (tkt(P)). Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn(2+) and Mg(2+). Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni(2+), ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKT(C): kcat/KM: 264 s(-1) mM(-1); TKT(P): kcat/KM: 231 s(-1) mM) and ribulose 5-phosphate (TKT(C): kcat/KM: 109 s(-1) mM; TKT(P): kcat/KM: 84 s(-1) mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKT(C): kcat/KM: 108 s(-1) mM; TKT(P): kcat/KM: 71 s(-1) mM) and fructose 6-phosphate (TKT(C) kcat/KM: 115 s(-1) mM; TKT(P): kcat/KM: 448 s(-1) mM). Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt(P), but not of tkt(C) during growth with methanol [J Bacteriol 188:3063-3072, 2006] argues for TKT(P) being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed.

Effects of dietary cis and trans unsaturated and saturated fatty acids on the glucose metabolites and enzymes of rats.

PubMed

Bernal, Claudio A; Rovira, Jordi; Colandré, María E; Cussó, Roser; Cadefau, Joan A

2006-05-01

The aim of the present study was to examine whether the level of dietary cis fatty acid (cFA), or the isomers (trans or cis) and/or the saturation of the fatty acids at high dietary fat levels altered the intracellular glucose metabolites and certain regulatory enzyme activities in the skeletal muscle and liver of rats. The animals were fed for 30 d on either a recommended control diet (7 % cFA, w/w) or a high-fat diet (20 % fatty acids, w/w). The high-fat diet was enriched with either cFA, trans fatty acid (tFA), a moderate proportion of saturated fatty acid (MSFA), or a high proportion of saturated fatty acid (HSFA). The most striking findings were observed in the gastrocnemius muscle with a HSFA diet. There was a significant increase in glucose-6-phosphate (306 %), glucose-1-phosphate (245 %), fructose-6-phosphate (400 %), fructose-1,6-bisphosphate (86 %), glyceraldehyde-3-phosphate (38 %), pyruvate (341 %), lactate (325 %), citrate (79 %) and the bisphosphorylated sugars as compared with the cFA diet. These changes were paralleled by an increase in muscle triacylglycerol content (49 %) and a decrease in glucose (39 %). In addition, the amount of cFA and the other types of fatty acid (i.e. tFA and MSFA) led to no great differences in glucose metabolism as compared with the respective control group. These data support the hypothesis that glucose changes induced by a HSFA diet are a multifaceted abnormality. Glucose and lactate transport and intracellular glucose metabolism could be the key biochemical defects involved in this detrimental effect on glucose metabolism.

Cerebral metabolic intermediate response following severe canine intrauterine growth retardation.

PubMed

Kliegman, R M

1986-07-01

The effect of intrauterine growth retardation and neonatal hypoglycemia on cerebral metabolic intermediates were determined in newborn dogs subjected to 5 days of maternal canine starvation (MCS) before birth. Birth weight was reduced 23% (232 +/- 6 versus 300 +/- 10 g). Circulating blood glucose was reduced after 3 h of neonatal fasting in MCS pups (2.7 +/- 0.4 +/- versus 5.7 +/- 1.1 mM). Cerebral cortical levels of glucose were also reduced at this time. Cerebral glucose-6-phosphate was not altered; nonetheless fructose-6-phosphate was lower in MCS pups at 6 and 9 h, while fructose 1,6-diphosphate appeared elevated at 3 h. These data suggest that cerebral glycolytic activity may be increased by increased activity of phosphofructokinase. Cerebral glutamine appeared reduced in fasting MCS pups at 3, 6, and 8 h of age. A considerable disturbance of the adenine nucleotide pool was noted between 3-9 h in MCS pups; while the cerebral energy reserve was diminished in MCS pups between 3-24 h. The data of reduced cerebral energy status and reserve suggest that cerebral energy production was diminished. Although glucose levels were low at 3 h, subsequent recovery was not immediate as adenine-nucleotides remained low beyond the period of hypoglycemia. The combined effects of intrauterine growth retardation and transient neonatal hypoglycemia appear to result in reduced cerebral oxidative metabolism; this occurs despite an apparent enhanced utilization of alternate fuels.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: II. Assimilation of Ammonium by Anaerobic Cells.

PubMed

Vanlerberghe, G C; Turpin, D H

1990-11-01

The green alga Selenastrum minutum (Naeg.) Collins is able to assimilate NH(4) (+) in the dark under anaerobic conditions (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). In the present study, analysis of metabolites following addition of NH(4) (+) to cells acclimated to anaerobic conditions has shown the following. There was a transient decline in adenylate energy charge from 0.6 to 0.4 followed by a recovery back to ~0.6. This was accompanied by a rapid increase in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also an increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase. These changes resulted in an increase in the rate of anaerobic starch breakdown. Anaerobic NH(4) (+) assimilation also resulted in a two-fold increase in the rate of production of the major fermentative end-products in this alga, d-lactate and ethanol. There was no change in the rate of accumulation of the fermentative end product succinate but malate accumulated under anoxia during NH(4) (+) assimilation. A rapid increase in Gln and decline in Glu indicates that primary NH(4) (+) assimilation under anoxia was via glutamine synthetase-glutamate synthase. Almost all N assimilated under these conditions was sequestered in alanine. These results allow us to propose a model for the regulation of carbon metabolism during anaerobic NH(4) (+) assimilation.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum1

PubMed Central

Vanlerberghe, Greg C.; Turpin, David H.

1990-01-01

The green alga Selenastrum minutum (Naeg.) Collins is able to assimilate NH4+ in the dark under anaerobic conditions (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). In the present study, analysis of metabolites following addition of NH4+ to cells acclimated to anaerobic conditions has shown the following. There was a transient decline in adenylate energy charge from 0.6 to 0.4 followed by a recovery back to ~0.6. This was accompanied by a rapid increase in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also an increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase. These changes resulted in an increase in the rate of anaerobic starch breakdown. Anaerobic NH4+ assimilation also resulted in a two-fold increase in the rate of production of the major fermentative end-products in this alga, d-lactate and ethanol. There was no change in the rate of accumulation of the fermentative end product succinate but malate accumulated under anoxia during NH4+ assimilation. A rapid increase in Gln and decline in Glu indicates that primary NH4+ assimilation under anoxia was via glutamine synthetase-glutamate synthase. Almost all N assimilated under these conditions was sequestered in alanine. These results allow us to propose a model for the regulation of carbon metabolism during anaerobic NH4+ assimilation. PMID:16667806

Silencing leaf sorbitol synthesis alters long-distance partitioning and apple fruit quality

PubMed Central

Teo, Gianni; Suzuki, Yasuo; Uratsu, Sandie L.; Lampinen, Bruce; Ormonde, Nichole; Hu, William K.; DeJong, Ted M.; Dandekar, Abhaya M.

2006-01-01

Sorbitol and sucrose are major products of photosynthesis distributed in apple trees (Malus domestica Borkh. cv. “Greensleeves”) that affect quality in fruit. Transgenic apple plants were silenced or up-regulated for sorbitol-6-phosphate dehydrogenase by using the CaMV35S promoter to define the role of sorbitol distribution in fruit development. Transgenic plants with suppressed sorbitol-6-phosphate dehydrogenase compensated by accumulating sucrose and starch in leaves, and morning and midday net carbon assimilation rates were significantly lower. The sorbitol to sucrose ratio in leaves was reduced by ≈90% and in phloem exudates by ≈75%. The fruit accumulated more glucose and less fructose, starch, and malic acid, with no overall differences in weight and firmness. Sorbitol dehydrogenase activity was reduced in silenced fruit, but activities of neutral invertase, vacuolar invertase, cell wall-bound invertase, fructose kinase, and hexokinase were unaffected. Analyses of transcript levels and activity of enzymes involved in carbohydrate metabolism throughout fruit development revealed significant differences in pathways related to sorbitol transport and breakdown. Together, these results suggest that sorbitol distribution plays a key role in fruit carbon metabolism and affects quality attributes such as sugar–acid balance and starch accumulation. PMID:17132742

Studies on the Development of Mouse Embyros in Vitro

PubMed Central

Brinster, Ralph L.

2016-01-01

The effect of various possible energy sources on the development in vitro of two-cell mouse ova into blastocysts was examined. Energy for development of two-cell mouse ova could be supplied by lactate, pyruvate, oxaloacetate, or phosphoenolpyruvate. Compounds such as glucose, fructose, ribose, glucose-6-phosphate, fructose-1, 6-phosphate, acetate, citrate, α-ketoglutarate, succinate, fumarate, and malate could not provide energy for development of two-cell mouse ova. The optimum concentrations at pH 7.38 for those compounds which would supply energy was 5.00 × 10−2 M lactate, 3.16 × 10−4 M pyruvate, 3.16 × 10−4 M oxaloacetate, and 1.00 × 10−2 M phosphoenolpyruvate. The possibility that interactions existed between the effects of osmolarity, pH, and energy source was examined in several experiments. There was no interaction between the effects of osmolarity and pH or osmolarity and the four possible energy sources. However, there was a significant interaction between energy source and pH. The result of this is that an increase in pH of the medium results in an increase in the optimum concentration of the compound supplying energy to the developing ova. PMID:14299682

Aqueous seed extract of Hunteria umbellata (K. Schum.) Hallier f. (Apocynaceae) palliates hyperglycemia, insulin resistance, dyslipidemia, inflammation and oxidative stress in high-fructose diet-induced metabolic syndrome in rats.

PubMed

Ajiboye, T O; Hussaini, A A; Nafiu, B Y; Ibitoye, O B

2017-02-23

Hunteria umbellata is used in the management and treatment of diabetes and obesity in Nigeria. This study evaluates the effect of aqueous seed extract of Hunteria umbellata on insulin resistance, dyslipidemia, inflammation and oxidative stress in high-fructose diet-induced metabolic syndrome MATERIALS AND METHODS: Rats were randomized into seven groups (A-G). Control (group A) and group C rats received control diet for nine weeks while rats in groups B, D - G were placed on high-fructose diet for 9 weeks. In addition to the diets, groups C - F rats orally received 400, 100, 200 and 400mg/kg body weight aqueous seed extract of Hunteria umbellata for 3 weeks starting from 6th - 9th week. High-fructose diet (when compared to control rats) mediated a significant (p<0.05) increase in body weight, body mass index and abdominal circumference. Similarly, levels of blood glucose, insulin, leptin, adiponectin and insulin resistance were increased. It also caused a significant increase in the levels of cholesterol, triglycerides, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, atherogenic index, cardiac index and coronary artery index while high-density lipoprotein cholesterol was decreased significantly. Levels of proinflammatory factor, tumour necrosis factor-α, interleukin-6 and 8 were also increased by the high fructose diet. Moreover, it mediated decrease in activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase and level of glutathione reduced. Conversely, levels of malondialdehyde, conjugated dienes, lipid hydroperoxides, protein carbonyl and fragmented DNA were elevated. Aqueous seed extract of Hunteria umbellata significantly ameliorated the high fructose diet-mediated alterations. From this study, it is concluded that aqueous seed extract of Hunteria umbellata possesses hypoglycemic, hypolipidemic and antioxidants abilities as evident from its capability to extenuate insulin resistance, dyslipidemia, inflammation and oxidative stress in high-fructose diet-induced metabolic syndrome rats. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from M. tuberculosis

PubMed Central

Pegan, Scott D.; Rukseree, Kamolchanok; Capodagli, Glenn C.; Baker, Erica A; Krasnykh, Olga; Franzblau, Scott G; Mesecar, Andrew D

2014-01-01

Class II fructose 1,6-bisphosphate aldolases (FBA; E.C. 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff-base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs has been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies on class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation/deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI/DHAP bound form of the enzyme and determined the X-ray structure of MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information plus site-directed mutagenesis and kinetic studies conducted on a series of residues within the active-site loop revealed that E169 facilitates a water mediated deprotonation/protonation step of the MtFBA reaction mechanism. Also, secondary isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form. PMID:23298222

Identification and expression of fructose-1,6-bisphosphate aldolase genes and their relations to oil content in developing seeds of tea oil tree (Camellia oleifera).

PubMed

Zeng, Yanling; Tan, Xiaofeng; Zhang, Lin; Jiang, Nan; Cao, Heping

2014-01-01

Tea oil tree (Camellia oleifera, Co) provides a fine edible oil source in China. Tea oil from the seeds is very beneficial to human health. Fructose-1,6-bisphosphate aldolase (FBA) hydrolyzes fructose-1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, two critical metabolites for oil biosynthesis. The objectives of this study were to identify FBA genes and investigate the relationship between FBA gene expression and oil content in developing seeds of tea oil tree. In this paper, four developmentally up-regulated CoFBA genes were identified in Camellia oleifera seeds based on the transcriptome from two seed developmental stages corresponding to the initiation and peak stages of lipid biosynthesis. The expression of CoFBA genes, along with three key oil biosynthesis genes CoACP, CoFAD2 and CoSAD were analyzed in seeds from eight developmental stages by real-time quantitative PCR. The oil content and fatty acid composition were also analyzed. The results showed that CoFBA and CoSAD mRNA levels were well-correlated with oil content whereas CoFAD2 gene expression levels were correlated with fatty acid composition in Camellia seeds. We propose that CoFBA and CoSAD are two important factors for determining tea oil yield because CoFBA gene controls the flux of key intermediates for oil biosynthesis and CoSAD gene controls the synthesis of oleic acid, which accounts for 80% of fatty acids in tea oil. These findings suggest that tea oil yield could be improved by enhanced expression of CoFBA and CoSAD genes in transgenic plants.

Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis.

PubMed

Pegan, Scott D; Rukseree, Kamolchanok; Capodagli, Glenn C; Baker, Erica A; Krasnykh, Olga; Franzblau, Scott G; Mesecar, Andrew D

2013-02-05

Class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation-deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation-protonation step of the MtFBA reaction mechanism. Also, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.

Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pegan, Scott D.; Rukseree, Kamolchanok; Capodagli, Glenn C.

The class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprises one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported,more » the structure of the active site loop responsible for catalyzing the protonation–deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA–PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation–protonation step of the MtFBA reaction mechanism. Furthermore, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.« less

The methylotrophic Bacillus methanolicus MGA3 possesses two distinct fructose 1,6-bisphosphate aldolases.

PubMed

Stolzenberger, Jessica; Lindner, Steffen N; Wendisch, Volker F

2013-08-01

The thermotolerant Gram-positive methylotroph Bacillus methanolicus is able to grow with methanol, glucose or mannitol as a sole carbon and energy source. Fructose 1,6-bisphosphate aldolase (FBA), a key enzyme of glycolysis and gluconeogenesis, is encoded in the genome of B. methanolicus by two putative fba genes, the chromosomally located fba(C) and fba(P) on the naturally occurring plasmid pBM19. Their amino acid sequences share 75 % identity and suggest a classification as class II aldolases. Both enzymes were purified from recombinant Escherichia coli and were found to be active as homotetramers. Both enzymes were activated by either manganese or cobalt ions, and inhibited by ADP, ATP and EDTA. The kinetic parameters allowed us to distinguish the chromosomally encoded FBA(C) from the plasmid encoded FBA(P), since FBA(C) showed higher affinity towards fructose 1,6-bisphosphate (Km of 0.16±0.01 mM as compared to 2±0.08 mM) as well as higher glycolytic catalytic efficiency (31.3 as compared to 0.8 s(-1) mM(-1)) than FBA(P). However, FBA(P) exhibited a higher catalytic efficiency in gluconeogenesis (50.4 as compared to 1.4 s(-1) mM(-1) with dihydroxyacetone phosphate and 4 as compared to 0.4 s(-1) mM(-1) with glyceraldehyde 3-phosphate as limiting substrate). The aldolase-negative Corynebacterium glutamicum mutant Δfda could be complemented with both FBA genes from B. methanolicus. Based on the kinetic data, we propose that FBA(C) acts as major aldolase in glycolysis, whereas FBA(P) acts as major aldolase in gluconeogenesis in B. methanolicus.

Genetic analysis of fructose-1,6-bisphosphatase (FBPase) deficiency in nine consanguineous Pakistani families.

PubMed

Ijaz, Sadaqat; Zahoor, Muhammad Yasir; Imran, Muhammad; Ramzan, Khushnooda; Bhinder, Munir Ahmad; Shakeel, Hussain; Iqbal, Muhammad; Aslam, Asim; Shehzad, Wasim; Cheema, Huma Arshad; Rehman, Habib

2017-10-26

Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inherited metabolic disorder characterized by recurrent episodes of hypoglycemia, ketosis and lactic acidosis. FBPase is encoded by FBP1 gene and catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate in the last step of gluconeogenesis. We report here FBP1 mutations in nine consanguineous Pakistani families affected with FBPase deficiency. Nine families having one or two individuals affected with FBPase deficiency were enrolled over a period of 3 years. All FBP1 exonic regions including splicing sites were PCR-amplified and sequenced bidirectionally. Familial cosegregation of mutations with disease was confirmed by direct sequencing and PCR-RFLP analysis. Three different FBP1 mutations were identified. Each of two previously reported mutations (c.472C>T (p.Arg158Trp) and c.841G>A (p.Glu281Lys)) was carried by four different families. The ninth family carried a novel 4-bp deletion (c.609_612delAAAA), which is predicted to result in frameshift (p.Lys204Argfs*72) and loss of FBPase function. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. FBPase deficiency is often fatal in the infancy and early childhood. Early diagnosis and prompt treatment is therefore crucial to preventing early mortality. We recommend the use of c.472C>T and c.841G>A mutations as first choice genetic markers for molecular diagnosis of FBPase deficiency in Pakistan.

The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

PubMed Central

2011-01-01

Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1+Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1. PMID:21609467

Synthesis of carbohydrates in a continuous flow reactor by immobilized phosphatase and aldolase.

PubMed

Babich, Lara; Hartog, Aloysius F; van Hemert, Lieke J C; Rutjes, Floris P J T; Wever, Ron

2012-12-01

Herein, we report a new flow process with immobilized enzymes to synthesize complex chiral carbohydrate analogues from achiral inexpensive building blocks in a three-step cascade reaction. The first reactor contained immobilized acid phosphatase, which phosphorylated dihydroxyacetone to dihydroxyacetone phosphate using pyrophosphate as the phosphate donor. The second flow reactor contained fructose-1,6-diphosphate aldolase (RAMA, rabbit muscle aldolase) or rhamnulose-1-phosphate aldolase (RhuA from Thermotoga maritima) and acid phosphatase. The immobilized aldolases coupled the formed dihydroxyacetone phosphate to aldehydes, resulting in phosphorylated carbohydrates. A final reactor containing acid phosphatase that dephosphorylated the phosphorylated product yielded the final product. Different aldehydes were used to synthesize carbohydrates on a gram scale. To demonstrate the feasibility of the flow systems, we synthesized 0.6 g of the D-fagomine precursor. By using immobilized aldolase RhuA we were also able to obtain other stereoisomers of the D-fagomine precursor. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Beneficial effects of calcitriol on hypertension, glucose intolerance, impairment of endothelium-dependent vascular relaxation, and visceral adiposity in fructose-fed hypertensive rats.

PubMed

Chou, Chu-Lin; Pang, Cheng-Yoong; Lee, Tony J F; Fang, Te-Chao

2015-01-01

Besides regulating calcium homeostasis, the effects of vitamin D on vascular tone and metabolic disturbances remain scarce in the literature despite an increase intake with high-fructose corn syrup worldwide. We investigated the effects of calcitriol, an active form of vitamin D, on vascular relaxation, glucose tolerance, and visceral fat pads in fructose-fed rats. Male Wistar-Kyoto rats were divided into 4 groups (n = 6 per group). Group Con: standard chow diet for 8 weeks; Group Fru: high-fructose diet (60% fructose) for 8 weeks; Group Fru-HVD: high-fructose diet as Group Fru, high-dose calcitriol treatment (20 ng / 100 g body weight per day) 4 weeks after the beginning of fructose feeding; and Group Fru-LVD: high-fructose diet as Group Fru, low-dose calcitriol treatment (10 ng / 100 g body weight per day) 4 weeks after the beginning of fructose feeding. Systolic blood pressure was measured twice a week by the tail-cuff method. Blood was examined for serum ionized calcium, phosphate, creatinine, glucose, triglycerides, and total cholesterol. Intra-peritoneal glucose intolerance test, aortic vascular reactivity, the weight of visceral fat pads, adipose size, and adipose angiotensin II levels were analyzed at the end of the study. The results showed that the fructose-fed rats significantly developed hypertension, impaired glucose tolerance, heavier weight and larger adipose size of visceral fat pads, and raised adipose angiotensin II expressions compared with the control rats. High- and low-dose calcitriol reduced modestly systolic blood pressure, increased endothelium-dependent aortic relaxation, ameliorated glucose intolerance, reduced the weight and adipose size of visceral fat pads, and lowered adipose angiotensin II expressions in the fructose-fed rats. However, high-dose calcitriol treatment mildly increased serum ionized calcium levels (1.44 ± 0.05 mmol/L). These results suggest a protective role of calcitriol treatment on endothelial function, glucose tolerance, and visceral adiposity in fructose-fed rats.

Quantification of sugars and sugar phosphates in Arabidopsis thaliana tissues using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry.

PubMed

Antonio, Carla; Larson, Tony; Gilday, Alison; Graham, Ian; Bergström, Ed; Thomas-Oates, Jane

2007-11-23

This work reports the development and optimisation of a negative ion mode on-line LC-ESI-MS/MS method for the sensitive targeted analysis of the key glycolytic intermediates, sugars and sugar phosphates from plants, using a porous graphitic carbon (PGC) stationary phase and an MS compatible mobile phase. Using this newly developed method, separation and detection of a solution of standard compounds is achieved in less than 20min. Target metabolite compounds were identified in plant extracts from their characteristic retention times, and product ion spectra. This on-line PGC-ESI-MS/MS method shows good linearity over the concentration range 0-100microM, selectivity, short analysis time, and limits of detection of 0.1microM for disaccharides trehalose (Tre), sucrose (Suc), and maltose, and 1.5microM for hexose phosphates fructose-6-phosphate (Fru6P), glucose-1-phosphate (Glc1P), and glucose-6-phosphate (Glc6P), and phosphoenolpyruvate (PEP). This paper describes details of our method and its application to the simultaneous quantitative analysis of soluble sugars and sugar phosphates from Arabidopsis thaliana tissues. We have demonstrated the utility of our method for the analysis of biological samples by applying it to the simultaneous quantitation of changes in soluble sugars and sugar phosphates in A. thaliana Columbia-0 (Col-0) and its starchless phosphoglucomutase (pgm) mutant over a 12-h light/12-h dark growth cycle.

²H enrichment distribution of hepatic glycogen from ²H₂O reveals the contribution of dietary fructose to glycogen synthesis.

PubMed

Delgado, Teresa C; Martins, Fátima O; Carvalho, Filipa; Gonçalves, Ana; Scott, Donald K; O'Doherty, Robert; Macedo, M Paula; Jones, John G

2013-02-15

Dietary fructose can benefit or hinder glycemic control, depending on the quantity consumed, and these contrasting effects are reflected by alterations in postprandial hepatic glycogen synthesis. Recently, we showed that ²H enrichment of glycogen positions 5 and 2 from deuterated water (²H₂O) informs direct and indirect pathway contributions to glycogenesis in naturally feeding rats. Inclusion of position 6(S) ²H enrichment data allows indirect pathway sources to be further resolved into triose phosphate and Krebs cycle precursors. This analysis was applied to six rats that had fed on standard chow (SC) and six rats that had fed on SC plus 35% sucrose in their drinking water (HS). After 2 wk, hepatic glycogenesis sources during overnight feeding were determined by ²H₂O administration and postmortem analysis of glycogen ²H enrichment at the conclusion of the dark period. Net overnight hepatic glycogenesis was similar between SC and HS rodents. Whereas direct pathway contributions were similar (403 ± 71 μmol/g dry wt HS vs. 578 ± 76 μmol/g dry wt SC), triose phosphate contributions were significantly higher for HS compared with SC (382 ± 61 vs. 87 ± 24 μmol/g dry wt, P < 0.01) and Krebs cycle inputs lower for HS compared with SC (110 ± 9 vs. 197 ± 32 μmol/g dry wt, P < 0.05). Analysis of plasma glucose ²H enrichments at the end of the feeding period also revealed a significantly higher fractional contribution of triose phosphate to plasma glucose levels in HS vs. SC. Hence, the ²H enrichment distributions of hepatic glycogen and glucose from ²H₂O inform the contribution of dietary fructose to hepatic glycogen and glucose synthesis.

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements1[OPEN

PubMed Central

Morisaki, Keiko; Sawada, Yuji; Sano, Ryosuke; Yamamoto, Atsushi; Kurata, Tetsuya; Suzuki, Shiro; Matsuda, Mami; Hasunuma, Tomohisa; Hirai, Masami Yokota

2016-01-01

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell’s metabolic activity for the biosynthesis of secondary wall polymers. PMID:27600813

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gowda, Giri; Sagurthi, Someswar Rao; Savithri, H. S.

2008-02-01

The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1},more » with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.« less

The Path of Carbon in Photosynthesis XVIII. The Identification of Nucleotide Coenzymes

DOE R&D Accomplishments Database

Buchanan, J. G.; Lynch, V. H.; Benson, A. A.; Calvin, M.; Bradley, D. F.

1953-01-19

The radioactive compounds to be observed when algae or green leaves are allowed to photosynthesize in C{sup 14}O{sub 2} for short periods are almost all phosphorylated derivatives of sugars. Of these, phosphate esters of trioses, sedoheptulose and fructose are the first to incorporate C{sup 14} followed closely by ribulose diphosphate, glucose-6-phosphate and a phosphate of mannose. It has been noted, in earlier papers of this series, that on radiograms of the products of photosynthesis, a dark area appeared in a position occupied by no known sugar phosphate and which gave glucose on acid hydrolysis or on treatment with a phosphatase preparation. This has hitherto been referred to as an 'unknown glucose phosphate'. It was found that this substance was more labile to acid than glucose-l-phosphate, itself a readily hydrolysable phosphate, and furthermore that other labile glucose derivatives were formed as intermediates during the acid hydrolysis. Accumulation of labeled glucose in this area precedes that in sucrose and suggests its synthetic relationship to sucrose phosphate synthesis.

Liver glucose metabolism in humans

PubMed Central

Adeva-Andany, María M.; Pérez-Felpete, Noemi; Fernández-Fernández, Carlos; Donapetry-García, Cristóbal; Pazos-García, Cristina

2016-01-01

Information about normal hepatic glucose metabolism may help to understand pathogenic mechanisms underlying obesity and diabetes mellitus. In addition, liver glucose metabolism is involved in glycosylation reactions and connected with fatty acid metabolism. The liver receives dietary carbohydrates directly from the intestine via the portal vein. Glucokinase phosphorylates glucose to glucose 6-phosphate inside the hepatocyte, ensuring that an adequate flow of glucose enters the cell to be metabolized. Glucose 6-phosphate may proceed to several metabolic pathways. During the post-prandial period, most glucose 6-phosphate is used to synthesize glycogen via the formation of glucose 1-phosphate and UDP–glucose. Minor amounts of UDP–glucose are used to form UDP–glucuronate and UDP–galactose, which are donors of monosaccharide units used in glycosylation. A second pathway of glucose 6-phosphate metabolism is the formation of fructose 6-phosphate, which may either start the hexosamine pathway to produce UDP-N-acetylglucosamine or follow the glycolytic pathway to generate pyruvate and then acetyl-CoA. Acetyl-CoA may enter the tricarboxylic acid (TCA) cycle to be oxidized or may be exported to the cytosol to synthesize fatty acids, when excess glucose is present within the hepatocyte. Finally, glucose 6-phosphate may produce NADPH and ribose 5-phosphate through the pentose phosphate pathway. Glucose metabolism supplies intermediates for glycosylation, a post-translational modification of proteins and lipids that modulates their activity. Congenital deficiency of phosphoglucomutase (PGM)-1 and PGM-3 is associated with impaired glycosylation. In addition to metabolize carbohydrates, the liver produces glucose to be used by other tissues, from glycogen breakdown or from de novo synthesis using primarily lactate and alanine (gluconeogenesis). PMID:27707936

Prevention of fructose-induced hypertension by dietary vitamins.

PubMed

Vasdev, Sudesh; Longerich, Linda; Gill, Vicki

2004-01-01

Essential hypertension in humans may develop through a combination of genetic and environmental factors. Diet has long been under investigation as a potential effector of blood pressure. A diet high in sucrose or fructose can give rise to hyperlipidemia, insulin resistance and hypertension. Insulin resistance, glucose intolerance and oxidative stress are common features of hypertension. If glucose metabolism through the glycolytic pathway is impaired, as in insulin resistance, there will be a build-up of glyceraldehyde, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate with further metabolism to methylglyoxal, a highly reactive ketoaldehyde. Excess aldehydes can bind sulfhydryl groups of membrane proteins, altering membrane calcium channels, increasing cytosolic free calcium, peripheral vascular resistance and blood pressure. The presence of reactive aldehydes can also lead to oxidative stress. Dietary management through lower sucrose or fructose intake and increased consumption of vitamins improves glucose metabolism, lowers tissue aldehydes, increases anti-oxidant capacity and may also prevent hypertension.

Uric acid and transforming growth factor in fructose-induced production of reactive oxygen species in skeletal muscle

PubMed Central

Maarman, Gerald J.; Ojuka, Edward

2016-01-01

The consumption of fructose, a major constituent of the modern diet, has raised increasing concern about the effects of fructose on health. Research suggests that excessive intake of fructose (>50 g/d) causes hyperuricemia, insulin resistance, mitochondrial dysfunction, de novo lipogenesis by the liver, and increased production of reactive oxygen species (ROS) in muscle. In a number of tissues, uric acid has been shown to stimulate the production of ROS via activation of transforming growth factor β1 and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4. The role of uric acid in fructose-induced production of ROS in skeletal muscle, however, has not been investigated. This review examines the evidence for fructose-induced production of ROS in skeletal muscle, highlights proposed mechanisms, and identifies gaps in current knowledge. PMID:26946251

Dioscoreophyllum cumminsii (Stapf) Diels leaves halt high-fructose induced metabolic syndrome: Hyperglycemia, insulin resistance, inflammation and oxidative stress.

PubMed

Ajiboye, T O; Aliyu, H; Tanimu, M A; Muhammad, R M; Ibitoye, O B

2016-11-04

Dioscoreophyllum cumminsii is widely used in the management and treatment of diabetes and obesity in Nigeria. This study evaluates the effect of aqueous leaf extract of D. cumminsii on high-fructose diet-induced metabolic syndrome. Seventy male rats were randomized into seven groups. All rats were fed with high-fructose diet for 9 weeks except groups A and C rats, which received control diet. In addition to the diet treatment, groups A and B rats received distilled water for 3 weeks starting from the seventh week of the experimental period. Rats in groups C-F orally received 400, 100, 200 and 400mg/kg body weight of aqueous leaf extract of D. cumminsii respectively, while group G received 300mg/kg bodyweight of metformin for 3 weeks starting from the seventh week. There was significant (p<0.05) reduction in high-fructose diet-mediated increase in body weight, body mass index, abdominal circumference, blood glucose, insulin, leptin and insulin resistance by aqueous leaf extract of D. cumminsii. Conversely, high-fructose diet-mediated decrease in adiponectin was reversed by the extract. Increased levels of cholesterol, triglycerides, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, atherogenic index, cardiac index and coronary artery index were significantly lowered by the extract, while high-fructose diet mediated decrease in high-density lipoprotein cholesterol was increased by the extract. Tumour necrosis factor-α, interleukin-6 and interleukin-8 levels increased significantly in high-fructose diet-fed rats, which were significantly reversed by the extract. High-fructose mediated-decrease in superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase and glutathione reduced were significantly reversed by aqueous leaf extract of D. cumminsii. Conversely, elevated levels of malondialdehyde, conjugated dienes, lipid hydroperoxides, protein carbonyl and fragmented DNA were significantly lowered by the extract. Data generated in this study further laid credence to the hypoglycemic activity of aqueous leaf extract of D. cumminsii as evident from the reversal of hyperglycemia, insulin resistance, dyslipidemia, inflammation and oxidative stress in high-fructose diet-induced metabolic syndrome rats. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Involvement of glucocorticoid prereceptor metabolism and signaling in rat visceral adipose tissue lipid metabolism after chronic stress combined with high-fructose diet.

PubMed

Bursać, Biljana; Djordjevic, Ana; Veličković, Nataša; Milutinović, Danijela Vojnović; Petrović, Snježana; Teofilović, Ana; Gligorovska, Ljupka; Preitner, Frederic; Tappy, Luc; Matić, Gordana

2018-05-03

Both fructose overconsumption and increased glucocorticoids secondary to chronic stress may contribute to overall dyslipidemia. In this study we specifically assessed the effects and interactions of dietary fructose and chronic stress on lipid metabolism in the visceral adipose tissue (VAT) of male Wistar rats. We analyzed the effects of 9-week 20% high fructose diet and 4-week chronic unpredictable stress, separately and in combination, on VAT histology, glucocorticoid prereceptor metabolism, glucocorticoid receptor subcellular redistribution and expression of major metabolic genes. Blood triglycerides and fatty acid composition were also measured to assess hepatic Δ9 desaturase activity. The results showed that fructose diet increased blood triglycerides and Δ9 desaturase activity. On the other hand, stress led to corticosterone elevation, glucocorticoid receptor activation and decrease in adipocyte size, while phosphoenolpyruvate carboxykinase, adipose tissue triglyceride lipase, FAT/CD36 and sterol regulatory element binding protein-1c (SREBP-1c) were increased, pointing to VAT lipolysis and glyceroneogenesis. The combination of stress and fructose diet was associated with marked stimulation of fatty acid synthase and acetyl-CoA carboxylase mRNA level and with increased 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase protein levels, suggesting a coordinated increase in hexose monophosphate shunt and de novo lipogenesis. It however did not influence the level of peroxisome proliferator-activated receptor-gamma, SREBP-1c and carbohydrate responsive element-binding protein. In conclusion, our results showed that only combination of dietary fructose and stress increase glucocorticoid prereceptor metabolism and stimulates lipogenic enzyme expression suggesting that interaction between stress and fructose may be instrumental in promoting VAT expansion and dysfunction. Copyright © 2018 Elsevier B.V. All rights reserved.

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

DOE PAGES

Wolf, Nina M.; Gutka, Hiten J.; Movahedzadeh, Farahnaz; ...

2018-04-03

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus . This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and ismore » probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis ( Mt FBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of Mt FBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of Mt FBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.« less

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolf, Nina M.; Gutka, Hiten J.; Movahedzadeh, Farahnaz

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus . This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and ismore » probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis ( Mt FBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of Mt FBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of Mt FBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.« less

Effect of altered sink:source ratio on photosynthetic metabolism of source leaves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plaut, Z.; Mayoral, M.L.; Reinhold, L.

When seven crop species were grown under identical environmental conditions, decreased sink:source ratio led to a decreased photosynthetic rate within 1 to 3 days in Cucumis sativus L., Gossypium hirsutum L., and Raphanus sativus L., but not in Capsicum annuum L., Solanum melongena L., Phaseolus vulgaris L., or Ricinus communis L. The decrease was not associated with stomatal closure. In cotton and cucumbers, sink removal led to an increase in starch and sugar content, in glucose 6-phosphate and fructose 6-phosphate pools, and in the proportion of /sup 14/C detected in sugar phosphates and UDPglucose following /sup 14/CO/sub 2/ supply. Whenmore » mannose was supplied to leaf discs to sequester cytoplasmic inorganic phosphate, promotion of starch synthesis, and inhibition of CO/sub 2/ fixation, were observed in control discs, but not in discs from treated plants. Phosphate buffer reduced starch synthesis in the latter, but not the former discs. The findings suggest that sink removal led to a decreased ratio inorganic phosphate:phosphorylated compounds. In beans /sup 14/C in sugar phosphates increased following sink removal, but without sucrose accumulation, suggesting tighter feedback control of sugar level. Starch accumulated to higher levels than in the other plants, but CO/sub 2/ fixation rate was constant for several days.« less

Purification and partial biochemical-genetic characterization of trehalose 6-phosphate synthase from muscles of adult female Ascaris suum.

PubMed

Dmitryjuk, M; Dopieralska, M; Łopieńska-Biernat, E; Frączek, R J

2013-06-01

Trehalose 6-phosphate (T6P) synthase (TPS; EC 2.4.1.15) was isolated from muscles of Ascaris suum by ammonium sulphate fractionation, ion-exchange DEAE SEPHACEL(TM) anion exchanger column chromatography and Sepharose 6B gel filtration. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), 265-fold purified TPS exhibited a molecular weight of 66 kDa. The optimum pH and temperature of the purified enzyme were 3.8-4.2 and 35°C, respectively. The isoelectric point (pI) of TPS was pH 5.4. The studied TPS was not absolutely substrate specific. Besides glucose 6-phosphate, the enzyme was able to use fructose 6-phosphate as an acceptor of glucose. TPS was activated by 10 mM MgCl2, 10 mM CaCl2 and 10 mM NaCl. In addition, it was inhibited by ethylenediaminetetra-acetic acid (EDTA), KCl, FeCl3 and ZnCl2. Two genes encoding TPS were isolated and sequenced from muscles of the parasite. Complete coding sequences for tps1 (JF412033.2) and tps2 (JF412034.2) were 3917 bp and 3976 bp, respectively. Translation products (AEX60788.1 and AEX60787.1) showed expression to the glucosyltransferase-GTB-type superfamily.

Purification and characterization of fructose bisphosphate aldolase from the ground squirrel, Spermophilus lateralis: enzyme role in mammalian hibernation.

PubMed

MacDonald, Justin A; Storey, Kenneth B

2002-12-15

Fructose-1,6-bisphosphate (F1,6P(2)) aldolase was purified to homogeneity from skeletal muscle of the golden-mantled ground squirrel, Spermophilus lateralis. Enzyme properties were examined at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C); parallel studies assessed rabbit muscle aldolase for comparison. Kinetic properties of each enzyme were differentially affected by assay temperature. For example, the K(m) for F1,6P(2) of ground squirrel aldolase was 0.9+/-0.05 microM at 37 degrees C and 50% higher (1.45+/-0.04 microM) at 5 degrees C, whereas the K(m) of rabbit aldolase increased threefold over the same temperature range. The inhibitory effects of adenylates were similar at both temperatures for the ground squirrel enzyme, but inhibition by adenosine 5(')-diphosphate, adenosine 5(')-monophosphate, and inosine 5(')-monophosphate was substantially reduced at 5 degrees C for rabbit aldolase. Inhibition by inorganic phosphate increased at lower temperatures for both enzymes; for ground squirrel aldolase, the K(i) was 1.18+/-0.1mM at 37 degrees C and 0.23+/-0.05 mM at 5 degrees C. Inhibition of aldolase by inorganic phosphate could be one factor that helps to shut down glycolysis during hibernation. Thus, mammalian hibernators may exploit low-temperature characteristics of aldolase to benefit the metabolic needs of the hibernating state.

Temperature enhanced succinate production concurrent with increased central metabolism turnover in the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Hasunuma, Tomohisa; Matsuda, Mami; Kato, Yuichi; Vavricka, Christopher John; Kondo, Akihiko

2018-05-27

Succinate is a versatile petrochemical compound that can be produced by microorganisms, often from carbohydrate based carbon sources. Phototrophic cyanobacteria including Synechocystis sp. PCC 6803 can more efficiently produce organic acids such as succinate without sugar supplementation, via photosynthetic production of glycogen followed by glycogen utilization, typically under dark conditions. In this study, Synechocystis 6803 bioproduction of organic acids under dark anoxic conditions was found to increase with elevation of temperature from 30 °C to 37 °C. The further enhancement of succinate bioproduction by overexpression of the rate limiting enzyme phosphoenolpyruvate carboxylase resulted in improved glycogen utilization. To gain more insight into the mechanisms underlying the increased organic acid output, a novel temperature dependent metabolomics analysis was performed. Adenylate energy charge was found to decrease along with elevating temperature, while central metabolites glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glycerol 3-phosphate, malate, fumarate and succinate increased. Temperature dependent 13 C-labeling metabolomics analysis further revealed a glycolysis to TCA bottleneck, which could be overcome by addition of CO 2 , leading to even higher organic acid production. Optimization of initial cell concentration to 25 g-dry cell weight/L, in combination with 100 mM NaHCO 3 supplementation, afforded a succinate titer of over 1.8 g/L, the highest reported autotrophic succinate titer. Succinate titers remained high after additional knockout of ackA, resulting in the highest reported autotrophic D-lactate titer as well. The optimization of Synechocystis 6803 organic acid production therefore holds significant promise for CO 2 capture and utilization. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Uric Acid Stimulates Fructokinase and Accelerates Fructose Metabolism in the Development of Fatty Liver

PubMed Central

Lanaspa, Miguel A.; Sanchez-Lozada, Laura G.; Cicerchi, Christina; Li, Nanxing; Roncal-Jimenez, Carlos A.; Ishimoto, Takuji; Le, Myphuong; Garcia, Gabriela E.; Thomas, Jeffrey B.; Rivard, Christopher J.; Andres-Hernando, Ana; Hunter, Brandi; Schreiner, George; Rodriguez-Iturbe, Bernardo; Sautin, Yuri Y.; Johnson, Richard J.

2012-01-01

Excessive dietary fructose intake may have an important role in the current epidemics of fatty liver, obesity and diabetes as its intake parallels the development of these syndromes and because it can induce features of metabolic syndrome. The effects of fructose to induce fatty liver, hypertriglyceridemia and insulin resistance, however, vary dramatically among individuals. The first step in fructose metabolism is mediated by fructokinase (KHK), which phosphorylates fructose to fructose-1-phosphate; intracellular uric acid is also generated as a consequence of the transient ATP depletion that occurs during this reaction. Here we show in human hepatocytes that uric acid up-regulates KHK expression thus leading to the amplification of the lipogenic effects of fructose. Inhibition of uric acid production markedly blocked fructose-induced triglyceride accumulation in hepatocytes in vitro and in vivo. The mechanism whereby uric acid stimulates KHK expression involves the activation of the transcription factor ChREBP, which, in turn, results in the transcriptional activation of KHK by binding to a specific sequence within its promoter. Since subjects sensitive to fructose often develop phenotypes associated with hyperuricemia, uric acid may be an underlying factor in sensitizing hepatocytes to fructose metabolism during the development of fatty liver. PMID:23112875

Structural analysis of the wheat genes encoding NADH-dependent glutamine-2-oxoglutarate amidotransferases genes and correlation with grain protein content

USDA-ARS?s Scientific Manuscript database

Nitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT). Together with glutami...

Prediction of Metabolite Concentrations, Rate Constants and Post-Translational Regulation Using Maximum Entropy-Based Simulations with Application to Central Metabolism of Neurospora crassa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cannon, William; Zucker, Jeremy; Baxter, Douglas

We report the application of a recently proposed approach for modeling biological systems using a maximum entropy production rate principle in lieu of having in vivo rate constants. The method is applied in four steps: (1) a new ODE-based optimization approach based on Marcelin’s 1910 mass action equation is used to obtain the maximum entropy distribution, (2) the predicted metabolite concentrations are compared to those generally expected from experiment using a loss function from which post-translational regulation of enzymes is inferred, (3) the system is re-optimized with the inferred regulation from which rate constants are determined from the metabolite concentrationsmore » and reaction fluxes, and finally (4) a full ODE-based, mass action simulation with rate parameters and allosteric regulation is obtained. From the last step, the power characteristics and resistance of each reaction can be determined. The method is applied to the central metabolism of Neurospora crassa and the flow of material through the three competing pathways of upper glycolysis, the non-oxidative pentose phosphate pathway, and the oxidative pentose phosphate pathway are evaluated as a function of the NADP/NADPH ratio. It is predicted that regulation of phosphofructokinase (PFK) and flow through the pentose phosphate pathway are essential for preventing an extreme level of fructose 1, 6-bisphophate accumulation. Such an extreme level of fructose 1,6-bisphophate would otherwise result in a glassy cytoplasm with limited diffusion, dramatically decreasing the entropy and energy production rate and, consequently, biological competitiveness.« less

Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan,K.; Fedorov, A.; Almo, S.

2008-01-01

Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies ofmore » d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate group hydrogen bonds not only with the conserved motif but also with an active site loop following the sixth {beta}-strand, providing a potential structural mechanism for coupling substrate binding with catalysis.« less

Acute liver failure in neonates with undiagnosed hereditary fructose intolerance due to exposure from widely available infant formulas.

PubMed

Li, Hong; Byers, Heather M; Diaz-Kuan, Alicia; Vos, Miriam B; Hall, Patricia L; Tortorelli, Silvia; Singh, Rani; Wallenstein, Matthew B; Allain, Meredith; Dimmock, David P; Farrell, Ryan M; McCandless, Shawn; Gambello, Michael J

2018-04-01

Hereditary fructose intolerance (HFI) is an autosomal recessive disorder caused by aldolase B (ALDOB) deficiency resulting in an inability to metabolize fructose. The toxic accumulation of intermediate fructose-1-phosphate causes multiple metabolic disturbances, including postprandial hypoglycemia, lactic acidosis, electrolyte disturbance, and liver/kidney dysfunction. The clinical presentation varies depending on the age of exposure and the load of fructose. Some common infant formulas contain fructose in various forms, such as sucrose, a disaccharide of fructose and glucose. Exposure to formula containing fructogenic compounds is an important, but often overlooked trigger for severe metabolic disturbances in HFI. Here we report four neonates with undiagnosed HFI, all caused by the common, homozygous mutation c.448G>C (p.A150P) in ALDOB, who developed life-threatening acute liver failure due to fructose-containing formulas. These cases underscore the importance of dietary history and consideration of HFI in cases of neonatal or infantile acute liver failure for prompt diagnosis and treatment of HFI. Copyright © 2018 Elsevier Inc. All rights reserved.

Temporal metabolomic responses of cultured HepG2 liver cells to high fructose and high glucose exposures.

PubMed

Meissen, John K; Hirahatake, Kristin M; Adams, Sean H; Fiehn, Oliver

2015-06-01

High fructose consumption has been implicated with deleterious effects on human health, including hyperlipidemia elicited through de novo lipogenesis. However, more global effects of fructose on cellular metabolism have not been elucidated. In order to explore the metabolic impact of fructose-containing nutrients, we applied both GC-TOF and HILIC-QTOF mass spectrometry metabolomic strategies using extracts from cultured HepG2 cells exposed to fructose, glucose, or fructose + glucose. Cellular responses were analyzed in a time-dependent manner, incubated in media containing 5.5 mM glucose + 5.0 mM fructose in comparison to controls incubated in media containing either 5.5 mM glucose or 10.5 mM glucose. Mass spectrometry identified 156 unique known metabolites and a large number of unknown compounds, which revealed metabolite changes due to both utilization of fructose and high-carbohydrate loads independent of hexose structure. Fructose was shown to be partially converted to sorbitol, and generated higher levels of fructose-1-phosphate as a precursor for glycolytic intermediates. Differentially regulated ratios of 3-phosphoglycerate to serine pathway intermediates in high fructose media indicated a diversion of carbon backbones away from energy metabolism. Additionally, high fructose conditions changed levels of complex lipids toward phosphatidylethanolamines. Patterns of acylcarnitines in response to high hexose exposure (10.5 mM glucose or glucose/fructose combination) suggested a reduction in mitochondrial beta-oxidation.

Effect of salt nutrients on mannitol production by Lactobacillus intermedius NRRL B-3693.

PubMed

Saha, Badal C

2006-10-01

The effects of four salt nutrients (ammonium citrate, sodium phosphate, magnesium sulfate, and manganese sulfate) on the production of mannitol by Lactobacillus intermedius NRRL B-3693 in a simplified medium containing 300 g fructose, 5 g soy peptone, and 50 g corn steep liquor per liter in pH-controlled fermentation at 5.0 at 37 degrees C were evaluated using a fractional factorial design. Only manganese sulfate was found to be essential for mannitol production. Added manganese sulfate concentration of 0.033 g/l was found to support maximum production. The bacterium produced 200.6 +/- 0.2 g mannitol, 61.9 +/- 0.1 g lactic acid, and 40.4 +/- 0.3 g acetic acid from 300 g fructose per liter in 67 h.

Alterations in Energy/Redox Metabolism Induced by Mitochondrial and Environmental Toxins: A Specific Role for Glucose-6-Phosphate-Dehydrogenase and the Pentose Phosphate Pathway in Paraquat Toxicity

PubMed Central

2015-01-01

Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression. PMID:24937102

Stannous Fluoride Effects on Gene Expression of Streptococcus mutans and Actinomyces viscosus.

PubMed

Shi, Y; Li, R; White, D J; Biesbrock, A R

2018-02-01

A genome-wide transcriptional analysis was performed to elucidate the bacterial cellular response of Streptococcus mutans and Actinomyces viscosus to NaF and SnF 2 . The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of SnF 2 were predetermined before microarray study. Gene expression profiling microarray experiments were carried out in the absence (control) and presence (experimental) of 10 ppm and 100 ppm Sn 2+ (in the form of SnF 2 ) and fluoride controls for 10-min exposures (4 biological replicates/treatment). These Sn 2+ levels and treatment time were chosen because they have been shown to slow bacterial growth of S. mutans (10 ppm) and A. viscosus (100 ppm) without affecting cell viability. All data generated by microarray experiments were analyzed with bioinformatics tools by applying the following criteria: 1) a q value should be ≤0.05, and 2) an absolute fold change in transcript level should be ≥1.5. Microarray results showed SnF 2 significantly inhibited several genes encoding enzymes of the galactose pathway upon a 10-min exposure versus a negative control: lacA and lacB (A and B subunits of the galactose-6-P isomerase), lacC (tagatose-6-P kinase), lacD (tagatose-1,6-bP adolase), galK (galactokinase), galT (galactose-1-phosphate uridylyltransferase), and galE (UDP-glucose 4-epimerase). A gene fruK encoding fructose-1-phosphate kinase in the fructose pathway was also significantly inhibited. Several genes encoding fructose/mannose-specific enzyme IIABC components in the phosphotransferase system (PTS) were also downregulated, as was ldh encoding lactate dehydrogenase, a key enzyme involved in lactic acid synthesis. SnF 2 downregulated the transcription of most key enzyme genes involved in the galactose pathway and also suppressed several key genes involved in the PTS, which transports sugars into the cell in the first step of glycolysis.

Effect of dietary fiber from banana (Musa paradisiaca) on metabolism of carbohydrates in rats fed cholesterol free diet.

PubMed

Usha, V; Vijayammal, P L; Kurup, P A

1989-05-01

Effect of feeding isolated dietary fiber from M. paradisiaca on the metabolism of carbohydrates in the liver has been studied. Fiber fed rats showed significantly lower levels of fasting blood glucose and higher concentration of liver glycogen. Activity of glycogen phosphorylase, glucose-1-phosphate, uridyl transferase and glycogen synthase was significantly higher while phosphoglucomutase activity showed lower activity. Activity of some glycolytic enzymes, viz. hexokinase and pyruvic kinase was lower. Glucose-6-phosphatase showed higher activity while fructose 1-6 diphosphatase activity was not affected. Glucose-6-phosphate dehydrogenase on the other hand showed higher activity. The changes in these enzyme activities have been attributed due to the effect of higher concentration of bile acids produced in the liver as a result of feeding fiber. Evidence for this has been obtained by studying the in vitro effect of cholic acid and chenodeoxy cholic acid.

In Lactobacillus plantarum, Carbamoyl Phosphate Is Synthesized by Two Carbamoyl-Phosphate Synthetases (CPS): Carbon Dioxide Differentiates the Arginine-Repressed from the Pyrimidine-Regulated CPS

PubMed Central

Nicoloff, Hervé; Hubert, Jean-Claude; Bringel, Françoise

2000-01-01

Carbamoyl phosphate (CP) is an intermediate in pyrimidine and arginine biosynthesis. Carbamoyl-phosphate synthetase (CPS) contains a small amidotransferase subunit (GLN) that hydrolyzes glutamine and transfers ammonia to the large synthetase subunit (SYN), where CP biosynthesis occurs in the presence of ATP and CO2. Lactobacillus plantarum, a lactic acid bacterium, harbors a pyrimidine-inhibited CPS (CPS-P; Elagöz et al., Gene 182:37–43, 1996) and an arginine-repressed CPS (CPS-A). Sequencing has shown that CPS-A is encoded by carA (GLN) and carB (SYN). Transcriptional studies have demonstrated that carB is transcribed both monocistronically and in the carAB arginine-repressed operon. CP biosynthesis in L. plantarum was studied with three mutants (ΔCPS-P, ΔCPS-A, and double deletion). In the absence of both CPSs, auxotrophy for pyrimidines and arginine was observed. CPS-P produced enough CP for both pathways. In CO2-enriched air but not in ordinary air, CPS-A provided CP only for arginine biosynthesis. Therefore, the uracil sensitivity observed in prototrophic wild-type L. plantarum without CO2 enrichment may be due to the low affinity of CPS-A for its substrate CO2 or to regulation of the CP pool by the cellular CO2/bicarbonate level. PMID:10852872

Uric acid and transforming growth factor in fructose-induced production of reactive oxygen species in skeletal muscle.

PubMed

Madlala, Hlengiwe P; Maarman, Gerald J; Ojuka, Edward

2016-04-01

The consumption of fructose, a major constituent of the modern diet, has raised increasing concern about the effects of fructose on health. Research suggests that excessive intake of fructose (>50 g/d) causes hyperuricemia, insulin resistance, mitochondrial dysfunction, de novo lipogenesis by the liver, and increased production of reactive oxygen species (ROS) in muscle. In a number of tissues, uric acid has been shown to stimulate the production of ROS via activation of transforming growth factor β1 and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4. The role of uric acid in fructose-induced production of ROS in skeletal muscle, however, has not been investigated. This review examines the evidence for fructose-induced production of ROS in skeletal muscle, highlights proposed mechanisms, and identifies gaps in current knowledge. © The Author(s) 2016. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Sweet siblings with different faces: the mechanisms of FBP and F6P aldolase, transaldolase, transketolase and phosphoketolase revisited in light of recent structural data.

PubMed

Tittmann, Kai

2014-12-01

Nature has evolved different strategies for the reversible cleavage of ketose phosphosugars as essential metabolic reactions in all domains of life. Prominent examples are the Schiff-base forming class I FBP and F6P aldolase as well as transaldolase, which all exploit an active center lysine to reversibly cleave the C3-C4 bond of fructose-1,6-bisphosphate or fructose-6-phosphate to give two 3-carbon products (aldolase), or to shuttle 3-carbon units between various phosphosugars (transaldolase). In contrast, transketolase and phosphoketolase make use of the bioorganic cofactor thiamin diphosphate to cleave the preceding C2-C3 bond of ketose phosphates. While transketolase catalyzes the reversible transfer of 2-carbon ketol fragments in a reaction analogous to that of transaldolase, phosphoketolase forms acetyl phosphate as final product in a reaction that comprises ketol cleavage, dehydration and phosphorolysis. In this review, common and divergent catalytic principles of these enzymes will be discussed, mostly, but not exclusively, on the basis of crystallographic snapshots of catalysis. These studies in combination with mutagenesis and kinetic analysis not only delineated the stereochemical course of substrate binding and processing, but also identified key catalytic players acting at the various stages of the reaction. The structural basis for the different chemical fates and lifetimes of the central enamine intermediates in all five enzymes will be particularly discussed, in addition to the mechanisms of substrate cleavage, dehydration and ring-opening reactions of cyclic substrates. The observation of covalent enzymatic intermediates in hyperreactive conformations such as Schiff-bases with twisted double-bond linkages in transaldolase and physically distorted substrate-thiamin conjugates with elongated substrate bonds to be cleaved in transketolase, which probably epitomize a canonical feature of enzyme catalysis, will be also highlighted. Copyright © 2014 Elsevier Inc. All rights reserved.

Self-assembled block copolymer photonic crystal for selective fructose detection.

PubMed

Ayyub, Omar B; Ibrahim, Michael B; Briber, Robert M; Kofinas, Peter

2013-08-15

The use of one-dimensional photonic crystals fabricated from a self-assembled lamellar block copolymer as a sensitive and selective fructose sensor is investigated. The polystyrene-b-poly(2-vinyl pyridine) (PS-b-P2VP) films are functionalized with 2-(bromomethyl)phenylboronic acid. The boronic acid moiety confined within the lamellar morphology can reversibly bind to sugars such as fructose, imparting the photonic properties of the PS-b-P2VP film. The films exhibit a detection limit of 500 μM in water and 1mM in phosphate buffered saline. Exposure to a 50 mM solution of fructose invokes a highly visible color change from blue to orange. The films are also able to selectively recognize and respond to fructose in competitive studies in the presence of glucose, mannose and sucrose. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

PubMed

Iversen, L F; Brzozowski, M; Hastrup, S; Hubbard, R; Kastrup, J S; Larsen, I K; Naerum, L; Nørskov-Lauridsen, L; Rasmussen, P B; Thim, L; Wiberg, F C; Lundgren, K

1997-05-01

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

PubMed Central

Iversen, L. F.; Brzozowski, M.; Hastrup, S.; Hubbard, R.; Kastrup, J. S.; Larsen, I. K.; Naerum, L.; Nørskov-Lauridsen, L.; Rasmussen, P. B.; Thim, L.; Wiberg, F. C.; Lundgren, K.

1997-01-01

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand. PMID:9144768

Measurement of glucuronidation by isolated rat liver cells using [14C]fructose.

PubMed

Dawson, J; Knowles, R G; Pogson, C I

1992-03-03

We have developed a simple and sensitive method for the study of the relative rates of glucuronidation of compounds, in isolated liver cells, based on the incorporation of 14C from fructose into glucuronide conjugates. Liver cells from fasted rats are used to minimize any reduction of the specific activity by glycogenolysis. Although rates of glucuronidation are lower in isolated liver cells from fasted rats than in those from fed rats, because of a reduction in the concentration of UDP-glucuronic acid, it is possible to compare the rates of glucuronidation of different compounds. Radiolabelled glucuronides are separated from [14C]fructose and [14C]glucose, produced by the liver cells, by normal-phase HPLC on a polar amino-cyano column. The specific activity of the glucuronide was found to be approximately 50% of that of the [14C]fructose. Absolute amounts of glucuronide can be determined by measuring the specific activity of the [14C]glucose, also produced by liver cells from fructose, which reflects that of the glucose-6-phosphate and hence the UDP-glucuronic acid used for glucuronidation, although for the measurement of relative rates this would not be necessary. We have used this method to examine the kinetics of the glucuronidation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The method should be applicable to the study of the rates of glucuronidation of a range of aglycones and, unlike other methods, does not require glucuronide standards or radiolabelled aglycone.

Modulations in primary and secondary metabolic pathways and adjustment in physiological behaviour of Withania somnifera under drought stress.

PubMed

Singh, Ruchi; Gupta, Pankhuri; Khan, Furqan; Singh, Susheel Kumar; Sanchita; Mishra, Tripti; Kumar, Anil; Dhawan, Sunita Singh; Shirke, Pramod Arvind

2018-07-01

In general medicinal plants grown under water limiting conditions show much higher concentrations of secondary metabolites in comparison to control plants. In the present study, Withania somnifera plants were subjected to water stress and data related to drought tolerance phenomenon was collected and a putative mechanistic concept considering growth responses, physiological behaviour, and metabolite content and gene expression aspects is presented. Drought induced metabolic and physiological responses as well as drastic decrease in CO 2 uptake due to stomatal limitations. As a result, the consumption of reduction equivalents (NADPH 2+ ) for CO 2 assimilation via the calvin cycle declines significantly resulting in the generation of a large oxidative stress and an oversupply of antioxidant enzymes. Drought also results in the shifting of metabolic processes towards biosynthetic activities that consume reduction equivalents. Thus, biosynthesis of reduced compounds (isoprenoids, phenols and alkaloids) is enhanced. The dynamics of various metabolites have been discussed in the light of gene expression analysis of control and drought treated leaves. Gene encoding enzymes of pathways leading to glucose, fructose and fructan production, conversion of triose phosphates to hexoses and hexose phosphorylation were up-regulated in the drought stressed leaves. The down-regulated Calvin cycle genes were co-ordinately regulated with the down-regulation of chloroplast triosephosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Expression of gene encoding Squalene Synthase (SQS) was highly upregulated under drought stress which is responsible for the diversion of carbon flux towards withanolides biosynthesis from isoprenoid pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida*

PubMed Central

Chavarría, Max; Santiago, César; Platero, Raúl; Krell, Tino; Casasnovas, José M.; de Lorenzo, Víctor

2011-01-01

The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5′-TTAAACGTTTCA-3′ (KD = 26.3 ± 3.1 nm) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a KD of 209 ± 20 nm. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida. PMID:21239488

Identification and functional analysis of the gene cluster for fructan utilization in Prevotella intermedia.

