Assessing inflammatory liver injury in an acute CCl4 model using dynamic 3D metabolic imaging of hyperpolarized [1-(13)C]pyruvate.

PubMed

Josan, Sonal; Billingsley, Kelvin; Orduna, Juan; Park, Jae Mo; Luong, Richard; Yu, Liqing; Hurd, Ralph; Pfefferbaum, Adolf; Spielman, Daniel; Mayer, Dirk

2015-12-01

To facilitate diagnosis and staging of liver disease, sensitive and non-invasive methods for the measurement of liver metabolism are needed. This study used hyperpolarized (13)C-pyruvate to assess metabolic parameters in a CCl4 model of liver damage in rats. Dynamic 3D (13)C chemical shift imaging data from a volume covering kidney and liver were acquired from 8 control and 10 CCl4-treated rats. At 12 time points at 5 s temporal resolution, we quantified the signal intensities and established time courses for pyruvate, alanine, and lactate. These measurements were compared with standard liver histology and an alanine transaminase (ALT) enzyme assay using liver tissue from the same animals. All CCl4-treated but none of the control animals showed histological liver damage and elevated ALT enzyme levels. In agreement with these results, metabolic imaging revealed an increased alanine/pyruvate ratio in liver of CCl4-treated rats, which is indicative of elevated ALT activity. Similarly, lactate/pyruvate ratios were higher in CCl4-treated compared with control animals, demonstrating the presence of inflammation. No significant differences in metabolite ratios were observed in kidney or vasculature. Thus this work shows that metabolic imaging using (13)C-pyruvate can be a successful tool to non-invasively assess liver damage in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

l-Valine Production with Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum▿

PubMed Central

Blombach, Bastian; Schreiner, Mark E.; Holátko, Jiří; Bartek, Tobias; Oldiges, Marco; Eikmanns, Bernhard J.

2007-01-01

Corynebacterium glutamicum was engineered for the production of l-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum ΔaceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, l-alanine, and l-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum ΔaceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and l-alanine towards l-valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum ΔaceE(pJC4ilvBNCE) produced up to 210 mM l-valine with a volumetric productivity of 10.0 mM h−1 (1.17 g l−1 h−1) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose. PMID:17293513

Twenty-seven Years of Cerebral Pyruvate Recycling.

PubMed

Cerdán, Sebastián

2017-06-01

Cerebral pyruvate recycling is a metabolic pathway deriving carbon skeletons and reducing equivalents from mitochondrial oxaloacetate and malate, to the synthesis of mitochondrial and cytosolic pyruvate, lactate and alanine. The pathway allows both, to provide the tricarboxylic acid cycle with pyruvate molecules produced from alternative substrates to glucose and, to generate reducing equivalents necessary for the operation of NADPH requiring processes. At the cellular level, pyruvate recycling involves the activity of malic enzyme, or the combined activities of phosphoenolpyruvate carboxykinase and pyruvate kinase, as well as of those transporters of the inner mitochondrial membrane exchanging the corresponding intermediates. Its cellular localization between the neuronal or astrocytic compartments of the in vivo brain has been controversial, with evidences favoring either a primarily neuronal or glial localizations, more recently accepted to occur in both environments. This review provides a brief history on the detection and characterization of the pathway, its relations with the early developments of cerebral high resolution 13 C NMR, and its potential neuroprotective functions under hypoglycemic conditions or ischemic redox stress.

n-Octyl gallate as inhibitor of pyruvate carboxylation and lactate gluconeogenesis.

PubMed

Eler, Gabrielle Jacklin; Santos, Israel Souza; de Moraes, Amarilis Giaretta; Comar, Jurandir Fernando; Peralta, Rosane Marina; Bracht, Adelar

2015-04-01

The alkyl gallates are found in several natural and industrial products. In the latter products, these compounds are added mainly for preventing oxidation. In the present work, the potencies of methyl gallate, n-propyl gallate, n-pentyl gallate, and n-octyl gallate as inhibitors of pyruvate carboxylation and lactate gluconeogenesis were evaluated. Experiments were done with isolated mitochondria and the isolated perfused rat liver. The potency of the gallic acid esters as inhibitors of pyruvate carboxylation in isolated mitochondria obeyed the following decreasing sequence: n-octyl gallate > n-pentyl gallate > n-propyl gallate > methyl gallate. A similar sequence of decreasing potency for lactate gluconeogenesis inhibition in the perfused liver was found in terms of the portal venous concentration. Both actions correlate with the lipophilicity of the compounds. The effects are harmful at high concentrations. At appropriate concentrations, however, octyl gallate should act therapeutically because its inhibitory action on gluconeogenesis will contribute further to its proposed antihyperglycemic effects. © 2014 Wiley Periodicals, Inc.

Modeling non‐linear kinetics of hyperpolarized [1‐13C] pyruvate in the crystalloid‐perfused rat heart

PubMed Central

Mariotti, E.; Orton, M. R.; Eerbeek, O.; Ashruf, J. F.; Zuurbier, C. J.; Southworth, R.