PubMed

Fuse, Haruka; Fukamachi, Haruka; Inoue, Mitsuko; Igarashi, Takeshi

2013-02-25

Fructanase enzymes hydrolyze the β-2,6 and β-2,1 linkages of levan and inulin fructans, respectively. We analyzed the influence of fructan on the growth of Prevotella intermedia. The growth of P. intermedia was enhanced by addition of inulin, implying that P. intermedia could also use inulin. Based on this finding, we identified and analyzed the genes encoding a putative fructanase (FruA), sugar transporter (FruB), and fructokinase (FruK) in the genome of strain ATCC25611. Transcript analysis by RT-PCR showed that the fruABK genes were co-transcribed as a single mRNA and semi-quantitative analysis confirmed that the fruA gene was induced in response to fructose and inulin. Recombinant FruA and FruK were purified and characterized biochemically. FruA strongly hydrolyzed inulin, with slight degradation of levan via an exo-type mechanism, revealing that FruA is an exo-β-d-fructanase. FruK converted fructose to fructose-6-phosphate in the presence of ATP, confirming that FruK is an ATP-dependent fructokinase. These results suggest that P. intermedia can utilize fructan as a carbon source for growth, and that the fructanase, sugar transporter, and fructokinase proteins we identified are involved in this fructan utilization. Copyright © 2012 Elsevier B.V. All rights reserved.

Capillary electrophoresis-based assay of phosphofructokinase-1.

PubMed

Malina, Andrew; Bryant, Sherrisse K; Chang, Simon H; Waldrop, Grover L; Gilman, S Douglass

2014-02-15

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by the addition of Mg²⁺ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC₅₀ value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.

Capillary Electrophoresis-Based Assay of Phosphofructokinase-1

PubMed Central

Malina, Andrew; Bryant, Sherrisse K.; Chang, Simon H.; Waldrop, Grover L.; Gilman, S. Douglass

2013-01-01

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by UV absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by addition of Mg2+ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC50 value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors. PMID:24444856

DOE Office of Scientific and Technical Information (OSTI.GOV)

Various

This report covers the following titles: (1) Fertility and litter size of normally ovulated and artificially ovulated mice; (2) Further studies on sterility produced in male mice by deuterium oxide; (3) Planarian disaggregation; (4) Uptake of organic compounds by planarians. II; (5) Effects of environmental complexity and training on acetylcholinesterase and cholinesterase activity in rat brain; (6) Effects of environmental complexity and training on brain chemistry and anatomy among mature rats; (7) Improvements in paper chromatographic techniques for labeled cell extracts; (8) measurement and adjustment of pH in small volumes of solutions; (9) Carbon-14 and Nitrogen-15 tracer studies of aminomore » acid synthesis during photosynthesis by Chlorella Pyrenoidosa; (10) Photosynthesis of {sup 14}C-labeled protein from {sup 14}CO{sub 2} by Chlorella; (11) Further studies on carboxydismutase; (12) Electron microscopy of chlorophyll a crystals; (13) The possible role of chromanyl phosphates in oxidative and photosynthetic phosphorylation; (14) Oxidation-reductions of some coenzymes; (15) Preparation of some [{sup 14}C] labeled substances: glucose-6-phosphate, fructose-6-phosphate, 6-phosphogluconic acid, pyruvic acid, and succinic acid; (16) attempt to synthesize high molecular weight polynucleotides using Schramm's purely chemical method; and (17) Optical properties of some dye-polyanion complexes.« less

The production of (14C) oxalate during the metabolism of (14C) carbohydrates in isolated rat hepatocytes.

PubMed

Rofe, A M; James, H M; Bais, R; Edwards, J B; Conyers, R A

1980-04-01

Oxalate (14C) was produced during the metabolism of (U-14C) carbohydrates in hepatocytes isolated from normal rats. At 10 mM, the order of oxalate production was fructose > glycerol > xylitol > sorbitol greater than or equal to glucose in the ratio 10 : 4 : 3 : 1 : 1. This difference between oxalate production from fructose and glucose was reflected in their rates of utilisation, glucose being poorly metabolised in hepatocytes from fasted rats. Fructose was rapidly metabolised, producing glucose, lactate and pyruvate as the major metabolites. Glycerol, xylitol and sorbitol were metabolised at half the rate of fructose, the major metabolites being glucose, lactate and glycerophosphate. The marked similarity in the pattern of intermediary metabolites produced by these polyols was not, however, reflected in the rates of oxalate production. Hepatic polyol metabolism resulted in high levels of cytosolic NADH, as indicated by elevated lactate : pyruvate and glycerophosphate : dihydroxyacetone phosphate ratios. The artificial electron acceptor, phenazine methosulphate (PMS) stimulated oxalate production from the polyols, particularly xylitol. In the presence of PMS, the order of oxalate production was fructose greater than or equal to xylitol > glycerol > sorbitol in the ratio 10 : 10 : 6 : 2. The production of glucose, lactate and pyruvate from the polyols was also stimulated by PMS, whereas the general metabolism of fructose, including oxalate production, was little affected. Oxalate (14C) was produced from (1-14C), (2-14C) and (6-14C) but not (3,4-14C) glucose in hepatocytes isolated from non-fasted, pyridoxine-deficient rats. Whilst this labelling pattern is consistent with oxalate being produced by a number of pathways, it is suggested that metabolism via hydroxypyruvate is a major route for oxalate production from various carbohydrates, with perhaps the exception of xylitol, which appears to have an alternative mechanism for oxalate production. The observation that carbohydrates, particularly fructose, contribute to endogenous oxalate production lends support to the hypothesis that a high sucrose consumption contributes to the formation of renal oxalate stones in man.

(13)C metabolic flux analysis in neurons utilizing a model that accounts for hexose phosphate recycling within the pentose phosphate pathway.

PubMed

Gebril, Hoda M; Avula, Bharathi; Wang, Yan-Hong; Khan, Ikhlas A; Jekabsons, Mika B

2016-02-01

Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation-reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [(13)C]lactate labeling from metabolism of [1,2-(13)C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28 ± 2% and 7.7 ± 0.2% of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52 ± 6% of glucose was metabolized by glycolysis, compared to 19 ± 2% by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29 ± 8% by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62 ± 6% of glucose was converted to pyruvate, the metabolism of which resulted in 16 ± 1% of glucose oxidized by mitochondria and 46 ± 6% exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a minimal set of measurements. Copyright © 2015 Elsevier Ltd. All rights reserved.

The sucrose-trehalose 6-phosphate (Tre6P) nexus: specificity and mechanisms of sucrose signalling by Tre6P.

PubMed

Yadav, Umesh Prasad; Ivakov, Alexander; Feil, Regina; Duan, Guang You; Walther, Dirk; Giavalisco, Patrick; Piques, Maria; Carillo, Petronia; Hubberten, Hans-Michael; Stitt, Mark; Lunn, John Edward

2014-03-01

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, has a profound influence on plant metabolism, growth, and development. It has been proposed that Tre6P acts as a signal of sugar availability and is possibly specific for sucrose status. Short-term sugar-feeding experiments were carried out with carbon-starved Arabidopsis thaliana seedlings grown in axenic shaking liquid cultures. Tre6P increased when seedlings were exogenously supplied with sucrose, or with hexoses that can be metabolized to sucrose, such as glucose and fructose. Conditional correlation analysis and inhibitor experiments indicated that the hexose-induced increase in Tre6P was an indirect response dependent on conversion of the hexose sugars to sucrose. Tre6P content was affected by changes in nitrogen status, but this response was also attributable to parallel changes in sucrose. The sucrose-induced rise in Tre6P was unaffected by cordycepin but almost completely blocked by cycloheximide, indicating that de novo protein synthesis is necessary for the response. There was a strong correlation between Tre6P and sucrose even in lines that constitutively express heterologous trehalose-phosphate synthase or trehalose-phosphate phosphatase, although the Tre6P:sucrose ratio was shifted higher or lower, respectively. It is proposed that the Tre6P:sucrose ratio is a critical parameter for the plant and forms part of a homeostatic mechanism to maintain sucrose levels within a range that is appropriate for the cell type and developmental stage of the plant.

Carbon Dioxide Fixation in Isolated Kalanchoe Chloroplasts 1

PubMed Central

Levi, Carolyn; Gibbs, Martin

1975-01-01

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 μmoles of CO2 per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO2 fixation were primarily those of the photosynthetic carbon reduction cycle. PMID:16659249

Fructose suppresses uric acid excretion to the intestinal lumen as a result of the induction of oxidative stress by NADPH oxidase activation.

PubMed

Kaneko, Chihiro; Ogura, Jiro; Sasaki, Shunichi; Okamoto, Keisuke; Kobayashi, Masaki; Kuwayama, Kaori; Narumi, Katsuya; Iseki, Ken

2017-03-01

A high intake of fructose increases the risk for hyperuricemia. It has been reported that long-term fructose consumption suppressed renal uric acid excretion and increased serum uric acid level. However, the effect of single administration of fructose on excretion of uric acid has not been clarified. We used male Wistar rats, which were orally administered fructose (5g/kg). Those rats were used in each experiment at 12h after administration. Single administration of fructose suppressed the function of ileal uric acid excretion and had no effect on the function of renal uric acid excretion. Breast cancer resistance protein (BCRP) predominantly contributes to intestinal excretion of uric acid as an active homodimer. Single administration of fructose decreased BCRP homodimer level in the ileum. Moreover, diphenyleneiodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), recovered the suppression of the function of ileal uric acid excretion and the Bcrp homodimer level in the ileum of rats that received single administration of fructose. Single administration of fructose decreases in BCRP homodimer level, resulting in the suppression the function of ileal uric acid excretion. The suppression of the function of ileal uric acid excretion by single administration of fructose is caused by the activation of Nox. The results of our study provide a new insight into the mechanism of fructose-induced hyperuricemia. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae

PubMed Central

May, Meghan; Brown, Daniel R.

2008-01-01

We explored the genetic basis for intraspecific variation in mycoplasmal sialidase activity that correlates with virulence, and its potentially advantageous linkage to nutrient catabolism. Polymorphism in N-acetylneuraminate scavenging and degradation genes (sialidase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate epimerase, N-acetylglucosamine-6-phosphate deacetylase, and glucosamine-6-phosphate deaminase) was evident among eight strains of the avian pathogen Mycoplasma synoviae. Most differences were single nucleotide polymorphisms, ranging from 0.34 ± 0.04 substitutions per 100 bp for N-acetylmannosamine kinase to 0.65 ± 0.03 for the single-copy sialidase gene nanI. Missense mutations were twice as common as silent mutations in nanI; 26% resulted in amino acids dissimilar to consensus; and there was a 12-base deletion near the nanI promoter in strain WVU1853T, supporting a complex genetic basis for differences in sialidase activity. Two strains had identical frameshifts in the N-acetylneuraminate lyase gene nanA, resulting in nonsense mutations, and both had downstream deletions in nanA. Such genetic lesions uncouple extracellular liberation of sialic acid from generation of fructose-6-phosphate and pyruvate via intracellular N-acetylneuraminate degradation. Retention of nanI by such strains, but not others in the M. synoviae phylogenetic cluster, is evidence that sialidase has an important non-nutritional role in the ecology of M. synoviae and certain other mycoplasmas. PMID:18490131

The Mechanism of Starch Over-Accumulation in Chlamydomonas reinhardtii High-Starch Mutants Identified by Comparative Transcriptome Analysis

PubMed Central

Koo, Kwang M.; Jung, Sera; Lee, Beom S.; Kim, Jin-Baek; Jo, Yeong D.; Choi, Hong-Il; Kang, Si-Yong; Chung, Gook-H.; Jeong, Won-Joong; Ahn, Joon-Woo

2017-01-01

The focus of this study was the mechanism of starch accumulation in Chlamydomonas reinhardtii high-starch mutants. Three C. reinhardtii mutants showing high-starch content were generated using gamma irradiation. When grown under nitrogen-deficient conditions, these mutants had more than twice as much starch than a wild-type control. The mechanism of starch over-accumulation in these mutants was studied with comparative transcriptome analysis. In all mutants, induction of phosphoglucomutase 1 (PGM1) expression was detected; PGM1 catalyzes the inter-conversion of glucose 1-phosphate and glucose 6-phosphate in both starch biosynthetic and glycolytic pathway. Interestingly, transcript levels of phosphoglucose isomerase 1 (PGI1), fructose 1,6-bisphosphate aldolase 1 and 2 (FBA1 and FBA2) were down-regulated in all mutants; PGI1, FBA1, and FBA2 act on downstream of glucose 6-phosphate conversion in glycolytic pathway. Therefore, down-regulations of PGI1, FBA1, and FBA2 may lead to accumulation of upstream metabolites, notably glucose 6-phosphate, resulting in induction of PGM1 expression through feed-forward regulation and that PGM1 overexpression caused starch over-accumulation in mutants. These results suggest that PGI1, FBA1, FBA2, and PGM1 correlate with each other in terms of coordinated transcriptional regulation and play central roles for starch over-accumulation in C. reinhardtii. PMID:28588557

Ketohexokinase C blockade ameliorates fructose-induced metabolic dysfunction in fructose-sensitive mice.

PubMed

Lanaspa, Miguel A; Andres-Hernando, Ana; Orlicky, David J; Cicerchi, Christina; Jang, Cholsoon; Li, Nanxing; Milagres, Tamara; Kuwabara, Masanari; Wempe, Michael F; Rabinowitz, Joshua D; Johnson, Richard J; Tolan, Dean R

2018-04-23

Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of aldolase B results in the accumulation of intracellular phosphorylated fructose, leading to phosphate sequestration and depletion, increased adenosine triphosphate (ATP) turnover, and a plethora of conditions that lead to clinical manifestations such as fatty liver, hyperuricemia, Fanconi syndrome, and severe hypoglycemia. Unfortunately, there is currently no treatment for HFI, and avoiding sugar and fructose has become challenging in our society. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI. Herein we provide evidence for the first time to our knowledge of a potential therapeutic approach for HFI. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.

Suppressing the activity of trehalase with validamycin disrupts the trehalose and chitin biosynthesis pathways in the rice brown planthopper, Nilaparvata lugens.

PubMed

Tang, Bin; Yang, Mengmeng; Shen, Qida; Xu, Yanxia; Wang, Huijuan; Wang, Shigui

2017-04-01

Trehalase (TRE) is a key enzyme in trehalose degradation and has important functions in insect growth and chitin synthesis. Though validamycin has the potential for pest control by suppressing TRE activities, it is not known whether validamycin acts on both trehalose and chitin metabolism. TRE1 and TRE2 activities and glucose and glycogen contents decreased significantly after the injection of different doses of validamycin solution compared with the control group, while the trehalose content increased significantly. Overall, it showed that about 13 to 38% insects was appeared abnormal phenotypes, and 10 to 57% of insects died 48h after injection of solutions with different concentrations of validamycin; the chitin content also decreased significantly. Validamycin altered the relative expression levels of trehalose, glycogen and chitin metabolism-related genes by suppressing the activities of two TREs. We showed that the expression levels of three TRE and two trehalose-6-phosphate synthase (TPS) genes increased, while the expression levels of GP; CHS1 and its two transcripts, CHS1a, CHS1b; six chitinases, including Cht3, Cht4, Cht5, Cht6, Cht7, Cht9; and the HK, G6PI2, GFAT, GNPNA, PAGM1, UAP, VVL, CI and AP genes decreased significantly 48h after the injection of any validamycin concentration compared with the control group. These results demonstrate that by inhibiting the activities of two TREs, validamycin alters N. lugens chitin synthesis and degradation and affects trehalose and chitin metabolism-related gene expression. The development of TRE inhibitors may provide effective pest control in the future. Copyright © 2016 Elsevier Inc. All rights reserved.

Glucose-Specific Polymer Hydrogels—A Reassessment

PubMed Central

Fazal, Furqan M.; Hansen, David E.

2007-01-01

Polymer hydrogels synthesized by crosslinking poly(allylamine hydrochloride) with (±)-epichlorohydrin in the presence of D-glucose-6-phosphate monobarium salt do not show imprinting on the molecular level. A series of hydrogels were prepared using the following five templates: D-glucose-6-phosphate monobarium salt, D-glucose, L-glucose, barium hydrogen phosphate (BaHPO4), and D-gluconamide; a hydrogel was also prepared in the absence of a template. For all six hydrogels, batch binding studies were conducted with D-glucose, L-glucose, D-fructose and D-gluconamide. The extent of analyte sugar binding was determined using 1H-NMR. Each hydrogel shows approximately the same relative binding affinity for the different sugar derivatives, and none displays selectivity for either glucose enantiomer. The results of the binding studies correlate with the octanol-water partition coefficients of the sugars, indicative that differential solubilities in the bulk polymer account for the binding affinities observed. Thus, in contrast to templated hydrogels prepared using methacrylate- or acrylamide-based reagents, true imprinting does not occur in this novel, crosslinked-poly(allylamine hydrochloride) system. PMID:17035016

Glucose-specific poly(allylamine) hydrogels--a reassessment.

PubMed

Fazal, Furqan M; Hansen, David E

2007-01-01

Polymer hydrogels synthesized by crosslinking poly(allylamine hydrochloride) with (+/-)-epichlorohydrin in the presence of d-glucose-6-phosphate monobarium salt do not show imprinting on the molecular level. A series of hydrogels was prepared using the following five templates: d-glucose-6-phosphate monobarium salt, d-glucose, l-glucose, barium hydrogen phosphate (BaHPO(4)), and d-gluconamide; a hydrogel was also prepared in the absence of a template. For all six hydrogels, batch binding studies were conducted with d-glucose, l-glucose, d-fructose, and d-gluconamide. The extent of analyte sugar binding was determined using (1)H NMR. Each hydrogel shows approximately the same relative binding affinity for the different sugar derivatives, and none displays selectivity for either glucose enantiomer. The results of the binding studies correlate with the octanol-water partition coefficients of the sugars, indicative that differential solubilities in the bulk polymer account for the binding affinities observed. Thus, in contrast to templated hydrogels prepared using methacrylate- or acrylamide-based reagents, true imprinting does not occur in this novel, crosslinked-poly(allylamine hydrochloride) system.

Self-assembled phytosterol-fructose-chitosan nanoparticles as a carrier of anticancer drug.

PubMed

Qiu, Yeyan; Zhu, Jun; Wang, Jianting; Gong, Renmin; Zheng, Mingming; Huang, Fenghong

2013-08-01

Self-assembled nanoparticles were synthesized from water-soluble fructose-chitosan, substituted by succinyl linkages with phytosterols as hydrophobic moieties for self-assembly. The physicochemical properties of the prepared self-assembled nanoparticles were characterized by Fourier transform infrared spectroscopy, fluorescence spectroscopy, and transmission electron microscopy. Doxorubicin (DOX), as a model anticancer drug, was physically entrapped inside prepared self-assembled nanoparticles by the dialysis method. With increasing initial levels of the drug, the drug loading content increased, but the encapsulation efficiency decreased. The release profiles in vitro demonstrated that the DOX showed slow sustained released over 48 h, and the release rate in phosphate buffered saline (PBS) solution (pH 7.4) was much slower than in PBS solution (pH 5.5 and pH 6.5), indicating the prepared self-assembled nanoparticles had the potential to be used as a carrier for targeted delivery of hydrophobic anticancer drugs with declined cytotoxicity to normal tissues.

Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes.

PubMed

Van Schaftingen, E; Vandercammen, A

1989-01-15

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.

Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

PubMed Central

Bettenbrock, Katja; Siebers, Ulrike; Ehrenreich, Petra; Alpert, Carl-Alfred

1999-01-01

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different. PMID:9864334

Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose.

PubMed

Chin, Young-Wook; Park, Jin-Byung; Park, Yong-Cheol; Kim, Kyoung Heon; Seo, Jin-Ho

2013-06-01

Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (GDP)-L-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-L-fucose from GDP-D-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-L-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-D-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-L-fucose at the specific rate of 0.11 mg g cell(-1) h(-1). The specific GDP-L-fucose content reached 5.5 mg g cell(-1), which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.

Direct demonstration of the lectin activity of gp90MEL, a lymphocyte homing receptor

PubMed Central

1990-01-01

Considerable evidence implicates gp90MEL as a lymphocyte homing receptor mediating lymphocyte attachment to high endothelial venules of lymph nodes in mouse. The protein appears to function as a calcium- dependent, lectin-like receptor as inferred primarily by the ability of specific carbohydrates to block its function and by the presence of a calcium-type lectin domain in its primary sequence. An ELISA assay is described which provides the first demonstration that the isolated protein has lectin activity and allows a further definition of its carbohydrate specificity. In addition to the monosaccharides mannose-6- phosphate and fructose-1-phosphate, ligand activity is shown for the sulfated glycolipid, sulfatide, and for two sulfated fucose-containing polysaccharides (fucoidin and egg jelly coat) from nonmammalian sources. PMID:2202735

Export of carbon from chloroplasts at night

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schleucher, J.; Vanderveer, P.J.; Sharkey, T.D.

Hexose export from chloroplasts at night has been inferred in previous studies of mutant and transgenic plants. The authors have tested whether hexose export is the normal route of carbon export from chloroplasts at night. The authors used nuclear magnetic resonance to distinguish glucose (Glc) made from hexose export and Glc made from triose export. Glc synthesized in vitro from fructose-6-phosphate in the presence of deuterium-labeled water had deuterium incorporated at C-2, whereas synthesis from triose phosphates caused C-2 through C-5 to become deuterated. In both tomato (Lycopersicon esculentum L.) and bean (phaseolus vulgaris L.), Glc from sucrose made atmore » night in the presence of deuterium-enriched water was deuterated only in the C-2 position, indicating that >75% of carbon is exported as hexoses at night. In darkness the phosphate in the cytosol was 28 mM, whereas that in the chloroplasts was 5 mW, but hexose phosphates were 10-fold higher in the cytosol than in the chloroplasts. Therefore, hexose phosphates would not move out of chloroplasts without the input of energy. The authors conclude that most carbon leaves chloroplasts at night as Glc, maltose, or higher maltodextrins under normal conditions.« less

Characterization of Fructose-1,6-Bisphosphate Aldolase during Anoxia in the Tolerant Turtle, Trachemys scripta elegans: An Assessment of Enzyme Activity, Expression and Structure

PubMed Central

Storey, Kenneth B.

2013-01-01

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival. PMID:23874782

Characterization of fructose-1,6-bisphosphate aldolase during anoxia in the tolerant turtle, Trachemys scripta elegans: an assessment of enzyme activity, expression and structure.

PubMed

Dawson, Neal J; Biggar, Kyle K; Storey, Kenneth B

2013-01-01

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

Targeted mitochondrial therapy using MitoQ shows equivalent renoprotection to angiotensin converting enzyme inhibition but no combined synergy in diabetes.

PubMed

Ward, Micheal S; Flemming, Nicole B; Gallo, Linda A; Fotheringham, Amelia K; McCarthy, Domenica A; Zhuang, Aowen; Tang, Peter H; Borg, Danielle J; Shaw, Hannah; Harvie, Benjamin; Briskey, David R; Roberts, Llion A; Plan, Manuel R; Murphy, Michael P; Hodson, Mark P; Forbes, Josephine M

2017-11-09

Mitochondrial dysfunction is a pathological mediator of diabetic kidney disease (DKD). Our objective was to test the mitochondrially targeted agent, MitoQ, alone and in combination with first line therapy for DKD. Intervention therapies (i) vehicle (D); (ii) MitoQ (DMitoQ;0.6 mg/kg/day); (iii) Ramipril (DRam;3 mg/kg/day) or (iv) combination (DCoAd) were administered to male diabetic db/db mice for 12 weeks (n = 11-13/group). Non-diabetic (C) db/m mice were followed concurrently. No therapy altered glycaemic control or body weight. By the study end, both monotherapies improved renal function, decreasing glomerular hyperfiltration and albuminuria. All therapies prevented tubulointerstitial collagen deposition, but glomerular mesangial expansion was unaffected. Renal cortical concentrations of ATP, ADP, AMP, cAMP, creatinine phosphate and ATP:AMP ratio were increased by diabetes and mostly decreased with therapy. A higher creatine phosphate:ATP ratio in diabetic kidney cortices, suggested a decrease in ATP consumption. Diabetes elevated glucose 6-phosphate, fructose 6-phosphate and oxidised (NAD+ and NADP+) and reduced (NADH) nicotinamide dinucleotides, which therapy decreased generally. Diabetes increased mitochondrial oxygen consumption (OCR) at complex II-IV. MitoQ further increased OCR but decreased ATP, suggesting mitochondrial uncoupling as its mechanism of action. MitoQ showed renoprotection equivalent to ramipril but no synergistic benefits of combining these agents were shown.

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation.

PubMed

Sengupta, Shinjinee; Lahiri, Sagar; Banerjee, Shakri; Bashistha, Bipasha; Ghosh, Anil K

2011-12-01

Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC). An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37°C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V(max) and lowest K(m) values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors. Substrate specificity, V(max) and K(m) values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis. 2011 Elsevier B.V. All rights reserved.

Efficacy of azelaic acid on hepatic key enzymes of carbohydrate metabolism in high fat diet induced type 2 diabetic mice.

PubMed

Muthulakshmi, Shanmugam; Saravanan, Ramalingam

2013-06-01

Azelaic acid (AzA), a C9 linear α,ω-dicarboxylic acid, is found in whole grains namely wheat, rye, barley, oat seeds and sorghum. The study was performed to investigate whether AzA exerts beneficial effect on hepatic key enzymes of carbohydrate metabolism in high fat diet (HFD) induced type 2 diabetic C57BL/6J mice. C57BL/6J mice were fed high fat diet for 10 weeks and subjected to intragastric administration of various doses (20 mg, 40 mg and 80 mg/kg BW) of AzA daily for the subsequent 5 weeks. Rosiglitazone (RSG) was used as reference drug. Body weight, food intake, plasma glucose, plasma insulin, blood haemoglobin (Hb), blood glycosylated haemoglobin (HbA1c), liver glycolytic enzyme (hexokinase), hepatic shunt enzyme (glucose-6-phosphate dehydrogenase), gluconeogenic enzymes(glucose-6-phosphatase and fructose-1,6-bisphosphatase), liver glycogen, plasma and liver triglycerides were examined in mice fed with normal standard diet (NC), high fat diet (HFD), HFD with AzA (HFD + AzA) and HFD with rosiglitazone (HFD + RSG). Among the three doses, 80 mg/kg BW of AzA was able to positively regulate plasma glucose, insulin, blood HbA1c and haemoglobin levels by significantly increasing the activity of hexokinase and glucose-6-phosphate dehydrogenase and significantly decreasing the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase thereby increasing the glycogen content in the liver. From this study, we put forward that AzA could significantly restore the levels of plasma glucose, insulin, HbA1c, Hb, liver glycogen and carbohydrate metabolic key enzymes to near normal in diabetic mice and hence, AzA may be useful as a biomaterial in the development of therapeutic agents against high fat diet induced T2DM. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Mechanism for acivicin inactivation of triad glutamine amidotransferases.

PubMed

Chittur, S V; Klem, T J; Shafer, C M; Davisson, V J

2001-01-30

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.

Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia.

PubMed

Imahori, Taichiro; Hosoda, Kohkichi; Nakai, Tomoaki; Yamamoto, Yusuke; Irino, Yasuhiro; Shinohara, Masakazu; Sato, Naoko; Sasayama, Takashi; Tanaka, Kazuhiro; Nagashima, Hiroaki; Kohta, Masaaki; Kohmura, Eiji

2017-05-04

The metabolic pathophysiology underlying ischemic stroke remains poorly understood. To gain insight into these mechanisms, we performed a comparative metabolic and transcriptional analysis of the effects of cerebral ischemia on the metabolism of the cerebral cortex using middle cerebral artery occlusion (MCAO) rat model. Metabolic profiling by gas-chromatography/mass-spectrometry analysis showed clear separation between the ischemia and control group. The decreases of fructose 6-phosphate and ribulose 5-phosphate suggested enhancement of the pentose phosphate pathway (PPP) during cerebral ischemia (120-min MCAO) without reperfusion. Transcriptional profiling by microarray hybridization indicated that the Toll-like receptor and mitogen-activated protein kinase (MAPK) signaling pathways were upregulated during cerebral ischemia without reperfusion. In relation to the PPP, upregulation of heat shock protein 27 (HSP27) was observed in the MAPK signaling pathway and was confirmed through real-time polymerase chain reaction. Immunoblotting showed a slight increase in HSP27 protein expression and a marked increase in HSP27 phosphorylation at serine 85 after 60-min and 120-min MCAO without reperfusion. Corresponding upregulation of glucose 6-phosphate dehydrogenase (G6PD) activity and an increase in the NADPH/NAD + ratio were also observed after 120-min MCAO. Furthermore, intracerebroventricular injection of ataxia telangiectasia mutated (ATM) kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD upregulation after MCAO, but that of protein kinase D inhibitor (CID755673) did not affect HSP27 phosphorylation. Consequently, G6PD activation via ischemia-induced HSP27 phosphorylation by ATM kinase may be part of an endogenous antioxidant defense neuroprotection mechanism during the earliest stages of ischemia. These findings have important therapeutic implications for the treatment of stroke. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Caffeine inhibition of aflatoxin synthesis: probable site of action.

PubMed Central

Buchanan, R L; Lewis, D F

1984-01-01

Aflatoxin production by pregrown cultures of Aspergillus parasiticus was completely inhibited by incorporation of 2 mg of caffeine per ml into the medium. This was accompanied by a decrease in glucose utilization and an inhibition of oxygen uptake and carbon dioxide evolution. Enzyme analyses indicated no significant differences in specific activities on glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphofructokinase, fructose 1,6-diphosphatase, pyruvate kinase, or malate dehydrogenase. Glucose uptake kinetics indicated a linear dose-related inhibition of glucose uptake. It appears likely that caffeine inhibits aflatoxin synthesis by restricting the uptake of carbohydrates which are ultimately used by the mold to synthesize this family of mycotoxins. PMID:6331311

Control of Molecular Weight Distribution of the Biopolymer Pullulan Produced by the Fungus Aureobasidium Pullulans

DTIC Science & Technology

1987-10-01

Source ( 1 0%) [!H QH (%) (k) Fructose 5.44 3.76 27.0 1122 2.5 Sucrose 5.44 3.69 34.4 895 2.4 Maltose 5. 44 4.08 25.4 881 2. l Corn Syrup 5. 44 3.85...pullulan and found that the uptake of glucose at more acid pH was diverted to the synthesis of extra- cellular pullulan, and that high extracellular...at pH 5.5, pH 6.0, and pH 6.5, Phosphate concentrations of 0.2%-0,4% yielded high MW pullulan at the lower pH level. They reported that using

Dynamic modeling of lactic acid fermentation metabolism with Lactococcus lactis.

PubMed

Oh, Euhlim; Lu, Mingshou; Park, Changhun; Park, Changhun; Oh, Han Bin; Lee, Sang Yup; Lee, Jinwon

2011-02-01

A dynamic model of lactic acid fermentation using Lactococcus lactis was constructed, and a metabolic flux analysis (MFA) and metabolic control analysis (MCA) were performed to reveal an intensive metabolic understanding of lactic acid bacteria (LAB). The parameter estimation was conducted with COPASI software to construct a more accurate metabolic model. The experimental data used in the parameter estimation were obtained from an LC-MS/ MS analysis and time-course simulation study. The MFA results were a reasonable explanation of the experimental data. Through the parameter estimation, the metabolic system of lactic acid bacteria can be thoroughly understood through comparisons with the original parameters. The coefficients derived from the MCA indicated that the reaction rate of L-lactate dehydrogenase was activated by fructose 1,6-bisphosphate and pyruvate, and pyruvate appeared to be a stronger activator of L-lactate dehydrogenase than fructose 1,6-bisphosphate. Additionally, pyruvate acted as an inhibitor to pyruvate kinase and the phosphotransferase system. Glucose 6-phosphate and phosphoenolpyruvate showed activation effects on pyruvate kinase. Hexose transporter was the strongest effector on the flux through L-lactate dehydrogenase. The concentration control coefficient (CCC) showed similar results to the flux control coefficient (FCC).

Response of rats fed diets low in glucose and glucose precursors to low levels of glucose, starch and chemically modified starch.

PubMed

Chen, S C; Tsai, S; Nesheim, M C

1980-05-01

Several levels of glucose or starches were added to a basal diet that was free of available carbohydrate and low in carbohydrate precursors and fed to male, weanling rats. Rats fed such diets were highly responsive to dietary carbohydrate in growth rate, blood glucose levels and blood ketone bodies. There were no significant differences in the activities of pancreatic amylase, liver glucokinase, glucose-6-phosphatase and fructose-1,6-diphosphatase when dietary carbohydrate varied from 1.5 to 6% of the diet. Under these feeding conditions, a minimum of 6% by weight or 5.8% of the dietary calories has to be provided by carbohydrate to allow the rat an optimum rate of growth. Such diets that are low in glucose precursors were employed as an assay system for glucose availability from chemically cross-bonded starches with various degrees of phosphate crosslinkage. The data showed that introducing low levels of phosphate crosslinkages into the starch had little effect on the glucose availability from the starch.

Computational and experimental analysis identified 6-diazo-5-oxonorleucine as a potential agent for treating infection by Plasmodium falciparum.

PubMed

Plaimas, Kitiporn; Wang, Yulin; Rotimi, Solomon O; Olasehinde, Grace; Fatumo, Segun; Lanzer, Michael; Adebiyi, Ezekiel; König, Rainer

2013-12-01

Plasmodium falciparum (PF) is the most severe malaria parasite. It is developing resistance quickly to existing drugs making it indispensable to discover new drugs. Effective drugs have been discovered targeting metabolic enzymes of the parasite. In order to predict new drug targets, computational methods can be used employing database information of metabolism. Using this data, we performed recently a computational network analysis of metabolism of PF. We analyzed the topology of the network to find reactions which are sensitive against perturbations, i.e., when a single enzyme is blocked by drugs. We now used a refined network comprising also the host enzymes which led to a refined set of the five targets glutamyl-tRNA (gln) amidotransferase, hydroxyethylthiazole kinase, deoxyribose-phophate aldolase, pseudouridylate synthase, and deoxyhypusine synthase. It was shown elsewhere that glutamyl-tRNA (gln) amidotransferase of other microorganisms can be inhibited by 6-diazo-5-oxonorleucine. Performing a half maximal inhibitory concentration (IC50) assay, we showed, that 6-diazo-5-oxonorleucine is also severely affecting viability of PF in blood plasma of the human host. We confirmed this by an in vivo study observing Plasmodium berghei infected mice. Copyright © 2013 Elsevier B.V. All rights reserved.

Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus , Sulfolobus tokodaii and Methanocaldococcus jannaschii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Yuzo; Yanai, Hisaaki; Kanagawa, Mayumi

2016-07-27

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, fromThermus thermophilus,Sulfolobus tokodaiiandMethanocaldococcus jannaschiiwere determined and their structural characteristics were analyzed. For PurS fromT. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations ofmore » the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.« less

Decreased glycolytic and tricarboxylic acid cycle intermediates coincide with peripheral nervous system oxidative stress in a murine model of type 2 diabetes.

PubMed

Hinder, Lucy M; Vivekanandan-Giri, Anuradha; McLean, Lisa L; Pennathur, Subramaniam; Feldman, Eva L

2013-01-01

Diabetic neuropathy (DN) is the most common complication of diabetes and is characterized by distal-to-proximal loss of peripheral nerve axons. The idea of tissue-specific pathological alterations in energy metabolism in diabetic complications-prone tissues is emerging. Altered nerve metabolism in type 1 diabetes models is observed; however, therapeutic strategies based on these models offer limited efficacy to type 2 diabetic patients with DN. Therefore, understanding how peripheral nerves metabolically adapt to the unique type 2 diabetic environment is critical to develop disease-modifying treatments. In the current study, we utilized targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) to characterize the glycolytic and tricarboxylic acid (TCA) cycle metabolomes in sural nerve, sciatic nerve, and dorsal root ganglia (DRG) from male type 2 diabetic mice (BKS.Cg-m+/+Lepr(db); db/db) and controls (db/+). We report depletion of glycolytic intermediates in diabetic sural nerve and sciatic nerve (glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate (sural nerve only), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and lactate), with no significant changes in DRG. Citrate and isocitrate TCA cycle intermediates were decreased in sural nerve, sciatic nerve, and DRG from diabetic mice. Utilizing LC/electrospray ionization/MS/MS and HPLC methods, we also observed increased protein and lipid oxidation (nitrotyrosine; hydroxyoctadecadienoic acids) in db/db tissue, with a proximal-to-distal increase in oxidative stress, with associated decreased aconitase enzyme activity. We propose a preliminary model, whereby the greater change in metabolomic profile, increase in oxidative stress, and decrease in TCA cycle enzyme activity may cause distal peripheral nerves to rely on truncated TCA cycle metabolism in the type 2 diabetes environment.

Brucella abortus Depends on Pyruvate Phosphate Dikinase and Malic Enzyme but Not on Fbp and GlpX Fructose-1,6-Bisphosphatases for Full Virulence in Laboratory Models

PubMed Central

Zúñiga-Ripa, Amaia; Barbier, Thibault; Conde-Álvarez, Raquel; Martínez-Gómez, Estrella; Palacios-Chaves, Leyre; Gil-Ramírez, Yolanda; Grilló, María Jesús; Letesson, Jean-Jacques; Iriarte, Maite

2014-01-01

The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ΔppdK and Δmae mutants was severely impaired and growth of the double Δfbp-ΔglpX mutant was reduced. In macrophages, only the ΔppdK and Δmae mutants showed reduced multiplication, and studies with the ΔppdK mutant confirmed that it reached the replicative niche. Similarly, only the ΔppdK and Δmae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt. PMID:24936050

Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: The role of extra copies of glpK, glpX, and tktA genes

PubMed Central

2014-01-01

Background For the production of L-phenylalanine (L-Phe), two molecules of phosphoenolpyruvate (PEP) and one molecule erythrose-4-phosphate (E4P) are necessary. PEP stems from glycolysis whereas E4P is formed in the pentose phosphate pathway (PPP). Glucose, commonly used for L-Phe production with recombinant E. coli, is taken up via the PEP-dependent phosphotransferase system which delivers glucose-6-phosphate (G6P). G6P enters either glycolysis or the PPP. In contrast, glycerol is phosphorylated by an ATP-dependent glycerol kinase (GlpK) thus saving one PEP. However, two gluconeogenic reactions (fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, FBPase) are necessary for growth and provision of E4P. Glycerol has become an important carbon source for biotechnology and reports on production of L-Phe from glycerol are available. However, the influence of FBPase and transketolase reactions on L-Phe production has not been reported. Results L-Phe productivity of parent strain FUS4/pF81 (plasmid-encoded genes for aroF, aroB, aroL, pheA) was compared on glucose and glycerol as C sources. On glucose, a maximal carbon recovery of 0.19 mM CPhe/CGlucose and a maximal space-time-yield (STY) of 0.13 g l−1 h−1 was found. With glycerol, the maximal carbon recovery was nearly the same (0.18 mM CPhe/CGlycerol), but the maximal STY was higher (0.21 g l−1 h−1). We raised the chromosomal gene copy number of the genes glpK (encoding glycerol kinase), tktA (encoding transketolase), and glpX (encoding fructose-1,6-bisphosphatase) individually. Overexpression of glpK (or its feedback-resistant variant, glpKG232D) had little effect on growth rate; L-Phe production was about 30% lower than in FUS4/pF81. Whereas the overexpression of either glpX or tktA had minor effects on productivity (0.20 mM CPhe/CGlycerol; 0.25 g l−1 h−1 and 0.21 mM CPhe/CGlycerol; 0.23 g l−1 h−1, respectively), the combination of extra genes of glpX and tktA together led to an increase in maximal STY of about 80% (0.37 g l−1 h−1) and a carbon recovery of 0.26 mM CPhe/CGlycerol. Conclusions Enhancing the gene copy numbers for glpX and tktA increased L-Phe productivity from glycerol without affecting growth rate. Engineering of glycerol metabolism towards L-Phe production in E. coli has to balance the pathways of gluconeogenesis, glycolysis, and PPP to improve the supply of the precursors, PEP and E4P. PMID:25012491

Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: the role of extra copies of glpK, glpX, and tktA genes.

PubMed

Gottlieb, Katrin; Albermann, Christoph; Sprenger, Georg A

2014-07-11

For the production of L-phenylalanine (L-Phe), two molecules of phosphoenolpyruvate (PEP) and one molecule erythrose-4-phosphate (E4P) are necessary. PEP stems from glycolysis whereas E4P is formed in the pentose phosphate pathway (PPP). Glucose, commonly used for L-Phe production with recombinant E. coli, is taken up via the PEP-dependent phosphotransferase system which delivers glucose-6-phosphate (G6P). G6P enters either glycolysis or the PPP. In contrast, glycerol is phosphorylated by an ATP-dependent glycerol kinase (GlpK) thus saving one PEP. However, two gluconeogenic reactions (fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, FBPase) are necessary for growth and provision of E4P. Glycerol has become an important carbon source for biotechnology and reports on production of L-Phe from glycerol are available. However, the influence of FBPase and transketolase reactions on L-Phe production has not been reported. L-Phe productivity of parent strain FUS4/pF81 (plasmid-encoded genes for aroF, aroB, aroL, pheA) was compared on glucose and glycerol as C sources. On glucose, a maximal carbon recovery of 0.19 mM C(Phe)/C(Glucose) and a maximal space-time-yield (STY) of 0.13 g l(-1) h(-1) was found. With glycerol, the maximal carbon recovery was nearly the same (0.18 mM C(Phe)/C(Glycerol)), but the maximal STY was higher (0.21 g l(-1) h(-1)). We raised the chromosomal gene copy number of the genes glpK (encoding glycerol kinase), tktA (encoding transketolase), and glpX (encoding fructose-1,6-bisphosphatase) individually. Overexpression of glpK (or its feedback-resistant variant, glpK(G232D)) had little effect on growth rate; L-Phe production was about 30% lower than in FUS4/pF81. Whereas the overexpression of either glpX or tktA had minor effects on productivity (0.20 mM C(Phe)/C(Glycerol); 0.25 g l(-1) h(-1) and 0.21 mM C(Phe)/C(Glycerol); 0.23 g l(-1) h(-1), respectively), the combination of extra genes of glpX and tktA together led to an increase in maximal STY of about 80% (0.37 g l(-1) h(-1)) and a carbon recovery of 0.26 mM C(Phe)/C(Glycerol). Enhancing the gene copy numbers for glpX and tktA increased L-Phe productivity from glycerol without affecting growth rate. Engineering of glycerol metabolism towards L-Phe production in E. coli has to balance the pathways of gluconeogenesis, glycolysis, and PPP to improve the supply of the precursors, PEP and E4P.

Molecular Docking Studies of Catechin and Its Derivatives as Anti-bacterial Inhibitor for Glucosamine-6-Phosphate Synthase

NASA Astrophysics Data System (ADS)

Fikrika, H.; Ambarsari, L.; Sumaryada, T.

2016-01-01

Molecular docking simulation of catechin and its derivatives on Glucosamine-6- Phosphate Synthase (GlmS) has been performed in this research. GlmS inhibition by a particular ligand will suppress the production of bacterial cell wall and significantly reduce the population of invading bacteria. In this study, catechin derivatives i.e epicatechin, galloatechin and epigalloatechin were found to have stronger binding affinities as compared to natural ligand of GlmS, Fructose-6-Phosphate (F6P). Those three ligands were docked on the same pocket in GlmS target as F6P, with 70% binding sites similarity. Based on the docking results, gallocatechin turns out to be the most potent ligand for anti-bacterial agent with ΔG= -8.00 kcal/mol. The docking between GlmS and catechin derivatives are characterized by a constant present of a strong hydrogen bond between functional group O3 and Ser-349. This hydrogen bond most likely plays a significant role in the docking mechanism and binding modes selection. The surprising result is catechin itself exhibited a quite strong binding with GlmS (ΔG= -7.80 kcal.mol), but docked on a completely different pocket compared to other ligands. This results suggest that catechin might still have a curing effect but with a completely different pathway and mechanism as compared to its derivatives.

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

PubMed Central

Guerrero-Mendiola, Carlos; García-Trejo, José J.; Encalada, Rusely; Saavedra, Emma

2017-01-01

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. PMID:28686591

Alterations of hippocampal glucose metabolism by even versus uneven medium chain triglycerides

PubMed Central

McDonald, Tanya S; Tan, Kah Ni; Hodson, Mark P; Borges, Karin

2014-01-01

Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimer's disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of β-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain. PMID:24169853

Alterations of hippocampal glucose metabolism by even versus uneven medium chain triglycerides.

PubMed

McDonald, Tanya S; Tan, Kah Ni; Hodson, Mark P; Borges, Karin

2014-01-01

Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimer's disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of β-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain.

Synthesis of rare sugars with L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8.

PubMed

Li, Zijie; Cai, Li; Qi, Qingsheng; Styslinger, Thomas J; Zhao, Guohui; Wang, Peng George

2011-09-01

We report herein a one-pot four-enzyme approach for the synthesis of the rare sugars d-psicose, d-sorbose, l-tagatose, and l-fructose with aldolase FucA from a thermophilic source (Thermus thermophilus HB8). Importantly, the cheap starting material DL-GP (DL-glycerol 3-phosphate), was used to significantly reduce the synthetic cost. Copyright © 2011 Elsevier Ltd. All rights reserved.

Trehalose 6-phosphate signal is closely related to sorbitol in apple (Malus domestica Borkh. cv. Gala)

PubMed Central

Zhang, Wen; Lunn, John E.; Feil, Regina; Wang, Yufei; Zhao, Jingjing; Tao, Hongxia; Zhao, Zhengyang

2017-01-01

ABSTRACT Trehalose-6-phosphate (Tre6P) is a precursor of trehalose, which is widespread in nature and greatly influences plant growth and development. Tre6P acts as a signal of carbon availability in many plants, but little is known about the function of Tre6P in rosaceous plants, which have specific sorbitol biosynthesis and transportation pathways. In the present study, Tre6P levels and Sorbitol:Tre6P ratios were analyzed in apple (Malus domestica, Borkh. cv. Gala). Tre6P levels were positively correlated with sorbitol content but negatively correlated with sucrose, glucose, and fructose content in developing fruit. However, under sorbitol-limited conditions, Tre6P levels were positively correlated with both sorbitol and sucrose. In the presence of different exogenous sugar supply, Tre6P levels increased corresponding with sorbitol, but this was not the case with sucrose. In addition, Tre6P content and sorbitol:Tre6P ratios were more highly correlated with ADP-glucose levels under sorbitol-limited conditions and fruit development stages, respectively. These results suggest that Tre6P is more closely related to sorbitol than other soluble sugars and has an important role in influencing carbon metabolism in apple. PMID:28069587

Trehalose 6-phosphate signal is closely related to sorbitol in apple (Malus domestica Borkh. cv. Gala).

PubMed

Zhang, Wen; Lunn, John E; Feil, Regina; Wang, Yufei; Zhao, Jingjing; Tao, Hongxia; Guo, Yanping; Zhao, Zhengyang

2017-02-15

Trehalose-6-phosphate (Tre6P) is a precursor of trehalose, which is widespread in nature and greatly influences plant growth and development. Tre6P acts as a signal of carbon availability in many plants, but little is known about the function of Tre6P in rosaceous plants, which have specific sorbitol biosynthesis and transportation pathways. In the present study, Tre6P levels and Sorbitol:Tre6P ratios were analyzed in apple ( Malus domestica , Borkh. cv. Gala). Tre6P levels were positively correlated with sorbitol content but negatively correlated with sucrose, glucose, and fructose content in developing fruit. However, under sorbitol-limited conditions, Tre6P levels were positively correlated with both sorbitol and sucrose. In the presence of different exogenous sugar supply, Tre6P levels increased corresponding with sorbitol, but this was not the case with sucrose. In addition, Tre6P content and sorbitol:Tre6P ratios were more highly correlated with ADP-glucose levels under sorbitol-limited conditions and fruit development stages, respectively. These results suggest that Tre6P is more closely related to sorbitol than other soluble sugars and has an important role in influencing carbon metabolism in apple. © 2017. Published by The Company of Biologists Ltd.

Formation of 4-Hydroxy-2,5-Dimethyl-3[2H]-Furanone by Zygosaccharomyces rouxii: Identification of an Intermediate

PubMed Central

Hauck, Tobias; Brühlmann, Fredi; Schwab, Wilfried

2003-01-01

The formation of the important flavor compound 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDMF; Furaneol) from d-fructose-1,6-bisphosphate by the yeast Zygosaccharomyces rouxii was studied with regard to the identification of intermediates present in the culture medium. Addition of o-phenylenediamine, a trapping reagent for α-dicarbonyls, to the culture medium and subsequent analysis by high-pressure liquid chromatography with diode array detection revealed the formation of three quinoxaline derivatives derived from d-fructose-1,6-bisphosphate under the applied growth conditions (30°C; pH 4 to 5). Isolation and characterization of these compounds by tandem mass spectrometry and nuclear magnetic resonance spectroscopy led to the identification of phosphoric acid mono-(2,3,4-trihydroxy-4-quinoxaline-2-yl-butyl) ester (Q1), phosphoric acid mono-[2,3-dihydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q2), and phosphoric acid mono-[2-hydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q3). Q1 and Q2 were formed independently of Z. rouxii cells, whereas Q3 was detected only in incubation systems containing the yeast. Identification of Q2 demonstrated for the first time the chemical formation of 1-deoxy-2,3-hexodiulose-6-phosphate in the culture medium, a generally expected but never identified intermediate in the formation pathway of HDMF. Since HDMF was detected only in the presence of Z. rouxii cells, additional enzymatic steps were presumed. Incubation of periplasmic and cytosolic protein extracts obtained from yeast cells with d-fructose-1,6-bisphosphate led to the formation of HDMF, implying the presence of the required enzymes in both extracts. PMID:12839760

Regulatory functions of trehalose-6-phosphate synthase in the chitin biosynthesis pathway in Tribolium castaneum (Coleoptera: Tenebrionidae) revealed by RNA interference.

PubMed

Chen, Q W; Jin, S; Zhang, L; Shen, Q D; Wei, P; Wei, Z M; Wang, S G; Tang, B

2018-06-01

RNA interference (RNAi) is a very effective technique for studying gene function and may be an efficient method for controlling pests. Trehalose-6-phosphate synthase (TPS), which plays a key role in the synthesis of trehalose and insect development, was cloned in Tribolium castaneum (Herbst) (TcTPS) and the putative functions were studied using RNAi via the injection of double-stranded RNA (dsRNA) corresponding to conserved TPS and trehalose-6-phosphate phosphatase domains. Expression analyses show that TcTPS is expressed higher in the fat body, while quantitative real-time polymerase chain reaction results show that the expression of four trehalase isoforms was significantly suppressed by dsTPS injection. Additionally, the expression of six chitin synthesis-related genes, such as hexokinase 2 and glutamine-fructose-6-phosphate aminotransferase, was suppressed at 48 and 72 h post-dsTPS-1 and dsTPS-2 RNA injection, which were two dsTPS fragments that had been designed for two different locations in TcTPS open reading frame, and that trehalose content and trehalase 1 activity decreased significantly at 72 h post-dsRNA injection. Furthermore, T. castaneum injected with dsTPS-1 and dsTPS-2 RNA displayed significantly lower levels of chitin and could not complete the molting process from larvae to pupae, revealing abnormal molting phenotypes. These results demonstrate that silencing TPS gene leads to molting deformities and high mortality rates via regulation of gene expression in the chitin biosynthetic pathway, and may be a promising approach for pest control in the future.

The return of metabolism: biochemistry and physiology of the pentose phosphate pathway

PubMed Central

Stincone, Anna; Prigione, Alessandro; Cramer, Thorsten; Wamelink, Mirjam M. C.; Campbell, Kate; Cheung, Eric; Olin-Sandoval, Viridiana; Grüning, Nana-Maria; Krüger, Antje; Alam, Mohammad Tauqeer; Keller, Markus A.; Breitenbach, Michael; Brindle, Kevin M.; Rabinowitz, Joshua D.; Ralser, Markus

2015-01-01

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner–Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the ‘Warburg effect’ of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite infections, neurons, stem cell potency and cancer metabolism. PMID:25243985

Energy and Fructose From Beverages Sweetened With Sugar or High-Fructose Corn Syrup Pose a Health Risk for Some People12

PubMed Central

Bray, George A.

2013-01-01

Sugar intake in the United States has increased by >40 fold since the American Revolution. The health concerns that have been raised about the amounts of sugar that are in the current diet, primarily as beverages, are the subject of this review. Just less than 50% of the added sugars (sugar and high-fructose corn syrup) are found in soft drinks and fruit drinks. The intake of soft drinks has increased 5-fold between 1950 and 2000. Most meta-analyses have shown that the risk of obesity, diabetes, cardiovascular disease, and metabolic syndrome are related to consumption of beverages sweetened with sugar or high-fructose corn syrup. Calorically sweetened beverage intake has also been related to the risk of nonalcoholic fatty liver disease, and, in men, gout. Calorically sweetened beverages contribute to obesity through their caloric load, and the intake of beverages does not produce a corresponding reduction in the intake of other food, suggesting that beverage calories are “add-on” calories. The increase in plasma triglyceride concentrations by sugar-sweetened beverages can be attributed to fructose rather than glucose in sugar. Several randomized trials of sugar-containing soft drinks versus low-calorie or calorie-free beverages show that either sugar, 50% of which is fructose, or fructose alone increases triglycerides, body weight, visceral adipose tissue, muscle fat, and liver fat. Fructose is metabolized primarily in the liver. When it is taken up by the liver, ATP decreases rapidly as the phosphate is transferred to fructose in a form that makes it easy to convert to lipid precursors. Fructose intake enhances lipogenesis and the production of uric acid. By worsening blood lipids, contributing to obesity, diabetes, fatty liver, and gout, fructose in the amounts currently consumed is hazardous to the health of some people. PMID:23493538

Energy and fructose from beverages sweetened with sugar or high-fructose corn syrup pose a health risk for some people.

PubMed

Bray, George A

2013-03-01

Sugar intake in the United States has increased by >40 fold since the American Revolution. The health concerns that have been raised about the amounts of sugar that are in the current diet, primarily as beverages, are the subject of this review. Just less than 50% of the added sugars (sugar and high-fructose corn syrup) are found in soft drinks and fruit drinks. The intake of soft drinks has increased 5-fold between 1950 and 2000. Most meta-analyses have shown that the risk of obesity, diabetes, cardiovascular disease, and metabolic syndrome are related to consumption of beverages sweetened with sugar or high-fructose corn syrup. Calorically sweetened beverage intake has also been related to the risk of nonalcoholic fatty liver disease, and, in men, gout. Calorically sweetened beverages contribute to obesity through their caloric load, and the intake of beverages does not produce a corresponding reduction in the intake of other food, suggesting that beverage calories are "add-on" calories. The increase in plasma triglyceride concentrations by sugar-sweetened beverages can be attributed to fructose rather than glucose in sugar. Several randomized trials of sugar-containing soft drinks versus low-calorie or calorie-free beverages show that either sugar, 50% of which is fructose, or fructose alone increases triglycerides, body weight, visceral adipose tissue, muscle fat, and liver fat. Fructose is metabolized primarily in the liver. When it is taken up by the liver, ATP decreases rapidly as the phosphate is transferred to fructose in a form that makes it easy to convert to lipid precursors. Fructose intake enhances lipogenesis and the production of uric acid. By worsening blood lipids, contributing to obesity, diabetes, fatty liver, and gout, fructose in the amounts currently consumed is hazardous to the health of some people.

[The effect of halothane on the fructose metabolism in the liver].

PubMed

Götz, E; Scholz, R

1975-10-01

Glucose production from frutose (2 mmol) and fructolysis was studied in perfused rat liver. In the presence of halothane (0.5, 1.5, and 4.0 vol%) glucose production was inhibited, whereas lactate production was stimulated. Total fructose metabolism was unchanged. Since halogenated hydrocarbon compounds are known to inhibit the mitochondrial respiratory chain, it is concluded that glucose synthesis is inhibited due to decreased supply of energy-rich phosphates from oxidative phosphorylation. On the other hand, this depletion of energy may be partially compensated for by an increased extramitochondrial energy production due to fructolysis.

Aldolase as a Chirality Intersection of L-Amino Acids and D-Sugars

NASA Astrophysics Data System (ADS)

Munegumi, Toratane

2015-06-01

Aldolase plays an important role in glycolysis and gluconeogenesis to produce D-fructose-1,6-bisphosphate (D-FBP) from dihydroxyacetone phosphate (DHP) and D-glyceraldehyde-3-phosphate (D-GAP). This reaction is stereoselective and retains the D-GAP 2R configuration and yields D-FBP (with the configuration: 3S, 4S, 5R). The 3- and 4-position carbons are the newly formed chiral carbons because the 5-position carbon of D-FBP comes from the 2-position of D-GAP. Although four diastereomeric products, ( 3S, 4R, 5R), ( 3R, 4R, 5R), ( 3R, 4S, 5R), ( 3S, 4S, 5R), are expected in the nonenzymatic reaction, only the ( 3S, 4S, 5R) diastereomer (D-FBP) is obtained. Therefore, the chirality in the 3- and 4-positions is induced by the chirality of the enzyme composed of L-amino acid residues. D-Glucose-6-phosphate (D-G6P), which is generated from D-FBP in the gluconeogenesis pathway, produces D-ribose-5-phosphate (D-R5P) in the pentose phosphate pathway. D-R5P is converted to PRPP (5-phosphoribosyl-α-pyrophosphate), which is used for the de novo synthesis of nucleotides. Ribonucleic acid (RNA) uses the nucleotides as building blocks. The configurations of the 4R-carbon and of the 3S-carbon are retained. The stereochemical structure of RNA is based on 3S as well as 4R (D). The consideration above suggests that aldolase is a key enzyme that determines the 3S configuration in D-R5P. It is thus a chirality intersection between amino acids and sugars, because the sugar chirality is determined by the chiral environment of an L-amino acid protein, aldolase, to produce D-FBP.

Glutamyl-gamma-boronate inhibitors of bacterial Glu-tRNA(Gln) amidotransferase.

PubMed

Decicco, C P; Nelson, D J; Luo, Y; Shen, L; Horiuchi, K Y; Amsler, K M; Foster, L A; Spitz, S M; Merrill, J J; Sizemore, C F; Rogers, K C; Copeland, R A; Harpel, M R

2001-09-17

Analogues of glutamyl-gamma-boronate (1) were synthesized as mechanism-based inhibitors of bacterial Glu-tRNA(Gln) amidotransferase (Glu-AdT) and were designed to engage a putative catalytic serine nucleophile required for the glutaminase activity of the enzyme. Although 1 provides potent enzyme inhibition, structure-activity studies revealed a narrow range of tolerated chemical changes that maintained activity. Nonetheless, growth inhibition of organisms that require Glu-AdT by the most potent enzyme inhibitors appears to validate mechanism-based inhibitor design of Glu-AdT as an approach to antimicrobial development.

Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis

PubMed Central

French, Nigel G.; Scoble, Judith A.; Williams, Charlotte C.; Churches, Quentin I.; Frazer, Andrew R.; Taylor, Matthew C.; Coia, Greg; Simpson, Gregory; Turner, Nicholas J.; Scott, Colin

2017-01-01

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed. PMID:29112947

Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis.

PubMed

Hartley, Carol J; French, Nigel G; Scoble, Judith A; Williams, Charlotte C; Churches, Quentin I; Frazer, Andrew R; Taylor, Matthew C; Coia, Greg; Simpson, Gregory; Turner, Nicholas J; Scott, Colin

2017-01-01

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.

Expression of fructose-1,6-bisphosphatase mRNA isoforms in normal and basal forebrain cholinergic lesioned rat brain.

PubMed

Löffler, T; Al-Robaiy, S; Bigl, M; Eschrich, K; Schliebs, R

2001-06-01

Fructose-1,6-bisphosphatase is one of the key enzymes in the gluconeogenic pathway predominantly occurring in liver, kidney and muscle. In the brain, fructose-1,6-bisphosphatase has been suggested to be an astrocyte-specific enzyme but the functional importance of glyconeogenesis in the brain is still unclear. To further elucidate the cellular source of fructose-1,6-bisphosphatase in the brain, non-radioactive in situ hybridizations were performed using digoxigenin-labeled RNA probes based on the sequence of recently cloned rat liver and muscle fructose-1,6-bisphosphatase cDNAs. In situ hybridization using a riboprobe for the liver isoform revealed a location of the hybridization signal mainly in neurons, while rat muscle fructose-1,6-bisphosphatase mRNA was detected in both neurons and astrocytes in the hippocampal formation and in layer I of the cerebral cortex.RT-PCR using RNA preparations of rat astrocytes, neurons, and adult whole brain demonstrated a localization of liver fructose-1,6-bisphosphatase mRNA isoform in neurons but not in astrocytes. The muscle fructose-1,6-bisphosphatase mRNA isoform could be detected by RT-PCR in total rat brain, astrocytic, and neuronal mRNA preparations. The isoforms of fructose-1,6-bisphosphatase mRNA seemingly demonstrate a distinct cellular expression pattern in rat brain suggesting a role of glyconeogenesis in both neurons and glial cells.

Fructose 2,6-bisphosphate and the climacteric in bananas.

PubMed

Ball, K L; ap Rees, T

1988-11-15

This work was done to test the view that there is a marked rise in the content of fructose 2,6-bisphosphate during the climacteric of the fruit of banana (Musa cavendishii Lamb ex. Paxton). Bananas were ripened in the dark in a continuous stream of air in the absence of exogenous ethylene. CO2 production and the contents of fructose 2,6-bisphosphate and sucrose were monitored over a 15-day period. A range of extraction procedures for fructose 2,6-bisphosphate were compared. Recovery of fructose 2,6-bisphosphate added to samples of unripe fruit varied from poor to unmeasurable. Recoveries from samples of ripe fruit were high. It is argued that this differential recovery of fructose 2,6-bisphosphate undermines claims that the amount of this compound increases at the climacteric. When recoveries are taken into account, our data suggest that there is no major change in fructose 2,6-bisphosphate content during the onset of the climacteric in bananas.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

PubMed

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural basis for catalysis of a tetrameric, class IIa fructose 1,6-bisphosphate aldolase from M. tuberculosis

PubMed Central

Pegan, Scott D.; Rukseree, Kamolchanok; Franzblau, Scott G.; Mesecar, Andrew D.

2009-01-01

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one third of the world’s population in its latent form. The emergence of multidrug resistant strains, MDR-TB and XDR-TB has highlighted the need for new pharmacological targets within M. tuberculosis. The Class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since its upregulated in latent TB. Since the structure of MtFBA had not been determined and since there was little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering the pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetonephosphate (DHAP), glyceraldehyde-3-phosphate (G3P), and fructose 1,6-bisphosphate (FBP) at 1.5 Å, 2.1 Å, and 1.3 Å respectively. Through these structures it was discovered that MtFBA belongs to a novel tetrameric class of the type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for being able to better predict the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Not only were the substrates and products observed in geometries poised for catalysis, but unexpectedly the hydroxyl enolate intermediate of DHAP was also captured and resolved structurally. These concise new details provide a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes. PMID:19167403

Fructose-1,6-Bisphospate does not preserve ATP in hypoxic-ischemic neonatal cerebrocortical slices

PubMed Central

Liu, Jia; Hirai, Kiyoshi; Litt, Lawrence

2008-01-01

Fructose-1,6-bisphosphate (FBP), an endogenous intracellular metabolite in glycolysis, was found in many preclinical studies to be neuroprotective during hypoxia-ischemia (HI) when administered exogenously. We looked for HI neuroprotection from FBP in a neonatal rat brain slice model, using 14.1 Tesla 1H /31P/13C NMR spectroscopy of perchloric acid slice extracts to ask: 1) if FBP preserves high energy phosphates during HI; and 2) if exogenous [1-13C]FBP enters cells and is glycolytically metabolized to [3-13C]lactate. We also asked: 3) if substantial superoxide production occurs during and after HI, thinking such might be treatable by exogenous FBP's antioxidant effects. Superfused P7 rat cerebrocortical slices (350μm) were treated with 2 mM FBP before and during 30 min of HI, and then given four hours of recovery with an FBP-free oxygenated superfusate. Slices were removed before HI, at the end of HI, and at 1 and 4 hours after HI. FBP did not improve high energy phosphate levels or change 1H metabolite profiles. Large increases in [3-13C]lactate were seen with 13C NMR, but the lactate fractional enrichment was always (1.1±0.5)%, implying that all of lactate's 13C was natural abundance 13C, that none was from metabolism of 13C-FBP. FBP had no effect on the fluorescence of ethidium produced from superoxide oxidation of hydroethidine. Compared to control slices, ethidium fluorescence was 25% higher during HI and 50% higher at the end of recovery. Exogenous FBP did not provide protection or enter glycolysis. Its use as an antioxidant might be worth studying at higher FBP concentrations. PMID:18725216

[The low molecular weight carbohydrates and polyols in the cambial sap of beech (Fagus silvatica L.) and a number of other deciduous trees].

PubMed

Oesch, F

1969-12-01

The low molecular-weight carbohydrates sucrose, D-glucose, D-fructose, myoinositol, O-α-D-galactopyranosyl-(1→1)-myoinositol, raffinose, D,L-inositol, stachyose, α,α-trehalose and D-galactose (in decreasing order of percentage by weight) were isolated from the cambial sap of the beech (Fagus silvatica L.). The acidic compounds glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-diphosphate, malic acid and oxalic acid were also tentatively identified.Practically the only difference between the cambial saps of beech trees felled at different times during the period of most active secondary growth was in their content of myoinositol and an as yet unidentified extremely labile basic substance (BF 2). The content of both these substances increased greatly towards the end of the growing season. The cambial sap of any tree differed little from that of the xylem which had already begun to differentiate. On the other hand the cambial sap next to the bark and the phloem sap had a completely different composition. In the former sucrose constituted ca. 90% and in the latter practically 100% of the neutral, water-soluble fraction.A comparison of the cambial saps of beech and a number of other deciduous trees brought to light a number of characteristic differences that appeared to be chemotaxonomically significant. Trehalose and D,L-inositol were detected only in the genus Fagus and never in the closely related genus Nothofagus. The presence of large amounts of stachyose and mannitol was characteristic for Fraxinus, and a high content of 2-O-methyl-L-inositol was typical of Acer. Galactose was not detected in all trees studied.

Analysis of Two-Component Systems in Group B Streptococcus Shows That RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

PubMed Central

Faralla, Cristina; Metruccio, Matteo M.; De Chiara, Matteo; Mu, Rong; Patras, Kathryn A.; Muzzi, Alessandro; Grandi, Guido; Margarit, Immaculada; Doran, Kelly S.

2014-01-01

ABSTRACT Group B Streptococcus (GBS), in the transition from commensal organisms to pathogens, will encounter diverse host environments and, thus, require coordinated control of the transcriptional responses to these changes. This work was aimed at better understanding the role of two-component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knockout strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1% to 3% of the genome. Interestingly, two sugar phosphotransferase systems appeared to be differentially regulated in the TCS-16 knockout strain (TCS loci were numbered in order of their appearance on the chromosome), suggesting an involvement in monitoring carbon source availability. High-throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for the growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16, with concomitant dramatic upregulation of the adjacent operon, which encodes a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and the data also provide experimental evidence for TCS-17/RgfAC involvement in virulence. PMID:24846378

Effect of infliximab and tocilizumab on fructose-induced hyperinsulinemia and hypertension in rats.

PubMed

Abdelrahman, Aly M; Al Suleimani, Yousuf M; Ashique, Mohammed; Manoj, Priyadarsini; Ali, Badreldin H

2018-05-29

Fructose administration can induce hypertension, insulin resistance and hypertriglyceridemia. Here, we investigated the possible protective effect of infliximab (IFX), a tumor necrosis factor alpha (TNF-α) inhibitor, or tocilizumab (TOC), an interleukin-6 (IL6) inhibitor, on fructose-induced increase in blood pressure, insulin resistance and hyperlipidemia in rats. The animals were fed a 60% fructose diet in the absence or presence of IFX (5 mg/kg, i.p., once weekly) or TOC (8 mg/kg, i.p., once every two weeks). Fructose significantly increased blood pressure, heart rate and homeostatic model assessment of insulin resistance (HOMA-IR). Fructose also significantly raised the concentrations of fasting plasma insulin, triglycerides, total cholesterol, uric acid, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), malondialdhyde (MDA) and nitric oxide. Fructose also significantly decreased plasma superoxide dismutase (SOD) and catalase activities. In addition, fructose significantly increased aortic endothelin and nitric oxide concentrations. Both IFX and TOC attenuated the fructose-induced increase in blood pressure, insulin resistance, and the concentrations of uric acid, MDA and IL-6. TOC significantly reduced fructose-induced increase in triglycerides and cholesterol. In addition, IFX increased plasma SOD and catalase activities. Our results showed that both IFX and TOC were partially successful in reversing fructose - induced changes. Copyright © 2018. Published by Elsevier Masson SAS.

13C NMR study of the generation of C2- and C3-deuterated lactic acid by tumoral pancreatic islet cells exposed to D-[1-13C]-, D-[2-13C]- and D-[6-13C]-glucose in 2H2O.

PubMed

Willem, R; Biesemans, M; Kayser, F; Malaisse, W J

1994-03-01

Tumoral pancreatic islet cells of the RIN5mF line were incubated for 120 min in media prepared in 2H2O and containing D-[1-13C]glucose, D-[2-13C]glucose, and D-[6-13C]glucose. The generation of C2- and C3-deuterated lactic acid was assessed by 13C NMR. The interpretation of experimental results suggests that a) the efficiency of deuteration on the C1 of D-fructose 6-phosphate does not exceed about 47% and 4% in the phosphoglucoisomerase and phosphomannoisomerase reactions, respectively; b) approximately 38% of the molecules of D-glyceraldehyde 3-phosphate generated from D-glucose escape deuteration in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase; and c) about 41% of the molecules of pyruvate generated by glycolysis are immediately converted to lactate, the remaining 59% of pyruvate molecules undergoing first a single or double back-and-forth interconversion with L-alanine. It is proposed that this methodological approach, based on high resolution 13C NMR spectroscopy, may provide novel information on the regulation of back-and-forth interconversion of glycolytic intermediates in intact cells as modulated, for instance, by enzyme-to-enzyme tunneling.

Generation of C3- and C2-deuterated L-lactic acid by human erythrocytes exposed to D-[1-13C]glucose, D-[2-13C]glucose and D-[6-13C]glucose in the presence of D2O.

PubMed

Malaisse, W J; Biesemans, M; Willem, R

1994-05-01

1. The generation of C2- and C3-deuterated L-lactate was monitored by 13C NMR in human erythrocytes exposed to D-[1-13C]glucose, D-[2-13C]glucose or D-[6-13C]glucose and incubated in a medium prepared in D2O. 2. The results suggested that the deuteration of the C1 of D-fructose 6-phosphate in the phosphoglucoisomerase reaction, the deuteration of the C1 of D-glyceraldehyde-3-phosphate in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase and the deuteration of the C3 of pyruvate in the reaction catalyzed by pyruvate kinase were all lower than expected from equilibration with D2O. 3. Moreover, about 40% of the molecules of pyruvate generated by glycolysis apparently underwent deuteration on their C3 during interconversion of the 2-keto acid and L-alanine in the reaction catalyzed by glutamate-pyruvate transaminase. 4. The occurrence of the latter process was also documented in cells exposed to exogenous [3-13C]pyruvate. 5. This methodological approach is proposed to provide a new tool to assess in intact cells the extent of back-and-forth interconversion of selected metabolic intermediates.

Glucose respiration in the intact chloroplast of Chlamydomonas reinhardtii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Changguo Chen; Gibbs, M.

1991-01-01

Chloroplastic respiration was monitored by measuring {sup 14}CO{sub 2} from {sup 14}C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast, The patterns of {sup 14}CO{sub 2} evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolypyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K{sub m} for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of {sup 14}CO{sub 2} was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO{sub 2} evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1,more » C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO{sub 2} evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH{sub 4}Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolypyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to Co{sub 2} and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.« less

In vivo operation of the pentose phosphate pathway in frog oocytes is limited by NADP+ availability.

PubMed

Preller, A; Guixé, V; Ureta, T

1999-03-05

Evolution of CO2 from labelled glucose microinjected into frog oocytes in vivo may be ascribed to the pentose-P pathway, as measured by radioactive CO2 production from [1-(14)C] and [6-(14)C]glucose. Coinjection of NADP+ and [14C]glucose significantly stimulated 14CO2 production. The effect depends on the amount of NADP+ injected, half maximal stimulation being obtained at 0.13 mM. The increase in CO2 production was also observed with microinjected glucose-1-P, glucose-6-P or fructose-6-P used as substrates. Phenazine methosulfate, mimicked the effects of NADP+. A high NADPH/NADP+ ratio of 4.3 was found in the cells, the intracellular concentration of NADP+ being 19 microM.

The purification and properties of human liver ketohexokinase. A role for ketohexokinase and fructose-bisphosphate aldolase in the metabolic production of oxalate from xylitol.

PubMed Central

Bais, R; James, H M; Rofe, A M; Conyers, R A

1985-01-01

Ketohexokinase (EC 2.7.1.3) was purified to homogeneity from human liver, and fructose-bisphosphate aldolase (EC 4.1.2.13) was partially purified from the same source. Ketohexokinase was shown, by column chromatography and polyacrylamide-gel electrophoresis, to be a dimer of Mr 75000. Inhibition studies with p-chloromercuribenzoate and N-ethylmaleimide indicate that ketohexokinase contains thiol groups, which are required for full activity. With D-xylulose as substrate, ketohexokinase and aldolase can catalyse a reaction sequence which forms glycolaldehyde, a known precursor of oxalate. The distribution of both enzymes in human tissues indicates that this reaction sequence occurs mainly in the liver, to a lesser extent in the kidney, and very little in heart, brain and muscle. The kinetic properties of ketohexokinase show that this enzyme can phosphorylate D-xylulose as readily as D-fructose, except that higher concentrations of D-xylulose are required. The kinetic properties of aldolase show that the enzyme has a higher affinity for D-xylulose 1-phosphate than for D-fructose 1-phosphate. These findings support a role for ketohexokinase and aldolase in the formation of glycolaldehyde. The effect of various metabolites on the activity of the two enzymes was tested to determine the conditions that favour the formation of glycolaldehyde from xylitol. The results indicate that few of these metabolites affect the activity of ketohexokinase, but that aldolase can be inhibited by several phosphorylated compounds. This work suggests that, although the formation of oxalate from xylitol is normally a minor pathway, under certain conditions of increased xylitol metabolism oxalate production can become significant and may result in oxalosis. Images Fig. 1. PMID:2996495

Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

PubMed Central

Ramos, Ana; Boels, Ingeborg C.; de Vos, Willem M.; Santos, Helena

2001-01-01

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation. PMID:11133425

Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.

PubMed Central

Reznick, A Z; Rosenfelder, L; Shpund, S; Gershon, D

1985-01-01

A method has been developed that enables us to identify intracellular degradation intermediates of fructose-bisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This method is based on the use of antibody against thoroughly denatured purified aldolase. This antibody has been shown to recognize only denatured molecules, and it did not interact with "native" enzyme. supernatants (24,000 X g for 30 min) of liver and kidney homogenates were incubated with antiserum to denatured enzyme. The antigen-antibody precipitates thus formed were subjected to NaDodSO4/PAGE, followed by electrotransfer to nitrocellulose paper and immunodecoration with antiserum to denatured enzyme and 125I-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of the intact subunit) to 18,000, which cross-reacted antigenically with denatured fructose-bisphosphate aldolase, could be identified in liver. The longest three peptides were also present in kidney. The possibility that these peptides were artifacts of homogenization was ruled out as follows: 125I-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were than subjected to NaDodSO4/PAGE followed by autoradiography, and the labeled enzyme was shown to remain intact. This method is suggested for general use in the search for degradation products of other cellular proteins. Images PMID:3898080

Fructose downregulates miR-330 to induce renal inflammatory response and insulin signaling impairment: Attenuation by morin.

PubMed

Gu, Ting-Ting; Song, Lin; Chen, Tian-Yu; Wang, Xing; Zhao, Xiao-Juan; Ding, Xiao-Qin; Yang, Yan-Zi; Pan, Ying; Zhang, Dong-Mei; Kong, Ling-Dong

2017-08-01

Fructose induces insulin resistance with kidney inflammation and injury. MicroRNAs are emerged as key regulators of insulin signaling. Morin has insulin-mimetic effect with the improvement of insulin resistance and kidney injury. This study investigated the protective mechanisms of morin against fructose-induced kidney injury, with particular focus on miR-330 expression change, inflammatory response, and insulin signaling impairment. miR-330, sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptor (S1PR)1/3 signaling, nuclear factor-κB (NF-κB)/NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, and insulin signaling were detected in kidney cortex of fructose-fed rats and fructose-exposed HK-2 cells, respectively. Whether miR-330 mediated inflammatory response to affect insulin signaling was examined using SphK1 inhibitor, S1PR1/3 short interfering RNA, or miR-330 mimic/inhibitor, respectively. Fructose was found to downregulate miR-330 expression to increase SphK1/S1P/S1PR1/3 signaling, and then activate NF-κB/NLRP3 inflammasome to produce IL-1β, causing insulin signaling impairment. Moreover, morin upregulated miR-330 and partly attenuated inflammatory response and insulin signaling impairment to alleviate kidney injury. These findings suggest that morin protects against fructose-induced kidney insulin signaling impairment by upregulating miR-330 to reduce inflammatory response. Morin may be a potential therapeutic agent for the treatment of kidney injury associated with fructose-induced inflammation and insulin signaling impairment. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Respiratory metabolism in the embryonic axis of germinating pea seed exposed to cadmium.

PubMed

Smiri, Moêz; Chaoui, Abdelilah; El Ferjani, Ezzedine

2009-02-15

Seeds of pea (Pisum sativum L.) were germinated for 5d by soaking in distilled water or 5mM cadmium nitrate. The relationships among cadmium stress, germination rate, changes in respiratory enzyme activities and carbohydrates mobilization were studied. Two cell fractions were obtained from embryonic axis: (1) mitochondria, used to determine enzyme activities of citric acid cycle and electron transport chain, and (2) soluble, to measure some enzyme activities involved in fermentation and pentose phosphate pathway. Activities of malate- and succinate-dehydrogenases (MDH, SDH) and NADH- and succinate-cytochrome c reductases (NCCR, SCCR) were rapidly inhibited, while cytochrome c oxidase (CCO) was unaltered by cadmium treatment. However, this stimulated the NADPH-generating enzyme activities of the pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases (G6PDH, 6PGDH), as well as enzyme activity of fermentation, alcohol dehydrogenase (ADH), with concomitant inhibition in the capacity of enzyme inactivator (INADH). Moreover, Cd restricted carbohydrate mobilization in the embryonic axis. Almost no glucose and less than 7% of control fructose and total soluble sugars were available in the embryo tissues after 5d of exposure to cadmium. Cotyledonary invertase isoenzyme activity was also inhibited by Cd. The results indicate that cadmium induces disorder in the resumption of respiration in germinating pea seeds. The contribution of Cd-stimulated alternative metabolic pathways to compensate for the failure in mitochondrial respiration is discussed in relation to the delay in seed germination and embryonic axis growth.

Regulation of phosphate transport and AMPK signal pathway by lower dietary phosphorus of broilers

PubMed Central

Miao, Zhiqiang; Feng, Yan; Zhang, Junzhen; Tian, Wenxia; Li, Jianhui; Yang, Yu

2017-01-01

Lower available P (aP) was used as a base value in nutritional strategies for mitigating P pollution by animal excreta. We hypothesized that the mechanism regulating phosphate transport under low dietary P might be related with the AMPK signal pathway. A total of 144 one-day-old Arbor Acres Plus broilers were randomly allocated to control (HP) or trial (LP) diets, containing 0.45 and 0.23% aP, respectively. Growth performance, blood, intestinal, and renal samples were tested in 21-day-old broilers. Results shown that LP decreased body weight gain and feed intake. Higher serum Ca and fructose, but lower serum P and insulin were detected in LP-fed broilers. NaPi-IIb mRNA expression in intestine and NaPi-IIa mRNA expression in kidney were higher in the LP group. AMP: ATP, p-AMPK: total AMPK, and p-ACC: total ACC ratios in the duodenal mucosa were decreased in the LP group, whereas the p-mTOR: total mTOR ratio increased. These findings suggested that the increase in phosphate transport owing to LP diet might be regulated either directly by higher mTOR activity or indirectly by the suppressive AMPK signal, with corresponding changes in blood insulin and fructose content. A novel viewpoint on the regulatory mechanism underlying phosphate transport under low dietary P conditions was revealed, which might provide theoretical guidelines for reducing P pollution by means of nutritional regulation. PMID:29296204

Regulatory properties of 6-phosphofructokinase and control of glycolysis in boar spermatozoa.

PubMed

Kamp, G; Schmidt, H; Stypa, H; Feiden, S; Mahling, C; Wegener, G

2007-01-01

Glycolysis is crucial for sperm functions (motility and fertilization), but how this pathway is regulated in spermatozoa is not clear. This prompted to study the location and the regulatory properties of 6-phosphofructokinase (PFK, EC 2.7.1.11), the most important element for control of glycolytic flux. Unlike some other glycolytic enzymes, PFK showed no tight binding to sperm structures. It could readily be extracted from ejaculated boar spermatozoa by sonication and was then chromatographically purified. At physiological pH, the enzyme was allosterically inhibited by near-physiological concentrations of its co-substrate ATP, which induced co-operativity, i.e. reduced the affinity for the substrate fructose 6-phosphate. Inhibition by ATP was reinforced by citrate and H+. Above pH 8, PFK lost all its regulatory properties and showed maximum activity. However, in the physiological pH range, PFK activity was very sensitive to small changes in effectors. At near-physiological substrate concentrations, PFK activity requires activators (de-inhibitors) of which the combination of AMP and fructose 2,6-bisphosphate (F2,6P2) was most efficient as a result of synergistic effects. The kinetics of PFK suggest AMP, F2,6P2, H+, and citrate as allosteric effectors controlling PFK activity in boar spermatozoa. Using immunogold labeling, PFK was localized in the mid-piece and principal piece of the flagellum as well as in the acrosomal area at the top of the head and in the cytoplasmic droplets released from the mid-piece after ejaculation.

Dual probiotic strains suppress high fructose-induced metabolic syndrome

PubMed Central

Park, Do-Young; Ahn, Young-Tae; Huh, Chul-Sung; McGregor, Robin A; Choi, Myung-Sook

2013-01-01

AIM: To investigate the effect of novel probiotics on the clinical characteristics of high-fructose induced metabolic syndrome. METHODS: Male Wistar rats aged 4 wk were fed a 70% w/w high-fructose diet (n = 27) or chow diet (n = 9) for 3 wk to induce metabolic syndrome, the rats were then randomized into groups and administered probiotic [Lactobacillus curvatus (L. curvatus) HY7601 and Lactobacillus plantarum (L. plantarum) KY1032] at 109 cfu/d or 1010 cfu/d or placebo by oral gavage for 3 wk. Food intake and body weight were measured once a week. After 6 wk, the rats were fasted for 12 h, then anesthetized with diethyl ether and sacrificed. Blood samples were taken from the inferior vena cava for plasma analysis of glucose, insulin, C-peptide, total-cholesterol, triglycerides and thiobarbituric acid-reacting substances. Real-time polymerase chain reaction was performed using mouse-specific Taqman probe sets to assess genes related to fatty acid β-oxidation, lipogenesis and cholesterol metabolism in the liver. Target gene expression was normalized to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. RESULTS: Rodents fed a high-fructose diet developed clinical characteristics of the metabolic syndrome including increased plasma glucose, insulin, triglycerides, total cholesterol and oxidative stress levels, as well as increased liver mass and liver lipids compared to chow fed controls. Probiotic treatment (L. curvatus HY7601 and L. plantarum KY1032) at high (1010 cfu/d) or low dosage (109 cfu/d) lowered plasma glucose, insulin, triglycerides and oxidative stress levels. Only high-dose probiotic treatment reduced liver mass and liver cholesterol. Probiotic treatment reduced lipogenesis via down-regulation of SREBP1, FAS and SCD1 mRNA levels and increased β-oxidation via up-regulation of PPARα and CPT2 mRNA levels. CONCLUSION: Probiotic L. curvatus HY7601 and L. plantarum KY1032 combined suppressed the clinical characteristics of high-fructose-induced metabolic syndrome, therefore, may provide a natural alternative for the treatment of diet-induced metabolic syndrome. PMID:23345951

Recent advances in the synthesis of rare sugars using DHAP-dependent aldolases.

PubMed

Li, Aimin; Cai, Li; Chen, Zhou; Wang, Mayan; Wang, Ning; Nakanishi, Hideki; Gao, Xiao-Dong; Li, Zijie

2017-11-27

The occurrence rates of non-communicable diseases like obesity, diabetes and hyperlipidemia have increased remarkably due to excessive consumption of a high-energy diet. Rare sugars therefore have become increasingly attractive owing to their unique nutritional properties. In the past two decades, various rare sugars have been successfully prepared guided by the "Izumoring strategy". As a valuable complement to the Izumoring approach, the controllable dihydroxyacetone phosphate (DHAP)-dependent aldolases have generally predictable regio- and stereoselectivity, which makes them powerful tools in C-C bond construction and rare sugar production. However, the main disadvantage for this group of aldolases is their strict substrate specificity toward the donor molecule DHAP, a very expensive and relatively unstable compound. Among the current methods involving DHAP, the one that couples DHAP production from inexpensive starting materials (for instance, glycerol, DL-glycerol 3-phosphate, dihydroxyacetone, and glucose) with aldol condensation appears to be the most promising. This review thus focuses on recent advances in the application of L-rhamnulose-1-phosphate aldolase (RhaD), L-fuculose-1-phosphate aldolase (FucA), and D-fructose-1,6-bisphosphate aldolase (FruA) for rare sugar synthesis in vitro and in vivo, while illustrating strategies for supplying DHAP in efficient and economical ways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cyanogen induced phosphorylation of D-fructose. [prebiotic modeling

NASA Technical Reports Server (NTRS)

Degani, CH.; Kawatsuji, M.; Halmann, M.

1975-01-01

It has been demonstrated that a phosphorylated sugar, identified as alpha-D-fructopyranose, can be formed as the result of cyanogen-induced phosphorylation of D-fructose at pH 8.8. The product was isolated from barium and cyclohexylammonium salts and identified on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalyzed hydrolysis, and the results of periodate oxidation and optical rotatory measurements. These results support the suggestion that the cyanogen-induced phosphorylation of free sugars could be a possible process for formation of sugar phosphates under prebiotic conditions (Halman et al., 1969).

Ferrocene-Boronic Acid-Fructose Binding Based on Dual-Plate Generator-Collector Voltammetry and Square-Wave Voltammetry.

PubMed

Li, Meng; Xu, Su-Ying; Gross, Andrew J; Hammond, Jules L; Estrela, Pedro; Weber, James; Lacina, Karel; James, Tony D; Marken, Frank

2015-06-10

The interaction of ferrocene-boronic acid with fructose is investigated in aqueous 0.1 m phosphate buffer at pH 7, 8 and 9. Two voltammetric methods, based on 1) a dual-plate generator-collector micro-trench electrode (steady state) and 2) a square-wave voltammetry (transient) method, are applied and compared in terms of mechanistic resolution. A combination of experimental data is employed to obtain new insights into the binding rates and the cumulative binding constants for both the reduced ferrocene-boronic acid (pH dependent and weakly binding) and for the oxidised ferrocene-boronic acid (pH independent and strongly binding).

Molecular and biochemical characterizations of three fructose-1,6-bisphosphate aldolases from Clonorchis sinensis.

PubMed

Li, Shan; Bian, Meng; Wang, Xiaoyun; Chen, Xueqing; Xie, Zhizhi; Sun, Hengchang; Jia, Feifei; Liang, Pei; Zhou, Chenhui; He, Lei; Mao, Qiang; Huang, Bo; Liang, Chi; Wu, Zhongdao; Li, Xuerong; Xu, Jin; Huang, Yan; Yu, Xinbing

2014-01-01

Fructose-1,6-bisphosphate aldolase (FbA) is a ubiquitous enzyme in glycolysis. In the present study, we screened out three distinct genes encoding FbA isozymes (CsFbAs, CsFbA-1/2/3) from Clonorchis sinensis (C. sinensis) and characterized their sequences and structures profiles as well as biochemical properties. The amino acid sequences of CsFbAs shared homology with those of Class I FbAs from other species. The putative quaternary structures revealed that CsFbA-2 and CsFbA-3 were tetramers, while CsFbA-1 was dimer. Recombinant CsFbA-2 and CsFbA-3 (rCsFbA-2/3) were confirmed to be Class I FbAs for their stable enzymatic activities in the presence of EDTA or metal ions. However, recombinant CsFbA-1 (rCsFbA-1) did not show the catalytic activity, which might be due to the inappropriate fold and interaction between its subunits. Both rCsFbA-2 and rCsFbA-3 showed similar enzymatic properties such as optimal temperatures and broad pH ranges that similar to human FbA isozymes. They showed relatively higher affinities for fructose-1,6-bisphosphate (FBP) than fructose-1-phosphate (F-1-P). Their kcat ratios of FBP to F-1-P were in accordance with those of human FbA-A or C. In addition, CsFbAs were differentially transcribed in the developmental stages of C. sinensis, suggesting their essential roles throughout the life stages. Extensive distribution of CsFbAs in adult worms indicated that ubiquitous activities of CsFbAs took place in these organs. Collectively, these results suggested that long-term parasitic environment might adapt these isozymes similar to host FbAs for metabolic requirement. Our study will provide new insight into CsFbAs in the glycometabolism of C. sinensis and relationship between the host and the parasite. Copyright © 2014 Elsevier B.V. All rights reserved.

Disruption of the Candida albicans TPS1 gene encoding trehalose-6-phosphate synthase impairs formation of hyphae and decreases infectivity.

PubMed

Zaragoza, O; Blazquez, M A; Gancedo, C

1998-08-01

The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30 degreesC was indistinguishable from that of the wild type. However, at 42 degreesC it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37 degreesC, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42 degreesC, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 10(6) CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation.

Lacking Ketohexokinase-A Exacerbates Renal Injury in Streptozotocin-induced Diabetic Mice.

PubMed

Doke, Tomohito; Ishimoto, Takuji; Hayasaki, Takahiro; Ikeda, Satsuki; Hasebe, Masako; Hirayama, Akiyoshi; Soga, Tomoyoshi; Kato, Noritoshi; Kosugi, Tomoki; Tsuboi, Naotake; Lanaspa, Miguel A; Johnson, Richard J; Kadomatsu, Kenji; Maruyama, Shoichi

2018-03-28

Ketohexokinase (KHK), a primary enzyme in fructose metabolism, has two isoforms, namely, KHK-A and KHK-C. Previously, we reported that renal injury was reduced in streptozotocin-induced diabetic mice which lacked both isoforms. Although both isoforms express in kidney, it has not been elucidated whether each isoform plays distinct roles in the development of diabetic kidney disease (DKD). The aim of the study is to elucidate the role of KHK-A for DKD progression. Diabetes was induced by five consecutive daily intraperitoneal injections of streptozotocin (50 mg/kg) in C57BL/6 J wild-type mice, mice lacking KHK-A alone (KHK-A KO), and mice lacking both KHK-A and KHK-C (KHK-A/C KO). At 35 weeks, renal injury, inflammation, hypoxia, and oxidative stress were examined. Metabolomic analysis including polyol pathway, fructose metabolism, glycolysis, TCA (tricarboxylic acid) cycle, and NAD (nicotinamide adenine dinucleotide) metabolism in kidney and urine was done. Diabetic KHK-A KO mice developed severe renal injury compared to diabetic wild-type mice, and this was associated with further increases of intrarenal fructose, dihydroxyacetone phosphate (DHAP), TCA cycle intermediates levels, and severe inflammation. In contrast, renal injury was prevented in diabetic KHK-A/C KO mice compared to both wild-type and KHK-A KO diabetic mice. Further, diabetic KHK-A KO mice contained decreased renal NAD + level with the increase of renal hypoxia-inducible factor 1-alpha expression despite having increased renal nicotinamide (NAM) level. These results suggest that KHK-C might play a deleterious role in DKD progression through endogenous fructose metabolism, and that KHK-A plays a unique protective role against the development of DKD. Copyright © 2018. Published by Elsevier Inc.

Alloscardovia venturai sp. nov., a fructose 6-phosphate phosphoketolase-positive species isolated from the oral cavity of a guinea-pig (Cavia aperea f. porcellus).

PubMed

Sechovcová, Hana; Killer, Jiri; Pechar, Radko; Geigerová, Martina; Švejstil, Roman; Salmonová, Hana; Mekadim, Chahrazed; Rada, Vojtěch; Vlková, Eva; Kofroňová, Olga; Benada, Oldřich

2017-08-01

A slightly irregular, short rod-shaped bacterial strain, MOZIV/2T, showing activity of fructose 6-phosphate phosphoketolase was isolated from the oral cavity of a home-bred guinea-pig. Based on comparative 16S rRNA gene sequence analyses, its closest relatives were Alloscardovia omnicolens DSM 21503T and Alloscardovia criceti DSM 17774T with 96.0 and 95.6 % pairwise similarities, respectively. Completeness of the compared sequences was 97.3 and 96.9 %, respectively. Growth was found only under anaerobic conditions. Activities of α- and β-gluco(galacto)sidases were detected in strain MOZIV/2T, which is characteristic for almost all members of the family Bifidobacteriaceae. Sequencing of other molecular markers (fusA, gyrB and xfp) revealed low gene sequence similarities to A. omnicolens DSM 21503T ranging from 72.7 to 87.5 %. Strain MOZIV/2T differed from other species within the genus Alloscardovia by the presence of C18 : 1ω9t. In addition, much higher proportions of C8 : 0, C11 : 0, C12 : 0, C14 : 1, C16 : 1 and C17 : 0 fatty acids were found in cells of strain MOZIV/2T. The peptidoglycan structure was of type A4α [l-Lys(l-Orn)-d-Asp], which is consistent with its classification within the genus Alloscardovia. The DNA G+C content (45.8 mol%) was lower than those found in other alloscardovia. Phylogenetic studies and evaluation of phenotypic characteristics including the results of biochemical, physiological and chemotaxonomic analyses confirmed the novel species status for strain MOZIV/2T, for which the name Alloscardovia venturai sp. nov. is proposed. The type strain is MOZIV/2T (=DSM 100237T=CCM 8604T=LMG 28781T).

Fructose Synthesis and Transport at the Uterine-Placental Interface of Pigs: Cell-Specific Localization of SLC2A5, SLC2A8, and Components of the Polyol Pathway.

PubMed

Steinhauser, Chelsie B; Landers, McKinsey; Myatt, Louise; Burghardt, Robert C; Vallet, Jeffrey L; Bazer, Fuller W; Johnson, Greg A

2016-11-01

The fetal fluids and uterine flushings of pigs contain higher concentrations of fructose than glucose, but fructose is not detected in maternal blood. Fructose can be synthesized from glucose via enzymes of the polyol pathway, aldose reductase (AKR1B1) and sorbitol dehydrogenase (SORD), transported across cell membranes by solute carriers SLC2A5 and SLC2A8, and converted to fructose-1-phosphate by ketohexokinase (KHK). SLC2A8, SLC2A5, AKR1B1, SORD, and KHK mRNAs and proteins were analyzed using quantitative PCR and immunohistochemistry or in situ hybridization in endometria and placentae of cyclic and pregnant gilts, cyclic gilts injected with estrogen, and ovariectomized gilts injected with progesterone. Progesterone up-regulated SLC2A8 protein in uterine luminal (LE) and glandular epithelia during the peri-implantation period, and expression became exclusively placental, chorion and blood vessels, after Day 30. P4 up-regulated SLC2A5 mRNA in uterine LE and glandular epithelia after implantation, and the chorion expressed SLC2A5 between Days 30 and 85. AKR1B1 and SORD proteins localized to uterine LE during the peri-implantation period, but expression switched to chorion by Day 20 and was maintained through Day 85. Uterine expression of AKR1B1 mRNA was down-regulated by estrogen. KHK protein localized to trophectoderm/chorion throughout gestation. These results provide evidence that components for the conversion of glucose to fructose and for fructose transport are present at the uterine-placental interface of pigs. The shift in expression from LE to chorion during pregnancy suggests free-floating conceptuses are supported by fructose synthesized by the uterus, but after implantation, the chorion becomes self-sufficient for fructose synthesis and transport. © 2016 by the Society for the Study of Reproduction, Inc.

Lethality of a Heat- and Phosphate-Catalyzed Glucose By-Product to Escherichia coli O157:H7 and Partial Protection Conferred by the rpoS Regulon

PubMed Central

Byrd, Jeffrey J.; Cheville, Ann M.; Bose, Jeffrey L.; Kaspar, Charles W.

1999-01-01

A by-product of glucose produced during sterilization (121°C, 15 lb/in2, 15 min) at neutral pH and in the presence of phosphate (i.e., phosphate-buffered saline) was bactericidal to Escherichia coli O157:H7 (ATCC 43895). Other six-carbon (fructose and galactose) and five-carbon (arabinose, ribose, and xylose) reducing sugars also produced a toxic by-product under the same conditions. Fructose and the five-carbon sugars yielded the most bactericidal activity. Glucose concentrations of 1% (wt/vol) resulted in a 99.9% decline in the CFU of stationary-phase cells per milliliter in 2 days at 25°C. An rpoS mutant (pRR10::rpoS) of strain 43895 (FRIK 816-3) was significantly (P < 0.001) more sensitive to the glucose-phosphate by-product than the parent strain, as glucose concentrations from 0.05 to 0.25% resulted in a 2- to 3-log10 reduction in CFU per milliliter in 2 days at 25°C. Likewise, log-phase cells of the wild-type strain, 43895, were significantly more sensitive (P < 0.001) to the glucose-phosphate by-product than were stationary-phase cells, which is consistent with the stability of rpoS and the regulation of rpoS-regulated genes. The bactericidal effect of the glucose-phosphate by-product was reduced when strains ATCC 43895 and FRIK 816-3 were incubated at a low temperature (4°C). Also, growth in glucose-free medium (i.e., nutrient broth) did not alleviate the sensitivity to the glucose-phosphate by-product and excludes the possibility of substrate-accelerated death as the cause of the bactericidal effect observed. The glucose-phosphate by-product was also bactericidal to Salmonella typhimurium, Shigella dysenteriae, and a Klebsiella sp. Attempts to identify the glucose-phosphate by-product were unsuccessful. These studies demonstrate the production of a glucose-phosphate by-product bactericidal to E. coli O157:H7 and the protective effects afforded by rpoS-regulated gene products. Additionally, the detection of sublethally injured bacteria may be compromised by the presence of this by-product in recovery media. PMID:10347019

Gluconic acid production and phosphate solubilization by the plant growth-promoting bacterium Azospirillum spp.

NASA Astrophysics Data System (ADS)

Rodriguez, Hilda; Gonzalez, Tania; Goire, Isabel; Bashan, Yoav

2004-11-01

In vitro gluconic acid formation and phosphate solubilization from sparingly soluble phosphorus sources by two strains of the plant growth-promoting bacteria A. brasilense (Cd and 8-I) and one strain of A. lipoferum JA4 were studied. Strains of A. brasilense were capable of producing gluconic acid when grown in sparingly soluble calcium phosphate medium when their usual fructose carbon source is amended with glucose. At the same time, there is a reduction in pH of the medium and release of soluble phosphate. To a greater extent, gluconic acid production and pH reduction were observed for A. lipoferum JA4. For the three strains, clearing halos were detected on solid medium plates with calcium phosphate. This is the first report of in vitro gluconic acid production and direct phosphate solubilization by A. brasilense and the first report of P solubilization by A. lipoferum. This adds to the very broad spectrum of plant growth-promoting abilities of this genus.

Structure of a class I tagatose-1,6-bisphosphate aldolase: investigation into an apparent loss of stereospecificity.

PubMed

LowKam, Clotilde; Liotard, Brigitte; Sygusch, Jurgen

2010-07-02

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (alpha/beta)(8) fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys(205), different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2-C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.

Structure of a Class I Tagatose-1,6-bisphosphate Aldolase

PubMed Central

LowKam, Clotilde; Liotard, Brigitte; Sygusch, Jurgen

2010-01-01

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (α/β)8 fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys205, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2–C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase. PMID:20427286

Carbohydrate-based electrochemical biosensor for detection of a cancer biomarker in human plasma.

PubMed

Devillers, Marion; Ahmad, Lama; Korri-Youssoufi, Hafsa; Salmon, Laurent

2017-10-15

Autocrine motility factor (AMF) is a tumor-secreted cytokine that stimulates tumor cell motility in vitro and metastasis in vivo. AMF could be detected in serum or urine of cancer patients with worse prognosis. Reported as a cancer biomarker, AMF secretion into body fluids might be closely related to metastases formation. In this study, a sensitive and specific carbohydrate-based electrochemical biosensor was designed for the detection and quantification of a protein model of AMF, namely phosphoglucose isomerase from rabbit muscle (RmPGI). Indeed, RmPGI displays high homology with AMF and has been shown to have AMF activity. The biosensor was constructed by covalent binding of the enzyme substrate d-fructose 6-phosphate (F6P). Immobilization was achieved on a gold surface electrode following a bottom-up approach through an aminated surface obtained by electrochemical patterning of ethylene diamine and terminal amine polyethylene glycol chain to prevent non-specific interactions. Carbohydrate-protein interactions were quantified in a range of 10 fM to 100nM. Complex formation was analyzed through monitoring of the redox couple Fe 2+ /Fe 3+ by electrochemical impedance spectroscopy and square wave voltammetry. The F6P-biosensor demonstrates a detection limit of 6.6 fM and high selectivity when compared to other non-specific glycolytic proteins such as d-glucose-6-phosphate dehydrogenase. Detection of protein in spiked plasma was demonstrated and accuracy of 95% is obtained compared to result obtained in PBS (phosphate buffered saline). F6P-biosensor is a very promising proof of concept required for the design of a carbohydrate-based electrochemical biosensor using the enzyme substrate as bioreceptor. Such biosensor could be generalized to detect other protein biomarkers of interest. Copyright © 2017 Elsevier B.V. All rights reserved.

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Lili; Khusnutdinova, Anna; Nocek, Boguslaw

DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027Wmore » revealed a novel phosphatase active site with fructose 6-phosphate and Mg2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Lili; Shanklin, John; Khusnutdinova, Anna

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

A family of metal-dependent phosphatases implicated in metabolite damage-control

DOE PAGES

Huang, Lili; Shanklin, John; Khusnutdinova, Anna; ...

2016-06-20

DUF89 family proteins occur widely in pro- and eukaryotes but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), subfamily II being split into standalone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metaldependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed amore » novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.« less

2,5-Dimethyl-4-hydroxy-3(2H)-furanone as a secondary metabolite from D-fructose-1,6-diphosphate metabolism by Zygosaccharomyces rouxii.

PubMed

Dahlen, T; Hauck, T; Wein, M; Schwab, W

2001-01-01

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is an important aroma compound found in many fruits such as strawberries and pineapples and it is also produced by the soy-sauce-fermenting yeast Zygosaccharomyces rouxii after the addition of d-fructose-1,6-diphosphate to yeast-peptone-dextrose nutrient media. Dilute DMHF solutions exhibit a strawberry-like flavor while DMHF concentrates have a caramel-like aroma. In media containing D-fructose-1,6-diphosphate as the sole carbon source, growth of Z. rouxii and formation of DMHF were not observed. Although Z. rouxii cells grew in media with D-glucose as the sole carbon source, DMHF was only produced when media were supplemented with D-fructose-1,6-diphosphate. The DMHF concentration always correlated with the yeast cell count and D-fructose-1,6-diphosphate concentration. Addition of CaCl2 (up to 50 g.l(-1)) led to a higher DMHF concentration. Addition of Na2SO3 reduced the growth of Z. rouxii and inhibited DMHF formation. The amount of DMHF formed by Z. rouxii was not significantly affected by the addition of KH2PO4. DMHF concentrations of 5 and 10 g.l(-1) partially and completely inhibited the growth of Z. rouxii cells, respectively. Only the singly labeled furanone was formed after the addition of 1-13C-D-fructose-1,6-diphosphate to the medium. However, unlabeled DMHF was formed in the presence of (13)C(6)-D-glucose. Therefore, the carbons of the furanone originate exclusively from exogenously supplied D-fructose-1,6-diphosphate as no exchange with the internal pool of D-fructose-1,6-diphosphate occurs. This implies that DMHF is a secondary metabolite of Z. rouxii formed from D-fructose-1,6-diphosphate. We assume that at least the first step of the metabolism of D-fructose-1,6-diphosphate takes place in the cell wall or membrane of the yeast.

Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

PubMed

Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

1996-06-01

We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

Restoration of blood 2,3-diphosphoglycerate levels in multi-transfused patients: effect of organic and inorganic phosphate.

PubMed

Iapichino, G; Radrizzani, D; Solca, M; Franzosi, M G; Pallavicini, F B; Spina, G; Scherini, A

1984-01-01

Blood stored in acid-citrate-dextrose (ACD) shows a progressive decrease in 2,3-diphosphoglycerate (DPG) content. Since the decrease in DPG increases hemoglobin oxygen affinity, which in turn may reduce tissue and venous PO2 and peripheral oxygen delivery, many efforts have been made to preserve or restore DPG levels in stored blood. An in vivo rejuvenating technique, employing fructose-1,6-diphosphate (FDP) at a mean dosage of 1 mmol kg-1 day-1 of phosphate, to increase the DPG circulating level in multi-transfused patients is proposed. Eighteen patients, who received at least one-third of their estimated blood volume (3990 +/- 480 (SEM) ml of ACD stored blood) in blood transfusion, were treated: nine with inorganic phosphate, and nine with FDP. Basal DPG was very low in both groups: 12.61 +/- 1.34 (SEM) and 10.42 +/- 0.98 (SEM) mumol g-1, respectively (normal value is 14.5 mumol g-1, at pH 7.40). However, DPG values increased significantly and promptly in patients receiving FDP, whereas in cases of inorganic phosphate administration, it was not significantly raised over the basal value until the third day. Phosphatemia remained normal and constant with FDP, but it rose significantly on the third day of treatment with inorganic phosphate. FDP appears to consistently and rapidly increase DPG levels after transfusion with blood stored in ACD, and to be particularly safe.

Elevation of β-galactoside α2,6-sialyltransferase 1 in a fructose-responsive manner promotes pancreatic cancer metastasis

PubMed Central

Hsieh, Chi-Che; Shyr, Yi-Ming; Liao, Wen-Ying; Chen, Tien-Hua; Wang, Shin-E; Lu, Peir-Chuen; Lin, Pei-Yu; Chen, Yan-Bo; Mao, Wan-Yu; Han, Hsin-Ying; Hsiao, Michael; Yang, Wen-Bin; Li, Wen-Shan; Sher, Yuh-Pyng; Shen, Chia-Ning

2017-01-01

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of pancreatic cancer with clinical characteristics of local invasion and early metastasis. Recent cohort studies indicate high fructose intake is associated with an increase in pancreatic cancer risk. However, the mechanisms by which fructose promotes pancreatic tumorigenesis remain unclear. Herein, Kras+/LSLG12D mice were crossed with Elas-CreER transgenic mice to determine whether fructose intake directly contributes to tumor formation. Orthotopic tumor-xenograft experiments were performed to determine whether fructose substitution enhances the metastatic potential of PDAC cells. The mechanisms underlying the effects of fructose were explored by RNAseq analysis in combination with high-performance anion exchange chromatography. Dietary fructose was initially found to promote the development of aggressive pancreatic cancer in mice conditionally expressing KrasG12D in the adult pancreas. We further revealed that fructose substitution enhanced the metastatic potential of human PDAC cell via selective outgrowth of aggressive ABCG2-positive subpopulations and elevating N-acetylmannosamine levels that upregulated β-galactoside α2,6-sialyltransferase 1 (ST6Gal1), thereby promoting distant metastasis. Finally, we observed that PDAC patients expressing higher levels of ST6Gal1 and GLUT5 presented poorer prognosis compared to other groups. In conclusion, our findings have elucidated a crucial role of ST6Gal1 in regulating the invasiveness of PDACs in a fructose-responsive manner. PMID:28032597

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seegmiller, J.E.; Dixon, R.M.; Kemp, G.J.

The hyperuricemia responsible for the development of gouty arthritis results from a wide range of environmental factors and underlying genetically determined aberrations of metabolism. {sup 31}P magnetic resonance spectroscopy studies of children with hereditary fructose intolerance revealed a readily detectable rise in phosphomonoesters with a marked fall in inorganic phosphate in their liver in vivo and a rise in serum urate in response to very low doses of oral fructose. Parents and some family members heterozygous for this enzyme deficiency showed a similar pattern when given a substantially larger dose of fructose. Three of the nine heterozygotes thus identified alsomore » had clinical gout, suggesting the possibility of this defect being a fairly common cause of gout. In the present study this same noninvasive technology was used to identify the same spectral pattern in 2 of the 11 families studied with hereditary gout. In one family, the index patient's three brothers and his mother all showed the fructose-induced abnormality of metabolism, in agreement with the maternal inheritance of metabolism, in agreement with the maternal inheritance of the gout in this family group. The test dose of fructose used produced a significantly larger increment in the concentration of serum urate in the patients showing the changes in {sup 31}P magnetic resonance spectra than in the other patients with familial gout or in nonaffected members, thus suggesting a simpler method for initial screening for the defect.« less

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae.

PubMed

Ji, Shi-Qi; Wang, Bing; Lu, Ming; Li, Fu-Li

2016-02-01

Brown algae are one of the largest groups of oceanic primary producers for CO2 removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2(T). Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-d-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing D-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD(+), and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor

PubMed Central

Asención Diez, Matías D.; Peirú, Salvador; Demonte, Ana M.; Gramajo, Hugo

2012-01-01

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high Vmax in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (Vmax of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium. PMID:22210767

Fructose- and glucose-conditioned preferences in FVB mice: strain differences in post-oral sugar appetition

PubMed Central

Zukerman, Steven; Ackroff, Karen

2014-01-01

Recent studies indicate that, unlike glucose, fructose has little or no post-oral preference conditioning actions in C57BL/6J (B6) mice. The present study determined whether this is also the case for FVB mice, which overconsume fructose relative to B6 mice. In experiment 1, FVB mice strongly preferred a noncaloric 0.1% sucralose + 0.1% saccharin (S+S) solution to 8% fructose in a 2-day choice test but switched their preference to fructose after separate experience with the two sweeteners. Other FVB mice displayed a stronger preference for 8% glucose over S+S. In a second experiment, ad libitum-fed FVB mice trained 24 h/day acquired a significant preference for a flavor (CS+) paired with intragastric (IG) self-infusions of 16% fructose over a different flavor (CS−) paired with IG water infusions. IG fructose infusions also conditioned flavor preferences in food-restricted FVB mice trained 1 h/day. IG infusions of 16% glucose conditioned stronger preferences in FVB mice trained 24- or 1 h/day. Thus, fructose has post-oral flavor conditioning effects in FVB mice, but these effects are less pronounced than those produced by glucose. Further studies of the differential post-oral conditioning effects of fructose and glucose in B6 and FVB mice should enhance our understanding of the physiological processes involved in sugar reward. PMID:25320345

Fructose- and glucose-conditioned preferences in FVB mice: strain differences in post-oral sugar appetition.

PubMed

Sclafani, Anthony; Zukerman, Steven; Ackroff, Karen

2014-12-15

Recent studies indicate that, unlike glucose, fructose has little or no post-oral preference conditioning actions in C57BL/6J (B6) mice. The present study determined whether this is also the case for FVB mice, which overconsume fructose relative to B6 mice. In experiment 1, FVB mice strongly preferred a noncaloric 0.1% sucralose + 0.1% saccharin (S+S) solution to 8% fructose in a 2-day choice test but switched their preference to fructose after separate experience with the two sweeteners. Other FVB mice displayed a stronger preference for 8% glucose over S+S. In a second experiment, ad libitum-fed FVB mice trained 24 h/day acquired a significant preference for a flavor (CS+) paired with intragastric (IG) self-infusions of 16% fructose over a different flavor (CS-) paired with IG water infusions. IG fructose infusions also conditioned flavor preferences in food-restricted FVB mice trained 1 h/day. IG infusions of 16% glucose conditioned stronger preferences in FVB mice trained 24- or 1 h/day. Thus, fructose has post-oral flavor conditioning effects in FVB mice, but these effects are less pronounced than those produced by glucose. Further studies of the differential post-oral conditioning effects of fructose and glucose in B6 and FVB mice should enhance our understanding of the physiological processes involved in sugar reward. Copyright © 2014 the American Physiological Society.

CAST/EiJ and C57BL/6J Mice Differ in Their Oral and Postoral Attraction to Glucose and Fructose.

PubMed

Sclafani, Anthony; Vural, Austin S; Ackroff, Karen

2017-03-01

A recent study indicated that CAST/EiJ and C57BL/6J mice differ in their taste preferences for maltodextrin but display similar sucrose preferences. The present study revealed strain differences in preferences for the constituent sugars of sucrose. Whereas B6 mice preferred 8% glucose to 8% fructose in 2-day tests, the CAST mice preferred fructose to glucose. These preferences emerged with repeated testing which suggested post-oral influences. In a second experiment, 2-day choice tests were conducted with the sugars versus a sucralose + saccharin (SS) mixture which is highly preferred in brief access tests. B6 mice strongly preferred glucose but not fructose to the non-nutritive SS whereas CAST mice preferred SS to both glucose and fructose even when food restricted. This implied that CAST mice are insensitive to the postoral appetite stimulating actions of the 2 sugars. A third experiment revealed, however, that intragastric glucose and fructose infusions conditioned significant but mild flavor preferences in CAST mice, whereas in B6 mice glucose conditioned a robust preference but fructose was ineffective. Thus, unlike other mouse strains and rats, glucose is not more reinforcing than fructose in CAST mice. Their oral preference for fructose over glucose may be related to a subsensitive maltodextrin receptor or glucose-specific receptor which is stimulated by glucose but not fructose. The failure of CAST mice to prefer glucose to a non-nutritive sweetener distinguishes this strain from other mouse strains and rats. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Regulation of Acetate Kinase Isozymes and Its Importance for Mixed-Acid Fermentation in Lactococcus lactis

PubMed Central

Puri, Pranav; Goel, Anisha; Bochynska, Agnieszka

2014-01-01

Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The Km values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate. PMID:24464460

High fructose-mediated attenuation of insulin receptor signaling does not affect PDGF-induced proliferative signaling in vascular smooth muscle cells.

PubMed

Osman, Islam; Poulose, Ninu; Ganapathy, Vadivel; Segar, Lakshman

2016-11-15

Insulin resistance is associated with accelerated atherosclerosis. Although high fructose is known to induce insulin resistance, it remains unclear as to how fructose regulates insulin receptor signaling and proliferative phenotype in vascular smooth muscle cells (VSMCs), which play a major role in atherosclerosis. Using human aortic VSMCs, we investigated the effects of high fructose treatment on insulin receptor substrate-1 (IRS-1) serine phosphorylation, insulin versus platelet-derived growth factor (PDGF)-induced phosphorylation of Akt, S6 ribosomal protein, and extracellular signal-regulated kinase (ERK), and cell cycle proteins. In comparison with PDGF (a potent mitogen), neither fructose nor insulin enhanced VSMC proliferation and cyclin D1 expression. d-[ 14 C(U)]fructose uptake studies revealed a progressive increase in fructose uptake in a time-dependent manner. Concentration-dependent studies with high fructose (5-25mM) showed marked increases in IRS-1 serine phosphorylation, a key adapter protein in insulin receptor signaling. Accordingly, high fructose treatment led to significant diminutions in insulin-induced phosphorylation of downstream signaling components including Akt and S6. In addition, high fructose significantly diminished insulin-induced ERK phosphorylation. Nevertheless, high fructose did not affect PDGF-induced key proliferative signaling events including phosphorylation of Akt, S6, and ERK and expression of cyclin D1 protein. Together, high fructose dysregulates IRS-1 phosphorylation state and proximal insulin receptor signaling in VSMCs, but does not affect PDGF-induced proliferative signaling. These findings suggest that systemic insulin resistance rather than VSMC-specific dysregulation of insulin receptor signaling by high fructose may play a major role in enhancing atherosclerosis and neointimal hyperplasia. Copyright © 2016 Elsevier B.V. All rights reserved.

Increased diuresis, renal vascular reactivity, and blood pressure levels in young rats fed high sodium, moderately high fructose, or their association: a comparative evaluation.

PubMed

Da Silva, Rita de Cássia Vilhena A F; de Souza, Priscila; da Silva-Santos, José Eduardo

2016-12-01

Excessive intakes of sodium or fructose have been described as risk factors for hypertension. We hypothesized that even a moderately high fructose diet (6% fructose), either alone or in combination with high sodium (4% NaCl), may impair diuresis and renal and systemic vascular reactivity, contributing to the onset of high blood pressure in rats. Male Wistar rats were fed chow containing 4% NaCl (HS), 6% fructose (MHF), or both 4% NaCl and 6% fructose (HSMHF) for 6 weeks and had their diuresis, plasma creatinine, vascular reactivity of perfused kidneys and systemic arterial pressure evaluated. We found no differences in augmented diuresis among animals given HS, MHF, or HSMHF diets. After 6 weeks both the HS and HSMHF groups had increased weight in their left kidneys, but only the HSMHF group showed augmented plasma creatinine. The effects of phenylephrine on renal vascular perfusion pressure were similarly enhanced in kidneys from the HS, MHF, and HSMHF groups, but not on the systemic arterial pressure. Although when evaluated in anesthetized rats, only the HSMHF group presented augmented blood pressure, evaluation in conscious animals revealed that both the MHF and HSMHF diets, but not the HS alone, were able to induce tachycardia and hypertension. In conclusion, a MHF diet containing 6% fructose was enough to render the renal vascular bed hyperreactive to phenylephrine and to induce both hypertension and tachycardia. The combination of 6% fructose with 4% NaCl led to plasma accumulation of creatinine and accelerated the development of tachycardia.

Chronic High Fructose Intake Reduces Serum 1,25 (OH)2D3 Levels in Calcium-Sufficient Rodents

PubMed Central

Douard, Veronique; Patel, Chirag; Lee, Jacklyn; Tharabenjasin, Phuntila; Williams, Edek; Fritton, J. Christopher; Sabbagh, Yves; Ferraris, Ronaldo P.

2014-01-01

Excessive fructose consumption inhibits adaptive increases in intestinal Ca2+ transport in lactating and weanling rats with increased Ca2+ requirements by preventing the increase in serum levels of 1,25(OH)2D3. Here we tested the hypothesis that chronic fructose intake decreases 1,25(OH)2D3 levels independent of increases in Ca2+ requirements. Adult mice fed for five wk a high glucose-low Ca2+ diet displayed expected compensatory increases in intestinal and renal Ca2+ transporter expression and activity, in renal CYP27B1 (coding for 1α-hydroxylase) expression as well as in serum 1,25(OH)2D3 levels, compared with mice fed isocaloric glucose- or fructose-normal Ca2+ diets. Replacing glucose with fructose prevented these increases in Ca2+ transporter, CYP27B1, and 1,25(OH)2D3 levels induced by a low Ca2+ diet. In adult mice fed for three mo a normal Ca2+ diet, renal expression of CYP27B1 and of CYP24A1 (24-hydroxylase) decreased and increased, respectively, when the carbohydrate source was fructose instead of glucose or starch. Intestinal and renal Ca2+ transporter activity and expression did not vary with dietary carbohydrate. To determine the time course of fructose effects, a high fructose or glucose diet with normal Ca2+ levels was fed to adult rats for three mo. Serum levels of 1,25(OH)2D3 decreased and of FGF23 increased significantly over time. Renal expression of CYP27B1 and serum levels of 1,25(OH)2D3 still decreased in fructose- compared to those in glucose-fed rats after three mo. Serum parathyroid hormone, Ca2+ and phosphate levels were normal and independent of dietary sugar as well as time of feeding. Thus, chronically high fructose intakes can decrease serum levels of 1,25(OH)2D3 in adult rodents experiencing no Ca2+ stress and fed sufficient levels of dietary Ca2+. This finding is highly significant because fructose constitutes a substantial portion of the average diet of Americans already deficient in vitamin D. PMID:24718641

BALB/c and SWR inbred mice differ in post-oral fructose appetition as revealed by sugar versus non-nutritive sweetener tests.

PubMed

Kraft, Tamar T; Huang, Donald; Lolier, Melanie; Warshaw, Deena; LaMagna, Sam; Natanova, Elona; Sclafani, Anthony; Bodnar, Richard J

2016-01-01

Recent studies indicate that C57BL/6J (B6) and FVB inbred mouse strains differ in post-oral fructose conditioning. This was demonstrated by their differential flavor conditioning response to intragastric fructose and their preference for fructose versus a non-nutritive sweetener. The present study extended this analysis to SWR and BALB/c inbred strains which are of interest because they both show robust flavor conditioning responses to fructose. In the first experiment, ad-libitum fed mice were given a series of 2-day, two-bottle preference tests between 8% fructose and a more preferred, but non-nutritive 0.1% sucralose +0.1% saccharin (S+S) solution (tests 1 & 4), and fructose or S+S versus water (tests 2 and 3). In test 1, SWR mice preferred S+S to fructose, and in tests 2 and 3, they preferred both sweeteners to water. In test 4, SWR mice switched their preference and consumed more fructose than S+S. In contrast, ad-libitum fed BALB/c mice strongly preferred S+S to fructose in both tests 1 and 4, although they preferred both sweeteners to water in tests 2 and 3. Food-restricted BALB/c mice also preferred the non-nutritive S+S to fructose in tests 1 and 4. The experience-induced fructose preference reversal observed in SWR, but not BALB/c mice indicates that fructose has a post-oral reinforcing effect in SWR mice as in FVB mice. Because B6 and FVB mice prefer glucose to fructose based on the post-oral actions of the two sugars, the second experiment compared the preferences of SWR and BALB/c mice for 8% glucose and fructose solutions. Ad-libitum fed and food-restricted SWR mice strongly preferred glucose to fructose. In contrast, ad-libitum fed BALB/c mice were indifferent to the sugars, perhaps because of their overall low intakes. Food-restricted BALB/c mice, however, strongly preferred glucose. These findings indicate that SWR and BALB/c mice differ in their preference response to the post-oral actions of fructose. Copyright © 2015 Elsevier Inc. All rights reserved.

BALB/c and SWR inbred mice differ in post-oral fructose appetition as revealed by sugar versus non-nutritive sweetener tests

PubMed Central

Kraft, Tamar T.; Huang, Donald; Lolier, Melanie; Warshaw, Deena; LaMagna, Sam; Natanova, Elona; Sclafani, Anthony; Bodnar, Richard J.

2017-01-01

Recent studies indicate that C57BL/6J (B6) and FVB inbred mouse strains differ in post-oral fructose conditioning. This was demonstrated by their differential flavor conditioning response to intragastric fructose and their preference for fructose versus a non-nutritive sweetener. The present study extended this analysis to SWR and BALB/c inbred strains which are of interest because they both show robust flavor conditioning responses to fructose. In the first experiment, ad-libitum fed mice were given a series of 2-day, two-bottle preference tests between 8% fructose and a more preferred, but non-nutritive 0.1% sucralose +0.1% saccharin (S + S) solution (tests 1 & 4), and fructose or S + S versus water (tests 2 and 3). In test 1, SWR mice preferred S + S to fructose, and in tests 2 and 3, they preferred both sweeteners to water. In test 4, SWR mice switched their preference and consumed more fructose than S + S. In contrast, ad-libitum fed BALB/c mice strongly preferred S + S to fructose in both tests 1 and 4, although they preferred both sweeteners to water in tests 2 and 3. Food-restricted BALB/c mice also preferred the non-nutritive S + S to fructose in tests 1 and 4. The experience-induced fructose preference reversal observed in SWR, but not BALB/c mice indicates that fructose has a post-oral reinforcing effect in SWR mice as in FVB mice. Because B6 and FVB mice prefer glucose to fructose based on the post-oral actions of the two sugars, the second experiment compared the preferences of SWR and BALB/c mice for 8% glucose and fructose solutions. Ad-libitum fed and food-restricted SWR mice strongly preferred glucose to fructose. In contrast, ad-libitum fed BALB/c mice were indifferent to the sugars, perhaps because of their overall low intakes. Food-restricted BALB/c mice, however, strongly preferred glucose. These findings indicate that SWR and BALB/c mice differ in their preference response to the post-oral actions of fructose. PMID:26485292

Disruption of the Candida albicans TPS1 Gene Encoding Trehalose-6-Phosphate Synthase Impairs Formation of Hyphae and Decreases Infectivity†

PubMed Central

Zaragoza, Oscar; Blazquez, Miguel A.; Gancedo, Carlos

1998-01-01

The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomyces cerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or after heat shock. Growth of the tps1/tps1 disruptant at 30°C was indistinguishable from that of the wild type. However, at 42°C it did not grow on glucose or fructose but grew normally on galactose or glycerol. At 37°C, the yeast-hypha transition in the mutant in glucose-calf serum medium did not occur. During growth at 42°C, the mutant did not form hyphae in galactose or in glycerol. Some of the growth defects observed may be traced to an unbalanced sugar metabolism that reduces the cellular content of ATP. Mice inoculated with 106 CFU of the tps1/tps1 mutant did not show visible symptoms of infection 16 days after inoculation, while those similarly inoculated with wild-type cells were dead 12 days after inoculation. PMID:9683476

A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes

PubMed Central

Zhang, Guo-Qing; Wu, Ying-Ying; Ng, Tzi-Bun; Chen, Qing-Jun; Wang, He-Xiang

2013-01-01

A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM. PMID:23586045

Effect of trichloroethylene (TCE) toxicity on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in kidney and other rat tissues.

PubMed

Khan, Sheeba; Priyamvada, Shubha; Khan, Sara A; Khan, Wasim; Farooq, Neelam; Khan, Farah; Yusufi, A N K

2009-07-01

Trichloroethylene (TCE), an industrial solvent, is a major environmental contaminant. Histopathological examinations revealed that TCE caused liver and kidney toxicity and carcinogenicity. However, biochemical mechanism and tissue response to toxic insult are not completely elucidated. We hypothesized that TCE induces oxidative stress to various rat tissues and alters their metabolic functions. Male Wistar rats were given TCE (1000 mg/kg/day) in corn oil orally for 25 d. Blood and tissues were collected and analyzed for various biochemical and enzymatic parameters. TCE administration increased blood urea nitrogen, serum creatinine, cholesterol and alkaline phosphatase but decreased serum glucose, inorganic phosphate and phospholipids indicating kidney and liver toxicity. Activity of hexokinase, lactate dehydrogenase increased in the intestine and liver whereas decreased in renal tissues. Malate dehydrogenase and glucose-6-phosphatase and fructose-1, 6-bisphosphatase decreased in all tissues whereas increased in medulla. Glucose-6-phosphate dehydrogenase increased but NADP-malic enzyme decreased in all tissues except in medulla. The activity of BBM enzymes decreased but renal Na/Pi transport increased. Superoxide dismutase and catalase activities variably declined whereas lipid peroxidation significantly enhanced in all tissues. The present results indicate that TCE caused severe damage to kidney, intestine, liver and brain; altered carbohydrate metabolism and suppressed antioxidant defense system.

Fructose in Breast Milk Is Positively Associated with Infant Body Composition at 6 Months of Age.

PubMed

Goran, Michael I; Martin, Ashley A; Alderete, Tanya L; Fujiwara, Hideji; Fields, David A

2017-02-16

Dietary sugars have been shown to promote excess adiposity among children and adults; however, no study has examined fructose in human milk and its effects on body composition during infancy. Twenty-five mother-infant dyads attended clinical visits to the Oklahoma Health Sciences Center at 1 and 6 months of infant age. Infants were exclusively breastfed for 6 months and sugars in breast milk (i.e., fructose, glucose, lactose) were measured by Liquid chromatography-mass spectrometry (LC-MS/MS) and glucose oxidase. Infant body composition was assessed using dual-energy X-ray absorptiometry at 1 and 6 months. Multiple linear regression was used to examine associations between breast milk sugars and infant body composition at 6 months of age. Fructose, glucose, and lactose were present in breast milk and stable across visits (means = 6.7 μg/mL, 255.2 μg/mL, and 7.6 g/dL, respectively). Despite its very low concentration, fructose was the only sugar significantly associated with infant body composition. A 1-μg/mL higher breast milk fructose was associated with a 257 g higher body weight ( p = 0.02), 170 g higher lean mass ( p = 0.01), 131 g higher fat mass ( p = 0.05), and 5 g higher bone mineral content ( p = 0.03). In conclusion, fructose is detectable in human breast milk and is positively associated with all components of body composition at 6 months of age.

Synthesis of 6-phosphofructose aspartic acid and some related Amadori compounds.

PubMed

Hansen, Alexandar L; Behrman, Edward J

2016-08-05

We describe the synthesis and characterization of 6-phosphofructose-aspartic acid, an intermediate in the metabolism of fructose-asparagine by Salmonella. We also report improved syntheses of fructose-asparagine itself and of fructose-aspartic acid. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular basis of the fructose-2,6-bisphosphatase reaction of PFKFB3: Transition state and the C-terminal function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cavalier, Michael C.; Kim, Song-Gun; Neau, David

2012-03-22

The molecular basis of fructose-2,6-bisphosphatase (F-2,6-P{sub 2}ase) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho-enzyme intermediate state (PFKFB3-P {center_dot} F-6-P), in a transition state-analogous complex (PFKFB3 {center_dot} AlF{sub 4}), and in a complex with pyrophosphate (PFKFB3 {center_dot} PP{sub i}) at resolutions of 2.45, 2.2, and 2.3 {angstrom}, respectively. Trapping the PFKFB3-P {center_dot} F-6-P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3 {center_dot} AlF{sub 4} and PFKFB3 {center_dot} PP{sub i} complexes were obtained by soaking. The PFKFB3 {center_dot} AlF{sub 4} and PFKFB3 {center_dot} PP{sub i}more » complexes resulted in removing F-6-P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3-P formation and break down, the phosphoryl donor and the acceptor are located within {approx}5.1 {angstrom}, and the pivotal point 2-P is on the same line, suggesting an 'in-line' transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent N-P bond, breaking the 2O-P bond in the substrate. The resulting high reactivity of the leaving group, 2O of F-6-P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygen of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C-terminal domain (residues 440-446) was rearranged in PFKFB3 {center_dot} PP{sub i}, implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P{sub 2}ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction.« less

6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle.

PubMed

Le, Simone Balzer; Heggeset, Tonje Marita Bjerkan; Haugen, Tone; Nærdal, Ingemar; Brautaset, Trygve

2017-05-01

D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The B. methanolicus wild-type strain MGA3 possesses two putative rpe and pfk genes encoded on plasmid pBM19 (rpe1-MGA3 and pfk1-MGA3) and on the chromosome (rpe2-MGA3 and pfk2-MGA3). The wild-type strain PB1 also encodes putative rpe and pfk genes on plasmid pBM20 (rpe1-PB1 and pfk1-PB1*); however, it only harbours a chromosomal pfk gene (pfk2-PB1). Transcription of the plasmid-encoded genes was 10-fold to 15-fold upregulated in cells growing on methanol compared to mannitol, while the chromosomal genes were transcribed at similar levels under both conditions in both strains. All seven gene products were recombinantly produced in Escherichia coli, purified and biochemically characterized. All three RPEs were active as hexamers, catalytically stimulated by Mg 2+ and Mn 2+ and displayed similar K' values (56-75 μM) for ribulose 5-phosphate. Rpe2-MGA3 showed displayed 2-fold lower V max (49 U/mg) and a significantly reduced thermostability compared to the two Rpe1 proteins. Pfk1-PB1* was shown to be non-functional. The PFKs were active both as octamers and as tetramers, were catalytically stimulated by Mg 2+ and Mn 2+ , and displayed similar thermostabilities. The PFKs have similar K m values for fructose 6-phosphate (0.61-0.94 μM) and for ATP (0.38-0.82 μM), while Pfk1-MGA3 had a 2-fold lower V max (6.3 U/mg) compared to the two Pfk2 proteins. Our results demonstrate that MGA3 and PB1 exert alternative solutions to plasmid-dependent methylotrophy, including genetic organization, regulation, and biochemistry of RuMP cycle enzymes.

Fructose utilization during exercise in men: rapid conversion of ingested fructose to circulating glucose.

PubMed

Jandrain, B J; Pallikarakis, N; Normand, S; Pirnay, F; Lacroix, M; Mosora, F; Pachiaudi, C; Gautier, J F; Scheen, A J; Riou, J P

1993-05-01

The aim of the present study was to compare the metabolic fate of repeated doses of fructose or glucose ingested every 30 min during long-duration moderate-intensity exercise in men. Healthy volunteers exercised for 3 h on a treadmill at 45% of their maximal oxygen consumption rate. "Naturally labeled" [13C]glucose or [13C]fructose was given orally at 25-g doses every 30 min (total feeding: 150 g; n = 6 in each group). Substrate utilization was evaluated by indirect calorimetry, and exogenous sugar oxidation was measured by isotope ratio mass spectrometry on expired CO2. Results were corrected for baseline drift in 13C/12C ratio in expired air due to exercise alone. Fructose conversion to plasma glucose was measured combining gas chromatography and isotope ratio mass spectrometry. Most of the ingested glucose was oxidized: 81 +/- 4 vs. 57 +/- 2 g/3 h for fructose (2P < 0.005). Exogenous glucose covered 20.8 +/- 1.4% of the total energy need (+/- 6.7 MJ) compared with 14.0 +/- 0.6% for fructose (2P < 0.005). The contribution of total carbohydrates was significantly higher and that of lipids significantly lower with glucose than with fructose. The blood glucose response was similar in both protocols. From 90 to 180 min, 55-60% of circulating glucose was derived from ingested fructose. In conclusion, when ingested repeatedly during moderate-intensity prolonged exercise, fructose is metabolically less available than glucose, despite a high rate of conversion to circulating glucose.

ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58*

PubMed Central

Wichelecki, Daniel J.; Vetting, Matthew W.; Chou, Liyushang; Al-Obaidi, Nawar; Bouvier, Jason T.; Almo, Steven C.; Gerlt, John A.

2015-01-01

Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. PMID:26472925

Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias.

PubMed

Sanchez-Moreno, M; Lasztity, D; Coppens, I; Opperdoes, F R

1992-09-01

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.

Characterization of vascular complications in experimental model of fructose-induced metabolic syndrome.

PubMed

El-Bassossy, Hany M; Dsokey, Nora; Fahmy, Ahmed

2014-12-01

Vascular dysfunction is an important complication associated with metabolic syndrome (MS). Here we fully characterized vascular complications in a rat model of fructose-induced MS. MS was induced by adding fructose (10%) to drinking water to male Wistar rats of 6 weeks age. Blood pressure (BP) and isolated aorta responses phenylephrine (PE), KCl, acetylcholine (ACh), and sodium nitroprusside (SNP) were recorded after 6, 9, and 12 weeks of fructose administration. In addition, serum levels of glucose, insulin, uric acid, tumor necrosis factor α (TNFα), lipids, advanced glycation end products (AGEs), and arginase activity were determined. Furthermore, aortic reactive oxygen species (ROS) generation, hemeoxygenase-1 expression, and collagen deposition were examined. Fructose administration resulted in a significant hyperinslinemia after 6 weeks which continued for 12 weeks. It was also associated with a significant increase in BP after 6 weeks which was stable for 12 weeks. Aorta isolated from MS animals showed exaggerated contractility to PE and KCl and impaired relaxation to ACh compared with control after 6 weeks which were clearer at 12 weeks of fructose administration. In addition, MS animals showed significant increases in serum levels of lipids, uric acid, AGEs, TNFα, and arginase enzyme activity after 12 weeks of fructose administration. Furthermore, aortae isolated from MS animals were characterized by increased ROS generation and collagen deposition. In conclusion, adding fructose (10%) to drinking water produces a model of MS with vascular complications after 12 weeks that are characterized by insulin resistance, hypertension, disturbed vascular reactivity and structure, hyperuricemia, dyslipidemia, and low-grade inflammation.

The Path of Carbon in Photosynthesis IV. The Identity and Sequence of the Intermediates in Sucrose Synthesis

DOE R&D Accomplishments Database

Calvin, M.; Benson, A.

1948-12-14

The synthesis of sucrose from C{sup 14}0{sub 2} by green algae has been investigated and the intermediates separated by the method of paper chromatography. It is shown that sucrose is the first free sugar appearing during photosynthesis. It is apparently formed by condensation of the glucose-I-phosphate and a fructose phosphate. A series of radioautographs of paper chromatograms of extracts from plants which have photosynthesized for different periods of time has been prepared. The results indicate that 2-phosphoglyceric acid is the first product synthesized from C0{sub 2} during photosynthesis.

Sequence Analysis and Molecular Characterization of Clonorchis sinensis Hexokinase, an Unusual Trimeric 50-kDa Glucose-6-Phosphate-Sensitive Allosteric Enzyme

PubMed Central

Chen, Tingjin; Ning, Dan; Sun, Hengchang; Li, Ran; Shang, Mei; Li, Xuerong; Wang, Xiaoyun; Chen, Wenjun; Liang, Chi; Li, Wenfang; Mao, Qiang; Li, Ye; Deng, Chuanhuan; Wang, Lexun; Wu, Zhongdao; Huang, Yan; Xu, Jin; Yu, Xinbing

2014-01-01

Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors of CsHK to interfere with glycolysis in C. sinensis. PMID:25232723

GlpR is a direct transcriptional repressor of fructose metabolic genes in Haloferax volcanii.

PubMed

Martin, Jonathan H; Rawls, Katie Sherwood; Chan, Jou Chin; Hwang, Sungmin; Martinez-Pastor, Mar; McMillan, Lana J; Prunetti, Laurence; Schmid, Amy K; Maupin-Furlow, Julie A

2018-06-18

DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and occur in select archaea. In the model archaeon Haloferax volcanii , previous work implicated GlpR, a DeoR-type transcriptional regulator, in transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase ( pfkB ) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here we compared transcriptomes of wild type and Δ glpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that Hfx. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators. IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents broad use of archaea as microbial factories for industrial products. Here we characterize how sugar uptake and use is regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in this archaeal species functions using molecular mechanisms conserved with distantly related bacterial species. Copyright © 2018 American Society for Microbiology.

Chronic fructose intake accelerates non-alcoholic fatty liver disease in the presence of essential hypertension.

PubMed

Lírio, Layla Mendonça; Forechi, Ludimila; Zanardo, Tadeu Caliman; Batista, Hiago Martins; Meira, Eduardo Frizera; Nogueira, Breno Valentim; Mill, José Geraldo; Baldo, Marcelo Perim

2016-01-01

The growing epidemic of metabolic syndrome has been related to the increased use of fructose by the food industry. In fact, the use of fructose as an ingredient has increased in sweetened beverages, such as sodas and juices. We thus hypothesized that fructose intake by hypertensive rats would have a worse prognosis in developing metabolic disorder and non-alcoholic fatty liver disease. Male Wistar and SHR rats aged 6weeks were given water or fructose (10%) for 6weeks. Blood glucose was measured every two weeks, and insulin and glucose sensitivity tests were assessed at the end of the follow-up. Systolic blood pressure was measure by plethysmography. Lean mass and abdominal fat mass were collected and weighed. Liver tissue was analyzed to determine interstitial fat deposition and fibrosis. Fasting glucose increased in animals that underwent a high fructose intake, independent of blood pressure levels. Also, insulin resistance was observed in normotensive and mostly in hypertensive rats after fructose intake. Fructose intake caused a 2.5-fold increase in triglycerides levels in both groups. Fructose intake did not change lean mass. However, we found that fructose intake significantly increased abdominal fat mass deposition in normotensive but not in hypertensive rats. Nevertheless, chronic fructose intake only increased fat deposition and fibrosis in the liver in hypertensive rats. We demonstrated that, in normotensive and hypertensive rats, fructose intake increased triglycerides and abdominal fat deposition, and caused insulin resistance. However, hypertensive rats that underwent fructose intake also developed interstitial fat deposition and fibrosis in liver. Copyright © 2016 Elsevier Inc. All rights reserved.

Physical Activity Offsets the Negative Effects of a High Fructose Diet

PubMed Central

Bidwell, Amy J; Fairchild, Timothy J; Redmond, Jessica; Wang, Long; Keslacy, Stefan; Kanaley, Jill A

2014-01-01

Objective To determine the interaction between a high fructose diet and PA levels on postprandial lipidemia and inflammation in normal weight, recreationally active individuals. METHODS Twenty-two men and women (age: 21.2 ± 0.6 yrs; BMI = 22.5 ± 0.6 kg/m2) consumed an additional 75 g of fructose for 14 days on two separate occasions: high physical activity (~12,500 steps/day: FR+Active) and low PA (~ 4,500 steps/day; FR+Inactive). A fructose-rich test meal was given prior to and at the end of each intervention. Blood was sampled at baseline and for 6 h after the meal for triglycerides (TG), very-low density lipoproteins (VLDL), total cholesterol (TC), glucose, insulin, tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and c-reactive protein (CRP). RESULTS Log transformed TG AUC significantly increased from pre (10.1 ± 0.1 mg/dL x min for 6 h) to post (10.3 ± 0.08 mg/dL x min for 6 h; p = 0.04) in the FR+Inactive intervention with an 88% increase in Δpeak[TG] (p=0.009) and an 84% increase in Δpeak[VLDL] (p=0.002). Δpeak[IL-6] also increased by 116% after FR+Inactive intervention (p=0.009). Insulin tAUC significantly decreased after FR+Active intervention (p=0.04) with no change in AUC after the FR+Inactive intervention. No changes were observed in glucose, TNF-α and CRP concentrations (p>0.05). CONCLUSIONS Low physical activity during a period of high fructose intake augments fructose-induced postprandial lipidemia and inflammation while high PA minimizes these fructose-induced metabolic disturbances. Even within a young healthy population, maintenance of high PA (>12500 steps/day) decreases susceptibility to cardiovascular risk factors associated with elevated fructose consumption. PMID:24848492

Mode of action of α-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by α-chlorohydrin and location of block in glycolysis

PubMed Central

Brown-Woodman, Patricia D. C.; Mohri, Hideo; Mohri, Toshiko; Suter, Dai; White, Ian G.

1978-01-01

1. The effect of α-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by α-chlorohydrin (0.1–1.0mm) than lactate or pyruvate. Inhibition of glycolysis by α-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and α-chlorohydrin. A second, much less sensitive site, of α-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-α-chlorohydrin showed a `block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. α-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-α-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of α-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. α-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with α-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of α-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of α-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of α-chlorohydrin in vitro. PMID:629780

High-Fructose Corn-Syrup-Sweetened Beverage Intake Increases 5-Hour Breast Milk Fructose Concentrations in Lactating Women.

PubMed

Berger, Paige K; Fields, David A; Demerath, Ellen W; Fujiwara, Hideji; Goran, Michael I

2018-05-24

This study determined the effects of consuming a high-fructose corn syrup (HFCS)-sweetened beverage on breast milk fructose, glucose, and lactose concentrations in lactating women. At six weeks postpartum, lactating mothers ( n = 41) were randomized to a crossover study to consume a commercially available HFCS-sweetened beverage or artificially sweetened control beverage. At each session, mothers pumped a complete breast milk expression every hour for six consecutive hours. The baseline fasting concentrations of breast milk fructose, glucose, and lactose were 5.0 ± 1.3 µg/mL, 0.6 ± 0.3 mg/mL, and 6.8 ± 1.6 g/dL, respectively. The changes over time in breast milk sugars were significant only for fructose (treatment × time, p < 0.01). Post hoc comparisons showed the HFCS-sweetened beverage vs. control beverage increased breast milk fructose at 120 min (8.8 ± 2.1 vs. 5.3 ± 1.9 µg/mL), 180 min (9.4 ± 1.9 vs. 5.2 ± 2.2 µg/mL), 240 min (7.8 ± 1.7 vs. 5.1 ± 1.9 µg/mL), and 300 min (6.9 ± 1.4 vs. 4.9 ± 1.9 µg/mL) (all p < 0.05). The mean incremental area under the curve for breast milk fructose was also different between treatments (14.7 ± 1.2 vs. -2.60 ± 1.2 µg/mL × 360 min, p < 0.01). There was no treatment × time interaction for breast milk glucose or lactose. Our data suggest that the consumption of an HFCS-sweetened beverage increased breast milk fructose concentrations, which remained elevated up to five hours post-consumption.

Intestinal Barrier Function and the Gut Microbiome Are Differentially Affected in Mice Fed a Western-Style Diet or Drinking Water Supplemented with Fructose.

PubMed

Volynets, Valentina; Louis, Sandrine; Pretz, Dominik; Lang, Lisa; Ostaff, Maureen J; Wehkamp, Jan; Bischoff, Stephan C

2017-05-01

Background: The consumption of a Western-style diet (WSD) and high fructose intake are risk factors for metabolic diseases. The underlying mechanisms are largely unclear. Objective: To unravel the mechanisms by which a WSD and fructose promote metabolic disease, we investigated their effects on the gut microbiome and barrier function. Methods: Adult female C57BL/6J mice were fed a sugar- and fat-rich WSD or control diet (CD) for 12 wk and given access to tap water or fructose-supplemented water. The microbiota was analyzed with the use of 16S rRNA gene sequencing. Barrier function was studied with the use of permeability tests, and endotoxin, mucus thickness, and gene expressions were measured. Results: The WSD increased body weight gain but not endotoxin translocation compared with the CD. In contrast, high fructose intake increased endotoxin translocation 2.6- and 3.8-fold in the groups fed the CD + fructose and WSD + fructose, respectively, compared with the CD group. The WSD + fructose treatment also induced a loss of mucus thickness in the colon (-46%) and reduced defensin expression in the ileum and colon. The lactulose:mannitol ratio in the WSD + fructose mice was 1.8-fold higher than in the CD mice. Microbiota analysis revealed that fructose, but not the WSD, increased the Firmicutes:Bacteroidetes ratio by 88% for CD + fructose and 63% for WSD + fructose compared with the CD group. Bifidobacterium abundance was greater in the WSD mice than in the CD mice (63-fold) and in the WSD + fructose mice than in the CD + fructose mice (330-fold). Conclusions: The consumption of a WSD or high fructose intake differentially affects gut permeability and the microbiome. Whether these differences are related to the distinct clinical outcomes, whereby the WSD primarily promotes weight gain and high fructose intake causes barrier dysfunction, needs to be investigated in future studies. © 2017 American Society for Nutrition.

Glucose 6-Phosphate Dehydrogenase Deficiency Increases Redox Stress and Moderately Accelerates the Development of Heart Failure

PubMed Central

Hecker, Peter A.; Lionetti, Vincenzo; Ribeiro, Rogerio F.; Rastogi, Sharad; Brown, Bethany H.; O’Connell, Kelly A.; Cox, James W.; Shekar, Kadambari C.; Gamble, Dionna; Sabbah, Hani N.; Leopold, Jane A.; Gupte, Sachin A.; Recchia, Fabio A.; Stanley, William C.

2013-01-01

Background Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in the world. In failing hearts, G6PD is upregulated and generates NADPH that is used by the glutathione pathway to remove reactive oxygen species (ROS), but also as a substrate by ROS-generating enzymes. Therefore, G6PD deficiency might prevent heart failure by decreasing NADPH and ROS production. Methods and Results This hypothesis was evaluated in a mouse model of human G6PD deficiency (G6PDX mice, ~40% normal activity). Myocardial infarction with 3 months followup resulted in LV dilation and dysfunction in both WT and G6PDX mice, but significantly greater end diastolic volume and wall thinning in G6PDX mice. Similarly, pressure overload induced by transverse aortic constriction (TAC) for 6 weeks caused greater LV dilation in G6PDX mice than WT. We further stressed TAC mice by feeding a high fructose diet to increase flux through G6PD and ROS production, and again observed worse LV remodeling and a lower ejection fraction in G6PDX than WT mice. Tissue content of lipid peroxidation products was increased in G6PDX mice in response to infarction and aconitase activity was decreased with TAC, suggesting that G6PD deficiency increases myocardial oxidative stress and subsequent damage. Conclusions Contrary to our hypothesis, G6PD deficiency increased redox stress in response to infarction or pressure overload. However, we found only a modest acceleration of LV remodeling, suggesting that, in individuals with G6PD deficiency and concurrent hypertension or myocardial infarction, the risk for developing heart failure is higher, but limited by compensatory mechanisms. PMID:23170010

Pcal_0111, a highly thermostable bifunctional fructose-1,6-bisphosphate aldolase/phosphatase from Pyrobaculum calidifontis.

PubMed

Aziz, Iram; Rashid, Naeem; Ashraf, Raza; Bashir, Qamar; Imanaka, Tadayuki; Akhtar, Muhammad

2017-05-01

Pyrobaculum calidifontis genome harbors an open reading frame Pcal_0111 annotated as fructose bisphosphate aldolase. Although the gene is annotated as fructose bisphosphate aldolase, it exhibits a high homology with previously reported fructose-1,6-bisphosphate aldolase/phosphatase from Thermoproteus neutrophilus. To examine the biochemical properties of Pcal_0111, we have cloned and expressed the gene in Escherichia coli. Purified recombinant Pcal_0111 catalyzed both phosphatase and aldolase reactions with specific activity values of 4 U and 1.3 U, respectively. These values are highest among the fructose 1,6-bisphosphatases/aldolases characterized from archaea. The enzyme activity increased linearly with the increase in temperature until 100 °C. Recombinant Pcal_0111 is highly stable with a half-life of 120 min at 100 °C. There was no significant change in the circular dichroism spectra of the protein up to 90 °C. The enzyme activity was not affected by AMP but strongly inhibited by ATP with an IC 50 value of 0.75 mM and mildly by ADP. High thermostability and inhibition by ATP make Pcal_0111 a unique fructose 1,6-bisphosphatase/aldolase.

Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan

PubMed Central

Vieira, Diana; Figueiredo, Teresa A.; Verma, Anil; Sobral, Rita G.; Ludovice, Ana M.; de Lencastre, Hermínia; Trincao, Jose

2014-01-01

Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases. PMID:24817726

Effects of D-glucose upon D-fructose metabolism in rat hepatocytes: A 13C NMR study.

PubMed

Malaisse, W J; Ladrière, L; Verbruggen, I; Willem, R

2002-12-01

Isolated hepatocytes from fed rats were exposed for 120 min to D-glucose (10 mM) and either D-[1-13C]fructose, D-[2-13C]fructose or D-[6-13C]fructose (also 10 mM) in the presence of D2O. The identification and quantification of 13C-enriched D-fructose and its metabolites (D-glucose, L-lactate, L-alanine) in the incubation medium and the measurement of their deuterated isotopomers indicated, by comparison with a prior study conducted in the absence of exogenous D-glucose, that the major effects of the aldohexose were to increase the recovery of 13C-enriched D-fructose, decrease the production of 13C-enriched D-glucose, restrict the deuteration of the 13C-enriched isotopomers of D-glucose to those generated by cells exposed to D-[2-13C]fructose, and to accentuate the lesser deuteration of the C, (as compared to C5) of 13C-enriched D-glucose derived from D-[2-13C]fructose. The ratio between C2-deuterated and C2-hydrogenated L-lactate, as well as the relative amounts of the CH3-, CH2D-, CHD, and CD3- isotopomers of 13C-enriched L-lactate were not significantly different, however, in the absence or presence of exogenous D-glucose. These findings indicate that exogenous D-glucose suppressed the deuteration of the C1 of D-[I-13C]glucose generated by hepatocytes exposed to D-[1-13C]fructose or D-[6-13C]fructose, as otherwise attributable, in part at least, to gluconeogenesis from fructose-derived [3-13C]pyruvate, and apparently favoured the phosphorylation of D-fructose by hexokinase isoenzymes, probably through stimulation of D-fructose phosphorylation by glucokinase.

Molecular and biochemical characterization of mannitol-1-phosphate dehydrogenase from the model brown alga Ectocarpus sp.

PubMed

Bonin, Patricia; Groisillier, Agnès; Raimbault, Alice; Guibert, Anaïs; Boyen, Catherine; Tonon, Thierry

2015-09-01

The sugar alcohol mannitol is important in the food, pharmaceutical, medical and chemical industries. It is one of the most commonly occurring polyols in nature, with the exception of Archaea and animals. It has a range of physiological roles, including as carbon storage, compatible solute, and osmolyte. Mannitol is present in large amounts in brown algae, where its synthesis involved two steps: a mannitol-1-phosphate dehydrogenase (M1PDH) catalyzes a reversible reaction between fructose-6-phosphate (F6P) and mannitol-1-phosphate (M1P) (EC 1.1.1.17), and a mannitol-1-phosphatase hydrolyzes M1P to mannitol (EC 3.1.3.22). Analysis of the model brown alga Ectocarpus sp. genome provided three candidate genes for M1PDH activities. We report here the sequence analysis of Ectocarpus M1PDHs (EsM1PDHs), and the biochemical characterization of the recombinant catalytic domain of EsM1PDH1 (EsM1PDH1cat). Ectocarpus M1PDHs are representatives of a new type of modular M1PDHs among the polyol-specific long-chain dehydrogenases/reductases (PSLDRs). The N-terminal domain of EsM1PDH1 was not necessary for enzymatic activity. Determination of kinetic parameters indicated that EsM1PDH1cat displayed higher catalytic efficiency for F6P reduction compared to M1P oxidation. Both activities were influenced by NaCl concentration and inhibited by the thioreactive compound pHMB. These observations were completed by measurement of endogenous M1PDH activity and of EsM1PDH gene expression during one diurnal cycle. No significant changes in enzyme activity were monitored between day and night, although transcription of two out of three genes was altered, suggesting different levels of regulation for this key metabolic pathway in brown algal physiology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Coordinated Regulation of the EIIMan and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways

PubMed Central

Zeng, Lin; Chakraborty, Brinta; Farivar, Tanaz

2017-01-01

ABSTRACT The glucose/mannose-phosphotransferase system (PTS) permease EIIMan encoded by manLMN in the dental caries pathogen Streptococcus mutans has a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations in manL affect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of the manLMN and the fruRKI operons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EIIFru, respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EIIMan. Expression of genes for EIIMan and EIIFru was directly regulated by CcpA and CR, as evinced by in vivo and in vitro methods. Unexpectedly, not only was the fruRKI operon negatively regulated by FruR, but also so was manLMN. Carbohydrate transport by EIIMan had a negative influence on expression of manLMN but not fruRKI. In agreement with the proposed role of FruR in regulating these PTS operons, loss of fruR or fruK substantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion of fruK or fruR. Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases in S. mutans and reveal novel and important contributions of fructose metabolism to global regulation of gene expression. IMPORTANCE The ability of Streptococcus mutans and other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and express virulence-related traits. Mutants lacking the FruR regulator or a particular phosphofructokinase, FruK, display changes in expression of a large number of genes encoding transcriptional regulators, enzymes required for energy metabolism, biofilm development, biosynthetic and degradative processes, and tolerance of a spectrum of environmental stressors. Since fructose is a major component of the modern human diet, the results have substantial significance in the context of oral health and the development of dental caries. PMID:28821551

Structure of Toxoplasma gondii fructose-1,6-bisphosphate aldolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boucher, Lauren E.; Bosch, Jürgen, E-mail: jbosch@jhu.edu; Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205

The structure of T. gondii fructose-1,6-bisphosphate aldolase, a glycolytic enzyme and structural component of the invasion machinery, was determined to a resolution of 2.0 Å. The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-@@bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolasemore » has been crystallized in space group P22{sub 1}2{sub 1}, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0 Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.« less

[Discovery of the target genes inhibited by formic acid in Candida shehatae].

PubMed

Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

2014-01-04

At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

AMP-activated protein kinase-mediated glucose transport as a novel target of tributyltin in human embryonic carcinoma cells.

PubMed

Yamada, Shigeru; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2013-05-01

Organotin compounds such as tributyltin (TBT) are known to cause various forms of cytotoxicity, including developmental toxicity and neurotoxicity. However, the molecular target of the toxicity induced by nanomolar levels of TBT has not been identified. In the present study, we found that exposure to 100 nM TBT induced growth arrest in human pluripotent embryonic carcinoma cell line NT2/D1. Since glucose provides metabolic energy, we focused on the glycolytic system. We found that exposure to TBT reduced the levels of both glucose-6-phosphate and fructose-6-phosphate. To investigate the effect of TBT exposure on glycolysis, we examined glucose transporter (GLUT) activity. TBT exposure inhibited glucose uptake via a decrease in the level of cell surface-bound GLUT1. Furthermore, we examined the effect of AMP-activated protein kinase (AMPK), which is known to regulate glucose transport by facilitating GLUT translocation. Treatment with the potent AMPK activator, AICAR, restored the TBT-induced reduction in cell surface-bound GLUT1 and glucose uptake. In conclusion, these results suggest that exposure to nanomolar levels of TBT causes growth arrest by targeting glycolytic systems in human embryonic carcinoma cells. Thus, understanding the energy metabolism may provide new insights into the mechanisms of metal-induced cytotoxicity.

Fatal attraction in glycolysis: how Saccharomyces cerevisiae manages sudden transitions to high glucose

PubMed Central

Heerden, Johan H. v.; Wortel, Meike T.; Bruggeman, Frank J.; Heijnen, Joseph J.; Bollen, Yves J.; Planqué, Robert; Hulshof, Josephus; O’Toole, Tom G.; Wahl, S. A.; Teusink, Bas

2014-01-01

In the model eukaryote Saccharomyces cerevisiae, it has long been known that a functional trehalose pathway is indispensable for transitions to high glucose conditions. Upon addition of glucose, cells with a defect in trehalose 6-phosphate synthase (Tps1), the first committed step in the trehalose pathway, display what we have termed an imbalanced glycolytic state; in this state the flux through the upper part of glycolysis outpaces that through the lower part of glycolysis. As a consequence, the intermediate fructose 1,6-bisphosphate (FBP) accumulates at low concentrations of ATP and inorganic phosphate (Pi). Despite significant research efforts, a satisfactory understanding of the regulatory role that trehalose metabolism plays during such transitions has remained infamously unresolved. In a recent study, we demonstrate that the startup of glycolysis exhibits two dynamic fates: a proper, functional, steady state or the imbalanced state described above. Both states are stable, attracting states, and the probability distribution of initial states determines the fate of a yeast cell exposed to glucose. Trehalose metabolism steers the dynamics of glycolysis towards the proper functional state through its ATP hydrolysis activity; a mechanism that ensures that the demand and supply of ATP is balanced with Pi availability under dynamic conditions. [van Heerden et al. Science (2014), DOI: 10.1126/science.1245114.] PMID:28357229

Use of an Escherichia coli Recombinant Producing Thermostable Polyphosphate Kinase as an ATP Regenerator To Produce Fructose 1,6-Diphosphate▿ †

PubMed Central

Iwamoto, Seishi; Motomura, Kei; Shinoda, Yasuharu; Urata, Masaaki; Kato, Junichi; Takiguchi, Noboru; Ohtake, Hisao; Hirota, Ryuichi; Kuroda, Akio

2007-01-01

Heat-treated Escherichia coli producing Thermus polyphosphate kinase regenerated ATP by using exogenous polyphosphate. This recombinant could be used as a platform to produce valuable compounds in combination with thermostable phosphorylating or energy-requiring enzymes. In this work, we demonstrated the production of fructose 1,6-diphosphate from fructose and polyphosphate. PMID:17616610

N-acetylglucosamine, the building block of chitin, inhibits growth of Neurospora crassa.

PubMed

Gaderer, Romana; Seidl-Seiboth, Verena; de Vries, Ronald P; Seiboth, Bernhard; Kappel, Lisa

2017-10-01

N-acetylglucosamine (GlcNAc) is the monomer of the polysaccharide chitin, an essential structural component of the fungal cell wall and the arthropod exoskeleton. We recently showed that the genes encoding the enzymes for GlcNAc catabolism are clustered in several ascomycetes. In the present study we tested these fungi for growth on GlcNAc and chitin. All fungi, containing the GlcNAc gene cluster, could grow on GlcNAc with the exception of four independent Neurospora crassa wild-type isolates, which were however able to grow on chitin. GlcNAc even inhibited their growth in the presence of other carbon sources. Genes involved in GlcNAc catabolism were strongly upregulated in the presence of GlcNAc, but during growth on chitin their expression was not increased. Deletion of hxk-3 (encoding the first catabolic enzyme, GlcNAc-hexokinase) and ngt-1 (encoding the GlcNAc transporter) improved growth of N. crassa on GlcNAc in the presence of glycerol. A crucial step in GlcNAc catabolism is enzymatic conversion from glucosamine-6-phosphate to fructose-6-phosphate, catalyzed by the glucosamine-6-phosphate deaminase, DAM-1. To assess, if DAM-1 is compromised in N. crassa, the orthologue from Trichoderma reesei, Trdam1, was expressed in N. crassa. Trdam1 expression partially alleviated the negative effects of GlcNAc in the presence of a second carbon source, but did not fully restore growth on GlcNAc. Our results indicate that the GlcNAc-catabolism pathway is bypassed during growth of N. crassa on chitin by use of an alternative pathway, emphasizing the different strategies that have evolved in the fungal kingdom for chitin utilization. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI) by high-throughput screening of existing drugs.

PubMed

Eltahan, Rana; Guo, Fengguang; Zhang, Haili; Xiang, Lixin; Zhu, Guan

2018-04-01

Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (K m  = 0.309 mM, V max  = 31.72 nmol/μg/min). We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC 50  = 8.33 μM; K i  = 36.33 μM), while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC 50  = 165 μM) at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC 50 on HCT-8 cells = 700 μM). Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Biosynthesis of rare ketoses through constructing a recombination pathway in an engineered Corynebacterium glutamicum.

PubMed

Yang, Jiangang; Zhu, Yueming; Li, Jitao; Men, Yan; Sun, Yuanxia; Ma, Yanhe

2015-01-01

Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars. © 2014 Wiley Periodicals, Inc.

Development of metal-chelating inhibitors for the Class II fructose 1,6-bisphosphate (FBP) aldolase.

PubMed

Labbé, Geneviève; Krismanich, Anthony P; de Groot, Sarah; Rasmusson, Timothy; Shang, Muhong; Brown, Matthew D R; Dmitrienko, Gary I; Guillemette, J Guy

2012-07-01

It has long been suggested that the essential and ubiquitous enzyme fructose 1,6-bisphosphate (FBP) aldolase could be a good drug target against bacteria and fungi, since lower organisms possess a metal-dependant (Class II) FBP aldolase, as opposed to higher organisms which possess a Schiff-base forming (Class I) FBP aldolase. We have tested the capacity of derivatives of the metal-chelating compound dipicolinic acid (DPA), as well a thiol-containing compound, to inhibit purified recombinant Class II FBP aldolases from Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus cereus, Bacillus anthracis, and from the Rice Blast causative agent Magnaporthe grisea. The aldolase from M. tuberculosis was the most sensitive to the metal-chelating inhibitors, with an IC(50) of 5.2 μM with 2,3-dimercaptopropanesulfonate (DMPS) and 28 μM with DPA. DMPS and the synthesized inhibitor 6-(phosphonomethyl)picolinic acid inhibited the enzyme in a time-dependent, competitive fashion, with second order rate constants of 273 and 270 M(-1) s(-1) respectively for the binding of these compounds to the M. tuberculosis aldolase's active site in the presence of the substrate FBP (K(M) 27.9 μM). The most potent first generation inhibitors were modeled into the active site of the M. tuberculosis aldolase structure, with results indicating that the metal chelators tested cannot bind the catalytic zinc in a bidentate fashion while it remains in its catalytic location, and that most enzyme-ligand interactions involve the phosphate binding pocket residues. Copyright © 2012 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salminen, S.O.; Streeter, J.G.

Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with /sup 14/C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O/sub 2/ (2% in the gas phase). Uptake and conversion of /sup 14/C to CO/sub 2/ were measured at intervals up to 90 minutes. Of the five compounds studied, UDP-glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO/sub 2/, and fructose was respired at a rate of at least double that of glucose. Sucrose and glucose were converted to CO/sub 2/ at a very low but measurable rate (<0.1more » nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO/sub 2/ at a rate 30 times greater than the conversion of carbon Number 6 to CO/sub 2/, indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase.« less

Enzymatic synthesis of rare sugars with L-rhamnulose-1-phosphate aldolase from Thermotoga maritima MSB8.

PubMed

Li, Zijie; Wu, Xiaoru; Cai, Li; Duan, Shenglin; Liu, Jia; Yuan, Peng; Nakanishi, Hideki; Gao, Xiao-Dong

2015-09-15

L-Rhamnulose-1-phosphate aldolase from a thermophilic source (Thermotoga maritima MSB8) (RhaDT.mari) was heterologously overexpressed in Escherichia coli and the stereoselectivity of this enzyme with or without Nus tag was investigated. We also applied this enzyme to the synthesis of rare sugars D-psicose, D-sorbose, L-tagatose and L-fructose using our one-pot four-enzyme system. To the best of our knowledge, this is the first use of RhaD from a thermophilic source for rare sugar synthesis and the temperature tolerance of this enzyme paves the path for large scale fermentation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bifidobacterium mongoliense sp. nov., from airag, a traditional fermented mare's milk product from Mongolia.

PubMed

Watanabe, Koichi; Makino, Hiroshi; Sasamoto, Masae; Kudo, Yuko; Fujimoto, Junji; Demberel, Shirchin

2009-06-01

Two novel micro-organisms, designated strains YIT 10443(T) and YIT 10738, were isolated from airag, a traditional fermented mare's milk from Mongolia. The two strains were Gram-positive-staining, non-motile, asporogenous, catalase-negative, facultatively anaerobic rods of various shapes. Comparative analyses of 16S rRNA and ClpC ATPase (clpC) gene sequences and the presence of fructose-6-phosphate phosphoketolase (F6PPK) demonstrated that the novel strains were members of the genus Bifidobacterium. On the basis of 16S rRNA gene sequence similarity, the type strains of Bifidobacterium minimum (96.6 %) and Bifidobacterium psychraerophilum (95.7 %) were the closest neighbours of the novel strains, and DNA-DNA reassociation values with these strains were found to be lower than 15 %. The phenotypic and genotypic features demonstrated that the two strains represent a single, novel Bifidobacterium species, for which the name Bifidobacterium mongoliense sp. nov. is proposed. The type strain is YIT 10443(T) (=JCM 15461(T) =DSM 21395(T)).

Up-regulation of aldolase A and methylglyoxal production in adipocytes.

PubMed

Liu, Jianghai; Desai, Kaushik; Wang, Rui; Wu, Lingyun

2013-04-01

We previously reported that up-regulation of aldolase B, a key enzyme in fructose metabolism, was mainly responsible for vascular methylglyoxal (MG) overproduction under different pathological conditions. Here we investigated whether aldolase A, an enzyme of the glycolytic pathway, also caused MG overproduction in insulin-sensitive adipocytes. The relative contributions of different metabolic pathways or enzymes to MG generation were evaluated in cultured 3T3-L1 adipocytes. Glucose (25 mM) had no effect on aldolase A gene expression, but insulin (100 nM) up-regulated aldolase A mRNA and protein levels in the absence or presence of 25 mM glucose in adipocytes. Treatment with insulin increased levels of basal or glucose (25 mM)-induced MG and glucose 6-phosphate. However, insulin, glucose (25 mM) or their combination had no effect on cellular levels of sorbitol and fructose, but down-regulated gene expression of aldolase B to a similar extent, when compared with the control group. Incubation of 3T3-L1 adipocytes with fructose, acetone, acetol, threonine or glycine (25 mM), with or without insulin did not alter cellular MG levels. The elevated MG levels induced by insulin, glucose (25 mM) or their combination in adipocytes was completely reduced by siRNA knock down of aldolase A or application of 2-deoxy-D-glucose (a non-specific inhibitor of glucose uptake and glycolysis), but not by knock down of aldolase B. Insulin enhanced MG overproduction in insulin-sensitive adipocytes by up-regulating aldolase A, a mechanism that could be involved in the development of insulin resistance and obesity. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

The Path of Carbon in Photosynthesis XVI. Kinetic Relationships of the Intermediates in Steady State Photosynthesis

DOE R&D Accomplishments Database

Benson, A. A.; Kawaguchi, S.; Hayes, P.; Calvin, M.

1952-06-05

A kinetic study of the accumulation of C{sup 14} in the intermediates of steady state photosynthesis in C{sup 14}O{sub 2} provides information regarding the sequence of reactions involved. The work described applied the radio-chromatographic technique for analysis of the labeled early products. The simultaneous carboxylation reaction resulting in malic acid as well as phosphoglycerate is demonstrated in experiments at high light intensity. A comparison of radioactivities in a number of phosphorylated sugars as a function of time reveals concurrent synthesis of fructose and sedoheptulose phosphates followed by that of ribulose phosphates and later by that of glucose phosphates. The possibility that the cleavage of C{sub 4} compounds to C{sub 2} carbon dioxide acceptors may involve C{sub 7} and C{sub 5} sugars and evidence for this mechanism is presented.

Inborn Errors of Fructose Metabolism. What Can We Learn from Them?

PubMed

Tran, Christel

2017-04-03

Fructose is one of the main sweetening agents in the human diet and its ingestion is increasing globally. Dietary sugar has particular effects on those whose capacity to metabolize fructose is limited. If intolerance to carbohydrates is a frequent finding in children, inborn errors of carbohydrate metabolism are rare conditions. Three inborn errors are known in the pathway of fructose metabolism; (1) essential or benign fructosuria due to fructokinase deficiency; (2) hereditary fructose intolerance; and (3) fructose-1,6-bisphosphatase deficiency. In this review the focus is set on the description of the clinical symptoms and biochemical anomalies in the three inborn errors of metabolism. The potential toxic effects of fructose in healthy humans also are discussed. Studies conducted in patients with inborn errors of fructose metabolism helped to understand fructose metabolism and its potential toxicity in healthy human. Influence of fructose on the glycolytic pathway and on purine catabolism is the cause of hypoglycemia, lactic acidosis and hyperuricemia. The discovery that fructose-mediated generation of uric acid may have a causal role in diabetes and obesity provided new understandings into pathogenesis for these frequent diseases.

Inborn Errors of Fructose Metabolism. What Can We Learn from Them?

PubMed Central

Tran, Christel

2017-01-01

Fructose is one of the main sweetening agents in the human diet and its ingestion is increasing globally. Dietary sugar has particular effects on those whose capacity to metabolize fructose is limited. If intolerance to carbohydrates is a frequent finding in children, inborn errors of carbohydrate metabolism are rare conditions. Three inborn errors are known in the pathway of fructose metabolism; (1) essential or benign fructosuria due to fructokinase deficiency; (2) hereditary fructose intolerance; and (3) fructose-1,6-bisphosphatase deficiency. In this review the focus is set on the description of the clinical symptoms and biochemical anomalies in the three inborn errors of metabolism. The potential toxic effects of fructose in healthy humans also are discussed. Studies conducted in patients with inborn errors of fructose metabolism helped to understand fructose metabolism and its potential toxicity in healthy human. Influence of fructose on the glycolytic pathway and on purine catabolism is the cause of hypoglycemia, lactic acidosis and hyperuricemia. The discovery that fructose-mediated generation of uric acid may have a causal role in diabetes and obesity provided new understandings into pathogenesis for these frequent diseases. PMID:28368361

Longitudinal Associations of High-Fructose Diet with Cardiovascular Events and Potential Risk Factors: Tehran Lipid and Glucose Study.

PubMed

Bahadoran, Zahra; Mirmiran, Parvin; Tohidi, Maryam; Azizi, Fereidoun

2017-08-21

The relationship between fructose and cardiovascular disease (CVD) remains controversial. In this study, we aimed to assess possible association of dietary intakes of fructose with the risk of CVD events in a prospective population-based study. Participants without CVD ( n = 2369) were recruited from the Tehran Lipid and Glucose Study and followed a mean of 6.7 years. Dietary data were collected using a validated 168 item semi-quantitative food frequency questionnaire. Dietary total fructose (TF) intake was calculated by sum of natural fructose (NF) in fruits and vegetables and added fructose (AF) in commercial foods. Multivariate Cox proportional hazard regression models, adjusted for potential confounders, were used to estimate the risk of CVD across tertiles of dietary fructose. Linear regression models were used to indicate association of fructose intakes with changes of CVD risk factors over the study period. The mean age of participants (43.5% men) was 38.1 ± 13.3 years at baseline. During an average of 6.7 ± 1.4 years of follow-up, 79 participants experienced CVD outcomes. The mean daily intake of TF was 6.4 ± 3.7% of total energy (3.6 ± 2.0 from AF and 2.7 ± 1.8 from NF). Higher consumption of TF (≥7.4% vs. <4.5% of total energy) was accompanied with an increased risk of CVD (HR = 1.81, 95% CI = 1.04-3.15); higher energy intake from AF was also related to incidence of CVD (HR = 1.80, 95% CI = 1.04-3.12), whereas NF was not associated with the risk of CVD outcomes. Both AF and TF were also related to changes of systolic and diastolic blood pressures, waist circumference, serum insulin and creatinine levels, as well as HDL-C. Our data provides further evidence regarding undesirable effects of fructose intake in relation to risk of CVD events.

Computer Simulation of the Virulome of Bacillus anthracis Using Proteomics

DTIC Science & Technology

2006-07-31

hypothetical protein gi|47526566 spermidine /putrescine ABC transporter, spermidine /putrescine-binding protein gi|47526625 oligoendopeptidase F, putative gi...glutamyl-trna(gln) amidotransferase, a subunit x gi|50196927 aspartate aminotransferase x gi|50196970 spermidine synthase x

Noninvasive Measurement of Murine Hepatic Acetyl-CoA 13C-Enrichment Following Overnight Feeding with 13C-Enriched Fructose and Glucose

PubMed Central

Carvalho, Filipa; Duarte, Joao; Simoes, Ana Rita; Cruz, Pedro F.; Jones, John G.

2013-01-01

The 13C-isotopomer enrichment of hepatic cytosolic acetyl-CoA of overnight-fed mice whose drinking water was supplemented with [U-13C]fructose, and [1-13C]glucose and p-amino benzoic acid (PABA) was quantified by 13C NMR analysis of urinary N-acetyl-PABA. Four mice were given normal chow plus drinking water supplemented with 5% [1-13C]glucose, 2.5% [U-13C]fructose, and 2.5% fructose (Solution 1) overnight. Four were given chow and water containing 17.5% [1-13C]glucose, 8.75% [U-13C]fructose and 8.75% fructose (Solution 2). PABA (0.25%) was present in both studies. Urinary N-acetyl-PABA was analyzed by 13C NMR. In addition to [2-13C]- and [1,2-13C]acetyl isotopomers from catabolism of [U-13C]fructose and [1-13C]glucose to acetyl-CoA, [1-13C]acetyl was also found indicating pyruvate recycling activity. This precluded precise estimates of [1-13C]glucose contribution to acetyl-CoA while that of [U-13C]fructose was unaffected. The fructose contribution to acetyl-CoA from Solutions 1 and 2 was 4.0 ± 0.4% and 10.6 ± 0.6%, respectively, indicating that it contributed to a minor fraction of lipogenic acetyl-CoA under these conditions. PMID:23841082

Combined, Functional Genomic-Biochemical Approach to Intermediary Metabolism: Interaction of Acivicin, a Glutamine Amidotransferase Inhibitor, with Escherichia coli K-12

PubMed Central

Smulski, Dana R.; Huang, Lixuan L.; McCluskey, Michael P.; Reeve, Mary Jane Gladnick; Vollmer, Amy C.; Van Dyk, Tina K.; LaRossa, Robert A.

2001-01-01

Acivicin, a modified amino acid natural product, is a glutamine analog. Thus, it might interfere with metabolism by hindering glutamine transport, formation, or usage in processes such as transamidation and translation. This molecule prevented the growth of Escherichia coli in minimal medium unless the medium was supplemented with a purine or histidine, suggesting that the HisHF enzyme, a glutamine amidotransferase, was the target of acivicin action. This enzyme, purified from E. coli, was inhibited by low concentrations of acivicin. Acivicin inhibition was overcome by the presence of three distinct genetic regions when harbored on multicopy plasmids. Comprehensive transcript profiling using DNA microarrays indicated that histidine biosynthesis was the predominant process blocked by acivicin. The response to acivicin, however, was quite complex, suggesting that acivicin inhibition resonated through more than a single cellular process. PMID:11344143

A dual-specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans.

PubMed

Salazar, J C; Zúñiga, R; Raczniak, G; Becker, H; Söll, D; Orellana, O

2001-07-06

The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA(Gln) amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA(Gln) to Gln-tRNA(Gln) and Asp-tRNA(Asn) to Asn-tRNA(Asn). Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

DOE PAGES

Price, J. Vincent; Chen, Long; Whitaker, W. Brian; ...

2016-10-24

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, J. Vincent; Chen, Long; Whitaker, W. Brian

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

PubMed Central

Price, J. Vincent; Chen, Long; Whitaker, W. Brian; Papoutsakis, Eleftherios; Chen, Wilfred

2016-01-01

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. PMID:27791059

Absence of renal enlargement in fructose-fed proximal-tubule-select insulin receptor (IR), insulin-like-growth factor receptor (IGF1R) double knockout mice.

PubMed

Li, Lijun; Byrd, Marcus; Doh, Kwame; Dixon, Patrice D; Lee, Hwal; Tiwari, Swasti; Ecelbarger, Carolyn M

2016-12-01

The major site of fructose metabolism in the kidney is the proximal tubule (PT). To test whether insulin and/or IGF1 signaling in the PT is involved in renal structural/functional responses to dietary fructose, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) in PT by Cre-lox recombination, using a γ-glutamyl transferase promoter. KO mice had slightly (~10%) reduced body and kidney weights, as well as, a reduction in mean protein-to-DNA ratio in kidney cortex suggesting smaller cell size. Under control diet, IR and IGF1R protein band densities were 30-50% (P < 0.05) lower than WT, and the relative difference was greater in male animals. Male, but not female KO, also had significantly reduced band densities for Akt (protein kinase B), phosphorylated Akt T308 and IR Y 1162/1163 A high-fructose diet (1-month) led to a significant increase in kidney weight in WT males (12%), but not in KO males or in either genotype of female mice. Kidney enlargement in the WT males was accompanied by a small, insignificant fall in protein-to-DNA ratio, supporting hyperplasia rather than hypertrophy. Fructose feeding of male WT mice led to significantly higher sodium bicarbonate exchanger (NBCe1), sodium hydrogen exchanger (NHE3), sodium phosphate co-transporter (NaPi-2), and transforming growth factor-β (TGF-β) abundances, as compared to male KO, suggesting elevated transport capacity and an early feature of fibrosis may have accompanied the renal enlargement. Overall, IR and/or IGF1R appear to have a role in PT cell size and enlargement in response to high-fructose diet. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Phosphoketolase pathway contributes to carbon metabolism in cyanobacteria.

PubMed

Xiong, Wei; Lee, Tai-Chi; Rommelfanger, Sarah; Gjersing, Erica; Cano, Melissa; Maness, Pin-Ching; Ghirardi, Maria; Yu, Jianping

2015-12-07

Central carbon metabolism in cyanobacteria comprises the Calvin-Benson-Bassham (CBB) cycle, glycolysis, the pentose phosphate (PP) pathway and the tricarboxylic acid (TCA) cycle. Redundancy in this complex metabolic network renders the rational engineering of cyanobacterial metabolism for the generation of biomass, biofuels and chemicals a challenge. Here we report the presence of a functional phosphoketolase pathway, which splits xylulose-5-phosphate (or fructose-6-phosphate) to acetate precursor acetyl phosphate, in an engineered strain of the model cyanobacterium Synechocystis (ΔglgC/xylAB), in which glycogen synthesis is blocked, and xylose catabolism enabled through the introduction of xylose isomerase and xylulokinase. We show that this mutant strain is able to metabolise xylose to acetate on nitrogen starvation. To see whether acetate production in the mutant is linked to the activity of phosphoketolase, we disrupted a putative phosphoketolase gene (slr0453) in the ΔglgC/xylAB strain, and monitored metabolic flux using (13)C labelling; acetate and 2-oxoglutarate production was reduced in the light. A metabolic flux analysis, based on isotopic data, suggests that the phosphoketolase pathway metabolises over 30% of the carbon consumed by ΔglgC/xylAB during photomixotrophic growth on xylose and CO2. Disruption of the putative phosphoketolase gene in wild-type Synechocystis also led to a deficiency in acetate production in the dark, indicative of a contribution of the phosphoketolase pathway to heterotrophic metabolism. We suggest that the phosphoketolase pathway, previously uncharacterized in photosynthetic organisms, confers flexibility in energy and carbon metabolism in cyanobacteria, and could be exploited to increase the efficiency of cyanobacterial carbon metabolism and photosynthetic productivity.

Some characteristics of fructose 1,6-diphosphatase activity in rat liver

NASA Technical Reports Server (NTRS)

Ashman, P. U.; Lampkin, S. L.; Dillon, L.; Parks, R.

1974-01-01

A reliable assay for hepatic fructose 1,6-diphosphatase in the rat was developed. It was found that the greatest enzymic activity and highest protein levels were eluted from the colored portion of the homogenate. When the substrate concentration was 0.01M, the enzyme had optimal activity when incubated with 0.01M MgSO4 for 10 min. at 37 C in 0.05M Tris-HC1 buffer, pH 7.5. Specificity for the substrate, fructose 1,6-diphosphate, was obtained at substrate concentration of 0.01M.

Endurance Training with or without Glucose-Fructose Ingestion: Effects on Lactate Metabolism Assessed in a Randomized Clinical Trial on Sedentary Men.

PubMed

Rosset, Robin; Lecoultre, Virgile; Egli, Léonie; Cros, Jérémy; Rey, Valentine; Stefanoni, Nathalie; Sauvinet, Valérie; Laville, Martine; Schneiter, Philippe; Tappy, Luc

2017-04-20

Glucose-fructose ingestion increases glucose and lactate oxidation during exercise. We hypothesized that training with glucose-fructose would induce key adaptations in lactate metabolism. Two groups of eight sedentary males were endurance-trained for three weeks while ingesting either glucose-fructose (GF) or water (C). Effects of glucose-fructose on lactate appearance, oxidation, and clearance were measured at rest and during exercise, pre-training, and post-training. Pre-training, resting lactate appearance was 3.6 ± 0.5 vs. 3.6 ± 0.4 mg·kg -1 ·min -1 in GF and C, and was increased to 11.2 ± 1.4 vs. 8.8 ± 0.7 mg·kg -1 ·min -1 by exercise (Exercise: p < 0.01). Lactate oxidation represented 20.6% ± 1.0% and 17.5% ± 1.7% of lactate appearance at rest, and 86.3% ± 3.8% and 86.8% ± 6.6% during exercise (Exercise: p < 0.01) in GF and C, respectively. Training with GF increased resting lactate appearance and oxidation (Training × Intervention: both p < 0.05), but not during exercise (Training × Intervention: both p > 0.05). Training with GF and C had similar effects to increase lactate clearance during exercise (+15.5 ± 9.2 and +10.1 ± 5.9 mL·kg -1 ·min -1 ; Training: p < 0.01; Training × Intervention: p = 0.97). The findings of this study show that in sedentary participants, glucose-fructose ingestion leads to high systemic lactate appearance, most of which is disposed non-oxidatively at rest and is oxidized during exercise. Training with or without glucose-fructose increases lactate clearance, without altering lactate appearance and oxidation during exercise.

Endurance Training with or without Glucose-Fructose Ingestion: Effects on Lactate Metabolism Assessed in a Randomized Clinical Trial on Sedentary Men

PubMed Central

Rosset, Robin; Lecoultre, Virgile; Egli, Léonie; Cros, Jérémy; Rey, Valentine; Stefanoni, Nathalie; Sauvinet, Valérie; Laville, Martine; Schneiter, Philippe; Tappy, Luc

2017-01-01

Glucose-fructose ingestion increases glucose and lactate oxidation during exercise. We hypothesized that training with glucose-fructose would induce key adaptations in lactate metabolism. Two groups of eight sedentary males were endurance-trained for three weeks while ingesting either glucose-fructose (GF) or water (C). Effects of glucose-fructose on lactate appearance, oxidation, and clearance were measured at rest and during exercise, pre-training, and post-training. Pre-training, resting lactate appearance was 3.6 ± 0.5 vs. 3.6 ± 0.4 mg·kg−1·min−1 in GF and C, and was increased to 11.2 ± 1.4 vs. 8.8 ± 0.7 mg·kg−1·min−1 by exercise (Exercise: p < 0.01). Lactate oxidation represented 20.6 ± 1.0% and 17.5 ± 1.7% of lactate appearance at rest, and 86.3 ± 3.8% and 86.8 ± 6.6% during exercise (Exercise: p < 0.01) in GF and C, respectively. Training with GF increased resting lactate appearance and oxidation (Training × Intervention: both p < 0.05), but not during exercise (Training × Intervention: both p > 0.05). Training with GF and C had similar effects to increase lactate clearance during exercise (+15.5 ± 9.2 and +10.1 ± 5.9 mL·kg−1·min−1; Training: p < 0.01; Training × Intervention: p = 0.97). The findings of this study show that in sedentary participants, glucose-fructose ingestion leads to high systemic lactate appearance, most of which is disposed non-oxidatively at rest and is oxidized during exercise. Training with or without glucose-fructose increases lactate clearance, without altering lactate appearance and oxidation during exercise. PMID:28425966

Cytoprotection by fructose and other ketohexoses during bile salt-induced apoptosis of hepatocytes.

PubMed

Zeid, I M; Bronk, S F; Fesmier, P J; Gores, G J

1997-01-01

Toxic bile salts cause hepatocyte necrosis at high concentrations and apoptosis at lower concentrations. Although fructose prevents bile salt-induced necrosis, the effect of fructose on bile salt-induced apoptosis is unclear. Our aim was to determine if fructose also protects against bile salt-induced apoptosis. Fructose inhibited glycochenodeoxycholate (GCDC)-induced apoptosis in a concentration-dependent manner with a maximum inhibition of 72% +/- 10% at 10 mmol/L. First, we determined if fructose inhibited apoptosis by decreasing adenosine triphosphate (ATP) and intracellular pH (pHi). Although fructose decreased ATP to <25% of basal values, oligomycin (an ATP synthase inhibitor) did not inhibit apoptosis despite decreasing ATP to similar values. Fructose (10 mmol/L) decreased intracellular pH (pHi) by 0.2 U. However, extracellular acidification (pH 6.8), which decreased hepatocyte pHi 0.35 U and is known to inhibit necrosis, actually potentiated apoptosis 1.6-fold. Fructose cytoprotection also could not be explained by induction of bcl-2 transcription or metal chelation. Because we could not attribute fructose cytoprotection to metabolic effects, alterations in the expression of bcl-2, or metal chelation, we next determined if the poorly metabolized ketohexoses, tagatose and sorbose, also inhibited apoptosis; unexpectedly, both ketohexoses inhibited apoptosis. Because bile salt-induced apoptosis and necrosis are inhibited by fructose, these data suggest that similar processes initiate bile salt-induced hepatocyte necrosis and apoptosis. In contrast, acidosis, which inhibits necrosis, potentiates apoptosis. Thus, ketohexose-sensitive pathways appear to initiate both bile salt-induced cell apoptosis and necrosis, whereas dissimilar, pH-sensitive, effector mechanisms execute these two different cell death processes.

A novel untargeted metabolomics correlation-based network analysis incorporating human metabolic reconstructions

PubMed Central

2013-01-01

Background Metabolomics has become increasingly popular in the study of disease phenotypes and molecular pathophysiology. One branch of metabolomics that encompasses the high-throughput screening of cellular metabolism is metabolic profiling. In the present study, the metabolic profiles of different tumour cells from colorectal carcinoma and breast adenocarcinoma were exposed to hypoxic and normoxic conditions and these have been compared to reveal the potential metabolic effects of hypoxia on the biochemistry of the tumour cells; this may contribute to their survival in oxygen compromised environments. In an attempt to analyse the complex interactions between metabolites beyond routine univariate and multivariate data analysis methods, correlation analysis has been integrated with a human metabolic reconstruction to reveal connections between pathways that are associated with normoxic or hypoxic oxygen environments. Results Correlation analysis has revealed statistically significant connections between metabolites, where differences in correlations between cells exposed to different oxygen levels have been highlighted as markers of hypoxic metabolism in cancer. Network mapping onto reconstructed human metabolic models is a novel addition to correlation analysis. Correlated metabolites have been mapped onto the Edinburgh human metabolic network (EHMN) with the aim of interlinking metabolites found to be regulated in a similar fashion in response to oxygen. This revealed novel pathways within the metabolic network that may be key to tumour cell survival at low oxygen. Results show that the metabolic responses to lowering oxygen availability can be conserved or specific to a particular cell line. Network-based correlation analysis identified conserved metabolites including malate, pyruvate, 2-oxoglutarate, glutamate and fructose-6-phosphate. In this way, this method has revealed metabolites not previously linked, or less well recognised, with respect to hypoxia before. Lactate fermentation is one of the key themes discussed in the field of hypoxia; however, malate, pyruvate, 2-oxoglutarate, glutamate and fructose-6-phosphate, which are connected by a single pathway, may provide a more significant marker of hypoxia in cancer. Conclusions Metabolic networks generated for each cell line were compared to identify conserved metabolite pathway responses to low oxygen environments. Furthermore, we believe this methodology will have general application within metabolomics. PMID:24153255

Structure of Toxoplasma gondii fructose-1,6-bisphosphate aldolase.

PubMed

Boucher, Lauren E; Bosch, Jürgen

2014-09-01

The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolase has been crystallized in space group P22121, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0 Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.

Effect of Restriction of Foods with High Fructose Corn Syrup Content on Metabolic Indices and Fatty Liver in Obese Children.

PubMed

Ibarra-Reynoso, Lorena Del Rocio; López-Lemus, Hilda Lissette; Garay-Sevilla, Ma Eugenia; Malacara, Juan Manuel

2017-01-01

We examined the effect of restriction of foods with high fructose content in obese school children. In a clinical study, we selected 54 obese children 6 to 11 years old with high fructose consumption (>70 g/day) in order indicate dietary fructose restriction (<20 g/day) for 6 weeks. Anthropometry, liver ultrasound as well as glucose, insulin, lipids, leptin, IGFBP1, and RBP4 serum levels were collected. The group of children had 80% adherence and reported decreased fructose consumption (110 ± 38.6 to 11.4 ± 12.0 g/day) and also a significant decrease in caloric (2,384 ± 568 to 1,757 ± 387 kcal/day) and carbohydrate consumption (302 ± 80.4 to 203 ± 56.0 g/day). The severity of steatosis improved significantly after fructose restriction (p < 0.000001). However, no changes in BMI, systolic blood pressure, or diastolic blood pressure were found. Only triglyceride levels decreased (1.44 ± 0.43 to 1.31 ± 0.38 mmol/l), High-densitiy lipoprotein cholesterol showed a marginal increase (1.45 ± 0.19 to 1.56 ± 0.44 mmol/l). Insulin resistance and RBP4 did not change. In school children, the restriction of high fructose foods with a decrease of caloric and carbohydrate intake at 6 weeks did not induce weight loss; however, triglyceride levels and hepatic steatosis decreased. Differences with other studies in regard to weight loss may be explained by adaptive changes on metabolic expenditure. © 2017 The Author(s) Published by S. Karger GmbH, Freiburg.

Effect of Restriction of Foods with High Fructose Corn Syrup Content on Metabolic Indices and Fatty Liver in Obese Children

PubMed Central

Ibarra-Reynoso, Lorena del Rocio; López-Lemus, Hilda Lissette; Garay-Sevilla, Ma Eugenia; Malacara, Juan Manuel

2017-01-01

Objective We examined the effect of restriction of foods with high fructose content in obese school children. Methods In a clinical study, we selected 54 obese children 6 to 11 years old with high fructose consumption (>70 g/day) in order indicate dietary fructose restriction (<20 g/day) for 6 weeks. Anthropometry, liver ultrasound as well as glucose, insulin, lipids, leptin, IGFBP1, and RBP4 serum levels were collected. Results The group of children had 80% adherence and reported decreased fructose consumption (110 ± 38.6 to 11.4 ± 12.0 g/day) and also a significant decrease in caloric (2,384 ± 568 to 1,757 ± 387 kcal/day) and carbohydrate consumption (302 ± 80.4 to 203 ± 56.0 g/day). The severity of steatosis improved significantly after fructose restriction (p < 0.000001). However, no changes in BMI, systolic blood pressure, or diastolic blood pressure were found. Only triglyceride levels decreased (1.44 ± 0.43 to 1.31 ± 0.38 mmol/l), High-densitiy lipoprotein cholesterol showed a marginal increase (1.45 ± 0.19 to 1.56 ± 0.44 mmol/l). Insulin resistance and RBP4 did not change. Conclusions In school children, the restriction of high fructose foods with a decrease of caloric and carbohydrate intake at 6 weeks did not induce weight loss; however, triglyceride levels and hepatic steatosis decreased. Differences with other studies in regard to weight loss may be explained by adaptive changes on metabolic expenditure. PMID:28787728

Substitution of drinking water by fructose solution induces hyperinsulinemia and hyperglycemia in hamsters.

PubMed

Barros, Carlos Magno M R; Lessa, Rosane Q; Grechi, Mauricio P; Mouço, Tanial L M; Souza, Maria das Graças C; Wiernsperger, Nicolas; Bouskela, Eliete

2007-06-01

To test the possibility of obtaining a practical and stable model of hyperinsulinemia and hyperglycemia in hamsters, substituting the drinking water by 10% or 20% fructose solutions for a period of 2, 4, or 6 months. Male hamsters were divided into 3 main groups, further divided in 3 subgroups: Two months: Group Ia control (n = 51) received filtered water, Group Ib (n = 49) received 10% fructose solution instead of water, Group Ic (n=8) received 20% fructose solution instead of water. Four months: Group IIa control (n=8), Group IIb 10% fructose (n = 7), Group IIc 20% fructose (FIIc, n = 7). Six months: Group IIIa control (n = 6), Group IIIb 10% Fructose (n = 6), Group IIIc 20% Fructose (n = 5). All groups were fed with the same laboratory diet. The animals were weighed every 2 weeks during the study period. On the final day of each experiment (61st, 121st, and 181st day after the beginning of the study, respectively), the animals were weighed and anesthetized for blood collection to determine plasma glucose and insulin after at least a 12-h fast. Ten animals of group Ia and 10 of group Ib were evaluated to determine changes in macromolecular permeability induced by ischemia/reperfusion as measured in the cheek pouch microcirculation. Compared to controls, the animals that drank the 10% or 20% fructose solution had significantly greater weight gain (P < .001), fasting plasma glucose (P < .001) Reperfusion, after 30 min ischemia, resulted in an immediate but reversible increase in postcapillary leakage (L) of 89.0 +/- 2.0 L/cm(2) (group Ia - controls), and 116.5 +/- 4.8 L/cm(2) (group Ib 10% fructose), P < .001. These results suggest that chronic administration of either 10% or 20% fructose solutions could be used to experimentally induce a stable hamster model of hyperinsulinemia and hyperglycemia. The model might facilitate the study of basic mechanisms of hyperglycemia and hyperinsulinemia affecting the microvasculature as demonstrated by the findings regarding ischemia/reperfusion after only 2 months of treatment.

Fructose content in popular beverages made with and without high-fructose corn syrup.

PubMed

Walker, Ryan W; Dumke, Kelly A; Goran, Michael I

2014-01-01

Excess fructose consumption is hypothesized to be associated with risk for metabolic disease. Actual fructose consumption levels are difficult to estimate because of the unlabeled quantity of fructose in beverages. The aims of this study were threefold: 1) re-examine the fructose content in previously tested beverages using two additional assay methods capable of detecting other sugars, especially maltose, 2) compare data across all methods to determine the actual free fructose-to-glucose ratio in beverages made either with or without high-fructose corn syrup (HFCS), and 3) expand the analysis to determine fructose content in commonly consumed juice products. Sugar-sweetened beverages (SSBs) and fruit juice drinks that were either made with or without HFCS were analyzed in separate, independent laboratories via three different methods to determine sugar profiles. For SSBs, the three independent laboratory methods showed consistent and reproducible results. In SSBs made with HFCS, fructose constituted 60.6% ± 2.7% of sugar content. In juices sweetened with HFCS, fructose accounted for 52.1% ± 5.9% of sugar content, although in some juices made from 100% fruit, fructose concentration reached 65.35 g/L accounting for 67% of sugars. Our results provide evidence of higher than expected amounts of free fructose in some beverages. Popular beverages made with HFCS have a fructose-to-glucose ratio of approximately 60:40, and thus contain 50% more fructose than glucose. Some pure fruit juices have twice as much fructose as glucose. These findings suggest that beverages made with HFCS and some juices have a sugar profile very different than sucrose, in which amounts of fructose and glucose are equivalent. Current dietary analyses may underestimate actual fructose consumption. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Effects of high fructose corn syrup and sucrose on the pharmacokinetics of fructose and acute metabolic and hemodynamic responses in healthy subjects

PubMed Central

Le, MyPhuong T.; Frye, Reginald F.; Rivard, Christopher J.; Cheng, Jing; McFann, Kim K.; Segal, Mark S.; Johnson, Richard J.; Johnson, Julie A.

2011-01-01

Objective It is unclear whether high fructose corn syrup (HFCS), which contains a higher amount of fructose and provides an immediate source of free fructose, induces greater systemic concentrations of fructose as compared to sucrose. It is also unclear whether exposure to higher levels of fructose leads to increased fructose-induced adverse effects. The objective was to prospectively compare the effects of HFCS- versus sucrose-sweetened soft drinks on acute metabolic and hemodynamic effects. Materials/Methods Forty men and women consumed 24 oz of HFCS- or sucrose-sweetened beverages in a randomized crossover design study. Blood and urine samples were collected over 6 hr. Blood pressure, heart rate, fructose, and a variety of other metabolic biomarkers were measured. Results Fructose area under the curve and maximum concentration, dose normalized glucose area under the curve and maximum concentration, relative bioavailability of glucose, changes in postprandial concentrations of serum uric acid, and systolic blood pressure maximum levels were higher when HFCS-sweetened beverages were consumed as compared to sucrose-sweetened beverages. Conclusions Compared to sucrose, HFCS leads to greater fructose systemic exposure and significantly different acute metabolic effects. PMID:22152650

Effects of high-fructose corn syrup and sucrose on the pharmacokinetics of fructose and acute metabolic and hemodynamic responses in healthy subjects.

PubMed

Le, Myphuong T; Frye, Reginald F; Rivard, Christopher J; Cheng, Jing; McFann, Kim K; Segal, Mark S; Johnson, Richard J; Johnson, Julie A

2012-05-01

It is unclear whether high-fructose corn syrup (HFCS), which contains a higher amount of fructose and provides an immediate source of free fructose, induces greater systemic concentrations of fructose as compared with sucrose. It is also unclear whether exposure to higher levels of fructose leads to increased fructose-induced adverse effects. The objective was to prospectively compare the effects of HFCS- vs sucrose-sweetened soft drinks on acute metabolic and hemodynamic effects. Forty men and women consumed 24 oz of HFCS- or sucrose-sweetened beverages in a randomized crossover design study. Blood and urine samples were collected over 6 hours. Blood pressure, heart rate, fructose, and a variety of other metabolic biomarkers were measured. Fructose area under the curve and maximum concentration, dose-normalized glucose area under the curve and maximum concentration, relative bioavailability of glucose, changes in postprandial concentrations of serum uric acid, and systolic blood pressure maximum levels were higher when HFCS-sweetened beverages were consumed as compared with sucrose-sweetened beverages. Compared with sucrose, HFCS leads to greater fructose systemic exposure and significantly different acute metabolic effects. Copyright © 2012 Elsevier Inc. All rights reserved.

Coordinated Regulation of the EIIMan and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways.

PubMed

Zeng, Lin; Chakraborty, Brinta; Farivar, Tanaz; Burne, Robert A

2017-11-01

The glucose/mannose-phosphotransferase system (PTS) permease EII Man encoded by manLMN in the dental caries pathogen Streptococcus mutans has a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations in manL affect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of the manLMN and the fruRKI operons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EII Fru , respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EII Man Expression of genes for EII Man and EII Fru was directly regulated by CcpA and CR, as evinced by in vivo and in vitro methods. Unexpectedly, not only was the fruRKI operon negatively regulated by FruR, but also so was manLMN Carbohydrate transport by EII Man had a negative influence on expression of manLMN but not fruRKI In agreement with the proposed role of FruR in regulating these PTS operons, loss of fruR or fruK substantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion of fruK or fruR Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases in S. mutans and reveal novel and important contributions of fructose metabolism to global regulation of gene expression. IMPORTANCE The ability of Streptococcus mutans and other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and express virulence-related traits. Mutants lacking the FruR regulator or a particular phosphofructokinase, FruK, display changes in expression of a large number of genes encoding transcriptional regulators, enzymes required for energy metabolism, biofilm development, biosynthetic and degradative processes, and tolerance of a spectrum of environmental stressors. Since fructose is a major component of the modern human diet, the results have substantial significance in the context of oral health and the development of dental caries. Copyright © 2017 American Society for Microbiology.

Three Months of High-Fructose Feeding Fails to Induce Excessive Weight Gain or Leptin Resistance in Mice

PubMed Central

Tillman, Erik J.; Morgan, Donald A.; Rahmouni, Kamal; Swoap, Steven J.

2014-01-01

High-fructose diets have been implicated in obesity via impairment of leptin signaling in humans and rodents. We investigated whether fructose-induced leptin resistance in mice could be used to study the metabolic consequences of fructose consumption in humans, particularly in children and adolescents. Male C57Bl/6 mice were weaned to a randomly assigned diet: high fructose, high sucrose, high fat, or control (sugar-free, low-fat). Mice were maintained on their diets for at least 14 weeks. While fructose-fed mice regularly consumed more kcal and expended more energy, there was no difference in body weight compared to control by the end of the study. Additionally, after 14 weeks, both fructose-fed and control mice displayed similar leptin sensitivity. Fructose-feeding also did not change circulating glucose, triglycerides, or free fatty acids. Though fructose has been linked to obesity in several animal models, our data fail to support a role for fructose intake through food lasting 3 months in altering of body weight and leptin signaling in mice. The lack of impact of fructose in the food of growing mice on either body weight or leptin sensitivity over this time frame was surprising, and important information for researchers interested in fructose and body weight regulation. PMID:25211467

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity by the Activase System in Lysed Spinach Chloroplasts

PubMed Central

Parry, Martin A. J.; Keys, Alfred J.; Foyer, Christine H.; Furbank, Robert T.; Walker, David A.

1988-01-01

Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach (Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO2 and Mg2+ by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of ΔpH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between ΔpH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO2 and Mg2+. PMID:16666184

In Silico Modeling of Crabtree Effect.

PubMed

Ghosh, Debraj; De, Rajat K

2017-01-01

Glycolytic activity during Crabtree effect is similar to that in tumor cells. Research regarding Crabtree effect is very much crucial. The mechanism of metabolic activities in glycolysis pathway and oxidative phosphorylation pathway in regards to Crabtree effect in Saccharomyces cerevisiae was studied in this paper. We also explored the effects of hexose phosphates in the activities of respiratory chain complexes (III and IV) in inhibition of respiration. Besides, the enhancement of fermentation in response to excess glucose concentration was studied. We discussed the significance of Crabtree effect in mammalian cancer in terms of Crabtree effect in a Crabtree positive organism, as it is similar to cancer metabolism in mammalian cells. We developed an in silico model of Crabtree effect. A comparative study was performed with laboratory experiments regarding inhibitory role of fructose 1,6-bisphosphate on metabolic respiration. The model was simulated for different concentration levels of glucose and hexose phosphates using COPASI and SNOOPY tools. We have shown that a hike in glucose concentration increases ethanol concentration and leads glycolytic activity towards fermentation. This phenomenon occurs during Crabtree effect. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Moderate (20%) fructose-enriched diet stimulates salt-sensitive hypertension with increased salt retention and decreased renal nitric oxide.

PubMed

Gordish, Kevin L; Kassem, Kamal M; Ortiz, Pablo A; Beierwaltes, William H

2017-04-01

Previously, we reported that 20% fructose diet causes salt-sensitive hypertension. In this study, we hypothesized that a high salt diet supplemented with 20% fructose (in drinking water) stimulates salt-sensitive hypertension by increasing salt retention through decreasing renal nitric oxide. Rats in metabolic cages consumed normal rat chow for 5 days (baseline), then either: (1) normal salt for 2 weeks, (2) 20% fructose in drinking water for 2 weeks, (3) 20% fructose for 1 week, then fructose + high salt (4% NaCl) for 1 week, (4) normal chow for 1 week, then high salt for 1 week, (5) 20% glucose for 1 week, then glucose + high salt for 1 week. Blood pressure, sodium excretion, and cumulative sodium balance were measured. Systolic blood pressure was unchanged by 20% fructose or high salt diet. 20% fructose + high salt increased systolic blood pressure from 125 ± 1 to 140 ± 2 mmHg ( P  < 0.001). Cumulative sodium balance was greater in rats consuming fructose + high salt than either high salt, or glucose + high salt (114.2 ± 4.4 vs. 103.6 ± 2.2 and 98.6 ± 5.6 mEq/Day19; P  < 0.05). Sodium excretion was lower in fructose + high salt group compared to high salt only: 5.33 ± 0.21 versus 7.67 ± 0.31 mmol/24 h; P  < 0.001). Nitric oxide excretion was 2935 ± 256  μ mol/24 h in high salt-fed rats, but reduced by 40% in the 20% fructose + high salt group (2139 ± 178  μ mol /24 hrs P  < 0.01). Our results suggest that fructose predisposes rats to salt-sensitivity and, combined with a high salt diet, leads to sodium retention, increased blood pressure, and impaired renal nitric oxide availability. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Kefir peptides prevent high-fructose corn syrup-induced non-alcoholic fatty liver disease in a murine model by modulation of inflammation and the JAK2 signaling pathway.

PubMed

Chen, H L; Tsai, T C; Tsai, Y C; Liao, J W; Yen, C C; Chen, C M

2016-12-12

In recent years, people have changed their eating habits, and high-fructose-containing bubble tea has become very popular. High-fructose intake has been suggested to be a key factor that induces non-alcoholic fatty liver disease (NAFLD). Kefir, a fermented milk product composed of microbial symbionts, has demonstrated numerous biological activities, including antibacterial, antioxidant and immunostimulating effects. The present study aims to evaluate the effects of kefir peptides on high-fructose-induced hepatic steatosis and the possible molecular mechanism. An animal model of 30% high-fructose-induced NAFLD in C57BL/6J mice was established. The experiment is divided into the following six groups: (1) normal: H 2 O drinking water; (2) mock: H 2 O+30% fructose; (3) KL: low-dose kefir peptides (50 mg kg -1 )+30% fructose; (4) KM: medium-dose kefir peptides (100 mg kg -1 )+30% fructose; (5) KH: high-dose kefir peptides (150 mg kg -1 )+30% fructose; and (6) CFM: commercial fermented milk (100 mg kg -1 )+30% fructose. The results show that kefir peptides improve fatty liver syndrome by decreasing body weight, serum alanine aminotransferase, triglycerides, insulin and hepatic triglycerides, cholesterol, and free fatty acids as well as the inflammatory cytokines (TNF-α, IL-6 and IL-1β) that had been elevated in fructose-induced NAFLD mice. In addition, kefir peptides markedly increased phosphorylation of AMPK to downregulate its targeted enzymes, ACC (acetyl-CoA carboxylase) and SREBP-1c (sterol regulatory element-binding protein 1), and inhibited de novo lipogenesis. Furthermore, kefir peptides activated JAK2 to stimulate STAT3 phosphorylation, which can translocate to the nucleus, and upregulated several genes, including the CPT1 (carnitine palmitoyltransferase-1) involved in fatty acid oxidation. Our data have demonstrated that kefir peptides can improve the symptoms of NAFLD, including body weight, energy intake, inflammatory reaction and the formation of fatty liver by activating JAK2 signal transduction through the JAK2/STAT3 and JAK2/AMPK pathways in the high-fructose-induced fatty liver animal model. Therefore, kefir peptides may have the potential for clinical application for the prevention or treatment of clinical metabolic syndrome.

Kefir peptides prevent high-fructose corn syrup-induced non-alcoholic fatty liver disease in a murine model by modulation of inflammation and the JAK2 signaling pathway

PubMed Central

Chen, H L; Tsai, T C; Tsai, Y C; Liao, J W; Yen, C C; Chen, C M

2016-01-01

Objective: In recent years, people have changed their eating habits, and high-fructose-containing bubble tea has become very popular. High-fructose intake has been suggested to be a key factor that induces non-alcoholic fatty liver disease (NAFLD). Kefir, a fermented milk product composed of microbial symbionts, has demonstrated numerous biological activities, including antibacterial, antioxidant and immunostimulating effects. The present study aims to evaluate the effects of kefir peptides on high-fructose-induced hepatic steatosis and the possible molecular mechanism. Results: An animal model of 30% high-fructose-induced NAFLD in C57BL/6J mice was established. The experiment is divided into the following six groups: (1) normal: H2O drinking water; (2) mock: H2O+30% fructose; (3) KL: low-dose kefir peptides (50 mg kg−1)+30% fructose; (4) KM: medium-dose kefir peptides (100 mg kg−1)+30% fructose; (5) KH: high-dose kefir peptides (150 mg kg−1)+30% fructose; and (6) CFM: commercial fermented milk (100 mg kg−1)+30% fructose. The results show that kefir peptides improve fatty liver syndrome by decreasing body weight, serum alanine aminotransferase, triglycerides, insulin and hepatic triglycerides, cholesterol, and free fatty acids as well as the inflammatory cytokines (TNF-α, IL-6 and IL-1β) that had been elevated in fructose-induced NAFLD mice. In addition, kefir peptides markedly increased phosphorylation of AMPK to downregulate its targeted enzymes, ACC (acetyl-CoA carboxylase) and SREBP-1c (sterol regulatory element-binding protein 1), and inhibited de novo lipogenesis. Furthermore, kefir peptides activated JAK2 to stimulate STAT3 phosphorylation, which can translocate to the nucleus, and upregulated several genes, including the CPT1 (carnitine palmitoyltransferase-1) involved in fatty acid oxidation. Conclusion: Our data have demonstrated that kefir peptides can improve the symptoms of NAFLD, including body weight, energy intake, inflammatory reaction and the formation of fatty liver by activating JAK2 signal transduction through the JAK2/STAT3 and JAK2/AMPK pathways in the high-fructose-induced fatty liver animal model. Therefore, kefir peptides may have the potential for clinical application for the prevention or treatment of clinical metabolic syndrome. PMID:27941940

Erythrocyte osmotic fragility and general health status of adolescent Sprague Dawley rats supplemented with Hibiscus sabdariffa aqueous calyx extracts as neonates followed by a high-fructose diet post-weaning.

PubMed

Ibrahim, K G; Lembede, B W; Chivandi, E; Erlwanger, K

2018-02-01

High-fructose diets (HFD) can cause oxidative damage to tissues including erythrocyte cell membranes. Hibiscus sabdariffa (HS) has protective antioxidant properties. Rats were used to investigate whether the consumption of HS by neonates would result in long-term effects on their erythrocyte osmotic fragility (EOF) and general health when later fed a high-fructose diet post-weaning through adolescence. Eighty of four-day-old Sprague Dawley rat pups were divided randomly into three treatment groups. The controls (n = 27) received distilled water at 10 ml/kg b. w, while the other groups received either 50 mg/kg (n = 28) or 500 mg/kg (n = 25) of an HS aqueous calyx extract orally till post-natal day 14. The rats in each group were weaned and divided into two subgroups; one continued on normal rat chow, and the other received fructose (20% w/v) in their drinking water for 30 days. Blood was collected in heparinised tubes and added to serially diluted (0.0-0.85%) phosphate-buffered saline to determine the EOF. Clinical markers of health status were determined with an automated chemical analyser. HS extracts did not programme metabolism in the growing rats to alter their general health and EOF in response to the HFD. © 2017 Blackwell Verlag GmbH.

Oral Fructose Absorption in Obese Children with Non-Alcoholic Fatty Liver Disease

PubMed Central

Sullivan, Jillian S; Le, MyPhuong T; Pan, Zhaoxing; Rivard, Christopher; Love-Osborne, Kathryn; Robbins, Kristen; Johnson, Richard J; Sokol, Ronald J; Sundaram, Shikha S

2014-01-01

Background Fructose intake is associated with NAFLD (Non-Alcoholic Fatty Liver Disease) development. Objective To measure fructose absorption/metabolism in pediatric NAFLD compared to obese and lean controls. Methods Children with histologically proven NAFLD, and obese and lean controls received oral fructose (1 gm/kg ideal body weight). Serum glucose, insulin, uric acid, and fructose, urine uric acid, urine fructose, and breath hydrogen levels were measured at baseline and multiple points until 360 minutes after fructose ingestion. Results Nine NAFLD (89% Hispanic, mean age 14.3 years, mean BMI 35.3 kg/m2), 6 Obese Controls (67% Hispanic, mean age 12.7 years, mean BMI 31.0 kg/m2), and 9 Lean Controls (44% Hispanic, mean age 14.3 years, mean BMI 19.4 kg/m2) were enrolled. Following fructose ingestion, NAFLD vs. Lean Controls had elevated serum glucose, insulin, and uric acid (p<0.05), higher urine uric acid (p=0.001) but lower fructose excretion (p=0.002) and lower breath hydrogen 180-min AUC (p=0.04). NAFLD vs. Obese Controls had similar post-fructose serum glucose, insulin, urine uric acid, and breath hydrogen, but elevated serum uric acid (p<0.05) and lower urine fructose excretion (p=0.02). Conclusions Children with NAFLD absorb and metabolize fructose more effectively than lean subjects, associated with an exacerbated metabolic profile following fructose ingestion. PMID:24961681

Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium.

PubMed

Jacobsen, Jacob H; Frigaard, Niels-Ulrik

2014-01-01

D-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO2 as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol L(-1) and a production rate of 0.15 g mannitol L(-1) day(-1). This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO2 in cyanobacteria. © 2013 International Metabolic Engineering Society Published by International Metabolic Engineering Society All rights reserved.

High expression of fructose-bisphosphate aldolase A induces progression of renal cell carcinoma.

PubMed

Huang, Zhengkai; Hua, Yibo; Tian, Ye; Qin, Chao; Qian, Jian; Bao, Meiling; Liu, Yiyang; Wang, Shangqian; Cao, Qiang; Ju, Xiaobing; Wang, Zengjun; Gu, Min

2018-06-01

Aldolase A (fructose-bisphosphate aldolase A, ALDOA) is a glycolytic enzyme that catalyzes reversible conversion of fructose‑1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. ALDOA has been revealed to be related with many carcinomas, but its expression and function in renal cell carcinoma (RCC) remain unknown. This study aimed to detect expression of ALDOA in human RCC tissue samples and to explore its function in RCC cell lines. Reverse transcription-polymerase chain reaction was used to quantify ALDOA in human RCC samples. A total of 139 RCC tissue samples obtained after surgery were analyzed in tissue microarray for ALDOA immunohistochemistry-based protein expression. Assays for cell cycle, viability, migration, and invasion were performed to assess phenotypic changes in RCC cells after ALDOA knockdown by small interfering RNA-mediated gene silencing approach and ALDOA upregulation by overexpression plasmids. Western blot analysis was used to identify alterations in markers for epithelial-mesenchymal transition (EMT), which affects metastasis and the Wnt/β‑catenin signaling pathway that influences RCC cell growth. ALDOA was upregulated in RCC samples and RCC cell lines (P<0.01). Expression of ALDOA was significantly associated with metastasis (P=0.020) and survival (P=0.0341). Downregulation of ALDOA suppressed proliferation (P<0.05) by triggering G0/G1 cell cycle arrest (P<0.05) and also inhibited migration (P<0.05) and invasion (P<0.01). Upregulation of ALDOA promoted proliferation (P<0.05) and enhanced migration (P<0.001) and invasion (P<0.001). Low expression of ALDOA could reverse EMT and inactivate the Wnt/β‑catenin signaling pathway. Our data revealed that ALDOA functions as a tumor promoter, plays a prominent role in proliferation, migration, and invasion of RCC cells with high expression, and may promote EMT and activate the Wnt/β‑catenin signaling pathway.

Combined Inoculation with Multiple Arbuscular Mycorrhizal Fungi Improves Growth, Nutrient Uptake and Photosynthesis in Cucumber Seedlings.

PubMed

Chen, Shuangchen; Zhao, Hongjiao; Zou, Chenchen; Li, Yongsheng; Chen, Yifei; Wang, Zhonghong; Jiang, Yan; Liu, Airong; Zhao, Puyan; Wang, Mengmeng; Ahammed, Golam J

2017-01-01

Mycorrhizal inoculation stimulates growth, photosynthesis and nutrient uptake in a wide range of host plants. However, the ultimate effects of arbuscular mycorrhyzal (AM) symbiosis vary with the plants and fungal species involved in the association. Therefore, identification of the appropriate combinations of AM fungi (AMF) that interact synergistically to improve their benefits is of high significance. Here, three AM fungal compositions namely VT ( Claroideoglomus sp., Funneliformis sp., Diversispora sp., Glomus sp., and Rhizophagus sp.) and BF ( Glomus intraradices , G. microageregatum BEG and G. Claroideum BEG 210), and Funneliformis mosseae (Fm) were investigated with respect to the growth, gas exchange parameters, enzymes activities in Calvin cycles and related gene expression in cucumber seedlings. The results showed that VT, BF and Fm could successfully colonize cucumber root to a different degree with the colonization rates 82.38, 74.65, and 70.32% at 46 days post inoculation, respectively. The plant height, stem diameter, dry weight, root to shoot ratio of cucumber seedlings inoculated with AMF increased significantly compared with the non-inoculated control. Moreover, AMF colonization greatly increased the root activity, chlorophyll content, net photosynthetic rate, light saturated rate of the CO 2 assimilation ( A sat), maximum carboxylation rate ( V cmax ) and maximum ribulose-1,5-bis-phosphate (RuBP) regeneration rate ( J max), which were increased by 52.81, 30.75, 58.76, 47.00, 69.15, and 65.53% when inoculated with VT, respectively. The activities of some key enzymes such RuBP carboxylase/oxygenase (RuBisCO), D-fructose-1,6-bisphosphatase (FBPase), D-fructose-6-phosphatase (F6P) and ribulose-5-phosphate kinase (Ru5PK), and related gene expression involved in the Calvin cycle including RCA , FBPase , FBPA , SBPase , rbcS and rbcL were upregulated by AMF colonization. AMF inoculation also improved macro- and micro nutrient contents such as N, P, K, S, Ca, Cu, Fe, Mn, Mg, and Zn in roots. Further analysis revealed that inoculation with VT had relatively better effect on growth of cucumber seedling followed by BF and Fm, indicating that AMF composition consisting of distant AMF species may have a better effect than a single or closely related AMF spp. This study advances the understanding of plant responses to different AM fungi toward development of strategies on AMF-promoted vegetable production.

The acute action of ammonia on rat brain metabolism in vivo.

PubMed

Hawkins, R A; Miller, A L; Nielsen, R C; Veech, R L

1973-08-01

1. Acute NH(4) (+) toxicity was studied by using a new apparatus that removes and freezes the brains of conscious rats within 1s. 2. Brains were removed and frozen 5min after intraperitoneal injection of ammonium acetate (2-3min before the onset of convulsions). Arterial [NH(4) (+)] rose from less than 0.01 to 1.74mm at 4-5min. The concentrations of all glycolytic intermediates measured, except glucose 6-phosphate, were increased by the indicated percentage above the control value as follows: glucose (by 41%), fructose 1,6-diphosphate (by 133%), dihydroxyacetone phosphate (by 164%), alpha-glycerophosphate (by 45%), phosphoenolpyruvate (by 67%) and pyruvate (by 26%). 4. Citrate and alpha-oxoglutarate concentrations were unchanged and that of malate was increased (by 17%). 5. Adenine nucleotides and P(i) concentrations were unchanged but the concentration of creatine phosphate decreased slightly (by 6%). 6. Brain [NH(4) (+)] increased from 0.2 to 1.53mm. Net glutamine synthesis occurred at an average rate of 0.33mumol/min per g. 7. The rate of brain glucose utilization was measured in vivo as 0.62mumol/min per g in controls and 0.81mumol/min per g after NH(4) (+) injection. 8. The arteriovenous difference of glucose and O(2) increased by 35%. 9. No significant arteriovenous differences of glutamate or glutamine were detected. Thus, although much NH(4) (+) was incorporated into glutamine the latter was not rapidly released from the brain to the circulation. 10. Plasma [K(+)] increased from 3.3 to 5.4mm. 11. The results indicate that NH(4) (+) stimulates oxidative metabolism but does not interfere with brain energy balance. The increased rate of oxidative metabolism could not be accounted for only on the basis of glutamine synthesis. We suggest that increased extracellular [NH(4) (+)] and [K(+)] decreased the resting transmembrane potential and stimulated Na(+),K(+)-stimulated adenosine triphosphatase activity thus accounting for the increased metabolic rate.

Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile†

PubMed Central

Bera, Aloke Kumar; Chen, Sihong; Smith, Janet L.; Zalkin, Howard

2000-01-01

Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus were expressed in Escherichia coli, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90°C, with half-lives of 65 min for GPAT and 60 h for GARS at 80°C. GPAT activity is known to depend upon channeling of NH3 from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH3 for synthesis of PRA was found to increase from 34% at 37°C to a maximum of 84% at 80°C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90°C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50°C, and remained relatively constant up to 90°C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion. PMID:10850988

Temperature-dependent function of the glutamine phosphoribosylpyrophosphate amidotransferase ammonia channel and coupling with glycinamide ribonucleotide synthetase in a hyperthermophile.

PubMed

Bera, A K; Chen, S; Smith, J L; Zalkin, H

2000-07-01

Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus were expressed in Escherichia coli, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90 degrees C, with half-lives of 65 min for GPAT and 60 h for GARS at 80 degrees C. GPAT activity is known to depend upon channeling of NH(3) from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH(3) for synthesis of PRA was found to increase from 34% at 37 degrees C to a maximum of 84% at 80 degrees C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90 degrees C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50 degrees C, and remained relatively constant up to 90 degrees C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.

Hexosamine Biosynthetic Pathway Mutations Cause Neuromuscular Transmission Defect

PubMed Central

Senderek, Jan; Müller, Juliane S.; Dusl, Marina; Strom, Tim M.; Guergueltcheva, Velina; Diepolder, Irmgard; Laval, Steven H.; Maxwell, Susan; Cossins, Judy; Krause, Sabine; Muelas, Nuria; Vilchez, Juan J.; Colomer, Jaume; Mallebrera, Cecilia Jimenez; Nascimento, Andres; Nafissi, Shahriar; Kariminejad, Ariana; Nilipour, Yalda; Bozorgmehr, Bita; Najmabadi, Hossein; Rodolico, Carmelo; Sieb, Jörn P.; Steinlein, Ortrud K.; Schlotter, Beate; Schoser, Benedikt; Kirschner, Janbernd; Herrmann, Ralf; Voit, Thomas; Oldfors, Anders; Lindbergh, Christopher; Urtizberea, Andoni; von der Hagen, Maja; Hübner, Angela; Palace, Jacqueline; Bushby, Kate; Straub, Volker; Beeson, David; Abicht, Angela; Lochmüller, Hanns

2011-01-01

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general. PMID:21310273

Protective and Anti-Pathology Effects of Sm Fructose-1,6-Bisphosphate Aldolase-Based DNA Vaccine against Schistosoma mansoni by Changing Route of Injection

PubMed Central

Saber, Mohamed; Hammam, Olft; Karim, Amr; Medhat, Amina; Khela, Mamdouh; El-Dabaa, Ehab

2013-01-01

This study aimed to evaluate the efficacy of fructose-1,6-bis phosphate aldolase (SMALDO) DNA vaccination against Schistosoma mansoni infection using different routes of injection. The SMALDO has been cloned into the eukaryotic expression vector pcDNA3.1/V5-His TOPO-TA and was used in injecting Swiss albino mice intramuscularly (IM), subcutaneously (SC), or intraperitoneally (IP) (50 µg/mouse). Mice vaccinated with non-recombinant pcDNA3.1 served as controls. Each group was immunized 4 times at weeks 0, 2, 4, and 6. Two weeks after the last booster dose, all mice groups were infected with 80 S. mansoni cercariae via tail immersion. At week 8 post-infection, animals were sacrificed for assessment of parasitological and histopathological parameters. High anti-SMALDO IgG antibody titers were detected in sera of all vaccinated groups (P<0.01) compared to the control group. Both the IP and SC vaccination routes resulted in a significant reduction in worm burden (46.2% and 28.9%, respectively, P<0.01). This was accompanied by a significant reduction in hepatic and intestinal egg counts (41.7% and 40.2%, respectively, P<0.01) in the IP group only. The number of dead eggs was significantly increased in both IP and IM groups (P<0.01). IP vaccination recorded the highest significant reduction in granuloma number and diameter (54.7% and 29.2%, respectively, P<0.01) and significant increase in dead miracidia (P<0.01). In conclusion, changing the injection route of SMALDO DNA vaccination significantly influenced the efficacy of vaccination. SMALDO DNA vaccination via IP route could be a promising protective and anti-pathology vaccine candidate against S. mansoni infection. PMID:23710082

Protective and anti-pathology effects of Sm fructose-1,6-bisphosphate aldolase-based DNA vaccine against schistosoma mansoni by changing route of injection.

PubMed

Saber, Mohamed; Diab, Tarek; Hammam, Olft; Karim, Amr; Medhat, Amina; Khela, Mamdouh; El-Dabaa, Ehab

2013-04-01

This study aimed to evaluate the efficacy of fructose-1,6-bis phosphate aldolase (SMALDO) DNA vaccination against Schistosoma mansoni infection using different routes of injection. The SMALDO has been cloned into the eukaryotic expression vector pcDNA3.1/V5-His TOPO-TA and was used in injecting Swiss albino mice intramuscularly (IM), subcutaneously (SC), or intraperitoneally (IP) (50 µg/mouse). Mice vaccinated with non-recombinant pcDNA3.1 served as controls. Each group was immunized 4 times at weeks 0, 2, 4, and 6. Two weeks after the last booster dose, all mice groups were infected with 80 S. mansoni cercariae via tail immersion. At week 8 post-infection, animals were sacrificed for assessment of parasitological and histopathological parameters. High anti-SMALDO IgG antibody titers were detected in sera of all vaccinated groups (P<0.01) compared to the control group. Both the IP and SC vaccination routes resulted in a significant reduction in worm burden (46.2% and 28.9%, respectively, P<0.01). This was accompanied by a significant reduction in hepatic and intestinal egg counts (41.7% and 40.2%, respectively, P<0.01) in the IP group only. The number of dead eggs was significantly increased in both IP and IM groups (P<0.01). IP vaccination recorded the highest significant reduction in granuloma number and diameter (54.7% and 29.2%, respectively, P<0.01) and significant increase in dead miracidia (P<0.01). In conclusion, changing the injection route of SMALDO DNA vaccination significantly influenced the efficacy of vaccination. SMALDO DNA vaccination via IP route could be a promising protective and anti-pathology vaccine candidate against S. mansoni infection.

Fructose increases corticosterone production in association with NADPH metabolism alterations in rat epididymal white adipose tissue.

PubMed

Prince, Paula D; Santander, Yanina A; Gerez, Estefania M; Höcht, Christian; Polizio, Ariel H; Mayer, Marcos A; Taira, Carlos A; Fraga, Cesar G; Galleano, Monica; Carranza, Andrea

2017-08-01

Metabolic syndrome is an array of closely metabolic disorders that includes glucose intolerance/insulin resistance, central obesity, dyslipidemia, and hypertension. Fructose, a highly lipogenic sugar, has profound metabolic effects in adipose tissue, and has been associated with the etiopathology of many components of the metabolic syndrome. In adipocytes, the enzyme 11 β-HSD1 amplifies local glucocorticoid production, being a key player in the pathogenesis of central obesity and metabolic syndrome. 11 β-HSD1 reductase activity is dependent on NADPH, a cofactor generated by H6PD inside the endoplasmic reticulum. Our focus was to explore the effect of fructose overload on epididymal white adipose tissue (EWAT) machinery involved in glucocorticoid production and NADPH and oxidants metabolism. Male Sprague-Dawley rats fed with a fructose solution (10% (w/v) in tap water) during 9 weeks developed some characteristic features of metabolic syndrome, such as hypertriglyceridemia, and hypertension. In addition, high levels of plasma and EWAT corticosterone were detected. Activities and expressions of H6PD and 11 β-HSD1, NAPDH content, superoxide anion production, expression of NADPH oxidase 2 subunits, and indicators of oxidative metabolism were measured. Fructose overloaded rats showed an increased potential in oxidant production respect to control rats. In parallel, in EWAT from fructose overloaded rats we found higher expression/activity of H6PD and 11 β-HSD1, and NADPH/NADP + ratio. Our in vivo results support that fructose overload installs in EWAT conditions favoring glucocorticoid production through higher H6PD expression/activity supplying NADPH for enhanced 11 β-HSD1 expression/activity, becoming this tissue a potential extra-adrenal source of corticosterone under these experimental conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation.

PubMed

Troncoso-Ponce, M A; Rivoal, J; Dorion, S; Moisan, M-C; Garcés, R; Martínez-Force, E

2011-03-01

A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 μmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation. Copyright © 2010 Elsevier GmbH. All rights reserved.

The metabolism of carbohydrates and lipid peroxidation in lead-exposed workers.

PubMed

Kasperczyk, Aleksandra; Dobrakowski, Michal; Ostałowska, Alina; Zalejska-Fiolka, Jolanta; Birkner, Ewa

2015-12-01

The present study was undertaken to estimate the effect of occupational exposure to lead on the blood concentration of glucose and several enzymes involved in glycolysis, the citric acid cycle, and the pentose phosphate pathway. To estimate the degree of lipid peroxidation, the concentrations of conjugated dienes were determined. The examined group included 145 healthy male employees of lead-zinc works. Taking into account the mean blood lead levels, the examined group was divided into two subgroups. The control group was composed of 36 healthy male administrative workers. The markers of lead exposure were significantly elevated in both subgroups when compared with the controls. There were no significant changes in fasting glucose concentration and fructose-1,6-bisphosphate aldolase activity in the study population. The concentration of conjugated dienes was significantly higher in both subgroups, whereas the activity of malate dehydrogenase was significantly higher only in the group with higher exposure. The activities of lactate dehydrogenase and sorbitol dehydrogenase were significantly decreased in the examined subgroups. The activity of glucose-6-phosphate dehydrogenase decreased significantly in the group with higher exposure and could be the cause of the elevated concentrations of conjugated dienes. It is possible to conclude that lead interferes with carbohydrate metabolism, but compensatory mechanisms seem to be efficient, as glucose homeostasis in lead-exposed workers was not disturbed. © The Author(s) 2013.

Industrial, not fruit fructose intake is associated with the severity of liver fibrosis in genotype 1 chronic hepatitis C patients.

PubMed

Petta, Salvatore; Marchesini, Giulio; Caracausi, Linda; Macaluso, Fabio Salvatore; Cammà, Calogero; Ciminnisi, Stefania; Cabibi, Daniela; Porcasi, Rossana; Craxì, Antonio; Di Marco, Vito

2013-12-01

Unhealthy food intake, specifically fructose, has been associated with metabolic alterations and with the severity of liver fibrosis in patients with non-alcoholic fatty liver disease. In a cohort of patients with genotype 1 chronic hepatitis C (G1 CHC), we tested the association of fructose intake with the severity of liver histology. Anthropometric and metabolic factors, including waist circumference (WC), waist-to-hip ratio (WHR), dorso-cervical lipohypertrophy and HOMA were assessed in 147 consecutive biopsy-proven G1 CHC patients. Food intake, namely industrial and fruit fructose, was investigated by a three-day structured interview and a computed database. All biopsies were scored by an experienced pathologist for staging and grading (Scheuer classification), and graded for steatosis, which was considered moderate-severe if ≥ 20%. Features of non-alcoholic steatohepatitis (NASH) in CHC were also assessed (Bedossa classification). Mean daily intake of total, industrial and fruit fructose was 18.0±8.7g, 6.0±4.7g, and 11.9±7.2g, respectively. Intake of industrial, not fruit fructose, was independently associated with higher WHR (p=0.02) and hypercaloric diet (p<0.001). CHC patients with severe liver fibrosis (⩾F3) reported a significantly higher intake of total (20.8±10.2 vs. 17.2±8.1g/day; p=0.04) and industrial fructose (7.8±6.0 vs. 5.5±4.2; p=0.01), not fruit fructose (12.9±8.0 vs. 11.6±7.0; p=0.34). Multivariate logistic regression analysis showed that older age (OR 1.048, 95% CI 1.004-1.094, p=0.03), severe necroinflammatory activity (OR 3.325, 95% CI 1.347-8.209, p=0.009), moderate-severe steatosis (OR 2.421, 95% CI 1.017-6.415, p=0.04), and industrial fructose intake (OR 1.147, 95% CI 1.047-1.257, p=0.003) were independently linked to severe fibrosis. No association was found between fructose intake and liver necroinflammatory activity, steatosis, and the features of NASH. The daily intake of industrial, not fruit fructose is a risk factor for metabolic alterations and the severity of liver fibrosis in patients with G1 CHC. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Direct renal effects of a fructose-enriched diet: interaction with high salt intake

PubMed Central

Ares, Gustavo R.

2015-01-01

Consumption of fructose has increased during the last 50 years. Excessive fructose consumption has a detrimental effect on mammalian health but the mechanisms remain unclear. In humans, a direct relationship exists between dietary intake of added sugars and increased risk for cardiovascular disease mortality (52). While the causes for this are unclear, we recently showed that fructose provided in the drinking water induces a salt-dependent increase in blood pressure in Sprague-Dawley rats in a matter of days (6). However, little is known about the effects of fructose in renal salt handling and whether combined intake of high fructose and salt can lead to salt-sensitive hypertension before the development of metabolic abnormalities. The long-term (more than 4 wk) adverse effects of fructose intake on renal function are not just due to fructose but are also secondary to alterations in metabolism which may have an impact on renal function. This minireview focuses on the acute effect of fructose intake and its effect on salt regulation, as they affect blood pressure. PMID:26447210

The protective effect of juglanin on fructose-induced hepatitis by inhibiting inflammation and apoptosis through TLR4 and JAK2/STAT3 signaling pathways in fructose-fed rats.

PubMed

Zhou, Guang-Yao; Yi, Yong-Xiang; Jin, Ling-Xiang; Lin, Wei; Fang, Pei-Pei; Lin, Xiu-Zheng; Zheng, Yi; Pan, Chen-Wei

2016-07-01

High fructose-feeding is an essential causative factor leading to the development and progression of hepatitis associated with high levels of endotoxin (LPS). Juglanin, as a natural compound extracted from the crude Polygonum aviculare, displayed inhibitory activity against inflammation response and cancer growth. However, researches about its role on anti-inflammation and apoptosis are far from available. Here, it is the first time that juglanin was administrated to investigate whether it inhibits fructose-feeding-induced hepatitis in rats and to elucidate the possible mechanism by which juglanin might recover it. Fructose-feeding rats were orally administrated with juglanin of 5, 10 and 20mg/kg for 6 weeks, respectively. Juglanin exerted prevention of fructose-feeding-stimulated increased LPS levels, accelerated alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) and up-regulated inflammatory cytokines expression in serum, mainly including tumor necrosis factor-alpha (TNF-a), Interleukin 1beta (IL-1β), Interleukin 6 (IL-6) and Interleukin 18 (IL-18). Meanwhile, toll-like receptor 4 (TLR4)-modulated mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB) and apoptosis-related Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway are involved in the progression of hepatic injury and inflammation. And juglanin was found to suppress fructose-feeding-induced activation of these signaling pathways compared with the model group administrated only with fructose. These results indicate that juglanin represses inflammatory response and apoptosis via TLR4-regulated MAPK/NF-κB and JAK2/STAT3 signaling pathway respectively in rats with hepatitis induced by LPS for fructose-feeding. Treatment of juglanin might be an effective therapeutic strategy for preventing hepatitis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Contribution of galactose and fructose to glucose homeostasis

USDA-ARS?s Scientific Manuscript database

To determine the contributions of galactose and fructose to glucose formation, 6 subjects (26 +/- 2 years old; body mass index, 22.4 +/-0.2 kg/m2) (mean +/- SE) were studied during fasting conditions. Three subjects received a primed constant intravenous infusion of[6,6-2H2] glucose for 3 hours foll...

Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

PubMed Central

Chavarría, Max; Kleijn, Roelco J.; Sauer, Uwe; Pflüger-Grau, Katharina; de Lorenzo, Víctor

2012-01-01

ABSTRACT Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFru or PTSNtr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtr protein of the PTSNtr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. PMID:22434849

Dietary fructose but not starch is responsible for hyperlipidemia associated with copper deficiency in rats: effect of high-fat diet.

PubMed

Fields, M; Lewis, C G

1999-02-01

To test the hypothesis that copper deficiency in rats may be hyperlipidemic only when the diets consumed contain nutrients which contribute to blood lipids such as fructose and high fat. Weanling male Sprague Dawley rats were fed diets which contained either starch or fructose as their sole carbohydrate source. The diets were either inadequate (0.6 microg Cu/g) or adequate (6.0 microg Cu/g) in copper and contained either high (300 g/kg) or low (60 g/kg) fat. At the end of the 4th week the rats were killed. Livers were analyzed for copper content. Plasma was analyzed for cholesterol and triglyceride concentrations. High-fat diet did not increase blood lipids in rats fed a copper-deficient diet containing starch. In contrast, the combination of high-fat diet with fructose increased blood triglycerides and fructose with copper deficiency resulted in a significant increases in blood cholesterol. Hyperlipidemia of copper deficiency in rats is dependent on synergistic effects between dietary fructose and copper deficiency and fructose and amount of dietary fat. Hyperlipidemia does not develop if starch is the main source of dietary carbohydrate in a copper-deficient diet even if a high-fat diet is fed.

High fructose consumption in pregnancy alters the perinatal environment without increasing metabolic disease in the offspring.

PubMed

Lineker, Christopher; Kerr, Paul M; Nguyen, Patricia; Bloor, Ian; Astbury, Stuart; Patel, Nikhil; Budge, Helen; Hemmings, Denise G; Plane, Frances; Symonds, Michael E; Bell, Rhonda C

2016-10-01

Maternal carbohydrate intake is one important determinant of fetal body composition, but whether increased exposure to individual sugars has long-term adverse effects on the offspring is not well established. Therefore, we examined the effect of fructose feeding on the mother, placenta, fetus and her offspring up to 6 months of life when they had been weaned onto a standard rodent diet and not exposed to additional fructose. Dams fed fructose were fatter, had raised plasma insulin and triglycerides from mid-gestation and higher glucose near term. Maternal resistance arteries showed changes in function that could negatively affect regulation of blood pressure and tissue perfusion in the mother and development of the fetus. Fructose feeding had no effect on placental weight or fetal metabolic profiles, but placental gene expression for the glucose transporter GLUT1 was reduced, whereas the abundance of sodium-dependent neutral amino acid transporter-2 was raised. Offspring born to fructose-fed and control dams were similar at birth and had similar post-weaning growth rates, and neither fat mass nor metabolic profiles were affected. In conclusion, raised fructose consumption during reproduction results in pronounced maternal metabolic and vascular effects, but no major detrimental metabolic effects were observed in offspring up to 6 months of age.

An in Situ NMR Study of the Mechanism for the Catalytic Conversion of Fructose to 5-Hydroxymethylfurfural and then to Levulinic Acid Using 13 C Labeled d -Fructose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jing; Weitz, Eric

The pathways for the formation of 5-hydroxymethylfurfural (HMF) by dehydration of d-fructose and for the formation of levulinic acid and formic acid from HMF by rehydration were investigated by in situ13C and 1H NMR using both unlabeled and 13C-labeled fructose. Water or DMSO was used as the solvent with Amberlyst 70, PO43–/niobic acid, or sulfuric acid as catalysts. Only HMF is observed using NMR for fructose dehydration in DMSO with any of the three catalysts or without a catalyst. For each system, results with 13C-labeled fructose indicate that the first carbon (C-1) or sixth carbon (C-6) of fructose maps ontomore » the corresponding carbons of HMF. For fructose dehydration in H2O with a PO43–/niobic acid catalyst, in addition to HMF, furfural was observed as a product. However, we show that furfural is not a reaction product deriving from HMF under our conditions. Rather our data indicate that there is a parallel reaction pathway open to fructose when the reaction takes place in H2O with a PO43–/niobic acid catalyst. The corresponding 13C-labeled results show that the first carbon in fructose maps onto the first carbon (aldehyde carbon) in furfural. Using 13C-enriched HMF formed from dehydration of 13C-labeled fructose in DMSO or H2O, we investigated the pathway for HMF rehydration to levulinic and formic acid. The data in different solvents and with different catalysts are consistent with a common mechanism for HMF rehydration, which results in the C-1 and C-6 carbon of HMF being transformed to the carbon of formic acid and methyl carbon (C-5) of levulinic acid, respectively.« less

Identification of TP53-induced glycolysis and apoptosis regulator (TIGAR) as the phosphoglycolate-independent 2,3-bisphosphoglycerate phosphatase.

PubMed

Gerin, Isabelle; Noël, Gaëtane; Bolsée, Jennifer; Haumont, Olivier; Van Schaftingen, Emile; Bommer, Guido T

2014-03-15

The p53-induced protein TIGAR [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator] is considered to be a F26BPase (fructose-2,6-bisphosphatase) with an important role in cancer cell metabolism. The reported catalytic efficiency of TIGAR as an F26BPase is several orders of magnitude lower than that of the F26BPase component of liver or muscle PFK2 (phosphofructokinase 2), suggesting that F26BP (fructose 2,6-bisphosphate) might not be the physiological substrate of TIGAR. We therefore set out to re-evaluate the biochemical function of TIGAR. Phosphatase activity of recombinant human TIGAR protein was tested on a series of physiological phosphate esters. The best substrate was 23BPG (2,3-bisphosphoglycerate), followed by 2PG (2-phosphoglycerate), 2-phosphoglycolate and PEP (phosphoenolpyruvate). In contrast the catalytic efficiency for F26BP was approximately 400-fold lower than that for 23BPG. Using genetic and shRNA-based cell culture models, we show that loss of TIGAR consistently leads to an up to 5-fold increase in the levels of 23BPG. Increases in F26BP levels were also observed, albeit in a more limited and cell-type dependent manner. The results of the present study challenge the concept that TIGAR acts primarily on F26BP. This has significant implications for our understanding of the metabolic changes downstream of p53 as well as for cancer cell metabolism in general. It also suggests that 23BPG might play an unrecognized function in metabolic control.

Oxidative Inactivation of Liver Mitochondria in High Fructose Diet-Induced Metabolic Syndrome in Rats: Effect of Glycyrrhizin Treatment.

PubMed

Sil, Rajarshi; Chakraborti, Abhay Sankar

2016-09-01

Metabolic syndrome is a serious health problem in the present world. Glycyrrhizin, a triterpenoid saponin of licorice (Glycyrrhiza glabra) root, has been reported to ameliorate the primary complications and hepatocellular damage in rats with the syndrome. In this study, we have explored metabolic syndrome-induced changes in liver mitochondrial function and effect of glycyrrhizin against the changes. Metabolic syndrome was induced in rats by high fructose (60%) diet for 6 weeks. The rats were then treated with glycyrrhizin (50 mg/kg body weight) by single intra-peritoneal injection. After 2 weeks of the treatment, the rats were sacrificed to collect liver tissue. Elevated mitochondrial ROS, lipid peroxidation and protein carbonyl, and decreased reduced glutathione content indicated oxidative stress in metabolic syndrome. Loss of mitochondrial inner membrane cardiolipin was observed. Mitochondrial complex I activity did not change but complex IV activity decreased significantly. Mitochondrial MTT reduction ability, membrane potential, phosphate utilisation and oxygen consumption decreased in metabolic syndrome. Reduced mitochondrial aconitase activity and increased aconitase carbonyl content suggested oxidative damage of the enzyme. Elevated Fe(2+) ion level in mitochondria might be associated with increased ROS generation in metabolic syndrome. Glycyrrhizin effectively attenuated mitochondrial oxidative stress and aconitase degradation, and improved electron transport chain activity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Hyperthyroidism results in increased glycolytic capacity in the rat heart. A 31P-NMR study.

PubMed

Seymour, A M; Eldar, H; Radda, G K

1990-11-12

We have investigated the metabolic adaptations that occur in the thyroxine-treated rat heart. Rats were made hyperthyroid by daily intra-peritoneal injections of thyroxine (35 micrograms/100 g body weight) over seven days. 31P-NMR investigations of isolated glucose-perfused isometric hearts showed that thyroxine treatment caused an increase in Pi (from 4.9 mumols.(g dry wt.)-1 in control hearts to 11.7 mumols.(g dry wt.)-1 in hyperthyroid hearts), a decrease in phosphocreatine (from 36.5 mumols.(g dry wt.)-1 to 21.8 mumols.(g dry wt.)-1) with no change in ATP or ADP concentrations under the same conditions of cardiac work. The unidirectional exchange flux Pi----ATP was measured by saturation transfer NMR in hyperthyroid rat hearts. This exchange (which has been shown to contain a significant glycolytic component) increased by 2.2-fold in thyroxine-treated hearts in comparison to control hearts (to 3.6 mumols.(g dry wt.)-1.s-1, from 1.6 mumols.(g dry wt.)-1.s-1). In parallel experiments, NMR analysis of extracts from hyperthyroid rat hearts showed significantly elevated levels of glucose 6-phosphate, and fructose 6-phosphate. Measurements of enzyme activities isolated from hyperthyroid and control tissue showed a 40% increase in phosphofructokinase activity. These data together with the increased concentration of Pi show that both glycolytic and glycogenolytic fluxes are increased in the hyperthyroid rat heart. This metabolic adaptation may be necessary to cope with the increased number and activity of Na+/K(+)-ATPase pumps that occur in response to thyroxine treatment.

Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa.

PubMed

Acero-Navarro, Kevin E; Jiménez-Ramírez, Mariella; Villalobos, Miguel A; Vargas-Martínez, Rocío; Perales-Vela, Hugo V; Velasco-García, Roberto

2018-02-01

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl 2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional specialization of one copy of glutamine phosphoribosyl pyrophosphate amidotransferase in ureide production from symbiotically fixed nitrogen in Phaseolus vulgaris.

PubMed

Coleto, Inmaculada; Trenas, Almudena T; Erban, Alexander; Kopka, Joachim; Pineda, Manuel; Alamillo, Josefa M

2016-08-01

Purines are essential molecules formed in a highly regulated pathway in all organisms. In tropical legumes, the nitrogen fixed in the nodules is used to generate ureides through the oxidation of de novo synthesized purines. Glutamine phosphoribosyl pyrophosphate amidotransferase (PRAT) catalyses the first committed step of de novo purine synthesis. In Phaseolus vulgaris there are three genes coding for PRAT. The three full-length sequences, which are intron-less genes, were cloned, and their expression levels were determined under conditions that affect the synthesis of purines. One of the three genes, PvPRAT3, is highly expressed in nodules and protein amount and enzymatic activity in these tissues correlate with nitrogen fixation activity. Inhibition of PvPRAT3 gene expression by RNAi-silencing and subsequent metabolomic analysis of the transformed roots shows that PvPRAT3 is essential for the synthesis of ureides in P. vulgaris nodules. © 2016 John Wiley & Sons Ltd.

Effects of glucose metabolism pathways on sperm motility and oxidative status during long-term liquid storage of goat semen.

PubMed

Qiu, Jian-Hua; Li, You-Wei; Xie, Hong-Li; Li, Qing; Dong, Hai-Bo; Sun, Ming-Ju; Gao, Wei-Qiang; Tan, Jing-He

2016-08-01

Although great efforts were made to prolong the fertility of liquid-stored semen, limited improvements have been achieved in different species. Although it is expected that energy supply and the redox potential will play an essential role in sperm function, there are few reports on the impact of specific energy substrates on spermatozoa during liquid semen storage. Furthermore, although it is accepted that glucose metabolism through glycolysis provides energy, roles of pentose phosphate pathway (PPP) and tricarboxylic acid cycle remain to be unequivocally found in spermatozoa. We have studied the pathways by which spermatozoa metabolize glucose during long-term liquid storage of goat semen. The results indicated that among the substrates tested, glucose and pyruvate were better than lactate in maintaining goat sperm motility. Although both glycolysis and PPP were essential, PPP was more important than glycolysis to maintain sperm motility. Pentose phosphate pathway reduced oxidative stress and provided glycolysis with more intermediate products such as fructose-6-phosphate. Pyruvate entered goat spermatozoa through monocarboxylate transporters and was oxidized by the tricarboxylic acid cycle and electron transfer to sustain sperm motility. Long-term liquid semen storage can be used as a good model to study sperm glucose metabolism. The data are important for an optimal control of sperm survival during semen handling and preservation not only in the goat but also in other species. Copyright © 2016 Elsevier Inc. All rights reserved.

TRITIUM-LABELED COMPOUNDS VII. ISOTOPE EFFECTS IN THE OXIDATION OF d- MANNITOLS-C$sup 14$ AND d-MANNITOLS-t TO d-FRUCTOSES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sniegoski, L.T.; Frush, H.L.; Isbell, H.S.

1961-09-01

D-Mannitols, labeled either with carbon-14 at C1, C2, or C3, or with tritium attached to C1, C2, or C3, were prepared. After oxidation by Acetobacter suboxydans, the distribution of radioactivity in each of the resulting labeled D- fructoses was determined. Labeled D-mannitol is unique among the hexitols in that it may be oxidized by A. suboxydans in either the labeled or the unlabeled part of the molecule. Except in the oxidation of D-mannitol-2-t, the competing reactions result in the formation of a mixture of D-fructoses, each having radioactivity in one of two different positions. Hence, the isotope effect, k*/ k,more » (where k* and k are, respectively, the rate constants for oxidation in the labeled and in the unlabeled part of the labeled emannitol molecule) is the ratio of the activities at the two positions of the product, D-fructose. The following isotope effects were found for the bacterial oxidation of labeled D-mannitols: for D-mannitol-2-C/sup 14/, k*/k = 0.93; for Dmannitol-2-t, k*/k = 0.23; and for D-mannitol-3-t, k*/k = 0.70. For D-mannitols labeled at other positions, no isotope effect was detected, since k*/k was unity. The large isotope effect for D-mannitol-2-t is indicative of rupture of the C2-H bond in the rate determining process. It is suggested that the secondary isotope effect for tritium at C3 indicates hyperconjugation of the C3 hydrogen atom in the activated enzyme-- substrate complex; the lack of such effect for tritium at C1 may be due to unfavorable steric conditions for hyperconjugation of the C1 hydrogen atoms in the complex. The following substances were prepared and their isotopic distributions determined: D-fructose1,6-C/sup 14/ and D-fructose-1,6-t (from 1- labeled D-mannitols); D-fructose-2,5-C/sup 14/ and D-fructose-5-t (from 2-labeled e-mannitols); and D-fructose-3,4-C/sup 14/ and D-fructose-3,4-t (from 3-labeled D- mannitols). A procedure, employing D-fructose-1,6-C/sup 14/ as an internal standard, was devised for the analysis of D-fructose-3,4-t. (auth)« less

Dietary fructose and glucose differentially affect lipid and glucose homeostasis.

PubMed

Schaefer, Ernst J; Gleason, Joi A; Dansinger, Michael L

2009-06-01

Absorbed glucose and fructose differ in that glucose largely escapes first-pass removal by the liver, whereas fructose does not, resulting in different metabolic effects of these 2 monosaccharides. In short-term controlled feeding studies, dietary fructose significantly increases postprandial triglyceride (TG) levels and has little effect on serum glucose concentrations, whereas dietary glucose has the opposite effects. When dietary glucose and fructose have been directly compared at approximately 20-25% of energy over a 4- to 6-wk period, dietary fructose caused significant increases in fasting TG and LDL cholesterol concentrations, whereas dietary glucose did not, but dietary glucose did increase serum glucose and insulin concentrations in the postprandial state whereas dietary fructose did not. When fructose at 30-60 g ( approximately 4-12% of energy) was added to the diet in the free-living state, there were no significant effects on lipid or glucose biomarkers. Sucrose and high-fructose corn syrup (HFCS) contain approximately equal amounts of fructose and glucose and no metabolic differences between them have been noted. Controlled feeding studies at more physiologic dietary intakes of fructose and glucose need to be conducted. In our view, to decrease the current high prevalence of obesity, dyslipidemia, insulin resistance, and diabetes, the focus should be on restricting the intake of excess energy, sucrose, HFCS, and animal and trans fats and increasing exercise and the intake of vegetables, vegetable oils, fish, fruit, whole grains, and fiber.

The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism.

PubMed

Iizuka, Katsumi

2017-02-22

Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP) is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase), fructolysis (Glut5, ketohexokinase), and lipogenesis (acetyl CoA carboxylase, fatty acid synthase). ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp -/- mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome.

The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism

PubMed Central

Iizuka, Katsumi

2017-01-01

Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP) is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase), fructolysis (Glut5, ketohexokinase), and lipogenesis (acetyl CoA carboxylase, fatty acid synthase). ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp−/− mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome. PMID:28241431

Evaluation of 13CO2 breath tests for the detection of fructose malabsorption.

PubMed

Hoekstra, J H; van den Aker, J H; Kneepkens, C M; Stellaard, F; Geypens, B; Ghoos, Y F

1996-03-01

Breath hydrogen (H2) studies have made clear that small intestinal absorption of fructose is limited, especially in toddlers. Malabsorption of fructose may be a cause of recurrent abdominal pain and chronic nonspecific diarrhea (toddler's diarrhea). Fructose absorption is facilitated by equimolar doses of glucose and, as we have found, amino acids (especially L-alanine); the mechanism underlying this effect remains unclear. To study fructose absorption in a more direct way, we combined breath H2 studies with breath 13CO2 studies. Gastric emptying was studied by using L-glycine-1-13C in 4 children from 12.1 to 16.0 years of age. After 25 gm of fructose and 27.5 gm of glucose, when given together, gastric emptying was significantly (p<0.05) slower than with either sugar alone. In a second series of experiments, 5 children from 12.0 to 15.9 years of age were tested with 25 gm of fructose, alone and with equimolar doses of glucose and L-alanine, and 4 younger children from 3.1 to 6.1 years of age were tested with 2 gm/kg (max 37.5 gm) fructose, alone or with an equimolar dose of L-alanine. All fructose solutions were enriched with 15 mg of D-fructose-13C-6. In all 9 children, fructose was malabsorbed as judged by breath H2 increases > or = 20 ppm, and the addition of glucose or L-alanine resulted in significantly lower breath H2 increases (p < or = 0.005 for glucose, p < or = 0.001 for alanine). In contrast, the addition of alanine or glucose did not change the pattern of breath 13CO2 excretion in the 5 older children, whereas in the 4 younger children (with relatively higher doses), L-alanine addition resulted in significantly lower increases in breath 13CO2. In the latter group, for each time point, breath H2 and 13CO2 concentrations after fructose were compared with those after fructose plus L-alanine; in 20 out of 24 points, both H2 and 13CO2 were higher after fructose. These results suggest that 13CO2 not only originated from the oxidation of absorbed substrate but also, at least in part, from colonic bacterial metabolism. For the detection of [correction of or] fructose malabsorption--as opposed to, for instance, lactose--the 13CO2 breath test seems to be of limited value.

Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Kenichiro; Ito, Sohei; Shimizu-Ibuka, Akiko

2005-08-01

This report describes the crystallization and X-ray diffraction data collection of three types (wild-type, W416F/V435W and C9S/C268S) of B. stearothermophilus. Crystals of C9S/C268S belonged to space group P6{sub 2}22 and diffracted to a resolution of 2.4 Å. Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. Amore » crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6{sub 2}22, with unit-cell parameters a = b = 145.97, c = 118.03 Å.« less

Fructose levels are markedly elevated in cerebrospinal fluid compared to plasma in pregnant women.

PubMed

Hwang, Janice J; Johnson, Andrea; Cline, Gary; Belfort-DeAguiar, Renata; Snegovskikh, Denis; Khokhar, Babar; Han, Christina S; Sherwin, Robert S

2015-01-01

Fructose, unlike glucose, promotes feeding behavior in rodents and its ingestion exerts differential effects in the human brain. However, plasma fructose is typically 1/1000 th of glucose levels and it is unclear to what extent fructose crosses the blood-brain barrier. We investigated whether local endogenous central nervous system (CNS) fructose production from glucose via the polyol pathway (glucose → sorbitol → fructose) contributes to brain exposure to fructose. In this observational study, fasting glucose, sorbitol and fructose concentrations were measured using gas-chromatography-liquid mass spectroscopy in cerebrospinal fluid (CSF), maternal plasma, and venous cord blood collected from 25 pregnant women (6 lean, 10 overweight/obese, and 9 T2DM/gestational DM) undergoing spinal anesthesia and elective cesarean section. As expected, CSF glucose was ~ 60% of plasma glucose levels. In contrast, fructose was nearly 20-fold higher in CSF than in plasma (p < 0.001), and CSF sorbitol was ~ 9-times higher than plasma levels (p < 0.001). Moreover, CSF fructose correlated positively with CSF glucose (ρ 0.45, p = 0.02) and sorbitol levels (ρ 0.75, p < 0.001). Cord blood sorbitol was also ~ 7-fold higher than maternal plasma sorbitol levels (p = 0.001). There were no differences in plasma, CSF, and cord blood glucose, fructose, or sorbitol levels between groups. These data raise the possibility that fructose may be produced endogenously in the human brain and that the effects of fructose in the human brain and placenta may extend beyond its dietary consumption.

Fructose Levels Are Markedly Elevated in Cerebrospinal Fluid Compared to Plasma in Pregnant Women

PubMed Central

Hwang, Janice J.; Johnson, Andrea; Cline, Gary; Belfort-DeAguiar, Renata; Snegovskikh, Denis; Khokhar, Babar; Han, Christina S.; Sherwin, Robert S.

2015-01-01

Background Fructose, unlike glucose, promotes feeding behavior in rodents and its ingestion exerts differential effects in the human brain. However, plasma fructose is typically 1/1000th of glucose levels and it is unclear to what extent fructose crosses the blood-brain barrier. We investigated whether local endogenous central nervous system (CNS) fructose production from glucose via the polyol pathway (glucose→sorbitol→fructose) contributes to brain exposure to fructose. Methods In this observational study, fasting glucose, sorbitol and fructose concentrations were measured using gas-chromatography-liquid mass spectroscopy in cerebrospinal fluid (CSF), maternal plasma, and venous cord blood collected from 25 pregnant women (6 lean, 10 overweight/obese, and 9 T2DM/gestational DM) undergoing spinal anesthesia and elective cesarean section. Results As expected, CSF glucose was ~60% of plasma glucose levels. In contrast, fructose was nearly 20-fold higher in CSF than in plasma (p < 0.001), and CSF sorbitol was ~9-times higher than plasma levels (p < 0.001). Moreover, CSF fructose correlated positively with CSF glucose (ρ 0.45, p = 0.02) and sorbitol levels (ρ 0.75, p < 0.001). Cord blood sorbitol was also ~7-fold higher than maternal plasma sorbitol levels (p = 0.001). There were no differences in plasma, CSF, and cord blood glucose, fructose, or sorbitol levels between groups. Conclusions These data raise the possibility that fructose may be produced endogenously in the human brain and that the effects of fructose in the human brain and placenta may extend beyond its dietary consumption. PMID:26035307

Sensitive and fast detection of fructose in complex media via symmetry breaking and signal amplification using surface-enhanced Raman spectroscopy.

PubMed

Sun, Fang; Bai, Tao; Zhang, Lei; Ella-Menye, Jean-Rene; Liu, Sijun; Nowinski, Ann K; Jiang, Shaoyi; Yu, Qiuming

2014-03-04

A new strategy is proposed to sensitively and rapidly detect analytes with weak Raman signals in complex media using surface-enhanced Raman spectroscopy (SERS) via detecting the SERS signal changes of the immobilized probe molecules on SERS-active substrates upon binding of the analytes. In this work, 4-mercaptophenylboronic acid (4-MPBA) was selected as the probe molecule which was immobilized on the gold surface of a quasi-three-dimensional plasmonic nanostructure array (Q3D-PNA) SERS substrate to detect fructose. The molecule of 4-MPBA possesses three key functions: molecule recognition and reversible binding of the analyte via the boronic acid group, amplification of SERS signals by the phenyl group and thus shielding of the background noise of complex media, and immobilization on the surface of SERS-active substrates via the thiol group. Most importantly, the symmetry breaking of the 4-MPBA molecule upon fructose binding leads to the change of area ratio between totally symmetric 8a ring mode and nontotally symmetric 8b ring mode, which enables the detection. The detection curves were obtained in phosphate-buffered saline (PBS) and in undiluted artificial urine at clinically relevant concentrations, and the limit of detection of 0.05 mM was achieved.

The Henry reaction of (1R)-(1,4:3,6-dianhydro-D-mannitol-2-yl)-1,4:3,6-dianhydro-D-fructose 5,5'-dinitrate. Different reactive features of nitromethane to nitroethane.

PubMed

Liu, Feng-Wu; Wang, Zhen-Ji; Song, Xiao-Ping; Zhang, Sai-Yang; Liu, Hong-Min

2009-12-14

Henry reactions of a novel higher sugar derivative, (1R)-(1,4:3,6-dianhydro-D-mannitol-2-yl)-1,4:3,6-dianhydro-D-fructose 5,5'-dinitrate (Alternate nomenclature: (1R)-(isomannid-2-yl)-1,4:3,6-dianhydro-D-fructose 5,5'-dinitrate), with nitromethane and nitroethane were studied. The kinetic and thermodynamic reactions with nitromethane under different conditions were carried out to afford (2S)- and (2R)-beta-nitroalcohols, respectively. But when using nitroethane the reaction gave a (2S)-beta-nitroalcohol with an inverted configuration at vicinal carbon C-1. Two stereogenic centers were generated, and one was altered in the reaction.

Replacing dietary glucose with fructose increases ChREBP activity and SREBP-1 protein in rat liver nucleus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koo, Hyun-Young; Miyashita, Michio; Department of Pediatrics, Nihon University School of Medicine, Itabashi, Tokyo

2009-12-11

Diets high in fructose cause hypertriglyceridemia and insulin resistance in part due to simultaneous induction of gluconeogenic and lipogenic genes in liver. We investigated the mechanism underlying the unique pattern of gene induction by dietary fructose. Male Sprague-Dawley rats (n = 6 per group) were meal-fed (4 h/d) either 63% (w/w) glucose or 63% fructose diet. After two weeks, animals were killed at the end of the last meal. Nuclear SREBP-1 was 2.2 times higher in fructose-fed rats than glucose-fed rats. Nuclear FoxO1 was elevated 1.7 times in fructose group, but did not reach significance (P = 0.08). Unexpectedly, nomore » difference was observed in nuclear ChREBP between two groups. However, ChREBP DNA binding was 3.9x higher in fructose-fed animals without an increase in xylulose-5-phospate, a proposed ChREBP activator. In conclusion, the gene induction by dietary fructose is likely to be mediated in part by simultaneously increased ChREBP activity, SREBP-1 and possibly FoxO1 protein in nucleus.« less

Rat liver uncoupling protein 2: changes induced by a fructose-rich diet.

PubMed

Castro, María C; Massa, María L; Del Zotto, Héctor; Gagliardino, Juan J; Francini, Flavio

2011-10-24

To evaluate the role of uncoupling protein 2 (UCP2) and peroxisome proliferator-activated receptors (PPARs) in the response of liver to glycoxidative stress triggered by administration of a fructose-rich diet (FRD). We assessed blood glucose in the fasting state and after a glucose load (glucose-oxidase method), serum triglyceride (enzymatic measurement), insulin (radioimmunoassay), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (colorimetric kits) in control and FRD animals. In liver, we measured UCP2, PPARα, PPARδ and PPARγ gene (real-time PCR) and protein (Western blot) expression, fatty acid synthase (FAS) and glycerol-3-phosphate acyltransferase (GPAT) gene expression, as well as triglyceride content. Blood glucose, serum insulin and triglyceride levels, homeostasis model assessment of insulin resistance (HOMA-IR) indexes and impaired glucose tolerance were higher in FRD rats. Whereas UCP2 and PPARδ gene and protein expression increased in these animals; PPARγ levels were lower and those of PPARα remained unchanged. FRD also increased the mRNA expression of PPARδ target genes FAS and GPAT. Our results suggest that a) the increased UCP2 gene and protein expression measured in FRD rats could be part of a compensatory mechanism to reduce reactive oxygen species production induced by the fructose overload, and b) PPARs expression participates actively in the regulation of UCP2 expression, and under the metabolic condition tested, PPARδ played a key role. This knowledge would help to better understand the mechanisms involved in liver adaptation to fructose-induced glycoxidative stress, and to develop appropriate prevention strategies in obesity and type 2 diabetes. Copyright © 2011 Elsevier Inc. All rights reserved.

Substitution of soy protein for casein prevents oxidative modification and inflammatory response induced in rats fed high fructose diet.

PubMed

Sreeja, S; Geetha, Rajagopalan; Priyadarshini, Emayavaramban; Bhavani, Krishnamoorthy; Anuradha, Carani Venkatraman

2014-01-01

Fructose-rich diet is known to cause metabolic dysregulation, oxidative stress, and inflammation. We aimed to compare the effects of two dietary proteins of animal and plant origins on fructose-induced oxidative stress and inflammatory changes in liver. Wistar rats were fed either starch or fructose (60%) diet with casein or soy protein (20%) as the protein source for 8 weeks. Glucose and insulin, glycated hemoglobin and fructosamine, AOPP, and FRAP were determined in circulation. Intracellular ROS, oxidatively modified proteins (4-HNE and 3-NT adducts), adiponectin, TNF- α , IL-6 and PAI-1 mRNA expression, phosphorylation and activation of JNK and IKK β , and NF- κ B binding activity were assayed in liver. In comparison with starch fed group, fructose + casein group registered significant decline in antioxidant potential and increase in plasma glucose, insulin, and glycated proteins. Increased ROS production, 4-HNE and 3-NT modified proteins, JNK and IKK β activation, and NF- κ B binding activity were observed in them along with increased gene expression of PAI-1, IL-6, and TNF- α and decreased adiponectin expression. Substitution of soy protein for casein reduced oxidative modification and inflammatory changes in fructose-fed rats. These data suggest that soy protein but not casein can avert the adverse effects elicited by chronic consumption of fructose.

The metabolic enzyme fructose-1,6-bisphosphate aldolase acts as a transcriptional regulator in pathogenic Francisella.

PubMed

Ziveri, Jason; Tros, Fabiola; Guerrera, Ida Chiara; Chhuon, Cerina; Audry, Mathilde; Dupuis, Marion; Barel, Monique; Korniotis, Sarantis; Fillatreau, Simon; Gales, Lara; Cahoreau, Edern; Charbit, Alain

2017-10-11

The enzyme fructose-bisphosphate aldolase occupies a central position in glycolysis and gluconeogenesis pathways. Beyond its housekeeping role in metabolism, fructose-bisphosphate aldolase has been involved in additional functions and is considered as a potential target for drug development against pathogenic bacteria. Here, we address the role of fructose-bisphosphate aldolase in the bacterial pathogen Francisella novicida. We demonstrate that fructose-bisphosphate aldolase is important for bacterial multiplication in macrophages in the presence of gluconeogenic substrates. In addition, we unravel a direct role of this metabolic enzyme in transcription regulation of genes katG and rpoA, encoding catalase and an RNA polymerase subunit, respectively. We propose a model in which fructose-bisphosphate aldolase participates in the control of host redox homeostasis and the inflammatory immune response.The enzyme fructose-bisphosphate aldolase (FBA) plays central roles in glycolysis and gluconeogenesis. Here, Ziveri et al. show that FBA of the pathogen Francisella novicida acts, in addition, as a transcriptional regulator and is important for bacterial multiplication in macrophages.

Medium pH, carbon and nitrogen concentrations modulate the phosphate solubilization efficiency of Penicillium purpurogenum through organic acid production.

PubMed

Scervino, J M; Papinutti, V L; Godoy, M S; Rodriguez, M A; Della Monica, I; Recchi, M; Pettinari, M J; Godeas, A M

2011-05-01

To study phosphate solubilization in Penicillium purpurogenum as function of medium pH, and carbon and nitrogen concentrations. Tricalcium phosphate (CP) solubilization efficiency of P. purpurogenum was evaluated at acid or alkaline pH using different C and N sources. Glucose- and (NH(4) )(2) SO(4) -based media showed the highest P solubilization values followed by fructose. P. purpurogenum solubilizing ability was higher in cultures grown at pH 6·5 than cultures at pH 8·5. Organic acids were detected in both alkaline and neutral media, but the relative percentages of each organic acid differed. Highest P release coincided with the highest organic acids production peak, especially gluconic acid. When P. purpurogenum grew in alkaline media, the nature and concentration of organic acids changed at different N and C concentrations. A factorial categorical experimental design showed that the highest P-solubilizing activity, coinciding with the highest organic acid production, corresponded to the highest C concentration and lowest N concentration. The results described in the present study show that medium pH and carbon and nitrogen concentrations modulate the P solubilization efficiency of P. purpurogenum through the production of organic acids and particularly that of gluconic acid. In the P solubilization optimization studies, glucose and (NH(4) )(2) SO(4) as C and N sources allowed a higher solubilization efficiency at high pH. This organism is a potentially proficient soil inoculant, especially in P-poor alkaline soils where other P solubilizers fail to release soluble P. Further work is necessary to elucidate whether these results can be extrapolated to natural soil ecosystems, where different pH values are present. Penicillium purpurogenum could be used to develop a bioprocess for the manufacture of phosphatic fertilizer with phosphate calcium minerals. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Assessment of insulin resistance in fructose-fed rats with 125I-6-deoxy-6-iodo-D-glucose, a new tracer of glucose transport.

PubMed

Perret, Pascale; Slimani, Lotfi; Briat, Arnaud; Villemain, Danièle; Halimi, Serge; Demongeot, Jacques; Fagret, Daniel; Ghezzi, Catherine

2007-05-01

Insulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state that has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats using (125)I-6-deoxy-6-iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo. Two protocols were performed, a hyperinsulinaemic-euglycaemic clamp and a normoinsulinaemic-normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood was assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs. Insulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p < 0.01; muscle, p<0.05; heart, p<0.001), whereas insulin did not stimulate 6DIG uptake in insulin-resistant fructose-fed rats. Moreover, in these tissues, the fractional transfer coefficients of entrance were significantly increased with insulin in control rats (basal vs insulin: diaphragm, p<0.001; muscle, p<0.001; heart, p<0.01) whereas no significant changes were observed in fructose-fed rats. This study sets the stage for the future use of 6DIG as a non-invasive means for the evaluation of insulin resistance by nuclear imaging.

Assessment of insulin resistance in fructose-fed rats with 125I-6-deoxy-6-iodo-D-glucose, a new tracer of glucose transport

PubMed Central

Perret, Pascale; Slimani, Lotfi; Briat, Arnaud; Villemain, Danièle; Halimi, Serge; Demongeot, Jacques; Fagret, Daniel; Ghezzi, Catherine

2007-01-01

Purpose Insulin resistance, characterised by an insulin-stimulated glucose transport defect, is an important feature of the pre-diabetic state and it has been observed in numerous pathological disorders. The purpose of this study was to assess variations in glucose transport in rats with 125I-6-Deoxy-6-Iodo-D-glucose (6DIG), a new tracer of glucose transport proposed as an imaging tool to assess insulin resistance in vivo. Methods Two protocols were performed, a hyperinsulinaemic-euglycaemic clamp and a normoinsulinaemic normoglycaemic protocol, in awake control and insulin-resistant fructose-fed rats. The tracer was injected at steady state, and activity in 11 tissues and the blood were assessed ex vivo at several time points. A multicompartmental mathematical model was developed to obtain fractional transfer coefficients of 6DIG from the blood to the organs. Results Insulin sensitivity of fructose-fed rats, estimated by the glucose infusion rate, was reduced by 40% compared with control rats. At steady-state, 6DIG uptake was significantly stimulated by insulin in insulin-sensitive tissues of control rats (basal versus insulin: diaphragm, p<0.01; muscle, p<0.05; heart, p<0.001), whereas insulin did not stimulate 6DIG uptake in insulin-resistant fructose-fed rats. Moreover, in these tissues, the fractional transfer coefficients of entrance were significantly increased with insulin in control rats (basal vs insulin: diaphragm, p<0.001; muscle, p<0.001; heart, p<0.01) and whereas no significant changes were observed in fructose-fed rats. Conclusion This study sets the stage for the future use of 6DIG as a non-invasive means for the evaluation of insulin resistance by nuclear imaging. PMID:17171359

Comparative genomics of Clavibacter michiganensis subspecies, pathogens of important agricultural crops.

PubMed

Tambong, James T

2017-01-01

Subspecies of Clavibacter michiganensis are important phytobacterial pathogens causing devastating diseases in several agricultural crops. The genome organizations of these pathogens are poorly understood. Here, the complete genomes of 5 subspecies (C. michiganensis subsp. michiganensis, Cmi; C. michiganensis subsp. sepedonicus, Cms; C. michiganensis subsp. nebraskensis, Cmn; C. michiganensis subsp. insidiosus, Cmi and C. michiganensis subsp. capsici, Cmc) were analyzed. This study assessed the taxonomic position of the subspecies based on 16S rRNA and genome-based DNA homology and concludes that there is ample evidence to elevate some of the subspecies to species-level. Comparative genomics analysis indicated distinct genomic features evident on the DNA structural atlases and annotation features. Based on orthologous gene analysis, about 2300 CDSs are shared across all the subspecies; and Cms showed the highest number of subspecies-specific CDS, most of which are mobile elements suggesting that Cms could be more prone to translocation of foreign genes. Cms and Cmi had the highest number of pseudogenes, an indication of potential degenerating genomes. The stress response factors that may be involved in cold/heat shock, detoxification, oxidative stress, osmoregulation, and carbon utilization are outlined. For example, the wco-cluster encoding for extracellular polysaccharide II is highly conserved while the sucrose-6-phosphate hydrolase that catalyzes the hydrolysis of sucrose-6-phosphate yielding glucose-6-phosphate and fructose is highly divergent. A unique second form of the enzyme is only present in Cmn NCPPB 2581. Also, twenty-eight plasmid-borne CDSs in the other subspecies were found to have homologues in the chromosomal genome of Cmn which is known not to carry plasmids. These CDSs include pathogenesis-related factors such as Endocellulases E1 and Beta-glucosidase. The results presented here provide an insight of the functional organization of the genomes of five core C. michiganensis subspecies, enabling a better understanding of these phytobacteria.

Comparative genomics of Clavibacter michiganensis subspecies, pathogens of important agricultural crops

PubMed Central

2017-01-01

Subspecies of Clavibacter michiganensis are important phytobacterial pathogens causing devastating diseases in several agricultural crops. The genome organizations of these pathogens are poorly understood. Here, the complete genomes of 5 subspecies (C. michiganensis subsp. michiganensis, Cmi; C. michiganensis subsp. sepedonicus, Cms; C. michiganensis subsp. nebraskensis, Cmn; C. michiganensis subsp. insidiosus, Cmi and C. michiganensis subsp. capsici, Cmc) were analyzed. This study assessed the taxonomic position of the subspecies based on 16S rRNA and genome-based DNA homology and concludes that there is ample evidence to elevate some of the subspecies to species-level. Comparative genomics analysis indicated distinct genomic features evident on the DNA structural atlases and annotation features. Based on orthologous gene analysis, about 2300 CDSs are shared across all the subspecies; and Cms showed the highest number of subspecies-specific CDS, most of which are mobile elements suggesting that Cms could be more prone to translocation of foreign genes. Cms and Cmi had the highest number of pseudogenes, an indication of potential degenerating genomes. The stress response factors that may be involved in cold/heat shock, detoxification, oxidative stress, osmoregulation, and carbon utilization are outlined. For example, the wco-cluster encoding for extracellular polysaccharide II is highly conserved while the sucrose-6-phosphate hydrolase that catalyzes the hydrolysis of sucrose-6-phosphate yielding glucose-6-phosphate and fructose is highly divergent. A unique second form of the enzyme is only present in Cmn NCPPB 2581. Also, twenty-eight plasmid-borne CDSs in the other subspecies were found to have homologues in the chromosomal genome of Cmn which is known not to carry plasmids. These CDSs include pathogenesis-related factors such as Endocellulases E1 and Beta-glucosidase. The results presented here provide an insight of the functional organization of the genomes of five core C. michiganensis subspecies, enabling a better understanding of these phytobacteria. PMID:28319117

LXRα Regulates Hepatic ChREBPα Activity and Lipogenesis upon Glucose, but Not Fructose Feeding in Mice

PubMed Central

Nørgaard, Rikke C.; Bindesbøll, Christian; Lucas, Christin; Dalen, Knut Tomas; Itkonen, Harri M.; Matthews, Jason; Nebb, Hilde I.; Grønning-Wang, Line M.

2017-01-01

Liver X receptors (LXRα/β) and carbohydrate response element-binding proteins (ChREBPα/β) are key players in the transcriptional control of hepatic de novo lipogenesis. LXRα/β double knockout (LXRα−/−/β−/−) mice have reduced feeding-induced nuclear O-linked N-acetylglucosamine (O-GlcNAc) signaling, ChREBPα activity, and lipogenic gene expression in livers, suggesting important roles for LXRs in linking hepatic glucose utilization to lipid synthesis. However, the role of LXRs in fructose-induced ChREBP activation and lipogenesis is currently unknown. In this study, we studied the effects of high fructose or high glucose feeding on hepatic carbohydrate metabolism and lipogenic gene expression in livers from fasted (24 h) and fasted-refed (12 h) wild type and LXRα knockout (LXRα−/−) mice. Hepatic lipogenic gene expression was reduced in glucose fed, but not fructose fed LXRα−/− mice. This was associated with lower expression of liver pyruvate-kinase (L-pk) and Chrebpβ, indicating reduced ChREBPα activity in glucose fed, but not fructose fed mice. Interestingly, ChREBP binding to the L-pk promoter was increased in fructose fed LXRα−/− mice, concomitant with increased glucose-6-phosphatase (G6pc) expression and O-GlcNAc modified LXRβ, suggesting a role for LXRβ in regulating ChREBPα activity upon fructose feeding. In conclusion, we propose that LXRα is an important regulator of hepatic lipogenesis and ChREBPα activity upon glucose, but not fructose feeding in mice. PMID:28661453

Succinic acid production from sucrose by Actinobacillus succinogenes NJ113.

PubMed

Jiang, Min; Dai, Wenyu; Xi, Yonglan; Wu, Mingke; Kong, Xiangping; Ma, Jiangfeng; Zhang, Min; Chen, Kequan; Wei, Ping

2014-02-01

In this study, sucrose, a reproducible disaccharide extracted from plants, was used as the carbon source for the production of succinic acid by Actinobacillus succinogenes NJ113. During serum bottle fermentation, the succinic acid concentration reached 57.1g/L with a yield of 71.5%. Further analysis of the sucrose utilization pathways revealed that sucrose was transported and utilized via a sucrose phosphotransferase system, sucrose-6-phosphate hydrolase, and a fructose PTS. Compared to glucose utilization in single pathway, more pathways of A. succinogenes NJ113 are dependent on sucrose utilization. By changing the control strategy in a fed-batch culture to alleviate sucrose inhibition, 60.5g/L of succinic acid was accumulated with a yield of 82.9%, and the productivity increased by 35.2%, reaching 2.16g/L/h. Thus utilization of sucrose has considerable potential economics and environmental meaning. Copyright © 2014 Elsevier Ltd. All rights reserved.

Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

PubMed Central

Buchanan, R L; Lewis, D F

1984-01-01

Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes. PMID:6091545

Impact of expression of EMP enzymes on glucose metabolism in Zymomonas mobilis.

PubMed

Chen, Rachel Ruizhen; Agrawal, Manoj; Mao, Zichao

2013-06-01

Zymomonas mobilis is the only known microorganism that utilizes the Entner-Doudoroff (ED) pathway anaerobically. In this work, we investigated whether the overexpression of a phosphofructokinase (PFK), the only missing Embden-Meyerhof-Parnas (EMP) pathway enzyme, could establish the pathway in this organism. Introduction of a pyrophosphate-dependent PFK, along with co-expression of homologous fructose-1,6-bisphosphate aldolase and triosephosphate isomerase, did not result in an EMP flux to any appreciable level. However, the metabolism of glucose was impacted significantly. Eight percent of glucose was metabolized to form a new metabolite, dihydroxyacetone. Reducing flux through the ED pathway by as much as 40 % through antisense of a key enzyme, ED aldolase, did not result in a fully functional EMP pathway, suggesting that the ED pathway, especially the lower arm, downstream from glyceraldehyde-3-phosphate, is very rigid, possibly due to redox balance.

Similarities between the Actions of Ethylene and Cyanide in Initiating the Climacteric and Ripening of Avocados 1

PubMed Central

Solomos, Theophanes; Laties, George G.

1974-01-01

A continuous exposure of intact avocados (Persea americana) to 400 μl/l of cyanide results in a rapid increase in the rate of respiration, followed by a rise in ethylene production, and eventual ripening. The pattern of changes in the glycolytic intermediates glucose 6-phosphate, fructose diphosphate, 3-phosphoglyceric acid, and phosphoenolpyruvate during the rapid rise in respiration in both ethylene and cyanide-treated fruits is similar to that found in fruits made anaerobic where a 2.3- to 3-fold increase in the rate of glycolysis is observed. It is suggested that both during the climacteric and in response to cyanide, glycolysis is enhanced. It is proposed that cyanide implements the diversion of electrons to the cyanide-resistant electron path through structural alterations which are independent of the simultaneous inhibition of cytochrome oxidase. PMID:16658918

Bifidobacterium aquikefiri sp. nov., isolated from water kefir.

PubMed

Laureys, David; Cnockaert, Margo; De Vuyst, Luc; Vandamme, Peter

2016-03-01

A novel Bifidobacterium , strain LMG 28769 T , was isolated from a household water kefir fermentation process. Cells were Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, oxidase-negative and facultatively anaerobic short rods. Analysis of its 16S rRNA gene sequence revealed Bifidobacterium crudilactis and Bifidobacterium psychraerophilum (97.4 and 97.1 % similarity towards the respective type strain sequences) as nearest phylogenetic neighbours. Its assignment to the genus Bifidobacterium was confirmed by the presence of fructose 6-phosphate phosphoketolase activity. Analysis of the hsp60 gene sequence revealed very low similarity with nucleotide sequences in the NCBI nucleotide database. The genotypic and phenotypic analyses allowed the differentiation of strain LMG 28769 T from all recognized Bifidobacterium species. Strain LMG 28769 T ( = CCUG 67145 T  = R 54638 T ) therefore represents a novel species, for which the name Bifidobacterium aquikefiri sp. nov. is proposed.

No differential effect of beverages sweetened with fructose, high-fructose corn syrup, or glucose on systemic or adipose tissue inflammation in normal-weight to obese adults: a randomized controlled trial.

PubMed

Kuzma, Jessica N; Cromer, Gail; Hagman, Derek K; Breymeyer, Kara L; Roth, Christian L; Foster-Schubert, Karen E; Holte, Sarah E; Weigle, David S; Kratz, Mario

2016-08-01

Sugar-sweetened beverage (SSB) consumption and low-grade chronic inflammation are both independently associated with type 2 diabetes and cardiovascular disease. Fructose, a major component of SSBs, may acutely trigger inflammation, which may be one link between SSB consumption and cardiometabolic disease. We sought to determine whether beverages sweetened with fructose, high-fructose corn syrup (HFCS), and glucose differentially influence systemic inflammation [fasting plasma C-reactive protein and interleukin-6 (IL-6) as primary endpoints] acutely and before major changes in body weight. Secondary endpoints included adipose tissue inflammation, intestinal permeability, and plasma fetuin-A as potential mechanistic links between fructose intake and low-grade inflammation. We conducted a randomized, controlled, double-blind, crossover design dietary intervention (the Diet and Systemic Inflammation Study) in 24 normal-weight to obese adults without fructose malabsorption. Participants drank 4 servings/d of fructose-, glucose-, or HFCS-sweetened beverages accounting for 25% of estimated calorie requirements while consuming a standardized diet ad libitum for three 8-d periods. Subjects consumed 116% of their estimated calorie requirement while drinking the beverages with no difference in total energy intake or body weight between groups as reported previously. Fasting plasma concentrations of C-reactive protein and IL-6 did not differ significantly at the end of the 3 diet periods. We did not detect a consistent differential effect of the diets on measures of adipose tissue inflammation except for adiponectin gene expression in adipose tissue (P = 0.005), which was lowest after the glucose phase. We also did not detect consistent evidence of a differential impact of these sugars on measures of intestinal permeability (lactulose:mannitol test, plasma zonulin, and plasma lipopolysaccharide-binding protein). Excessive amounts of fructose, HFCS, and glucose from SSBs consumed over 8 d did not differentially affect low-grade chronic systemic inflammation in normal-weight to obese adults. This trial was registered at clinicaltrials.gov as NCT01424306. © 2016 American Society for Nutrition.

No differential effect of beverages sweetened with fructose, high-fructose corn syrup, or glucose on systemic or adipose tissue inflammation in normal-weight to obese adults: a randomized controlled trial1

PubMed Central

Cromer, Gail; Breymeyer, Kara L; Roth, Christian L; Weigle, David S

2016-01-01

Background: Sugar-sweetened beverage (SSB) consumption and low-grade chronic inflammation are both independently associated with type 2 diabetes and cardiovascular disease. Fructose, a major component of SSBs, may acutely trigger inflammation, which may be one link between SSB consumption and cardiometabolic disease. Objective: We sought to determine whether beverages sweetened with fructose, high-fructose corn syrup (HFCS), and glucose differentially influence systemic inflammation [fasting plasma C-reactive protein and interleukin-6 (IL-6) as primary endpoints] acutely and before major changes in body weight. Secondary endpoints included adipose tissue inflammation, intestinal permeability, and plasma fetuin-A as potential mechanistic links between fructose intake and low-grade inflammation. Design: We conducted a randomized, controlled, double-blind, crossover design dietary intervention (the Diet and Systemic Inflammation Study) in 24 normal-weight to obese adults without fructose malabsorption. Participants drank 4 servings/d of fructose-, glucose-, or HFCS-sweetened beverages accounting for 25% of estimated calorie requirements while consuming a standardized diet ad libitum for three 8-d periods. Results: Subjects consumed 116% of their estimated calorie requirement while drinking the beverages with no difference in total energy intake or body weight between groups as reported previously. Fasting plasma concentrations of C-reactive protein and IL-6 did not differ significantly at the end of the 3 diet periods. We did not detect a consistent differential effect of the diets on measures of adipose tissue inflammation except for adiponectin gene expression in adipose tissue (P = 0.005), which was lowest after the glucose phase. We also did not detect consistent evidence of a differential impact of these sugars on measures of intestinal permeability (lactulose:mannitol test, plasma zonulin, and plasma lipopolysaccharide-binding protein). Conclusion: Excessive amounts of fructose, HFCS, and glucose from SSBs consumed over 8 d did not differentially affect low-grade chronic systemic inflammation in normal-weight to obese adults. This trial was registered at clinicaltrials.gov as NCT01424306. PMID:27357093

Sensitivity of fructose-1,6-biphosphatase from yeast, liver and skeletal muscle to fructose-2,6-biphosphate and 5'-adenosine monophosphate.

PubMed

von Herrath, M; Holzer, H

1988-05-01

As a prerequisite for future studies on the possible effect of sulphite, an anti-microbial agent, on gluconeogenesis in yeast, a comparative study of fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis, from yeast, liver and skeletal muscle is reported. In contrast to FBPase from yeast or liver, FBPase from skeletal muscle is approximately 1000-fold more sensitive to inhibition by 5' adenosine monophosphate and 30 to 250-fold less sensitive to inhibition by fructose-2,6-bisphosphate. The kinetic properties of the FBPases, determined by the ratios R(Mg2+/Mn2+) and R (pH 7/9) of the enzyme activities, measured at 10 mM Mg2+ and 2 mM Mn2+ and at pH 7.0 and 9.0, respectively, show a drastic difference between the skeletal muscle and the yeast or liver enzymes. The data support the idea that the enzymes from yeast and liver function in gluconeogenesis, whereas the enzyme from skeletal muscle is involved in other biological functions.

Synthesis of 5-hydroxymethylfurural from carbohydrates using large-pore mesoporous tin phosphate.

PubMed

Dutta, Arghya; Gupta, Dinesh; Patra, Astam K; Saha, Basudeb; Bhaumik, Asim

2014-03-01

A large-pore mesoporous tin phosphate (LPSnP-1) material has been synthesized hydrothermally by using Pluronic P123 as the structure-directing agent. The material is composed of aggregated nanoparticles of 10-15 nm in diameter and has a BET surface area of 216 m(2)  g(-1) with an average pore diameter of 10.4 nm. This pore diameter is twice as large as that of mesoporous tin phosphate materials synthesized through the surfactant-templating pathways reported previously. LPSnP-1 shows excellent catalytic activity for the conversion of fructose, glucose, sucrose, cellobiose, and cellulose to 5-hydroxymethylfurfural (HMF) in a water/methyl isobutyl ketone biphasic solvent to give maximum yields of HMF of 77, 50, 51, 39, and 32 mol %, respectively, under microwave-assisted heating at 423 K. Under comparable reaction conditions, LPSnP-1 gives 12 % more HMF yield than a small-pore mesoporous tin phosphate catalyst that has an identical framework composition. This confirms the beneficial role of large mesopores and nanoscale particle morphology in catalytic reactions that involve bulky natural carbohydrate molecules. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Contribution of the NADH-oxidase (Nox) to the aerobic life of Lactobacillus sanfranciscensis DSM20451T.

PubMed

Jänsch, André; Freiding, Simone; Behr, Jürgen; Vogel, Rudi F

2011-02-01

Lactobacillus sanfranciscensis is the key bacterium in traditional sourdough fermentation. The molecular background of its oxygen tolerance was investigated by comparison of wild type and NADH-oxidase (Nox) knock out mutants. The nox gene of L. sanfranciscensis DSM20451(T) coding for a NADH-oxidase (Nox) was inactivated by single crossover integration to yield strain L. sanfranciscensis DSM20451Δnox. By inactivation of the native NADH-oxidase gene, it was ensured that besides fructose, O(2) can react as an electron acceptor. In aerated cultures the mutant strain was only able to grow in MRS media supplemented with fructose as electron acceptor, whereas the wild type strain showed a fructose independent growth response. The use of oxygen as an external electron acceptor enables L. sanfranciscensis to shift from acetyl-phosphate into the acetate branch and gain an additionally ATP, while the reduced cofactors were regenerated by Nox-activity. In aerated cultures the wild type strain formed a fermentation ratio of lactate to acetate of 1.09 in MRS supplemented with fructose after 24 h of fermentation, while the mutant strain formed a fermentation ratio of 3.05. Additionally, L. sanfranciscensis showed manganese-dependent growth response in aerated cultures, the final OD and growth velocity was increased in media supplemented with manganese. The expression of two predicted Mn(2+)/Fe(2+) transporters MntH1 and MntH2 in L. sanfranciscensis DSM20451(T) was verified by amplification of a 318 bp fragment of MntH1 and a 239 bp fragment of MntH2 from cDNA library obtained from aerobically, exponentially growing cells of L. sanfranciscensis DSM20451(T) in MRS. Moreover, the mutant strain DSM20451Δnox was more sensitive to the superoxide generating agent paraquat and showed inhibition of growth on diamide-treated MRS-plates without fructose supplementation. Copyright © 2010 Elsevier Ltd. All rights reserved.

High-fructose diet during periadolescent development increases depressive-like behavior and remodels the hypothalamic transcriptome in male rats

PubMed Central

Harrell, Constance S.; Burgado, Jillybeth; Kelly, Sean D.; Johnson, Zachary P.; Neigh, Gretchen N.

2015-01-01

Fructose consumption, which promotes insulin resistance, hypertension, and dyslipidemia, has increased by over 25% since the 1970s. In addition to metabolic dysregulation, fructose ingestion stimulates the hypothalamic-pituitary-adrenal (HPA) axis leading to elevations in glucocorticoids. Adolescents are the greatest consumers of fructose, and adolescence is a critical period for maturation of the HPA axis. Repeated consumption of high levels of fructose during adolescence has the potential to promote long-term dysregulation of the stress response. Therefore, we determined the extent to which consumption of a diet high in fructose affected behavior, serum corticosterone, and hypothalamic gene expression using a whole-transcriptomics approach. In addition, we examined the potential of a high-fructose diet to interact with exposure to chronic adolescent stress. Male Wistar rats fed the periadolescent high-fructose diet showed increased anxiety-like behavior in the elevated plus maze and depressive-like behavior in the forced swim test in adulthood, irrespective of stress history. Periadolescent fructose-fed rats also exhibited elevated basal corticosterone concentrations relative to their chow-fed peers. These behavioral and hormonal responses to the high-fructose diet did not occur in rats fed fructose during adulthood only. Finally, rats fed the high-fructose diet throughout development underwent marked hypothalamic transcript expression remodeling, with 966 genes (5.6%) significantly altered and a pronounced enrichment of significantly altered transcripts in several pathways relating to regulation of the HPA axis. Collectively, the data presented herein indicate that diet, specifically one high in fructose, has the potential to alter behavior, HPA axis function, and the hypothalamic transcriptome in male rats. PMID:26356038

High-yield production of pure tagatose from fructose by a three-step enzymatic cascade reaction.

PubMed

Lee, Seon-Hwa; Hong, Seung-Hye; Kim, Kyoung-Rok; Oh, Deok-Kun

2017-08-01

To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. Fructose at 1 M (180 g l -1 ) was converted to 0.8 M (144 g l -1 ) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16 h with a productivity of 9 g l -1 h -1 . No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%. This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

Inhibitory effect of bofutsushosan (fang feng tong sheng san) on glucose transporter 5 function in vitro.

PubMed

Gao, Shengli; Satsu, Hideo; Makino, Toshiaki

2018-03-01

Bofutsushosan (BTS; fang feng tong sheng san in Chinese) is a formula in traditional Japanese Kampo medicine and Chinese medicine comprising eighteen crude drugs, and is used to treat obesity and metabolic syndrome. Fructose is contained in refreshing beverages as high-fructose corn syrup, and is associated with obesity. Fructose is absorbed via glucose transporter 5 (GLUT5) in the intestine. Therefore, the inhibition of GLUT5 is considered to be a target of obesity drugs. We evaluated the inhibitory effects of BTS extract and its constituents on fructose uptake using Chinese hamster ovary K1 cells, i.e., cells stably expressing GLUT5. Boiled water extract of BTS significantly suppressed GLUT5 function in a concentration-dependent manner without cytotoxicities. Among 18 components of BTS, the boiled water extracts of the rhizome of Zingiber officinale, the root and rhizome of Saposhnikovia divaricata, and the root of Platycodon grandiflorum exhibited significant inhibitory effects on fructose uptake with IC 50 values of 314, 119 and 475 µg/ml, respectively. Among the constituents of the rhizome of Z. officinale extract, 6-gingerol significantly inhibited GLUT5 with an IC 50 value of 39 µM, while 6-shogaol exhibited a significant but weak inhibition on GLUT5 at 100 µM. One of the mechanisms of action of BTS may be the inhibition of fructose absorption in the intestine, and one of the active components of BTS is the rhizome of Z. officinale and 6-gingerol.

Dual effects of fructose on ChREBP and FoxO1/3α are responsible for AldoB up-regulation and vascular remodelling.

PubMed

Cao, Wei; Chang, Tuanjie; Li, Xiao-Qiang; Wang, Rui; Wu, Lingyun

2017-02-01

Increased production of methylglyoxal (MG) in vascular tissues is one of the causative factors for vascular remodelling in different subtypes of metabolic syndrome, including hypertension and insulin resistance. Fructose-induced up-regulation of aldolase B (AldoB) contributes to increased vascular MG production but the underlying mechanisms are unclear. Serum levels of MG and fructose were determined in diabetic patients with hypertension. MG level had significant positive correlations with blood pressure and fructose level respectively. C57BL/6 mice were fed with control or fructose-enriched diet for 3 months and ultrasonographic and histologic analyses were performed to evaluate arterial structural changes. Fructose-fed mice exhibited hypertension and high levels of serum MG with normal glucose level. Fructose intake increased blood vessel wall thickness and vascular smooth muscle cell (VSMC) proliferation. Western blotting and real-time PCR analysis revealed that AldoB level was significantly increased in both the aorta of fructose-fed mice and the fructose-treated VSMCs, whereas aldolase A (AldoA) expression was not changed. The knockdown of AldoB expression prevented fructose-induced MG overproduction and VSMC proliferation. Moreover, fructose significantly increased carbohydrate-responsive element-binding protein (ChREBP), phosphorylated FoxO1/3α and Akt1 levels. Fructose induced translocation of ChREBP from the cytosol to nucleus and activated AldoB gene expression, which was inhibited by the knockdown of ChREBP. Meanwhile, fructose caused FoxO1/3α shuttling from the nucleus to cytosol and inhibited its binding to AldoB promoter region. Fructose-induced AldoB up-regulation was suppressed by Akt1 inhibitor but enhanced by FoxO1/3α siRNA. Collectively, fructose activates ChREBP and inactivates FoxO1/3α pathways to up-regulate AldoB expression and MG production, leading to vascular remodelling. © 2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Fructose consumption as a risk factor for non-alcoholic fatty liver disease.

PubMed

Ouyang, Xiaosen; Cirillo, Pietro; Sautin, Yuri; McCall, Shannon; Bruchette, James L; Diehl, Anna Mae; Johnson, Richard J; Abdelmalek, Manal F

2008-06-01

While the rise in non-alcoholic fatty liver disease (NAFLD) parallels the increase in obesity and diabetes, a significant increase in dietary fructose consumption in industrialized countries has also occurred. The increased consumption of high fructose corn syrup, primarily in the form of soft drinks, is linked with complications of the insulin resistance syndrome. Furthermore, the hepatic metabolism of fructose favors de novo lipogenesis and ATP depletion. We hypothesize that increased fructose consumption contributes to the development of NAFLD. A dietary history and paired serum and liver tissue were obtained from patients with evidence of biopsy-proven NAFLD (n=49) without cirrhosis and controls (n=24) matched for gender, age (+/-5 years), and body mass index (+/-3 points). Consumption of fructose in patients with NAFLD was nearly 2- to 3-fold higher than controls [365 kcal vs 170 kcal (p<0.05)]. In patients with NAFLD (n=6), hepatic mRNA expression of fructokinase (KHK), an important enzyme for fructose metabolism, and fatty acid synthase, an important enzyme for lipogenesis were increased (p=0.04 and p=0.02, respectively). In an AML hepatocyte cell line, fructose resulted in dose-dependent increase in KHK protein and activity. The pathogenic mechanism underlying the development of NAFLD may be associated with excessive dietary fructose consumption.

Active site remodeling during the catalytic cycle in metal-dependent fructose-1,6-bisphosphate aldolases.

PubMed

Jacques, Benoit; Coinçon, Mathieu; Sygusch, Jurgen

2018-03-28

Crystal structures of two bacterial metal (Zn) dependent D-fructose 1,6-bisphosphate (FBP) aldolases in complex with substrate, analogues, and triose-P reaction products were determined to 1.5-2.0 Å resolution. The ligand complexes cryotrapped in native or mutant H. pylori aldolase crystals enabled a novel mechanistic description of FBP C 3 -C 4 bond cleavage. The reaction mechanism uses active site remodelling during the catalytic cycle implicating relocation of the Zn cofactor that is mediated by conformational changes of active site loops. Substrate binding initiates conformational changes, triggered upon P 1 -phosphate binding, which liberates the Zn chelating His180, allowing it to act as a general base for the proton abstraction at the FBP C 4 -hydroxyl group. A second zinc chelating His83 hydrogen bonds the substrate C 4 - hydroxyl group and assists cleavage by stabilizing the developing negative charge during proton abstraction. Cleavage is concerted with relocation of the metal cofactor from an interior to a surface exposed site, thereby stabilizing the nascent enediolate form. Conserved residue Glu142 is essential for protonation of the enediolate form, prior to product release. A D-tagatose 1,6-bisphosphate enzymatic complex reveals how His180 mediated proton abstraction controls stereospecificity of the cleavage reaction. Recognition and discrimination of the reaction products, dihydroxyacetone-P and D-glyceraldehyde-3-P, occurs via charged hydrogen bonds between hydroxyl groups of the triose-Ps and conserved residues, Asp82 and Asp255, respectively, and are crucial aspects of the enzyme's role in gluconeogenesis. Conformational changes in mobile loops β5-α7 and β6-α8 (containing catalytic residues Glu142 and His180, respectively) drive active site remodelling enabling the relocation of the metal cofactor. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

The Expression of Glyceraldehyde-3-Phosphate Dehydrogenase Associated Cell Cycle (GACC) Genes Correlates with Cancer Stage and Poor Survival in Patients with Solid Tumors

PubMed Central

Wang, Dunrui; Moothart, Daniel R.; Lowy, Douglas R.; Qian, Xiaolan

2013-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is often used as a stable housekeeping marker for constant gene expression. However, the transcriptional levels of GAPDH may be highly up-regulated in some cancers, including non-small cell lung cancers (NSCLC). Using a publically available microarray database, we identified a group of genes whose expression levels in some cancers are highly correlated with GAPDH up-regulation. The majority of the identified genes are cell cycle-dependent (GAPDH Associated Cell Cycle, or GACC). The up-regulation pattern of GAPDH positively associated genes in NSCLC is similar to that observed in cultured fibroblasts grown under conditions that induce anti-senescence. Data analysis demonstrated that up-regulated GAPDH levels are correlated with aberrant gene expression related to both glycolysis and gluconeogenesis pathways. Down-regulation of fructose-1,6-bisphosphatase (FBP1) in gluconeogenesis in conjunction with up-regulation of most glycolytic genes is closely related to high expression of GAPDH in the tumors. The data presented demonstrate that up-regulation of GAPDH positively associated genes is proportional to the malignant stage of various tumors and is associated with an unfavourable prognosis. Thus, this work suggests that GACC genes represent a potential new signature for cancer stage identification and disease prognosis. PMID:23620736

Identifying novel genes and chemicals related to nasopharyngeal cancer in a heterogeneous network.

PubMed

Li, Zhandong; An, Lifeng; Li, Hao; Wang, ShaoPeng; Zhou, You; Yuan, Fei; Li, Lin

2016-05-05

Nasopharyngeal cancer or nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. The factors that induce nasopharyngeal cancer are still not clear. Additional information about the chemicals or genes related to nasopharyngeal cancer will promote a better understanding of the pathogenesis of this cancer and the factors that induce it. Thus, a computational method NPC-RGCP was proposed in this study to identify the possible relevant chemicals and genes based on the presently known chemicals and genes related to nasopharyngeal cancer. To extensively utilize the functional associations between proteins and chemicals, a heterogeneous network was constructed based on interactions of proteins and chemicals. The NPC-RGCP included two stages: the searching stage and the screening stage. The former stage is for finding new possible genes and chemicals in the heterogeneous network, while the latter stage is for screening and removing false discoveries and selecting the core genes and chemicals. As a result, five putative genes, CXCR3, IRF1, CDK1, GSTP1, and CDH2, and seven putative chemicals, iron, propionic acid, dimethyl sulfoxide, isopropanol, erythrose 4-phosphate, β-D-Fructose 6-phosphate, and flavin adenine dinucleotide, were identified by NPC-RGCP. Extensive analyses provided confirmation that the putative genes and chemicals have significant associations with nasopharyngeal cancer.

Identifying novel genes and chemicals related to nasopharyngeal cancer in a heterogeneous network

PubMed Central

Li, Zhandong; An, Lifeng; Li, Hao; Wang, ShaoPeng; Zhou, You; Yuan, Fei; Li, Lin

2016-01-01

Nasopharyngeal cancer or nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. The factors that induce nasopharyngeal cancer are still not clear. Additional information about the chemicals or genes related to nasopharyngeal cancer will promote a better understanding of the pathogenesis of this cancer and the factors that induce it. Thus, a computational method NPC-RGCP was proposed in this study to identify the possible relevant chemicals and genes based on the presently known chemicals and genes related to nasopharyngeal cancer. To extensively utilize the functional associations between proteins and chemicals, a heterogeneous network was constructed based on interactions of proteins and chemicals. The NPC-RGCP included two stages: the searching stage and the screening stage. The former stage is for finding new possible genes and chemicals in the heterogeneous network, while the latter stage is for screening and removing false discoveries and selecting the core genes and chemicals. As a result, five putative genes, CXCR3, IRF1, CDK1, GSTP1, and CDH2, and seven putative chemicals, iron, propionic acid, dimethyl sulfoxide, isopropanol, erythrose 4-phosphate, β-D-Fructose 6-phosphate, and flavin adenine dinucleotide, were identified by NPC-RGCP. Extensive analyses provided confirmation that the putative genes and chemicals have significant associations with nasopharyngeal cancer. PMID:27149165

Increased oxidative stress and apoptosis in peripheral blood mononuclear cells of fructose-fed rats.

PubMed

Porto, Marcella L; Lírio, Layla M; Dias, Ananda T; Batista, Alan T; Campagnaro, Bianca P; Mill, José G; Meyrelles, Silvana S; Baldo, Marcelo P

2015-12-01

Measuring of oxidative stress in peripheral blood mononuclear cells is a suitable model of dietary induced systemic oxidative stress. Thus, we aimed to evaluate whether a chronic high fructose intake could induce oxidative damage in peripheral blood and bone marrow mononuclear cells of rats. Animals were randomly assigned to the following groups: Control group (standard rat chow and tap water n=8), and Fructose group (standard rat chow and a 10% fructose solution in the drinking water n=8). Reactive oxygen species and cytokines were measure using flow cytometry in peripheral blood and bone-marrow mononuclear cells. Apoptotic cell death and the advanced oxidation protein products (AOPP) were also determined. We observed a significant increase in ROS production in peripheral blood mononuclear cells of fructose group as compared to control rats. Apoptosis and the AOPP were higher in those animals underwent high fructose intake. Serum levels of IL-6 and IL-12 were also increased after 12 weeks of high fructose intake. We concluded that fructose intake leads to systemic oxidative stress and pro-inflammatory condition which affect peripheral blood mononuclear cells and bone-marrow mononuclear cells viability. Copyright © 2015 Elsevier B.V. All rights reserved.

Sugar Allocation to Metabolic Pathways is Tightly Regulated and Affects the Virulence of Streptococcus mutans

PubMed Central

Kawada-Matsuo, Miki; Oogai, Yuichi; Komatsuzawa, Hitoshi

2016-01-01

Bacteria take up and metabolize sugar as a carbohydrate source for survival. Most bacteria can utilize many sugars, including glucose, sucrose, and galactose, as well as amino sugars, such as glucosamine and N-acetylglucosamine. After entering the cytoplasm, the sugars are mainly allocated to the glycolysis pathway (energy production) and to various bacterial component biosynthesis pathways, including the cell wall, nucleic acids and amino acids. Sugars are also utilized to produce several virulence factors, such as capsule and lipoteichoic acid. Glutamine-fructose-6-phosphate aminotransferase (GlmS) and glucosamine-6-phosphate deaminase (NagB) have crucial roles in sugar distribution to the glycolysis pathway and to cell wall biosynthesis. In Streptococcus mutans, a cariogenic pathogen, the expression levels of glmS and nagB are coordinately regulated in response to the presence or absence of amino sugars. In addition, the disruption of this regulation affects the virulence of S. mutans. The expression of nagB and glmS is regulated by NagR in S. mutans, but the precise mechanism underlying glmS regulation is not clear. In Staphylococcus aureus and Bacillus subtilis, the mRNA of glmS has ribozyme activity and undergoes self-degradation at the mRNA level. However, there is no ribozyme activity region on glmS mRNA in S. mutans. In this review article, we summarize the sugar distribution, particularly the coordinated regulation of GlmS and NagB expression, and its relationship with the virulence of S. mutans. PMID:28036052

Reducing properties, energy efficiency and carbohydrate metabolism in hyperhydric and normal carnation shoots cultured in vitro: a hypoxia stress?

PubMed

Saher, Shady; Fernández-García, Nieves; Piqueras, Abel; Hellín, Eladio; Olmos, Enrique

2005-06-01

Hyperhydricity is considered as a physiological disorder that can be induced by different stressing conditions. In the present work we have studied the metabolic and energetic states of hyperhydric carnation shoots. We have evaluated the hypothesis that hypoxia stress is the main factor affecting the metabolism of hyperhydric leaves. Our results indicate a low level of ATP in hyperhydric tissues, but only slight modifications in pyridine nucleotide contents. Concurrently, the glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49) activity in hyperhydric leaves was increased but glucokinase (GK; EC 2.7.1.2) activity was unchanged. We have observed that the metabolism of pyruvate was altered in hyperhydric tissues by the induction of pyruvate synthesis via NADP-dependent malic enzyme (EC 1.1.1.40). The enzymes of the fermentative metabolism pyruvate decarboxylase (PDC; EC 4.1.1.1) and alcohol dehydrogenase (ADH; EC 1.1.1.1) were highly increased in hyperhydric leaves. Sucrose metabolism was modified in hyperhydric leaves with a high increase in the activity of both synthesis and catabolic enzymes. The analysis of the sucrose, glucose and fructose contents indicated that all of these sugars were accumulated in hyperhydric leaves. However, the pinitol content was drastically decreased in hyperhydric leaves. We consider that these results suggest that hyperhydric leaves of carnation have adapted to hypoxia stress conditions by the induction of the oxidative pentose phosphate and fermentative pathways.

Fructose intake exacerbates the contractile response elicited by norepinephrine in mesenteric vascular bed of rats via increased endothelial prostanoids.

PubMed

Sousa, Glauciene J; Oliveira, Phablo Wendell C; Nogueira, Breno V; Melo, Antônio F; Faria, Thaís de Oliveira; Meira, Eduardo Frizera; Mill, José G; Bissoli, Nazaré S; Baldo, Marcelo P

2017-10-01

Chronic fructose intake induces major cardiovascular and metabolic disturbances and is associated with the development of hypertension due to changes in vascular function. We hypothesized that high fructose intake for 6 weeks would cause metabolic syndrome and lead to initial vascular dysfunction. Male Wistar rats were assigned to receive fructose (FRU, 10%) or drinking water (CON) for 6 weeks. Systolic blood pressure was evaluated by tail plethysmography. Fasting glucose, insulin and glucose tolerance were measured at the end of the follow-up. Mesenteric vascular bed reactivity was tested before and after pharmacological blockade. Western blot analysis was performed for iNOS, eNOS, Nox2 and COX-2. DHE staining was used for vascular superoxide anion detection. Vessel structure was evaluated by optical and electronic microscopy. Fructose intake did not alter blood pressure, but did increase visceral fat deposition and fasting glucose as well as impair insulin and glucose tolerance. Fructose increased NE-induced vasoconstriction compared with CON, and this difference was abrogated by indomethacin perfusion as well as endothelium removal. ACh-induced relaxation was preserved, and the NO modulation tested after L-NAME perfusion was similar between groups. SNP-induced relaxation was not altered. Inducible NOS was increased; however, there were no changes in eNOS, Nox2 or COX-2 protein expression. Basal or stimulated superoxide anion production was not changed by fructose intake. In conclusion, high fructose intake increased NE-induced vasoconstriction through the endothelial prostanoids even in the presence of a preserved endothelium-mediated relaxation. No major changes in vessel structure were detected. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of fructose and sucralose on flow-mediated vasodilatation in healthy, white European males.

PubMed

Memon, Muhammad Qasim; Simpson, Elizabeth Jane; Macdonald, Ian Andrew

2014-07-01

To assess how acute consumption of fructose affects flow-mediated dilatation in brachial artery. The randomised cross-over study was conducted at the University of Nottingham's Medical School, Nottingham, United Kingdom in July 2009. Ten healthy, white European males visited the laboratory twice, on separate mornings. On each visit, the volunteers consumed water (3 ml/kg bodyweight) and rested semi-supine on the bed. After 30 minutes, baseline diastolic brachial artery diameter and blood velocity was measured. At 60 minutes, blood velocity and five scans of brachial artery diameter were recorded before a blood pressure cuff was inflated on the forearm for 5 minutes and at 50-60-70-80 and 90 sec after cuff deflation. Fifteen minutes later, the volunteers consumed 500 ml of test-drink containing either fructose (0.75 g/kg bodyweight) or sucralose (sweetness-matched with fructose drink); 45 minutes later, baseline and flow-mediated dilatation was re-measured. Pre-drink and post-drink baseline values were similar on two occasions (p > 0.05). Brachial artery diameter increased (p < 0.05) by 7 +/- 3% pre-fructose and by 6.9 +/- 3% above baseline values post-fructose with no significant difference in these responses (p < 0.15). It increased (p < 0.05) by 5.9 +/- 3% above baseline before and by 6.7 +/- 2% (p < 0.01) after sucralose; a significant difference was noted in these flow-mediated dilatation responses (p < 0.02). Responses before and after sucralose were not different from those before and after fructose (p < 0.294). Acute ingestion of fructose or sucralose had no effect on flow-mediated dilatation measured at brachial artery.

High-sugar intake does not exacerbate metabolic abnormalities or cardiac dysfunction in genetic cardiomyopathy.

PubMed

Hecker, Peter A; Galvao, Tatiana F; O'Shea, Karen M; Brown, Bethany H; Henderson, Reney; Riggle, Heather; Gupte, Sachin A; Stanley, William C

2012-05-01

A high-sugar intake increases heart disease risk in humans. In animals, sugar intake accelerates heart failure development by increased reactive oxygen species (ROS). Glucose-6-phosphate dehydrogenase (G6PD) can fuel ROS production by providing reduced nicotinamide adenine dinucleotide phosphate (NADPH) for superoxide generation by NADPH oxidase. Conversely, G6PD also facilitates ROS scavenging using the glutathione pathway. We hypothesized that a high-sugar intake would increase flux through G6PD to increase myocardial NADPH and ROS and accelerate cardiac dysfunction and death. Six-week-old TO-2 hamsters, a non-hypertensive model of genetic cardiomyopathy caused by a δ-sarcoglycan mutation, were fed a long-term diet of high starch or high sugar (57% of energy from sucrose plus fructose). After 24 wk, the δ-sarcoglycan-deficient animals displayed expected decreases in survival and cardiac function associated with cardiomyopathy (ejection fraction: control 68.7 ± 4.5%, TO-2 starch 46.1 ± 3.7%, P < 0.05 for TO-2 starch versus control; TO-2 sugar 58.0 ± 4.2%, NS, versus TO-2 starch or control; median survival: TO-2 starch 278 d, TO-2 sugar 318 d, P = 0.133). Although the high-sugar intake was expected to exacerbate cardiomyopathy, surprisingly, there was no further decrease in ejection fraction or survival with high sugar compared with starch in cardiomyopathic animals. Cardiomyopathic animals had systemic and cardiac metabolic abnormalities (increased serum lipids and glucose and decreased myocardial oxidative enzymes) that were unaffected by diet. The high-sugar intake increased myocardial superoxide, but NADPH and lipid peroxidation were unaffected. A sugar-enriched diet did not exacerbate ventricular function, metabolic abnormalities, or survival in heart failure despite an increase in superoxide production. Copyright © 2012 Elsevier Inc. All rights reserved.

Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr.

PubMed

Datta, Prasun K; Deshmane, Satish; Khalili, Kamel; Merali, Salim; Gordon, John C; Fecchio, Chiara; Barrero, Carlos A

2016-09-01

HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages. We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation. The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages. Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS.

Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr

PubMed Central

Datta, Prasun K.; Deshmane, Satish; Khalili, Kamel; Merali, Salim; Gordon, John C.; Fecchio, Chiara; Barrero, Carlos A.

2016-01-01

ABSTRACT HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages. We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation. The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages. Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS. PMID:27245560

Comparison of the anti-diabetic effects of resveratrol, gliclazide and losartan in streptozotocin-induced experimental diabetes.

PubMed

Yazgan, Ümit Can; Taşdemir, Ezel; Bilgin, Hakkı Murat; Deniz Obay, Basra; Şermet, Abdurrahman; Elbey, Bilal

2015-01-01

The aim of this study was to compare the effect of the resveratrol with gliclazide and losartan in streptozotocin induced diabetic rats. Adult male Wistar albino rats were divided into five groups of seven rats each. Diabetes was induced with a single intraperitoneal (i.p.) injection of streptozotocin (55 mg/kg). Rats with blood glucose levels above 250 mg/dl after 48 h of streptozotocin injection were included in the diabetic group. Gliclazide and resveratrol were administered for 3 weeks at 5 mg/kg per day and losartan was administered for 3 weeks at 30 mg/kg per day in an oral aqueous suspension. At the end of the third week all rats were euthanized and fasting blood glucose, HbA1c and the metabolic activity of the hepatic enzymes hexokinase and glucose-6 phosphate dehydrogenase were measured in tail blood and liver specimens. All parameters were quantified using an ELISA plate reader. Resveratrol and gliclazide significantly reduced both blood glucose levels and HbA1c levels in diabetic rats (p < 0.001). However, losartan did not exhibit the same effects (p < 0.05). The enzymatic activity of the liver enzymes hexokinase, glucose-6 phosphate dehydrogenase, fructose 1,6-biphosphatase, pyruvate kinase and glucose-6 phosphatase were enhanced by resveratrol and gliclazide, while losartan treatment was not associated with significant changes in liver carbohydrate metabolism. Resveratrol was not effective in improving liver carbohydrate metabolism relative to gliclazide, a drug widely used to treat diabetes. Dose-response profile of resveratrol remains indeterminate and additional studies may be necessary to determine effective dosing in diabetes.

Role of Isradipine Loaded Solid Lipid Nanoparticles on the Pharmacodynamic Effect in Rats.

PubMed

Thirupathi, G; Swetha, E; Narendar, D

2017-03-01

Isradipine (ID), is an antihypertensive drug, having low oral bioavailability (15-24%) due to poor aqueous solubility (0.01 mg/mL) and also hepatic first-pass metabolism. Among various approaches, Solid lipid nanoparticles (SLNs) were developed using stearic acid, glyceryl monostearate as lipid matrices for improving the oral bioavailability of ID. ID-SLNs were prepared by using hot homogenization followed by ultrasonication. The prepared SLNs were characterized for size, PDI, zeta potential (ZP), entrapment efficiency (EE) and drug content. In vitro release studies were performed in 0.1NHCl and pH 6.8 phosphate buffer of by open tube method. Physical stability of the SLNs was observed at refrigerated temperature and room temperature for 90 days. Further, pharmacodynamic study was conducted in wistar rats. SLNs prepared with GMS having size of 188.6±3.6 nm, PDI of 0.273±0.052, ZP of - 21.8±2.7 mV with 86.86±0.75% EE were optimized. Differential scanning calorimetric (DSC) study revealed that no interaction between drug and lipid. In vitro release studies showed that more cumulative release of ID in pH 6.8 phosphate buffer than in 0.1NHCl during 24 h. The lyophilized SLN formulation was used in knowing morphology of SLNs, and was found to have spherical shape with increased polydispersity by Scanning electron microscopy. Pharmacodynamic study of SLNs in fructose induced hypertensive rats showed a decrease in systolic blood pressure for 36 h, when compared to suspension, which showed a decrease in systolic blood pressure for only 2 h. Thus, the results conclusively demonstrated the role of SLNs for a significant enhancement in pharmacodynamic effect of ID. © Georg Thieme Verlag KG Stuttgart · New York.

Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus.

PubMed

Brautaset, Trygve; Jakobsen M, Øyvind M; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E

2004-03-01

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50 degrees C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

PubMed Central

Brautaset, Trygve; Jakobsen, Øyvind M.; Flickinger, Michael C.; Valla, Svein; Ellingsen, Trond E.

2004-01-01

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50°C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs. PMID:14973041

An incubation medium for the elevation of adenosine triphosphate and 2,3-diphosphoglycerate in fresh and long-preserved human erythrocytes.

PubMed

Rubinstein, D; Warrendorf, E

1975-06-01

The levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate in freshly drawn human erythrocytes can be tripled by a 2 h incubation at 37 degrees C in a medium containing 21 mM glucose, 1.8 mM adenine, 5 mM pyruvate, 10 mM inosine, and 96 mM phosphate. Similar incubation conditions will restore the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood levels preserved for 12 and 15 weeks, respectively, to those of fresh cells. Omission of pyruvate from the incubation medium further increases the level of ATP slightly, but there is little elevation of 2,3-diphosphoglycerate. Under these conditions labelled pyruvate and lactate production from [14-C]glucose or [14-C]inosine is not diminished, but labelled fructose 1,6-diphosphate, rather than 2,3-diphosphoglycerate, accumulates. In addition, omission of pyruvate from the incubation medium, with a concomitant decrease in accumulation of 2,3-diphosphoglycerate, diminishes the concentration of inorganic phosphate required for optimal ATP elevation. A 5 h incubation in the glucose-adenine-pyruvate-inosine-phosphate medium elevates the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood preserved in the cold for 15 weeks to twice that of fresh cells, indicating that the cells retain their metabolic potential even after prolonged storage at 2 degrees C. The medium may provide a method of rejuvenating 10-12 week cold-preserved erythrocytes for transfusion purposes, by a 1 h incubation at 37 degrees C.

Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants

PubMed Central

Pujol, Claire; Bailly, Marc; Kern, Daniel; Maréchal-Drouard, Laurence; Becker, Hubert; Duchêne, Anne-Marie

2008-01-01

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNAGln is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNAGln is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNAGln, which is the only tRNAGln present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNAGln by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNAGln formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts. PMID:18441100

Signaling Role of Fructose Mediated by FINS1/FBP in Arabidopsis thaliana

PubMed Central

Cho, Young-Hee; Yoo, Sang-Dong

2011-01-01

Sugars are evolutionarily conserved signaling molecules that regulate the growth and development of both unicellular and multicellular organisms. As sugar-producing photosynthetic organisms, plants utilize glucose as one of their major signaling molecules. However, the details of other sugar signaling molecules and their regulatory factors have remained elusive, due to the complexity of the metabolite and hormone interactions that control physiological and developmental programs in plants. We combined information from a gain-of-function cell-based screen and a loss-of-function reverse-genetic analysis to demonstrate that fructose acts as a signaling molecule in Arabidopsis thaliana. Fructose signaling induced seedling developmental arrest and interacted with plant stress hormone signaling in a manner similar to that of glucose. For fructose signaling responses, the plant glucose sensor HEXOKINASE1 (HXK1) was dispensable, while FRUCTOSE INSENSITIVE1 (FINS1), a putative FRUCTOSE-1,6-BISPHOSPHATASE, played a crucial role. Interestingly, FINS1 function in fructose signaling appeared to be independent of its catalytic activity in sugar metabolism. Genetic analysis further indicated that FINS1–dependent fructose signaling may act downstream of the abscisic acid pathway, in spite of the fact that HXK1–dependent glucose signaling works upstream of hormone synthesis. Our findings revealed that multiple layers of controls by fructose, glucose, and abscisic acid finely tune the plant autotrophic transition and modulate early seedling establishment after seed germination. PMID:21253566

Influence of fenofibrate treatment on triacylglycerides, diacylglycerides and fatty acids in fructose fed rats.

PubMed

Kopf, Thomas; Schaefer, Hans-Ludwig; Troetzmueller, Martin; Koefeler, Harald; Broenstrup, Mark; Konovalova, Tatiana; Schmitz, Gerd

2014-01-01

Fenofibrate (FF) lowers plasma triglycerides via PPARα activation. Here, we analyzed lipidomic changes upon FF treatment of fructose fed rats. Three groups with 6 animals each were defined as control, fructose-fed and fructose-fed/FF treated. Male Wistar Unilever Rats were subjected to 10% fructose-feeding for 20 days. On day 14, fenofibrate treatment (100 mg/kg p.o.) was initiated and maintained for 7 days. Lipid species in serum were analyzed using mass spectrometry (ESI-MS/MS; LC-FT-MS, GC-MS) on days 0, 14 and 20 in all three groups. In addition, lipid levels in liver and intestine were determined. Short-chain TAGs increased in serum and liver upon fructose-feeding, while almost all TAG-species decreased under FF treatment. Long-chain unsaturated DAG-levels (36:1, 36:2, 36:4, 38:3, 38:4, 38:5) increased upon FF treatment in rat liver and decreased in rat serum. FAs, especially short-chain FAs (12:0, 14:0, 16:0) increased during fructose-challenge. VLDL secretion increased upon fructose-feeding and together with FA-levels decreased to control levels during FF treatment. Fructose challenge of de novo fatty acid synthesis through fatty acid synthase (FAS) may enhance the release of FAs ≤ 16:0 chain length, a process reversed by FF-mediated PPARα-activation.

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis.

PubMed

Narasaki, Craig T; Mertens, Katja; Samuel, James E

2011-01-01

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.

Consumption of Alcopops During Brain Maturation Period: Higher Impact of Fructose Than Ethanol on Brain Metabolism.

PubMed

El Hamrani, Dounia; Gin, Henri; Gallis, Jean-Louis; Bouzier-Sore, Anne-Karine; Beauvieux, Marie-Christine

2018-01-01

Alcopops are flavored alcoholic beverages sweetened by sodas, known to contain fructose. These drinks have the goal of democratizing alcohol among young consumers (12-17 years old) and in the past few years have been considered as fashionable amongst teenagers. Adolescence, however, is a key period for brain maturation, occurring in the prefrontal cortex and limbic system until 21 years old. Therefore, this drinking behavior has become a public health concern. Despite the extensive literature concerning the respective impacts of either fructose or ethanol on brain, the effects following joint consumption of these substrates remains unknown. Our objective was to study the early brain modifications induced by a combined diet of high fructose (20%) and moderate amount of alcohol in young rats by 13 C Nuclear Magnetic Resonance (NMR) spectroscopy. Wistar rats had isocaloric pair-fed diets containing fructose (HF, 20%), ethanol (Et, 0.5 g/day/kg) or both substrates at the same time (HFEt). After 6 weeks of diet, the rats were infused with 13 C-glucose and brain perchloric acid extracts were analyzed by NMR spectroscopy ( 1 H and 13 C). Surprisingly, the most important modifications of brain metabolism were observed under fructose diet. Alterations, observed after only 6 weeks of diet, show that the brain is vulnerable at the metabolic level to fructose consumption during late-adolescence throughout adulthood in rats. The main result was an increase in oxidative metabolism compared to glycolysis, which may impact lactate levels in the brain and may, at least partially, explain memory impairment in teenagers consuming alcopops.

Prior Exercise Does Not Reduce Postprandial Lipemia Following a Mixed Glucose Meal When Compared with a Mixed Fructose Meal.

PubMed

Rowe, James R; Biggerstaff, Kyle D; Ben-Ezra, Vic; Nichols, David L; DiMarco, Nancy

2016-10-01

This study examined the effect of prior exercise on postprandial lipemia (PPL) concentration following a mixed meal (MM) made with either glucose or fructose. Sedentary women completed four trials in random order: 1) Rest-Fructose: RF, 2) Rest-Glucose: RG, 3) Exercise-Fructose: EF, 4) Exercise-Glucose: EG. Exercise expended 500 kcal while walking at 70%VO 2max . Rest was 60 min of sitting. The morning after each trial, a fasting (12 hr) blood sample was collected followed by consumption of the MM. The MM was blended with whole milk and ice cream plus a glucose or fructose powder. Glucose and fructose powder accounted for 30% of the total kcal within the MM. Blood was collected periodically for 6 hr post-MM and analyzed for PPL. Magnitude of PPL over the 6 hr postmeal was quantified using the triglyceride incremental area under the curve (TG AUC I ). Significant differences (p < .05) between trials were determined using repeated-measures ANOVA and Bonferroni post hoc test. There was no significant difference in the TG AUC I between the four trials (p > .05). A significant trial by time interaction for TG concentration was reported (p < .05). Despite lack of change in the AUC I with prior exercise, the lower TG concentration at multiple time points in the EG trial does indicate that prior exercise has some desirable effect on PPL. This study suggests that replacing fructose with glucose sugars and incorporating exercise may minimize PPL following a mixed meal but exercise will need to elicit greater energy expenditure.

The Bacillus subtilis ywjI (glpX) gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the class III Fbp enzyme.

PubMed

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-05-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.

The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme▿

PubMed Central

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-01-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions. PMID:19270101

Flavor preference conditioning by different sugars in sweet ageusic Trpm5 knockout mice.

PubMed

Sclafani, Anthony; Ackroff, Karen

2015-03-01

Knockout (KO) mice missing the taste signaling protein Trpm5 have greatly attenuated sweetener preferences but develop strong preferences for glucose in 24-h tests, which is attributed to post-oral sugar conditioning. Trpm5 KO mice express mild preferences for galactose but no preferences for fructose in 24-h tests, which suggests that these sugars differ in their post-oral reinforcing effects. Here we investigated sugar-conditioned flavor preferences in Trpm5 KO and C57BL/6J wildtype (B6) mice. The mice were trained to consume a flavored (CS+, e.g. grape) 8% sugar solution and flavored (CS-, e.g., cherry) water on alternating days followed by two-bottle choice tests with CS+ vs. CS- flavors in water and with unflavored sugar vs. water. The KO mice displayed strong preferences (>80%) for the CS+ glucose and CS+ galactose but not for the CS+ fructose flavor. They also preferred glucose and galactose, but not fructose to water. In contrast, the B6 mice preferred all three CS+ flavors to the CS- flavor, and all three sugars to water. In tests with the non-metabolizable sugar α-methyl-d-glucopyranoside (MDG), the KO and B6 mice preferred 8% MDG to water but did not prefer the CS+ 8% MDG to CS-. However, they preferred a CS+ flavor mixed with 4% MDG over the CS- flavor. Trpm5 KO mice also preferred galactose and MDG to fructose in direct choice tests. The Trpm5 KO data indicate that glucose and, to a lesser extent, galactose and MDG have post-oral reinforcing actions that stimulate intake and preference while fructose has a much weaker effect. The CS+ flavor and sugar preferences of B6 mice may be mediated by the sweet taste and/or post-oral actions of the various sugars. Glucose, galactose, and MDG, but not fructose, are ligands for the sodium-glucose transporter 1 (SGLT1) which is implicated in post-oral sugar conditioning in B6 mice. Copyright © 2014 Elsevier Inc. All rights reserved.

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

PubMed Central

De Diego, Nuria; Muñoz, Francisco J.; Baroja-Fernández, Edurne; Li, Jun; Ricarte-Bermejo, Adriana; Baslam, Marouane; Aranjuelo, Iker; Almagro, Goizeder; Humplík, Jan F.; Novák, Ondřej; Spíchal, Lukáš; Doležal, Karel; Pozueta-Romero, Javier

2015-01-01

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs. PMID:25811607

Effect of tangeretin, a polymethoxylated flavone on glucose metabolism in streptozotocin-induced diabetic rats.

PubMed

Sundaram, Ramalingam; Shanthi, Palanivelu; Sachdanandam, Panchanatham

2014-05-15

The present study was designed to evaluate the antihyperglycemic potential of tangeretin on the activities of key enzymes of carbohydrate and glycogen metabolism in control and streptozotocin induced diabetic rats. The daily oral administration of tangeretin (100mg/kg body weight) to diabetic rats for 30 days resulted in a significant reduction in the levels of plasma glucose, glycosylated hemoglobin (HbA1c) and increase in the levels of insulin and hemoglobin. The altered activities of the key enzymes of carbohydrate metabolism such as hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase and glycogen phosphorylase in liver of diabetic rats were significantly reverted to near normal levels by the administration of tangeretin. Further, tangeretin administration to diabetic rats improved hepatic glycogen content suggesting the antihyperglycemic potential of tangeretin in diabetic rats. The effect produced by tangeretin on various parameters was comparable to that of glibenclamide - a standard oral hypoglycemic drug. Thus, these results show that tangeretin modulates the activities of hepatic enzymes via enhanced secretion of insulin and decreases the blood glucose in streptozotocin induced diabetic rats by its antioxidant potential. Copyright © 2014 Elsevier GmbH. All rights reserved.

Modulatory effects of naringin on hepatic key enzymes of carbohydrate metabolism in high-fat diet/low-dose streptozotocin-induced diabetes in rats.

PubMed

Pari, Leelavinothan; Chandramohan, Ramasamy

2017-07-01

We evaluated the modulatory effects of naringin on altered hepatic key enzymes of carbohydrate metabolism in high-fat diet/low-dose streptozotocin-induced diabetic rats. Oral treatment of naringin at a doses of 20, 40 and 80 mg/kg body weight to diabetic rats for 30 days resulted in a significant reduction in the levels of plasma glucose, blood glycosylated hemoglobin and increase in the levels of plasma insulin and blood hemoglobin. The altered activities of the hepatic key enzymes of carbohydrate metabolism such as hexokinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase, glycogen phosphorylase and glycogen content of diabetic rats were significantly reverted to near normal levels by the treatment of naringin in a dose-dependent manner. Naringin at a dose of 80 mg/kg body weight showed the highest significant effect than the other two doses (20 and 40 mg/kg). Further, immunohistochemical observation of pancreas revealed that naringin-treated diabetic rats showed the increased number of insulin immunoreactive β-cells, which confirmed the biochemical findings. These findings revealed that naringin has potential antihyperglycemic activity in high-fat diet/low-dose streptozotocin-induced diabetic rats.

The genes and enzymes of sucrose metabolism in moderately thermophilic methanotroph Methylocaldum szegediense O12.

PubMed

But, Sergey Y; Solntseva, Natalia P; Egorova, Svetlana V; Mustakhimov, Ildar I; Khmelenina, Valentina N; Reshetnikov, Alexander; Trotsenko, Yuri A

2018-05-01

Four enzymes involved in sucrose metabolism: sucrose phosphate synthase (Sps), sucrose phosphate phosphatase (Spp), sucrose synthase (Sus) and fructokinase (FruK), were obtained as his-tagged proteins from the moderately thermophilic methanotroph Methylocaldum szegediense O12. Sps, Spp, FruK and Sus demonstrated biochemical properties similar to those of other bacterial counterparts, but the translated amino acid sequences of Sps and Spp displayed high divergence from the respective microbial enzymes. The Sus of M. szegediense O12 catalyzed the reversible reaction of sucrose cleavage in the presence of ADP or UDP and preferred ADP as a substrate, thus implying a connection between sucrose and glycogen metabolism. Sus-like genes were found only in a few methanotrophs, whereas amylosucrase was generally used in sucrose cleavage in this group of bacteria. Like other microbial fructokinases, FruK of M. szegediense O12 showed a high specificity to fructose.

The effect of a low fructose and low glycemic index/load (FRAGILE) dietary intervention on indices of liver function, cardiometabolic risk factors, and body composition in children and adolescents with nonalcoholic fatty liver disease (NAFLD).

PubMed

Mager, Diana R; Iñiguez, Ingrid Rivera; Gilmour, Susan; Yap, Jason

2015-01-01

Nonalcoholic fatty liver disease (NAFLD) is a common liver disease in obese children. Diets high in added fructose (high fructose corn syrup; HFCS) and glycemic index (GI)/glycemic load (GL) are associated with increased risk of NAFLD. Lifestyle modification is the main treatment, but no guidelines regarding specific dietary interventions for childhood NAFLD exist. We hypothesized that reductions in dietary fructose (total, free, and HFCS)/GI/GL over 6 months would result in improvements in body composition and markers of liver dysfunction and cardiometabolic risk in childhood NAFLD. Children and adolescents with NAFLD (n = 12) and healthy controls (n = 14) 7-18 years were studied at baseline and 3 and 6 months post-dietary intervention. Plasma markers of liver dysfunction (ALT, AST, γGT), cardiometabolic risk (TG, total cholesterol, LDL-HDL cholesterol, Apo-B100, Apo-B48, Apo-CIII, insulin, homeostasis model of assessment of insulin resistance [HOMA-IR]), inflammation (TNF-α, IL-6, IL-10), anthropometric, and blood pressure (BP) were studied using validated methodologies. Significant reductions in systolic BP (SBP), percentage body fat (BF), and plasma concentrations of ALT (P = .04), Apo-B100 (P < .001), and HOMA-IR were observed in children with NAFLD at 3 and 6 months (P < .05). Dietary reductions in total/free fructose/HFCS and GL were related to reductions in SBP (P = .01), ALT (P = .004), HOMA-IR (P = .03), and percentage BF in children with NAFLD. Reductions in dietary GI were associated with reduced plasma Apo-B100 (P = .02) in both groups. With the exception of Apo-B100, no changes in laboratory variables were observed in the control group. Modest reductions in fructose (total/free, HFCS) and GI/GL intake result in improvements of plasma markers of liver dysfunction and cardiometabolic risk in childhood NAFLD. © 2013 American Society for Parenteral and Enteral Nutrition.

Fructose-Glucose Composite Carbohydrates and Endurance Performance: Critical Review and Future Perspectives.

PubMed

Rowlands, David S; Houltham, S; Musa-Veloso, K; Brown, F; Paulionis, L; Bailey, D

2015-11-01

Sports beverages formulated with fructose and glucose composites enhance exogenous carbohydrate oxidation, gut comfort, and endurance performance, relative to single-saccharide formulations. However, a critical review of performance data is absent. We conducted a comprehensive literature review of the effect of fructose:glucose/maltodextrin (glucose or maltodextrin) composites versus glucose/maltodextrin on endurance performance. Mechanistic associations were drawn from effects on carbohydrate metabolism, gut, and other sensory responses. Overall, 14 studies contained estimates of 2.5-3.0-h endurance performance in men, mostly in cycling. Relative to isocaloric glucose/maltodextrin, the ingestion of 0.5-1.0:1-ratio fructose:glucose/maltodextrin beverages at 1.3-2.4 g carbohydrate·min(-1) produced small to moderate enhancements (1-9 %; 95 % confidence interval 0-19) in mean power. When 0.5:1-ratio composites were ingested at ≥1.7 g·min(-1), improvements were larger (4-9 %; 2-19) than at 1.4-1.6 g·min(-1) (1-3 %; 0-6). The effect sizes at higher ingestion rates were associated with increased exogenous carbohydrate oxidation rate, unilateral fluid absorption, and lower gastrointestinal distress, relative to control. Solutions containing a 0.7-1.0:1 fructose:glucose ratio were absorbed fastest; when ingested at 1.5-1.8 g·min(-1), a 0.8:1 fructose:glucose ratio conveyed the highest exogenous carbohydrate energy and endurance power compared with lower or higher fructose:glucose ratios. To conclude, ingesting 0.5-1.0:1-ratio fructose:glucose/maltodextrin beverages at 1.3-2.4 g·min(-1) likely benefits 2.5-3.0 h endurance power versus isocaloric single saccharide. Further ratio and dose-response research should determine if meaningful performance benefits of composites accrue with ingestion <1.3 g·min(-1), relative to higher doses. Effects should be established in competition, females, other food formats, and in heat-stress and ultra-endurance exercise where carbohydrate demands may differ from the current analysis.

Beraprost sodium, a prostacyclin analogue, reduces fructose-induced hepatocellular steatosis in mice and in vitro via the microRNA-200a and SIRT1 signaling pathway.

PubMed

Zhang, Pengyuan; Xu, Lijuan; Guan, Hongyu; Liu, Liehua; Liu, Juan; Huang, Zhimin; Cao, Xiaopei; Liao, Zhihong; Xiao, Haipeng; Li, Yanbing

2017-08-01

To determine whether beraprost sodium, a prostacyclin analogue, could reduce hepatic lipid accumulation induced by fructose in mice and cultured human hepatocytes, and to investigate the expression of microRNAs and the sirtuin 1 (SIRT1) pathway. Male C57BL/6JNju mice were divided into three groups and fed one of the following diets: a normal diet, a high fructose diet, or a high fructose diet with beraprost sodium treatment. In addition, human-derived HepG2 cells were cultured and treated with fructose (25mmol/L) with or without beraprost sodium (10μmol/L) for 24h, and transfected with small interfering RNA (siRNA) against SIRT1, miR-200a mimic, or miR-200a inhibitor for 48h. The miRNA microarray analysis was performed on the HepG2 cells, and the expression profiles of miRNAs were analyzed using Gene Cluster 3.0 and verified using qPCR. Beraprost sodium treatment attenuated hepatic steatosis, induced the transcription of genes involved in lipid metabolism in C57BL/6 mice (P<0.05), and increased the expression of hepatic SIRT1 and peroxisome proliferator activated receptor α (PPARα) in the cells treated with fructose. These effects were blocked in HepG2 cells after transfection with siRNA against SIRT1. MiR-200a was highly expressed during fructose treatment and was down regulated by beraprost sodium (P<0.05). A luciferase assay showed that miR-200a regulated SIRT1 by binding to the 3' UTR. Overexpression of miR-200a inhibited expression of hepatic SIRT1. Our study demonstrated that SIRT1 pathway mediated the effects of beraprost sodium on attenuation of hepatic lipid disorders induced by fructose and revealed the primary role of miR-200a in the regulation of hepatic SIRT1 by beraprost sodium. Our findings suggested that SIRT1 might be a therapeutic target of fructose-related metabolism disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

3D-QSAR studies and molecular docking on [5-(4-amino-1 H-benzoimidazol-2-yl)-furan-2-yl]-phosphonic acid derivatives as fructose-1,6-biphophatase inhibitors

NASA Astrophysics Data System (ADS)

Lan, Ping; Xie, Mei-Qi; Yao, Yue-Mei; Chen, Wan-Na; Chen, Wei-Min

2010-12-01

Fructose-1,6-biphophatase has been regarded as a novel therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). 3D-QSAR and docking studies were performed on a series of [5-(4-amino-1 H-benzoimidazol-2-yl)-furan-2-yl]-phosphonic acid derivatives as fructose-1,6-biphophatase inhibitors. The CoMFA and CoMSIA models using thirty-seven molecules in the training set gave r cv 2 values of 0.614 and 0.598, r 2 values of 0.950 and 0.928, respectively. The external validation indicated that our CoMFA and CoMSIA models possessed high predictive powers with r 0 2 values of 0.994 and 0.994, r m 2 values of 0.751 and 0.690, respectively. Molecular docking studies revealed that a phosphonic group was essential for binding to the receptor, and some key features were also identified. A set of forty new analogues were designed by utilizing the results revealed in the present study, and were predicted with significantly improved potencies in the developed models. The findings can be quite useful to aid the designing of new fructose-1,6-biphophatase inhibitors with improved biological response.

Fructose diet alleviates acetaminophen-induced hepatotoxicity in mice.

PubMed

Cho, Sungjoon; Tripathi, Ashutosh; Chlipala, George; Green, Stefan; Lee, Hyunwoo; Chang, Eugene B; Jeong, Hyunyoung

2017-01-01

Acetaminophen (APAP) is a commonly used analgesic and antipyretic that can cause hepatotoxicity due to production of toxic metabolites via cytochrome P450 (Cyp) 1a2 and Cyp2e1. Previous studies have shown conflicting effects of fructose (the major component in Western diet) on the susceptibility to APAP-induced hepatotoxicity. To evaluate the role of fructose-supplemented diet in modulating the extent of APAP-induced liver injury, male C57BL/6J mice were given 30% (w/v) fructose in water (or regular water) for 8 weeks, followed by oral administration of APAP. APAP-induced liver injury (determined by serum levels of liver enzymes) was decreased by two-fold in mice pretreated with fructose. Fructose-treated mice exhibited (~1.5 fold) higher basal glutathione levels and (~2 fold) lower basal (mRNA and activity) levels of Cyp1a2 and Cyp2e1, suggesting decreased bioactivation of APAP and increased detoxification of toxic metabolite in fructose-fed mice. Hepatic mRNA expression of heat shock protein 70 was also found increased in fructose-fed mice. Analysis of bacterial 16S rRNA gene amplicons from the cecal samples of vehicle groups showed that the fructose diet altered gut bacterial community, leading to increased α-diversity. The abundance of several bacterial taxa including the genus Anaerostipes was found to be significantly correlated with the levels of hepatic Cyp2e1, Cyp1a2 mRNA, and glutathione. Together, these results suggest that the fructose-supplemented diet decreases APAP-induced liver injury in mice, in part by reducing metabolic activation of APAP and inducing detoxification of toxic metabolites, potentially through altered composition of gut microbiota.

Intestinal multidrug resistance-associated protein 2 is down-regulated in fructose-fed rats.

PubMed

Londero, Ana Sofía; Arana, Maite Rocío; Perdomo, Virginia Gabriela; Tocchetti, Guillermo Nicolás; Zecchinati, Felipe; Ghanem, Carolina Inés; Ruiz, María Laura; Rigalli, Juan Pablo; Mottino, Aldo Domingo; García, Fabiana; Villanueva, Silvina Stella Maris

2017-02-01

Expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2) and glutathione-S-transferase (GST) were examined in fructose fed Wistar rats, an experimental model of metabolic syndrome. Animals were fed on (a) control diet or (b) control diet plus 10% w/vol fructose in the drinking water. Mrp2 and the α class of GST proteins as well as their corresponding mRNAs were decreased, suggesting a transcriptional regulation by fructose. Confocal microscopy studies reaffirmed down-regulation of Mrp2. Everted intestinal sacs were incubated with 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl- S-glutathione (DNP-SG; model Mrp2 substrate), was measured in the same compartment to estimate Mrp2 activity. Excretion of DNP-SG was substantially decreased by fructose treatment, consistent with simultaneous down-regulation of Mrp2 and GST. In addition, the effect of fructose on intestinal barrier function exerted by Mrp2 was evaluated in vivo using valsartan, a recognized Mrp2 substrate of therapeutic use. After intraduodenal administration as a bolus, intestinal absorption of valsartan was increased in fructose-drinking animals. Fructose administration also induced oxidative stress in intestinal tissue as demonstrated by significant increases of intestinal lipid peroxidation end products and activity of the antioxidant enzyme superoxide dismutase, by a decreased GSH/GSSG ratio. Moreover, fructose treatment conduced to increased intestinal levels of the proinflammatory cytokines IL-β1 and IL-6. Collectively, our results demonstrate that metabolic syndrome-like conditions, induced by a fructose-rich diet, result in down-regulation of intestinal Mrp2 expression and activity and consequently in an impairment of its barrier function. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of a high-fructose diet on glucose tolerance, plasma lipid and hemorheological parameters during oral contraceptive administration in female rats.

PubMed

Olatunji, Lawrence Aderemi; Oyeyipo, Ibukun Peter; Usman, Taofeek Oluwamayowa

2013-01-01

Oral contraceptive (OC) use and increased fructose feeding have been associated with altered cardiometabolic effects. The effect of increased dietary fructose during OC use on cardiometabolic parameters is unknown. We investigated the effects of a high-fructose diet on body weight gain, fasting blood glucose, glucose tolerance, plasma lipid and hemorheological parameters in female rats treated with a combination of OC steroids (norgestrel/ethinyl estradiol; NEE). Rats were given (p.o.) vehicle, high-dose NEE (10.0 μg norgestrel/1.0 μg ethinyl estradiol) or low-dose NEE (1.0 μg norgestrel/0.1 μg ethinyl estradiol) with or without high dietary fructose daily for 6 weeks. Results demonstrated that high-dose NEE but not low-dose NEE treatment led to significant increases in hematocrit, blood viscosity, and decreases in body weight gain, glucose tolerance, and plasma HDL-cholesterol level. Both NEE treatments resulted in significant increases in plasma viscosity and triglyceride. Increased dietary fructose without NEE treatment produced significant increases in fasting blood glucose, hematocrit, blood and plasma viscosities, while increased dietary fructose significantly potentiated the effects on blood and plasma viscosities observed during NEE treatment. Conversely, the effects of NEE treatment on body weight gain, glucose tolerance, plasma triglyceride and HDL-cholesterol were significantly attenuated. In conclusion, the results indicate that increase in dietary fructose may worsen abnormal blood rheology. The results also demonstrate that increased dietary fructose may not impact negatively on glucose and lipid metabolisms during OC use. The findings imply that fructose-enriched diet might be an important consideration during OC use regarding blood rheological properties.

Maternal high fructose and low protein consumption during pregnancy and lactation share some but not all effects on early-life growth and metabolic programming of rat offspring.

PubMed

Arentson-Lantz, Emily J; Zou, Mi; Teegarden, Dorothy; Buhman, Kimberly K; Donkin, Shawn S

2016-09-01

Maternal nutritional stress during pregnancy acts to program offspring metabolism. We hypothesized that the nutritional stress caused by maternal fructose or low protein intake during pregnancy would program the offspring to develop metabolic aberrations that would be exacerbated by a diet rich in fructose or fat during adult life. The objective of this study was to characterize and compare the fetal programming effects of maternal fructose with the established programming model of a low-protein diet on offspring. Male offspring from Sprague-Dawley dams fed a 60% starch control diet, a 60% fructose diet, or a low-protein diet throughout pregnancy and lactation were weaned onto either a 60% starch control diet, 60% fructose diet, or a 30% fat diet for 15 weeks. Offspring from low-protein and fructose-fed dam showed retarded growth (P<.05) at weaning (50.3, 29.6 vs 59.1±0.8 g) and at 18 weeks of age (420, 369 vs 464±10.9 g). At 18 weeks of age, offspring from fructose dams expressed greater quantities (P<.05) of intestinal Pgc1a messenger RNA compared with offspring from control or low-protein dams (1.31 vs 0.89, 0.85; confidence interval, 0.78-1.04). Similarly, maternal fructose (P=.09) and low-protein (P<.05) consumption increased expression of Pgc1a in offspring liver (7.24, 2.22 vs 1.22; confidence interval, 2.11-3.45). These data indicate that maternal fructose feeding is a programming model that shares some features of maternal protein restriction such as retarded growth, but is unique in programming of selected hepatic and intestinal transcripts. Copyright © 2016. Published by Elsevier Inc.

Comparative Study of 6-Mercaptopurine Metabolism in Human Leukemic Leukocytes and L1210 Cells

PubMed Central

Higuchi, Tomihiko; Nakamura, Toru; Uchino, Haruto; Wakisaka, Gyoichi

1977-01-01

Leukocytes from patients with leukemia and L1210 cells from mice were examined for the rate of formation and cellular concentration of phosphoribosylpyrophosphate, the rate of thioinosinic acid formation, and a number of selected enzymes involved in purine nucleotide synthesis. The amount of thioinosinic acid formed in L1210 cells was much higher than that in human leukemic leukocytes. In cell extracts, the synthesis of thioinosinic acid was similar in both cell types, and the amount of purine phosphoribosyltransferase was not rate limiting in either case. Much higher concentrations and rates of formation of phosphoribosylpyrophosphate were found in L1210 cells than in human leukemic leukocytes. The difference in response to 6-mercaptopurine between L1210 cells and human leukemic leukocytes might be attributed to their difference in supply of phosphoribosylpyrophosphate. Phosphoribosylpyrophosphate-amidotransferase was found to be high in L1210 cells, but was not detected in human leukemic leukocytes. PMID:921247

Acute metabolic and endocrine responses induced by glucose and fructose in healthy young subjects: A double-blinded, randomized, crossover trial.

PubMed

Cai, Wenwen; Li, Jie; Shi, Jiahui; Yang, Bo; Tang, Jun; Truby, Helen; Li, Duo

2018-04-01

A rise in fructose consumption has been implicated in the etiology of obesity, diabetes and cardiovascular disease. Serum uric acid (UA) elevates after fructose ingestion, increasing the risk of cardiovascular disease. However, the impact of fructose ingestion on nitric oxide (NO) has not yet been confirmed. The aim of this study was to investigate the postprandial metabolic and endocrine responses following an acute ingestion of fructose and glucose in healthy subjects. This was a double-blinded, randomized, crossover postprandial trial. Eighteen healthy young subjects (9 males and 9 females) with a mean age of 23.6 ± 2.3 years and mean BMI of 20.2 ± 1.5 kg/m 2 completed the experiment that was conducted in Hangzhou, China. Volunteers were randomized to two groups (A and B): after an 8-h overnight fast, volunteers either ingested 300 mL of 25% glucose (group A) or fructose (group B) solution at 0830 within 5 min. After a one-week washout period, volunteers were crossed over to receive the alternate test solution. Blood pressure was measured at 0 h, 1 h, 2 h and 3 h and venous blood was drawn at 0 h, 0.5 h, 1 h, 2 h and 3 h after ingestion of the test solution. Eighteen subjects completed the study. Serum NO level tended to be lower at 1 h (59.40 ± 3.10 μmol/L and 68.1 ± 3.40 μmol/L, respectively, p ≤ 0.05) and 2 h (62.70 ± 3.10 μmol/L and 70.10 ± 3.50 μmol/L, respectively, p ≤ 0.05) after fructose ingestion than after glucose. The 3-h AUC (area under curve) of NO was significantly lower after fructose ingestion than after glucose (p ≤ 0.05). UA level was higher at 1 h (512.17 ± 17.74 μmol/L and 372.11 ± 17.41 μmol/L, respectively, p ≤ 0.01) and 2 h (440.22 ± 16.07 μmol/L and 357.39 ± 14.80 μmol/L, respectively, p ≤ 0.05) after fructose ingestion than after glucose. The 3-h AUC of UA was significantly higher after fructose ingestion than after glucose (p ≤ 0.01). Correlation analyses revealed that NO was negatively associated with UA at T 0.5h (r = -0.62, p ≤ 0.01), T 1.0h (r = -0.69, p ≤ 0.001), T 2.0h (r = -0.86, p ≤ 0.001) and T 3.0h (r = -0.85, p ≤ 0.001) after fructose ingestion. SBP (systolic blood pressure) tended to be higher at 1 h (125.33 ± 1.95 mmHg and 112.06 ± 1.77 mmHg, respectively, p ≤ 0.05) after fructose ingestion than after glucose. The 3-h AUC of SBP was significantly higher after fructose ingestion than after glucose (p ≤ 0.05). The 3 h-AUC of TG, TC, HDL-C and LDL-C showed no differences between fructose and glucose. LDH (lactate dehydrogenase) level was higher at 1 h (195.00 ± 5.6 U/L and 177.67 ± 6.8 U/L, respectively, p ≤ 0.05) and 2 h (197.01 ± 6.32 U/L and 185.50 ± 7.37 U/L, respectively, p ≤ 0.05) after fructose ingestion than after glucose. The 3-h AUC of LDH was significantly higher after fructose ingestion than after glucose (p ≤ 0.05). AR was significantly higher at 1 h (19.86 ± 0.52 ng/mg Hb and 16.98 ± 0.29 ng/mg Hb, respectively, p ≤ 0.05) after fructose ingestion than after glucose. The 3-h AUC of AR (p ≤ 0.05) was significantly higher after fructose ingestion than after glucose (p ≤ 0.05). Ingestion of a 75 g fructose load led to acute but unfavorable changes in certain metabolic and endocrine responses including increased serum concentrations and 3 h-AUC of UA, AR and LDH, increased SBP, and decreased endothelial NO production when compared with the same amount of ingested glucose. Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

No difference between high-fructose and high-glucose diets on liver triacylglycerol or biochemistry in healthy overweight men.

PubMed

Johnston, Richard D; Stephenson, Mary C; Crossland, Hannah; Cordon, Sally M; Palcidi, Elisa; Cox, Eleanor F; Taylor, Moira A; Aithal, Guruprasad P; Macdonald, Ian A

2013-11-01

Diets high in fructose have been proposed to contribute to nonalcoholic fatty liver disease. We compared the effects of high-fructose and matched glucose intake on hepatic triacylglycerol (TAG) concentration and other liver parameters. In a double-blind study, we randomly assigned 32 healthy but centrally overweight men to groups that received either a high-fructose or high-glucose diet (25% energy). These diets were provided during an initial isocaloric period of 2 weeks, followed by a 6-week washout period, and then again during a hypercaloric 2-week period. The primary outcome measure was hepatic level of TAG, with additional assessments of TAG levels in serum and soleus muscle, hepatic levels of adenosine triphosphate, and systemic and hepatic insulin resistance. During the isocaloric period of the study, both groups had stable body weights and concentrations of TAG in liver, serum, and soleus muscle. The high-fructose diet produced an increase of 22 ± 52 μmol/L in the serum level of uric acid, whereas the high-glucose diet led to a reduction of 23 ± 25 μmol/L (P < .01). The high-fructose diet also produced an increase of 0.8 ± 0.9 in the homeostasis model assessment of insulin resistance, whereas the high-glucose diet produced an increase of only 0.1 ± 0.7 (P = .03). During the hypercaloric period, participants in the high-fructose and high-glucose groups had similar increases in weight (1.0 ± 1.4 vs 0.6 ± 1.0 kg; P = .29) and absolute concentration of TAG in liver (1.70% ± 2.6% vs 2.05% ± 2.9%; P = .73) and serum (0.36 ± 0.75 vs 0.33 ± 0.38 mmol/L; P = .91), and similar results in biochemical assays of liver function. Body weight changes were associated with changes in liver biochemistry and concentration of TAGs. In the isocaloric period, overweight men who were on a high-fructose or a high-glucose diet did not develop any significant changes in hepatic concentration of TAGs or serum levels of liver enzymes. However, in the hypercaloric period, both high-fructose and high-glucose diets produced significant increases in these parameters without any significant difference between the 2 groups. This indicates an energy-mediated, rather than a specific macronutrient-mediated, effect. Clinical trials.gov no: NCT01050140. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

Regulation of the lactose phosphotransferase system of Streptococcus bovis by glucose: independence of inducer exclusion and expulsion mechanisms.

PubMed

Cook, G M; Kearns, D B; Russell, J B; Reizer, J; Saier, M H

1995-09-01

Streptococcus bovis had a diauxic pattern of glucose and lactose utilization, and both of these sugars were transported by the sugar phosphotransferase system (PTS). Lactose catabolism was inducible, and S. bovis used the tagatose pathway to ferment lactose. Since a mutant that was deficient in glucose PTS activity transported lactose as fast as the wild-type, it appeared that S. bovis has separate enzyme IIs for glucose and lactose. The nonmetabolizable glucose analogue 2-deoxyglucose (2-DG) was a noncompetitive inhibitor of methyl beta-D-thiogalactopyranoside (TMG) transport, and cells that were provided with either glucose or 2-DG were unable to transport TMG or lactose. Because the glucose-PTS-deficient mutant could ferment glucose, but could not exclude TMG, it appeared that enzyme IIGlc rather than glucose catabolism per se was the critical feature of inducer exclusion. Cells that had accumulated TMG as TMG 6-phosphate expelled free TMG when glucose was added, but 2-DG was unable to cause TMG expulsion. The glucose-PTS-deficient mutant could still expel TMG in the presence of exogenous glucose. Membrane vesicles also exhibited glucose-dependent TMG exclusion and TMG expulsion. Membrane vesicles that were electroporated with phosphoenolpyruvate (PEP) and HPr retained TMG for more than 3 min, but vesicles that were electroporated with PEP plus HPr and fructose 1,6-diphosphate (FDP) (or glycerate 2-phosphate) lost their ability to retain TMG. Because FDP was able to trigger the ATP-dependent phosphorylation of HPr, it appeared that inducer expulsion was mediated by an FDP-activated protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of a mutant defective in triacylglycerol accumulation in nitrogen-starved Chlamydomonas reinhardtii.

PubMed

Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

2016-09-01

Triacylglycerol (TAG), a major source of biodiesel production, accumulates in nitrogen-starved Chlamydomonas reinhardtii. However, the metabolic pathway of starch-to-TAG conversion remains elusive because an enzyme that affects the starch degradation is unknown. Here, we isolated a new class of mutant bgal1, which expressed an overaccumulation of starch granules and defective photosynthetic growth. The bgal1 was a null mutant of a previously uncharacterized β-galactosidase-like gene (Cre02.g119700), which decreased total β-galactosidase activity 40% of the wild type. Upon nitrogen starvation, the bgal1 mutant showed decreased TAG accumulation mainly due to the reduced flux of de novo TAG biosynthesis evidenced by increased unsaturation of fatty acid composition in TAG and reduced TAG accumulation by additional supplementation of acetate to the culture media. Metabolomic analysis of the bgal1 mutant showed significantly reduced levels of metabolites following the hydrolysis of starch and substrates for TAG accumulation, whereas metabolites in TCA cycle were unaffected. Upon nitrogen starvation, while levels of glucose 6-phosphate, fructose 6-phosphate and acetyl-CoA remained lower, most of the other metabolites in glycolysis were increased but those in the TCA cycle were decreased, supporting TAG accumulation. We suggest that BGAL1 may be involved in the degradation of starch, which affects TAG accumulation in nitrogen-starved C. reinhardtii. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. Copyright © 2016 Elsevier B.V. All rights reserved.

Fructose Containing Sugars at Normal Levels of Consumption Do Not Effect Adversely Components of the Metabolic Syndrome and Risk Factors for Cardiovascular Disease

PubMed Central

Angelopoulos, Theodore J.; Lowndes, Joshua; Sinnett, Stephanie; Rippe, James M.

2016-01-01

The objective of the current study was to explore our hypothesis that average consumption of fructose and fructose containing sugars would not increase risk factors for cardiovascular disease (CVD) and the metabolic syndrome (MetS). A randomized, double blind, parallel group study was conducted where 267 individuals with BMI between 23 and 35 kg/m2 consumed low fat sugar sweetened milk, daily for ten weeks as part of usual weight-maintenance diet. One group consumed 18% of calories from high fructose corn syrup (HFCS), another group consumed 18% of calories from sucrose, a third group consumed 9% of calories from fructose, and the fourth group consumed 9% of calories from glucose. There was a small change in waist circumference (80.9 ± 9.5 vs. 81.5 ± 9.5 cm) in the entire cohort, as well as in total cholesterol (4.6 ± 1.0 vs. 4.7 ± 1.0 mmol/L, p < 0.01), triglycerides (TGs) (11.5 ± 6.4 vs. 12.6 ± 8.9 mmol/L, p < 0.01), and systolic (109.2 ± 10.2 vs. 106.1 ± 10.4 mmHg, p < 0.01) and diastolic blood pressure (69.8 ± 8.7 vs. 68.1 ± 9.7 mmHg, p < 0.01). The effects of commonly consumed sugars on components of the MetS and CVD risk factors are minimal, mixed and not clinically significant. PMID:27023594

Fructose Containing Sugars at Normal Levels of Consumption Do Not Effect Adversely Components of the Metabolic Syndrome and Risk Factors for Cardiovascular Disease.

PubMed

Angelopoulos, Theodore J; Lowndes, Joshua; Sinnett, Stephanie; Rippe, James M

2016-03-23

The objective of the current study was to explore our hypothesis that average consumption of fructose and fructose containing sugars would not increase risk factors for cardiovascular disease (CVD) and the metabolic syndrome (MetS). A randomized, double blind, parallel group study was conducted where 267 individuals with BMI between 23 and 35 kg/m² consumed low fat sugar sweetened milk, daily for ten weeks as part of usual weight-maintenance diet. One group consumed 18% of calories from high fructose corn syrup (HFCS), another group consumed 18% of calories from sucrose, a third group consumed 9% of calories from fructose, and the fourth group consumed 9% of calories from glucose. There was a small change in waist circumference (80.9 ± 9.5 vs. 81.5 ± 9.5 cm) in the entire cohort, as well as in total cholesterol (4.6 ± 1.0 vs. 4.7 ± 1.0 mmol/L, p < 0.01), triglycerides (TGs) (11.5 ± 6.4 vs. 12.6 ± 8.9 mmol/L, p < 0.01), and systolic (109.2 ± 10.2 vs. 106.1 ± 10.4 mmHg, p < 0.01) and diastolic blood pressure (69.8 ± 8.7 vs. 68.1 ± 9.7 mmHg, p < 0.01). The effects of commonly consumed sugars on components of the MetS and CVD risk factors are minimal, mixed and not clinically significant.

Metabolic Fate of Fructose Ingested with and without Glucose in a Mixed Meal

PubMed Central

Theytaz, Fanny; de Giorgi, Sara; Hodson, Leanne; Stefanoni, Nathalie; Rey, Valentine; Schneiter, Philippe; Giusti, Vittorio; Tappy, Luc

2014-01-01

Ingestion of pure fructose stimulates de novo lipogenesis and gluconeogenesis. This may however not be relevant to typical nutritional situations, where fructose is invariably ingested with glucose. We therefore assessed the metabolic fate of fructose incorporated in a mixed meal without or with glucose in eight healthy volunteers. Each participant was studied over six hours after the ingestion of liquid meals containing either 13C-labelled fructose, unlabeled glucose, lipids and protein (Fr + G) or 13C-labelled fructose, lipids and protein, but without glucose (Fr), or protein and lipids alone (ProLip). After Fr + G, plasma 13C-glucose production accounted for 19.0% ± 1.5% and 13CO2 production for 32.2% ± 1.3% of 13C-fructose carbons. After Fr, 13C-glucose production (26.5% ± 1.4%) and 13CO2 production (36.6% ± 1.9%) were higher (p < 0.05) than with Fr + G. 13C-lactate concentration and very low density lipoprotein VLDL 13C-palmitate concentrations increased to the same extent with Fr + G and Fr, while chylomicron 13C-palmitate tended to increase more with Fr + G. These data indicate that gluconeogenesis, lactic acid production and both intestinal and hepatic de novo lipogenesis contributed to the disposal of fructose carbons ingested together with a mixed meal. Co-ingestion of glucose decreased fructose oxidation and gluconeogenesis and tended to increase 13C-pamitate concentration in gut-derived chylomicrons, but not in hepatic-borne VLDL-triacylglycerol (TG). This trial was approved by clinicaltrial. gov. Identifier is NCT01792089. PMID:25029210

Dietary Fat, Eicosanoids and Breast Cancer Risk

DTIC Science & Technology

2005-04-01

Celery log 1Og log Peas 15g 15g 15g Garlic Powder/Salt/Pepper Dash Dash Dash Lemon Juice 2g 2g 2g Mayonnaise 25g 9g 0 Fat Free Miracle Whip 0 20g 30g...Sugar 0 4g 4g Oatmeal Walnut Cookie 30g 30g 30g Dinner: Beef Tenderloin 65g 60g 75g Mashed Potatoes 10Og 140g 140g w/Butter/Olive Oil 5g/4g 0 0 Corn 80g...Fat Free Gravy 40g 40g 40g w/ Safflower Oil/Flax Oil 7g (Safflower) 3g (Safflower) 3g (Flax) Mashed Potato 100g 140g 140g Green Beans 100g 100g �g

Potential application of Candida melibiosica in biofuel cells.

PubMed

Hubenova, Yolina; Mitov, Mario

2010-04-01

Various prokaryote species have been widely studied for microbial fuel cell (MFC) application. However, the information about yeast utilization into biofuel cells is still scanty. The aim of this investigation is to verify if Candida melibiosica 2491, a yeast strain, possessing high phytase activity, could be applied as a biocatalyst in a yeast biofuel cell. The microbiological requirements were coupled with the electrochemical ones tracing main biochemical pathway metabolites such as different carbohydrate and inorganic phosphates and their assimilation with time. The obtained results show that from the three carbohydrates investigated - glucose, fructose and sucrose, fructose is the most suitable for the yeast cultivation. The presence of yeast extract and peptone improves the performance into the biofuel cell. The relationship between the yeast cell amount and the biofuel cell characteristics was determined. Analyses showed that electricity was generated by the yeast culture even in the absence of an artificial mediator. The addition of methylene blue at concentrations higher than 0.1 mM improves the current and power density output. The obtained experimental results proved that C. melibiosica 2491 belongs to the electrogenic strains. 2009 Elsevier B.V. All rights reserved.

Glucokinase is an integral component of the insulin granules in glucose-responsive insulin secretory cells and does not translocate during glucose stimulation.

PubMed

Arden, Catherine; Harbottle, Andrew; Baltrusch, Simone; Tiedge, Markus; Agius, Loranne

2004-09-01

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

A Microplate Format Assay for Real-Time Screening for New Aldolases that Accept Aryl-Substituted Acceptor Substrates.

PubMed

Ma, Huan; Enugala, Thilak Reddy; Widersten, Mikael

2015-12-01

Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose 6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been reported to be a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with bulky acceptor substrates of this type is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an aldehyde reductase produced in vitro by directed evolution; it reduces the aldehyde product formed after cleavage of the aldol by FSA. The assay is suitable both for steady-state enzyme kinetics and for real-time activity screening in a 96-well format. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Current Status of the Congenital Myasthenic Syndromes

PubMed Central

Engel, Andrew G.

2011-01-01

Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Clinical, electrophysiologic, and morphologic studies have paved the way for detecting CMS-related mutations in proteins residing in the nerve terminal, the synaptic basal lamina, and in the postsynaptic region of the motor endplate. The disease proteins identified to date include choline acetyltransferase (ChAT), the endplate species of acetylcholinesterase (AChE), β2-laminin, the acetylcholine receptor (AChR), rapsyn, plectin, Nav1.4, the muscle specific protein kinase (MuSK), agrin, downstream of tyrosine kinase 7 (Dok-7), and glutamine-fructose-6-phosphate transaminase 1 (GFPT1). Myasthenic syndromes associated with centronuclear myopathies were recently recognized. Analysis of properties of expressed mutant proteins contributed to finding improved therapy for most CMS. Despite these advances, the molecular basis of some phenotypically characterized CMS remains elusive. Moreover, other types of CMS and disease genes likely exist and await discovery. PMID:22104196

Phosphomannose isomerase and phosphomannomutase gene disruptions in Streptomyces nodosus: impact on amphotericin biosynthesis and implications for glycosylation engineering.

PubMed

Nic Lochlainn, Laura; Caffrey, Patrick

2009-01-01

Streptomycetes synthesise several bioactive natural products that are modified with sugar residues derived from GDP-mannose. These include the antifungal polyenes, the antibacterial antibiotics hygromycin A and mannopeptimycins, and the anticancer agent bleomycin. Three enzymes function in biosynthesis of GDP-mannose from the glycolytic intermediate fructose 6-phosphate: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMPP). Synthesis of GDP-mannose from exogenous mannose requires hexokinase or phosphotransferase enzymes together with PMM and GMPP. In this study, a region containing genes for PMI, PMM and GMPP was cloned from Streptomyces nodosus, producer of the polyenes amphotericins A and B. Inactivation of the manA gene for PMI resulted in production of amphotericins and their aglycones, 8-deoxyamphoteronolides. A double mutant lacking the PMI and PMM genes produced 8-deoxyamphoteronolides in good yields along with trace levels of glycosylated amphotericins. With further genetic engineering these mutants may activate alternative hexoses as GDP-sugars for transfer to aglycones in vivo.

Serum uric acid concentrations and fructose consumption are independently associated with NASH in children and adolescents.

PubMed

Mosca, Antonella; Nobili, Valerio; De Vito, Rita; Crudele, Annalisa; Scorletti, Eleonora; Villani, Alberto; Alisi, Anna; Byrne, Christopher D

2017-05-01

Recent research has suggested that dietary fructose intake may increase serum uric acid (UA) concentrations. Both UA concentration and fructose consumption maybe also increase in NAFLD. It is not known whether dietary fructose consumption and UA concentration are independently associated with non-alcoholic steatohepatitis (NASH). Our aim was to investigate the factors associated with NASH in children and adolescents with proven NAFLD, and to test whether UA concentrations and fructose consumption are independently associated with NASH. Obese children with NAFLD were studied (n=271). NASH was diagnosed by a NAFLD activity score ⩾5 and the fatty liver inhibition of progression (FLIP) algorithm. Fructose consumption (g/day) was assessed by food frequency questionnaire, and UA (mg/dl) was measured in serum. Binary logistic regression with adjustment for covariates and potential confounders was undertaken to test factors independently associated with NASH. NASH occurred in 37.6% of patients. Hyperuricaemia (UA ⩾5.9mg/dl) was present in 47% of patients with NASH compared with 29.7% of non-NASH patients (p=0.003). Both UA concentration (OR=2.488, 95% CI: 1.87-2.83, p=0.004) and fructose consumption (OR=1.612, 95% CI 1.25-1.86, p=0.001) were independently associated with NASH, after adjustment for multiple (and all) measured confounders. Fructose consumption was independently associated with hyperuricaemia (OR=2.021, 95% CI: 1.66-2.78, p=0.01). These data were confirmed using the FLIP algorithm. Both dietary fructose consumption and serum UA concentrations are independently associated with NASH. Fructose consumption was the only factor independently associated with serum UA concentration. Currently, it is not known whether dietary fructose consumption and uric acid (UA) concentration are linked with non-alcoholic steatohepatitis (NASH) in children and adolescents. Our aim was to test whether UA concentrations and fructose consumption are independently associated with NASH in children and adolescents with proven non-alcoholic fatty liver disease (NAFLD). We show that both dietary fructose consumption and serum UA concentrations are independently associated with NASH and fructose consumption was independently linked with high serum UA concentrations. Copyright © 2017 European Association for the Study of the Liver. All rights reserved.

Fructose consumption during pregnancy and lactation induces fatty liver and glucose intolerance in rats

PubMed Central

Zou, Mi; Arentson, Emily J.; Teegarden, Dorothy; Koser, Stephanie L.; Onyskow, Laurie; Donkin, Shawn S.

2015-01-01

Nutritional insults during pregnancy and lactation are health risks for mother and offspring. Both fructose and low protein diets are linked to hepatic steatosis and insulin resistance in non-pregnant animals. We hypothesized that dietary fructose or low protein intake during pregnancy may exacerbate the already compromised glucose homeostasis to induce gestational diabetes and fatty liver. Therefore, we investigated and compared the effects of low protein or fructose intake on hepatic steatosis and insulin resistance in unmated controls and pregnant and lactating rats. Sprague-Dawley rats were fed either a control (CT), a 63% fructose (FR) or an 8% protein (LP) diet. Glucose tolerance test at day 17 of the study revealed greater (P < 0.05) blood glucose at 10 (75.6 vs. 64.0 ± 4.8 mg/dl) and 20 (72.4 vs. 58.6 ± 4.0 mg/dl) min after glucose dose and greater area under the curve (4302.3 vs. 3763.4 ± 263.6 mg·dl−1·min−1) for FR-fed dams compared with CT-fed dams. The rats were euthanized at 21 days postpartum. Both the FR- and LP-fed dams had enlarged (P < 0.05) livers (9.3, 7.1 vs. 4.8 ± 0.2 % body weight) and elevated (P < 0.05) liver triacylglycerol (216.0, 130.0 vs. 19.9 ± 12.6 mg/g liver weight) compared with CT-fed dams. FR induced fatty liver and glucose intolerance in pregnant and lactating rats, but not unmated control rats. The data demonstrate a unique physiological status response to diet resulting in the development of gestational diabetes coupled with hepatic steatosis in FR-fed dams, which is more severe than a LP diet. PMID:22935342

Oral solution of fructose promotes SREBP-1c high-expression in the hypothalamus of Wistar rats.

PubMed

Batista, Leandro Oliveira; Ramos, Viviane Wagner; Rosas Fernández, Mariana Alejandra; Concha Vilca, Carlos Marcelo; Albuquerque, Kelse Tibau de

2018-01-25

We evaluate whether the consumption of fructose for 8 weeks affects enzymes and transcription factors of the lipogenic and inflammatory pathways in the hypothalamus of Wistar rats. At 30 days, the animals were divided into groups: Control (C) and Fructose (F) and maintained with free access to feed and filtered water (C) or aqueous solution of purified fructose at 20% (F). RT-PCR and Western blotting were performed for the target genes and proteins. In F group, results showed a lower feed intake, an increase in glycemia (146.20 ± 6.09 vs. 102.32 ± 4.58; n: 9) and triacylglycerol (F: 191.65 ± 13.51 vs. C: 131.69 ± 6.49; n: 9) and there was no difference in water and energy consumption. We identified a higher content of acetyl-CoA carboxylase (ACC) (F: 133.93 ± 5.58 vs. C: 100 ± 0.0; n: 9-10) and NFκB (F: 125.5 ± 8.85 vs. C: 100 ± 0; n: 14) in group F, whereas fatty acid synthase (FAS) was lower (F: 85.90 ± 4.81 vs. C: 100 ± 0.0; n: 4-6). SREBP-1c gene expression was higher in F vs. C group (F: 4.08 ± 0.44 vs. C: 1.13 ± 0.15; n: 5-6), although we did not found difference between groups in the gene expression for ACC, SREBP-2, and NFκB. Dietary fructose can change important lipogenic and inflammatory factors in the hypothalamus of rats and it leads to regulation of transcription factors before changes in body mass are evident.

Increase in the activity of fructose-1,6-bisphosphatase in cytosol affects sugar partitioning and increases the lateral shoots in tobacco plants at elevated CO2 levels.

PubMed

Tamoi, Masahiro; Hiramatsu, Yoshie; Nedachi, Shigeki; Otori, Kumi; Tanabe, Noriaki; Maruta, Takanori; Shigeoka, Shigeru

2011-05-01

We generated transgenic tobacco plants with high levels of fructose-1,6-bisphosphatase expressing cyanobacterialfructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol. At ambient CO(2) levels (360 ppm), growth, photosynthetic activity, and fresh weight were unchanged but the sucrose/hexose/starch ratio was slightly altered in the transgenic plants compared with wild-type plants. At elevated CO(2) levels (1200 ppm), lateral shoot, leaf number, and fresh weight were significantly increased in the transgenic plants. Photosynthetic activity was also increased. Hexose accumulated in the upper leaves in the wild-type plants, while sucrose and starch accumulated in the lower leaves and lateral shoots in the transgenic plants. These findings suggest that cytosolic fructose-1,6-bisphosphatase contributes to the efficient conversion of hexose into sucrose, and that the change in carbon partitioning affects photosynthetic capacity and morphogenesis at elevated CO(2) levels.

Chronic Alcohol Consumption Causes Liver Injury in High-Fructose-Fed Male Mice Through Enhanced Hepatic Inflammatory Response

PubMed Central

Song, Ming; Chen, Theresa; Prough, Russell A.; Cave, Matthew C.; McClain, Craig J.

2017-01-01

Background Obesity and the metabolic syndrome occur in approximately one-third of patients with alcoholic liver disease (ALD). The increased consumption of fructose parallels the increased prevalence of obesity and the metabolic syndrome in the United States and worldwide. In this study, we investigated whether dietary high fructose potentiates chronic alcohol-induced liver injury, and explored potential mechanism(s). Methods Six-week-old male C57BL/6J mice were assigned to 4 groups: control, high fructose, chronic ethanol (EtOH), and high fructose plus chronic alcohol. The mice were fed either control diet or high-fructose diet (60%, w/w) for 18 weeks. Chronic alcohol-fed mice were given 20% (v/v) ethanol (Meadows-Cook model) ad libitum as the only available liquid from the 9th week through the 18th week. Liver injury, steatosis, hepatic inflammatory gene expression, and copper status were assessed. Results High-fructose diet and chronic alcohol consumption alone each induce hepatic fat accumulation and impair copper status. However, the combination of dietary high fructose plus chronic alcohol synergistically induced liver injury as evidenced by robustly increased plasma alanine aminotransferase and aspartate aminotransferase, but the combination did not exacerbate hepatic fat accumulation nor worsen copper status. Moreover, FE-fed mice were characterized by prominent microvesicular steatosis. High-fructose diet and chronic alcohol ingestion together led to a significant up-regulation of Kupffer cell (KC) M1 phenotype gene expression (e.g., tumor necrosis factor-a and monocyte chemoattractant protein-1), as well as Toll-like receptor 4 (TLR4) signaling gene expression, which is also associated with the up-regulation of KCs and activation marker gene expression, including Emr1, CD68, and CD163. Conclusions Our data suggest that dietary high fructose may potentiate chronic alcohol consumption-induced liver injury. The underlying mechanism might be due to the synergistic effect of dietary high fructose and alcohol on the activation of the TLR4 signaling pathway, which in turn leads to KC activation and phenotype switch toward M1 polarization. This study suggests that alcohol–fructose combination contributes to ALD progression. PMID:26858005

Effects of Fructose vs Glucose on Regional Cerebral Blood Flow in Brain Regions Involved With Appetite and Reward Pathways

PubMed Central

Page, Kathleen A.; Chan, Owen; Arora, Jagriti; Belfort-DeAguiar, Renata; Dzuira, James; Roehmholdt, Brian; Cline, Gary W.; Naik, Sarita; Sinha, Rajita; Constable, R. Todd; Sherwin, Robert S.

2014-01-01

Importance Increases in fructose consumption have paralleled the increasing prevalence of obesity, and high-fructose diets are thought to promote weight gain and insulin resistance. Fructose ingestion produces smaller increases in circulating satiety hormones compared with glucose ingestion, and central administration of fructose provokes feeding in rodents, whereas centrally administered glucose promotes satiety. Objective To study neurophysiological factors that might underlie associations between fructose consumption and weight gain. Design, Setting, and Participants Twenty healthy adult volunteers underwent 2 magnetic resonance imaging sessions at Yale University in conjunction with fructose or glucose drink ingestion in a blinded, random-order, crossover design. Main Outcome Measures Relative changes in hypothalamic regional cerebral blood flow (CBF) after glucose or fructose ingestion. Secondary outcomes included whole-brain analyses to explore regional CBF changes, functional connectivity analysis to investigate correlations between the hypothalamus and other brain region responses, and hormone responses to fructose and glucose ingestion. Results There was a significantly greater reduction in hypothalamic CBF after glucose vs fructose ingestion (–5.45 vs 2.84 mL/g per minute, respectively; mean difference, 8.3 mL/g per minute [95% CI of mean difference, 1.87-14.70]; P=.01). Glucose ingestion (compared with baseline) increased functional connectivity between the hypothalamus and the thalamus and striatum. Fructose increased connectivity between the hypothalamus and thalamus but not the striatum. Regional CBF within the hypothalamus, thalamus, insula, anterior cingulate, and striatum (appetite and reward regions) was reduced after glucose ingestion compared with baseline (P<.05 significance threshold, family-wise error [FWE] whole-brain corrected). In contrast, fructose reduced regional CBF in the thalamus, hippocampus, posterior cingulate cortex, fusiform, and visual cortex (P<.05 significance threshold, FWE whole-brain corrected). In whole-brain voxel-level analyses, there were no significant differences between direct comparisons of fructose vs glucose sessions following correction for multiple comparisons. Fructose vs glucose ingestion resulted in lower peak levels of serum glucose (mean difference, 41.0 mg/dL [95% CI, 27.7-54.5]; P<.001), insulin (mean difference, 49.6 μU/mL [95% CI, 38.2-61.1]; P<.001), and glucagon-like polypep-tide 1 (mean difference, 2.1 pmol/L [95% CI, 0.9-3.2]; P=.01). Conclusion and Relevance In a series of exploratory analyses, consumption of fructose compared with glucose resulted in a distinct pattern of regional CBF and a smaller increase in systemic glucose, insulin, and glucagon-like polypeptide 1 levels. PMID:23280226

D-tagatose 1,6-diphosphate aldolase from lactic streptococci: purification, properties, and use in measuring intracellular tagatose 1,6-diphosphate.

PubMed Central

Crow, V L; Thomas, T D

1982-01-01

Two D-ketohexose 1,6-diphosphate aldolases are present in Streptococcus cremoris E8 and S. lactis C10. One aldolase, which was induced by growth on either lactose or galactose, was active with both tagatose 1,6-diphosphate (TDP) and fructose 1,6-diphosphate (FDP), having a lower Km and a higher Vmax with TDP as the substrate. This enzyme, named TDP aldolase, had properties typical of a class I aldolase, being insensitive to EDTA and showing substrate-dependent inactivation by sodium borohydride. Sodium dodecyl sulfate-gel electrophoresis indicated a subunit molecular weight of 34,500. The amino acid composition of TDP aldolase is reported. When the enzyme was incubated with either triose phosphates or FDP, the equilibrium mixture contained an FDP/TDP ratio of 6.9:1. The other aldolase, which had properties typical of a class II aldolase, showed activity with FDP but not with TDP. The intracellular TDP concentration, measured with the purified TDP aldolase, was 0.4 to 4.0 mM in cells growing on lactose or galactose and was lower (0 to 1.0 mM) in cells growing on glucose. The intracellular concentration of FDP was always higher than that of TDP. The role of ketohexose diphosphates in the regulation of end product fermentation by lactic streptococci is discussed. PMID:6807956

Fructose 1,6-diphosphatase in striated muscle

PubMed Central

Krebs, H. A.; Woodford, Muriel

1965-01-01

1. The occurrence of fructose diphosphatase in muscle tissue was investigated with reference to the question whether lactate can be converted into glycogen in muscle, as postulated by Meyerhof (1930), fructose diphosphatase being one of the enzymes required for this conversion. 2. Fructose diphosphatase was found in skeletal muscle of man, dog, cat, rat, mouse, rabbit, guinea pig, cattle, sheep, pigeon, fowl and frog. Under the test conditions between 5 and 60 μmoles of substrate were split/g. fresh wt./hr. at 22°. 3. Like liver fructose diphosphatase, the muscle enzyme is inhibited by substrate concentrations above 0·1 mm, by AMP and by trace quantities of Zn2+, Fe2+ and Fe3+; it is `activated' by EDTA. Inhibitions by the above agents may account for the failure of previous authors to detect the enzyme. 4. Heart muscle of several vertebrate species and the smooth muscle of pigeon and fowl gizzard had no measurable activity. 5. The presence of fructose diphosphatase and the virtual absence of the enzyme systems converting pyruvate into phosphopyruvate means that lactate and pyruvate cannot be converted into glycogen in muscle, whereas the phosphorylated C3 compounds can. The reconversion into carbohydrate of lactate (which readily diffuses out of muscle) occurs in liver and kidney only. The reconversion of phosphorylated C3 intermediates (which cannot diffuse out of the tissue) can occur only within the muscle. 6. α-Glycerophosphate is probably the main intermediate requiring conversion into glycogen. The possible role of α-glycerophosphate formation in vertebrate muscle, already well established in insect muscle, is discussed. PMID:14346089

Genetic disorder in carbohydrates metabolism: hereditary fructose intolerance associated with celiac disease.

PubMed

Păcurar, Daniela; Leşanu, Gabriela; Dijmărescu, Irina; Ţincu, Iulia Florentina; Gherghiceanu, Mihaela; Orăşeanu, Dumitru

2017-01-01

Celiac disease (CD) has been associated with several genetic and immune disorders, but association between CD and hereditary fructose intolerance (HFI) is extremely rare. HFI is an autosomal recessive disease caused by catalytic deficiency of aldolase B (fructose-1,6-bisphosphate aldolase). We report the case of a 5-year-old boy suffering from CD, admitted with an initial diagnosis of Reye's-like syndrome. He presented with episodic unconsciousness, seizures, hypoglycemia, hepatomegaly and abnormal liver function. The patient has been on an exclusion diet for three years, but he still had symptoms: stunting, hepatomegaly, high transaminases, but tissue transglutaminase antibodies were negative. Liver biopsy showed hepatic steatosis and mitochondrial damage. The dietary history showed an aversion to fruits, vegetables and sweet-tasting foods. The fructose tolerance test was positive, revealing the diagnostic of hereditary fructose intolerance. Appropriate dietary management and precautions were recommended. The patient has been symptom-free and exhibited normal growth and development until 10 years of age.

Occurrence of Bifidobacteriaceae in human hypochlorhydria stomach

PubMed Central

Mattarelli, Paola; Brandi, Giovanni; Calabrese, Carlo; Fornari, Fabio; Prati, Gian Maria; Biavati, Bruno; Sgorbati, Barbara

2014-01-01

Background The human stomach, when healthy, is not a suitable host for microorganisms, but in pathological conditions such as gastritis, when gastric acid secretion is impaired, microbial overgrowth can be observed. Apart from Helicobacter pylori, the composition of microbiota, resident or exogenously introduced during neutral/high pH conditions, has not been investigated thoroughly. Thus, it is possible that Bifidobacteriaceae, important autochthonous and beneficial bacteria of human gastrointestinal microbiota, could over-colonize the stomach of hypochlorhydria patients suffering from autoimmune atrophic gastritis (AAG) or omeprazole-treated (OME) gastritis. This prompted us to characterize the Bifidobacteriaceae in such patients’ gastric microbiota and to study its abnormal colonization. Methods Samples of gastric juices, and antrum and corpus mucosa from 23 hypochlorhydria patients (13 AAG and 10 OME) and from 10 control volunteers with base-line normochlorhydria, were cultivated in Brain Heart Infusion (BHI) and selective Bifidobacterium-Tryptone-Phytone-Yeast extract (Bif-TPY) media. The isolates were characterized by the fructose-6-phosphate phosphoketolase (F6PPK) test, electrophoresis of cellular proteins, the fermentation test, guanine-cytosine% DNA content, and DNA–DNA hybridization. Negative F6PPK isolates were characterized by order-specific polymerase chain reaction (PCR). Results A total of 125 isolates, assigned to the Bifidobacteriaceae family on the basis of their morphology, were obtained from AAG and OME patients, but not from normal subjects. Of these isolates, 55 were assigned to the Bifidobacteriaceae family on the basis of their fructose-6-phosphoketolase (PPK) activity, PPK being the key taxonomic enzyme of this family. The remaining 70 isolates, which were PPK-negative, were attributed to the Actinomycetales order following specific primer PCR analysis. We observed a significantly higher abundance of Bifidobacteriaceae (Bifidobacterium dentium, Scardovia inopinata, and Parascardovia denticolens) in OME group than the AAG group. Furthermore, the Actinomycetales distribution was homogeneous for both hypochlorhydria patient groups. Conclusions This study suggests that the Bifidobacteriaceae species, typically found in the oral cavity, readily colonizes the hypochlorhydria stomach of OME patients. The clinical relevance and the mechanism underlying this Bifidobacteriaceae presence in OME gastritis requires further functional studies. PMID:24427111

Characterization of a thermostable recombinant l-rhamnose isomerase from Caldicellulosiruptor obsidiansis OB47 and its application for the production of l-fructose and l-rhamnulose.

PubMed

Chen, Ziwei; Xu, Wei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

2018-04-01

l-Hexoses are rare sugars that are important components and precursors in the synthesis of biological compounds and pharmaceutical drugs. l-Rhamnose isomerase (L-RI, EC 5.3.1.14) is an aldose-ketose isomerase that plays a significant role in the production of l-sugars. In this study, a thermostable, l-sugar-producing L-RI from the hyperthermophile Caldicellulosiruptor obsidiansis OB47 was characterized. The recombinant L-RI displayed maximal activity at pH 8.0 and 85 °C and was significantly activated by Co 2+ . It exhibited a relatively high thermostability, with measured half-lives of 24.75, 11.55, 4.15 and 3.30 h in the presence of Co 2+ at 70, 75, 80 and 85 °C, respectively. Specific activities of 277.6, 57.9, 13.7 and 9.6 U mg -1 were measured when l-rhamnose, l-mannose, d-allose and l-fructose were used as substrates, respectively. l-Rhamnulose was produced with conversion ratios of 44.0% and 38.6% from 25 and 50 g L -1 l-rhamnose, respectively. l-Fructose was also efficiently produced by the L-RI, with conversion ratios of 67.0% and 58.4% from 25 and 50 g L -1 l-mannose, respectively. The recombinant L-RI could effectively catalyze the formation of l-rhamnulose and l-fructose, suggesting that it was a promising candidate for industrial production of l-rhamnulose and l-fructose. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Acute effect of fructose intake from sugar-sweetened beverages on plasma uric acid: a randomised controlled trial.

PubMed

Carran, E L; White, S J; Reynolds, A N; Haszard, J J; Venn, B J

2016-09-01

Excessive fructose intake has been linked to hyperuricaemia. Our aim was to test whether 355 and 600 ml of commercial sugar-sweetened soft drinks would acutely raise plasma uric acid. Forty-one participants were randomised to a control group or an intervention group. The control group consumed 600 ml of fructose and 600 ml of glucose beverages. The soft drink group consumed 355 and 600 ml of beverages in random order. The control beverages were matched for fructose content with 600 ml of soft drink (26.7 g). Blood samples were collected at baseline, 30 and 60 min and analysed for plasma uric acid. Plasma uric acid concentrations were 13 (95% confidence interval: (CI): 3, 23) and 17 μmol/l (95% CI: 6, 28) higher 30 and 60 min after consumption of 600 ml of soft drink compared with the glucose control. The corresponding values for the fructose beverage were 22 (95% CI: 16, 29) and 23 μmol/l (95% CI: 14, 33). There was no significant difference in the increase in uric acid following the 600-ml soft drink compared with the fructose control at 30 min (6 μmol/l; 95% CI: -4, 15) or 60 min (5 μmol/l; 95% CI: -7, 17). There was no difference in the uric-acid-raising effect between the 355 and 600 ml volumes at 30 min (-1 μmol/l; 95% CI: -9, 6) or 60 min (-5 μmol/l; 95% CI: -10, 1). Small and transient increases in plasma uric acid are likely after consumption of sucrose-sweetened commercially available single-serve soft drinks in volumes as small as 355 ml.

Association of the widespread A149P hereditary fructose intolerance mutation with newly identified sequence polymorphisms in the aldolase B gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brooks, C.C.; Tolan, D.R.

1993-04-01

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease resulting from the catalytic deficiency of fructose 1-phosphate aldolase (aldolase B) in fructose-metabolizing tissues. The A149P mutation in exon 5 of the aldolase B gene, located on chromosome 9q2l.3-q22.2, is widespread and the most common HFI mutation, accounting for 57% of HFI chromosomes. The possible origin of this mutation was studied by linkage to polymorphisms within the aldolase B gene. DNA fragments of the aldolase B gene containing the polymorphic marker loci from HFI patients homozygous for the A149P allele were amplified by PCR. Absolute linkage to a commonmore » Pvull RFLP allele was observed in 10 A149P homozygotes. In a more informative study, highly heterozygous polymorphisms were detected by direct sequence determination of a PCR-amplified aldolase B gene fragment. Two two-allele, single-base-pair polymorphisms, themselves in absolute linkage disequilibrium, in intron 8 (C at nucleotide 84 and A at nucleotide 105, or T at 84 and G at 105) of the aldolase B gene were identified. Mendelian segregation of these polymorphisms was confirmed in three families. Allele-specific oligonucleotide (ASO) hybridizations with probes for both sequence polymorphisms showed that 47% of 32 unrelated individuals were heterozygous at these loci; the calculated PIC value was .37. Finally, ASO hybridizations of PCR-amplified DNA from 15 HFI patients homozygous for the A149P allele with probes for these sequence polymorphisms revealed absolute linkage disequilibrium between the A149P mutation and the 84T/105G allele. These results are consistent with a single origin of the A149P allele and subsequent spread by genetic drift. 32 refs., 4 figs., 3 tabs.« less

Central cavity of fructose-1,6-bisphosphatase and the evolution of AMP/fructose 2,6-bisphosphate synergism in eukaryotic organisms.

PubMed

Gao, Yang; Shen, Lu; Honzatko, Richard B

2014-03-21

The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P2 synergism in pFBPase is absent in eFBPase. Fru-2,6-P2 induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P2 synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser(45) → His substantially fills the central cavity of pFBPase, and the triple mutation Ser(45) → His, Thr(46) → Arg, and Leu(186) → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P2 on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P2 exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P2 complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P2 synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P2 synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P.

Modulation of brain insulin signaling in Alzheimer's disease: New insight on the protective role of green coffee bean extract.

PubMed

Mohamed, Hoda E; Asker, Mervat E; Younis, Nahla N; Shaheen, Mohamed A; Eissa, Rana G

2018-05-01

Alzheimer's disease (AD), a neurodegenerative disorder, involves brain insulin signaling cascades and insulin resistance (IR). Because of limited treatment options, new treatment strategies are mandatory. Green coffee bean extract (GCBE) was reported to attenuate IR and improve brain energy metabolism. We aimed to investigate the possible use of GCBE as a prophylactic strategy to delay the onset of AD or combined with pioglitazone (PIO) as a strategy to retard the progression of AD. Rats received 10% fructose in drinking water for 18 weeks to induce AD. GCBE-prophylactic group received GCBE for 22 weeks started 4 weeks prior to fructose administration. The PIO group treated with PIO for 6 weeks started on week 12 of fructose administration. The GCBE+PIO group received GCBE for 22 weeks started 4 weeks prior to fructose administration and treated with PIO for the last 6 weeks of fructose administration. Pretreatment with GCBE, either alone or combined with PIO, alleviated IR-induced AD changes. GCBE improved cognition, decreased serine phosphorylation of insulin receptor substrate, increased phosphoinositol-3 kinase (PI3K) activity and protein kinase B (Akt) gene expression, decreased glycogen synthase kinase-3β (GS3Kβ) gene expression and Tau hyperphosphorylation. GCBE exerted neuroprotective effects against IR-induced AD mediated by alleviating IR and modulating brain insulin signaling cascade.

Postoral Glucose Sensing, Not Caloric Content, Determines Sugar Reward in C57BL/6J Mice

PubMed Central

Zukerman, Steven; Ackroff, Karen

2015-01-01

Recent studies suggest that because of their energy value, sugars are more rewarding than non-caloric sweeteners. However, intragastric infusion data indicate that sugars differ in their postoral appetite-stimulating effects. We therefore compared the preference for isocaloric 8% sucrose, glucose, and fructose solutions with that of a non-caloric sweetener solution (0.8% sucralose) in C57BL/6J mice. Brief 2-bottle tests indicated that sucralose was isopreferred to sucrose but more preferred than glucose or fructose. Yet, in long-term tests, the mice preferred sucrose and glucose, but not fructose to sucralose. Additional experiments were conducted with a non-caloric 0.1% sucralose + 0.1% saccharin mixture (S + S), which does not have the postoral inhibitory effects of 0.8% sucralose. The S + S was preferred to fructose in brief and long-term choice tests. S + S was also preferred to glucose and sucrose in brief tests, but the sugars were preferred in long-term tests. In progressive ratio tests, non-deprived and food-deprived mice licked more for glucose but not fructose than for S + S. These findings demonstrate that the nutrient-specific postoral actions, not calories per se, determine the avidity for sugar versus non-caloric sweeteners. Furthermore, sweet taste intensity and potential postoral inhibitory actions must be considered in comparing non-caloric and caloric sweeteners. PMID:25715333

Effects of grain, fructose, and histidine on ruminal pH and fermentation products during an induced subacute acidosis protocol.

PubMed

Golder, H M; Celi, P; Rabiee, A R; Heuer, C; Bramley, E; Miller, D W; King, R; Lean, I J

2012-04-01

The effects of grain, fructose, and histidine on ruminal pH and fermentation products were studied in dairy cattle during an induced subacute acidosis protocol. Thirty Holstein heifers were randomly allocated to 5 treatment groups: (1) control (no grain); (2) grain [fed at a crushed triticale dry matter intake (DMI) of 1.2% of body weight (BW)]; (3) grain (0.8% of BW DMI)+fructose (0.4% of BW DMI); (4) grain (1.2% of BW DMI)+histidine (6 g/head); and (5) grain (0.8% of BW DMI)+fructose (0.4% of BW DMI)+histidine (6 g/head) in a partial factorial arrangement. Heifers were fed 1 kg of grain daily with ad libitum access to ryegrass silage and alfalfa hay for 10 d. Feed was withheld for 14 h before challenge day, on which heifers were fed 200 g of alfalfa hay and then the treatment diets immediately thereafter. Rumen samples were collected 5 min after diet ingestion, 60 min later, and at 3 subsequent 50-min intervals. Grain decreased ruminal pH and increased ammonia, total volatile fatty acid (VFA), acetate, butyrate, propionate, and valerate concentrations compared with controls. The addition of grain had no effect on ruminal D- and L-lactate concentrations. Fructose markedly decreased ruminal pH and markedly increased D- and L-lactate concentrations. Fructose increased total VFA and butyrate and decreased valerate concentrations. Although histidine did not have a marked effect on ruminal fermentation, increased concentrations of histamine were observed following feeding. This study demonstrates that the substitution of some grain for fructose can lower ruminal pH and increase VFA and lactate concentrations, warranting further investigation into the role of sugars on the risk of acidosis in dairy cattle. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Postexercise repletion of muscle energy stores with fructose or glucose in mixed meals.

PubMed

Rosset, Robin; Lecoultre, Virgile; Egli, Léonie; Cros, Jérémy; Dokumaci, Ayse Sila; Zwygart, Karin; Boesch, Chris; Kreis, Roland; Schneiter, Philippe; Tappy, Luc

2017-03-01

Background: Postexercise nutrition is paramount to the restoration of muscle energy stores by providing carbohydrate and fat as precursors of glycogen and intramyocellular lipid (IMCL) synthesis. Compared with glucose, fructose ingestion results in lower postprandial glucose and higher lactate and triglyceride concentrations. We hypothesized that these differences in substrate concentration would be associated with a different partition of energy stored as IMCLs or glycogen postexercise. Objective: The purpose of this study was to compare the effect of isocaloric liquid mixed meals containing fat, protein, and either fructose or glucose on the repletion of muscle energy stores over 24 h after a strenuous exercise session. Design: Eight male endurance athletes (mean ± SEM age: 29 ± 2 y; peak oxygen consumption: 66.8 ± 1.3 mL · kg -1 · min -1 ) were studied twice. On each occasion, muscle energy stores were first lowered by a combination of a 3-d controlled diet and prolonged exercise. After assessment of glycogen and IMCL concentrations in vastus muscles, subjects rested for 24 h and ingested mixed meals providing fat and protein together with 4.4 g/kg fructose (the fructose condition; FRU) or glucose (the glucose condition; GLU). Postprandial metabolism was assessed over 6 h, and glycogen and IMCL concentrations were measured again after 24 h. Finally, energy metabolism was evaluated during a subsequent exercise session. Results: FRU and GLU resulted in similar IMCL [+2.4 ± 0.4 compared with +2.0 ± 0.6 mmol · kg -1 wet weight · d -1 ; time × condition (mixed-model analysis): P = 0.45] and muscle glycogen (+10.9 ± 0.9 compared with +12.3 ± 1.9 mmol · kg -1 wet weight · d -1 ; time × condition: P = 0.45) repletion. Fructose consumption in FRU increased postprandial net carbohydrate oxidation and decreased net carbohydrate storage (estimating total, muscle, and liver glycogen synthesis) compared with GLU (+117 ± 9 compared with +135 ± 9 g/6 h, respectively; P < 0.01). Compared with GLU, FRU also resulted in lower plasma glucose concentrations and decreased exercise performance the next day. Conclusions: Mixed meals containing fat, protein, and either fructose or glucose elicit similar repletion of IMCLs and muscle glycogen. Under such conditions, fructose lowers whole-body glycogen synthesis and impairs subsequent exercise performance, presumably because of lower hepatic glycogen stores. This trial was registered at clinicaltrials.gov as NCT01866215. © 2017 American Society for Nutrition.

Kalpaamruthaa ameliorates mitochondrial and metabolic alterations in diabetes mellitus induced cardiovascular damage.

PubMed

Latha, Raja; Shanthi, Palanivelu; Sachdanandam, Panchanadham

2014-12-01

Efficacy of Kalpaamruthaa on the activities of lipid and carbohydrate metabolic enzymes, electron transport chain complexes and mitochondrial ATPases were studied in heart and liver of experimental rats. Cardiovascular damage (CVD) was developed in 8 weeks after type 2 diabetes mellitus induction with high fat diet (2 weeks) and low dose of streptozotocin (2 × 35 mg/kg b.w. i.p. in 24 hr interval). In CVD-induced rats, the activities of total lipase, cholesterol ester hydrolase and cholesterol ester synthetase were increased, while lipoprotein lipase and lecithin-cholesterol acyltransferase activities were decreased. The activities of lipid-metabolizing enzymes were altered by Kalpaamruthaa in CVD-induced rats towards normal. Kalpaamruthaa modulated the activities of glycolytic enzymes (hexokinase, phosphogluco-isomerase, aldolase and glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1, 6-bisphosphatase) and glycogenolytic enzyme (glycogen phosphorylase) along with increased glycogen content in the liver of CVD-induced rats. The activities of isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, α-ketoglutarate dehydrogenase, Complexes and ATPases (Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase) were decreased in CVD-induced rats, which were ameliorated by the treatment with Kalpaamruthaa. This study ascertained the efficacy of Kalpaamruthaa for the treatment of CVD in diabetes through the modulation of metabolizing enzymes and mitochondrial dysfunction.

Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana.

PubMed

Rojas-González, José A; Soto-Súarez, Mauricio; García-Díaz, Ángel; Romero-Puertas, María C; Sandalio, Luisa M; Mérida, Ángel; Thormählen, Ina; Geigenberger, Peter; Serrato, Antonio J; Sahrawy, Mariam

2015-05-01

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin-Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The role of glycolysis and gluconeogenesis in the cytoprotection of neuroblastoma cells against 1-methyl 4-phenylpyridinium ion toxicity.

PubMed

Mazzio, Elizabeth; Soliman, Karam F A

2003-01-01

1-Methyl-4-phenylpyridinium (MPP+) is a mitochondrial Complex I inhibitor and is frequently used to investigate the pathological degeneration of neurons associated with Parkinson's disease (PD). In vitro, extracellular concentration of glucose is one of the most critical factors in establishing the vulnerability of neurons to MPP+ toxicity. While glucose is the primary energy fuel for the brain, central nervous system (CNS) neurons can also take up and utilize other metabolic intermediates for energy. In this study, we compared various monosaccharides, disaccharides, nutritive/non-nutritive sugar alcohols, glycolytic and gluconeogenic metabolic intermediates for their cytoprotection against MPP+ in murine brain neuroblastoma cells. Several monosaccharides were effective against MMP+ (500 microM) including glucose, fructose and mannose, which restored cell viability to 109 +/- 5%, 70 +/- 5%, 99 +/- 3% of live controls, respectively. Slight protective effects were observed in the presence of 3-phosphoglyceric acid and glucose-6-phosphate; however, no protective effects were exhibited by galactose, sucrose, sorbitol, mannitol, glycerol or various gluconeogenic and ketogenic amino acids. On the other hand, fructose 1,6 bisphosphate and gluconeogenic energy intermediates [pyruvic acid, malic acid and phospho(enol)pyruvate (PEP)] were neuroprotective against MPP+. The gluconeogenic intermediates elevated intracellular levels of ATP and reduced propidium iodide (PI) nucleic acid staining to live controls, but did not alter the MPP(+)-induced loss of mitochondrial O2 consumption. These data indicate that malic acid, pyruvic acid and PEP contribute to anaerobic substrate level phosphorylation. The use of hydrazine sulfate to impede gluconeogenesis through PEP carboxykinase (PEPCK) inhibition heightened the protective effects of energy substrates possibly due to attenuated ATP demands from pyruvate carboxylase (PC) activity and pyruvate mitochondrial transport. It was concluded from these studies that several metabolic intermediates are effective in fueling anaerobic glycolysis during mitochondrial inhibition by MPP+.

Biosynthesis of l-Sorbose and l-Psicose Based on C—C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain

PubMed Central

Yang, Jiangang; Li, Jitao; Men, Yan; Zhu, Yueming; Zhang, Ying; Ma, Yanhe

2015-01-01

The property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when using l-glyceraldehyde and synthesized not only l-sorbose but also a high proportion of l-psicose. Among the aldolases tested, TagA from Bacillus licheniformis (BGatY) showed the highest enzyme activity with l-glyceraldehyde. Subsequently, a “one-pot” reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/liter l-psicose and 199 mg/liter l-sorbose from dihydroxyacetone-phosphate (DHAP) and l-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to produce l-sorbose/l-psicose from glucose and l-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed in Escherichia coli and Corynebacterium glutamicum strains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinant C. glutamicum strain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/g l-glyceraldehyde. Moreover, deletion of gene cgl0331, encoding the Zn-dependent alcohol dehydrogenase in C. glutamicum, was confirmed for the first time to significantly decrease conversion of l-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/liter l-sorbose and 2.3 g/liter l-psicose, with a yield of 0.61 g/g l-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize other l-sugars. PMID:25888171

Oxygen dependency of germinating Brassica seeds

NASA Astrophysics Data System (ADS)

Park, Myoung Ryoul; Hasenstein, Karl H.

2016-02-01

Establishing plants in space, Moon or Mars requires adaptation to altered conditions, including reduced pressure and composition of atmospheres. To determine the oxygen requirements for seed germination, we imbibed Brassica rapa seeds under varying oxygen concentrations and profiled the transcription patterns of genes related to early metabolism such as starch degradation, glycolysis, and fermentation. We also analyzed the activity of lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH), and measured starch degradation. Partial oxygen pressure (pO2) greater than 10% resulted in normal germination (i.e., protrusion of radicle about 18 hours after imbibition) but lower pO2 delayed and reduced germination. Imbibition in an oxygen-free atmosphere for three days resulted in no germination but subsequent transfer to air initiated germination in 75% of the seeds and the root growth rate was transiently greater than in roots germinated under ambient pO2. In hypoxic seeds soluble sugars degraded faster but the content of starch after 24 h was higher than at ambient oxygen. Transcription of genes related to starch degradation, α-amylase (AMY) and Sucrose Synthase (SUS), was higher under ambient O2 than under hypoxia. Glycolysis and fermentation pathway-related genes, glucose phosphate isomerase (GPI), 6-phosphofructokinase (PFK), fructose 1,6-bisphosphate aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate decarboxylase (PDC), LDH, and ADH, were induced by low pO2. The activity of LDH and ADH was the highest in anoxic seeds. Germination under low O2 conditions initiated ethanolic fermentation. Therefore, sufficient oxygen availability is important for germination before photosynthesis provides necessary oxygen and the determination of an oxygen carrying capacity is important for uniform growth in space conditions.

Increased prevalence of mutant null alleles that cause hereditary fructose intolerance in the American population.

PubMed

Coffee, Erin M; Yerkes, Laura; Ewen, Elizabeth P; Zee, Tiffany; Tolan, Dean R

2010-02-01

Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Delta4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Delta4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance.

Effect of Caffeic Acid Phenethyl Ester on Vascular Damage Caused by Consumption of High Fructose Corn Syrup in Rats

PubMed Central

Gun, Aburrahman; Bilgic, Sedat; Kocaman, Nevin; Ozan, Gonca

2016-01-01

Fructose corn syrup is cheap sweetener and prolongs the shelf life of products, but fructose intake causes hyperinsulinemia, hypertriglyceridemia, and hypertension. All of them are referred to as metabolic syndrome and they are risk factors for cardiovascular diseases. Hence, the harmful effects of increased fructose intake on health and their prevention should take greater consideration. Caffeic Acid Phenethyl Ester (CAPE) has beneficial effects on metabolic syndrome and vascular function which is important in the prevention of cardiovascular disease. However, there are no known studies about the effect of CAPE on fructose-induced vascular dysfunction. In this study, we examined the effect of CAPE on vascular dysfunction due to high fructose corn syrup (HFCS). HFCS (6 weeks, 30% fed with drinking water) caused vascular dysfunction, but treatment with CAPE (50 micromol/kg i.p. for the last two weeks) effectively restored this problem. Additionally, hypertension in HFCS-fed rats was also decreased in CAPE supplemented rats. CAPE supplements lowered HFCS consumption-induced raise in blood glucose, homocysteine, and cholesterol levels. The aorta tissue endothelial nitric oxide synthase (eNOS) production was decreased in rats given HFCS and in contrast CAPE supplementation efficiently increased its production. The presented results showed that HFCS-induced cardiovascular abnormalities could be prevented by CAPE treatment. PMID:27042260

Effect of Caffeic Acid Phenethyl Ester on Vascular Damage Caused by Consumption of High Fructose Corn Syrup in Rats.

PubMed

Gun, Aburrahman; Ozer, Mehmet Kaya; Bilgic, Sedat; Kocaman, Nevin; Ozan, Gonca

2016-01-01

Fructose corn syrup is cheap sweetener and prolongs the shelf life of products, but fructose intake causes hyperinsulinemia, hypertriglyceridemia, and hypertension. All of them are referred to as metabolic syndrome and they are risk factors for cardiovascular diseases. Hence, the harmful effects of increased fructose intake on health and their prevention should take greater consideration. Caffeic Acid Phenethyl Ester (CAPE) has beneficial effects on metabolic syndrome and vascular function which is important in the prevention of cardiovascular disease. However, there are no known studies about the effect of CAPE on fructose-induced vascular dysfunction. In this study, we examined the effect of CAPE on vascular dysfunction due to high fructose corn syrup (HFCS). HFCS (6 weeks, 30% fed with drinking water) caused vascular dysfunction, but treatment with CAPE (50 micromol/kg i.p. for the last two weeks) effectively restored this problem. Additionally, hypertension in HFCS-fed rats was also decreased in CAPE supplemented rats. CAPE supplements lowered HFCS consumption-induced raise in blood glucose, homocysteine, and cholesterol levels. The aorta tissue endothelial nitric oxide synthase (eNOS) production was decreased in rats given HFCS and in contrast CAPE supplementation efficiently increased its production. The presented results showed that HFCS-induced cardiovascular abnormalities could be prevented by CAPE treatment.

Fructose and Sucrose Intake Increase Exogenous Carbohydrate Oxidation during Exercise

PubMed Central

Trommelen, Jorn; Fuchs, Cas J.; Beelen, Milou; Lenaerts, Kaatje; Jeukendrup, Asker E.; Cermak, Naomi M.; van Loon, Luc J. C.

2017-01-01

Peak exogenous carbohydrate oxidation rates typically reach ~1 g·min−1 during exercise when ample glucose or glucose polymers are ingested. Fructose co-ingestion has been shown to further increase exogenous carbohydrate oxidation rates. The purpose of this study was to assess the impact of fructose co-ingestion provided either as a monosaccharide or as part of the disaccharide sucrose on exogenous carbohydrate oxidation rates during prolonged exercise in trained cyclists. Ten trained male cyclists (VO2peak: 65 ± 2 mL·kg−1·min−1) cycled on four different occasions for 180 min at 50% Wmax during which they consumed a carbohydrate solution providing 1.8 g·min−1 of glucose (GLU), 1.2 g·min−1 glucose + 0.6 g·min−1 fructose (GLU + FRU), 0.6 g·min−1 glucose + 1.2 g·min−1 sucrose (GLU + SUC), or water (WAT). Peak exogenous carbohydrate oxidation rates did not differ between GLU + FRU and GLU + SUC (1.40 ± 0.06 vs. 1.29 ± 0.07 g·min−1, respectively, p = 0.999), but were 46% ± 8% higher when compared to GLU (0.96 ± 0.06 g·min−1: p < 0.05). In line, exogenous carbohydrate oxidation rates during the latter 120 min of exercise were 46% ± 8% higher in GLU + FRU or GLU + SUC compared with GLU (1.19 ± 0.12, 1.13 ± 0.21, and 0.82 ± 0.16 g·min−1, respectively, p < 0.05). We conclude that fructose co-ingestion (0.6 g·min−1) with glucose (1.2 g·min−1) provided either as a monosaccharide or as sucrose strongly increases exogenous carbohydrate oxidation rates during prolonged exercise in trained cyclists. PMID:28230742

Fructose and Sucrose Intake Increase Exogenous  Carbohydrate Oxidation during Exercise.

PubMed

Trommelen, Jorn; Fuchs, Cas J; Beelen, Milou; Lenaerts, Kaatje; Jeukendrup, Asker E; Cermak, Naomi M; van Loon, Luc J C

2017-02-20

Peak exogenous carbohydrate oxidation rates typically reach ~1 g∙min-1 during exercise when ample glucose or glucose polymers are ingested. Fructose co-ingestion has been shown to further increase exogenous carbohydrate oxidation rates. The purpose of this study was to assess the impact of fructose co-ingestion provided either as a monosaccharide or as part of the disaccharide sucrose on exogenous carbohydrate oxidation rates during prolonged exercise in trained cyclists. Ten trained male cyclists (VO2peak: 65 ± 2 mL∙kg-1∙min-1) cycled on four different occasions for 180 min at 50% Wmax during which they consumed a carbohydrate solution providing 1.8 g∙min-1 of glucose (GLU), 1.2 g∙min-1 glucose + 0.6 g∙min-1 fructose (GLU + FRU), 0.6 g∙min-1 glucose + 1.2 g∙min-1 sucrose (GLU + SUC), or water (WAT). Peak exogenous carbohydrate oxidation rates did not differ between GLU + FRU and GLU + SUC (1.40 ± 0.06 vs. 1.29 ± 0.07 g∙min-1, respectively, p = 0.999), but were 46% ± 8% higher when compared to GLU (0.96 ± 0.06 g∙min-1: p < 0.05). In line, exogenous carbohydrate oxidation rates during the latter 120 min of exercise were 46% ± 8% higher in GLU + FRU or GLU + SUC compared with GLU (1.19 ± 0.12, 1.13 ± 0.21, and 0.82 ± 0.16 g∙min-1, respectively, p < 0.05). We conclude that fructose co-ingestion (0.6 g∙min-1) with glucose (1.2 g∙min-1) provided either as a monosaccharide or as sucrose strongly increases exogenous carbohydrate oxidation rates during prolonged exercise in trained cyclists.

Development of a simple, low cost chronoamperometric assay for fructose based on a commercial graphite-nanoparticle modified screen-printed carbon electrode.

PubMed

Nicholas, Phil; Pittson, Robin; Hart, John P

2018-02-15

This paper describes the development of a simple, low cost chronoamperometric assay, for the measurement of fructose, using a graphite-nanoparticle modified screen-printed electrode (SPCE-G-COOH). Cyclic voltammetry showed that the response of the SPCE-G-COOH enhanced the sensitivity and precision, towards the enzymatically generated ferrocyanide species, over a plain SPCE; therefore the former was employed in subsequent studies. Calibration studies were carried out using chronoamperometry with a 40µl mixture containing fructose, mediator and FDH, deposited onto the SPCE-G-COOH. The response was linear from 0.1mM to 1.0mM. A commercial fruit juice sample was analysed using the developed assay and the fructose concentration was calculated to be 477mM with a precision of 3.03% (n=5). Following fortification (477mM fructose) the mean recovery was found to be 97.12% with a coefficient of variation of 6.42% (n=5); consequently, the method holds promise for the analysis of commercial fruit juices. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose.

PubMed

Yoshida, Hiromi; Yamada, Mitsugu; Nishitani, Takeyori; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

2007-11-23

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 A, respectively. A subunit of P. cichoriid-TE adopts a (beta/alpha)(8) barrel structure, and a metal ion (Mn(2+)) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the beta-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn(2+), and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.

Content and Vacuole/Extravacuole Distribution of Neutral Sugars, Free Amino Acids, and Anthocyanin in Protoplasts 1

PubMed Central

Wagner, George J.

1979-01-01

Neutral sugar, free amino acid, and anthocyanin levels and vacuole/extravacuole distribution were determined for Hippeastrum and Tulipa petal and Tulipa leaf protoplasts. Glucose and fructose, the predominant neutral monosaccharides observed, were primarily vacuolar in location. Glutamine, the predominant free amino acid found, was primarily extravacuolar. γ-Methyleneglutamate was identified as a major constituent of Tulipa protoplasts. Qualitative characterization of Hippeastrum petal and vacuole organic acids indicated the presence of oxalic, malic, citric, and isocitric acids. Data are presented which indicate that vacuoles obtained by gentle osmotic shock of protoplasts in dibasic phosphate have good purity and retain their contents. Images PMID:16660921