2016-01-01

Hyperpolarized 13C MR measurements have the potential to display non‐linear kinetics. We have developed an approach to describe possible non‐first‐order kinetics of hyperpolarized [1‐13C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass of the hyperpolarized signal. Simultaneous fitting to a second‐order model for conversion of [1‐13C] pyruvate to bicarbonate, lactate and alanine was well described in the isolated rat heart perfused with Krebs buffer containing glucose as sole energy substrate, or glucose supplemented with pyruvate. Second‐order modeling yielded significantly improved fits of pyruvate–bicarbonate kinetics compared with the more traditionally used first‐order model and suggested time‐dependent decreases in pyruvate–bicarbonate flux. Second‐order modeling gave time‐dependent changes in forward and reverse reaction kinetics of pyruvate–lactate exchange and pyruvate–alanine exchange in both groups of hearts during the infusion of pyruvate; however, the fits were not significantly improved with respect to a traditional first‐order model. The mechanism giving rise to second‐order pyruvate dehydrogenase (PDH) kinetics was explored experimentally using surface fluorescence measurements of nicotinamide adenine dinucleotide reduced form (NADH) performed under the same conditions, demonstrating a significant increase of NADH during pyruvate infusion. This suggests a simultaneous depletion of available mitochondrial NAD+ (the cofactor for PDH), consistent with the non‐linear nature of the kinetics. NADH levels returned to baseline following cessation of the pyruvate infusion, suggesting this to be a transient effect. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. PMID:26777799

Performance during a strenuous swimming session is associated with high blood lactate: pyruvate ratio and hypoglycemia in fasted rats

PubMed Central

Travassos, P.B.; Godoy, G.; De Souza, H.M.; Curi, R.; Bazotte, R.B.

2018-01-01

The aim of this study was to investigate the effect of lactatemia elevation and glycemia reduction on strenuous swimming performance in fasted rats. Three rats were placed in a swimming tank at the same time. The first rat was removed immediately (control group) and the remaining ones were submitted to a strenuous swimming session. After the second rat was exhausted (Exh group), the third one was immediately removed from the water (Exe group). According to the period of time required for exhaustion, the rats were divided into four groups: low performance (3–7 min), low-intermediary performance (8–12 min), high-intermediary performance (13–17 min), and high performance (18–22 min). All rats were removed from the swimming tanks and immediately killed by decapitation for blood collection or anesthetized for liver perfusion experiments. Blood glucose, lactate, and pyruvate concentrations, blood lactate/pyruvate ratio, and liver lactate uptake and its conversion to glucose were evaluated. Exhaustion in low and low-intermediary performance were better associated with higher lactate/pyruvate ratio. On the other hand, exhaustion in high-intermediary and high performance was better associated with hypoglycemia. Lactate uptake and glucose production from lactate in livers from the Exe and Exh groups were maintained. We concluded that there is a time sequence in the participation of lactate/pyruvate ratio and hypoglycemia in performance during an acute strenuous swimming section in fasted rats. The liver had an important participation in preventing hyperlactatemia and hypoglycemia during swimming through lactate uptake and its conversion to glucose. PMID:29590261

Performance during a strenuous swimming session is associated with high blood lactate: pyruvate ratio and hypoglycemia in fasted rats.

PubMed

Travassos, P B; Godoy, G; De Souza, H M; Curi, R; Bazotte, R B

2018-03-26

The aim of this study was to investigate the effect of lactatemia elevation and glycemia reduction on strenuous swimming performance in fasted rats. Three rats were placed in a swimming tank at the same time. The first rat was removed immediately (control group) and the remaining ones were submitted to a strenuous swimming session. After the second rat was exhausted (Exh group), the third one was immediately removed from the water (Exe group). According to the period of time required for exhaustion, the rats were divided into four groups: low performance (3-7 min), low-intermediary performance (8-12 min), high-intermediary performance (13-17 min), and high performance (18-22 min). All rats were removed from the swimming tanks and immediately killed by decapitation for blood collection or anesthetized for liver perfusion experiments. Blood glucose, lactate, and pyruvate concentrations, blood lactate/pyruvate ratio, and liver lactate uptake and its conversion to glucose were evaluated. Exhaustion in low and low-intermediary performance were better associated with higher lactate/pyruvate ratio. On the other hand, exhaustion in high-intermediary and high performance was better associated with hypoglycemia. Lactate uptake and glucose production from lactate in livers from the Exe and Exh groups were maintained. We concluded that there is a time sequence in the participation of lactate/pyruvate ratio and hypoglycemia in performance during an acute strenuous swimming section in fasted rats. The liver had an important participation in preventing hyperlactatemia and hypoglycemia during swimming through lactate uptake and its conversion to glucose.

1H-NMR and Hyperpolarized 13C-NMR Assays of Pyruvate-Lactate Exhange: a comparative study

PubMed Central

Orton, Matthew R.; Tardif, Nicolas; Parkes, Harold G.; Robinson, Simon P.; Leach, Martin O.; Chung, Yuen-Li; Eykyn, Thomas R.

2015-01-01

Pyruvate-lactate exchange is mediated by the enzyme lactate dehydrogenase (LDH) and is central to the altered energy metabolism in cancer cells. Measurement of exchange kinetics using hyperpolarized 13C NMR has provided a biomarker of response to novel therapeutics. In this study we investigated an alternative in vitro 1H assay, using [3-13C]pyruvate, and compared the measured kinetics with a hyperpolarized 13C-NMR assay, using [1-13C]pyruvate, under the same conditions in human colorectal carcinoma SW1222 cells. The apparent forward reaction rate constants (kPL) derived from the two assays showed no significant difference, and both assays had similar reproducibility (kPL = 0.506 ± 0.054 and kPL = 0.441 ± 0.090 nmol/s/106 cells, (mean ± standard deviation, n = 3); 1H, 13C assays respectively). The apparent backward reaction rate constant (kLP) could only be measured with good reproducibility using the 1H-NMR assay (kLP = 0.376 ± 0.091 nmol/s/106 cells, (mean ± standard deviation, n = 3)). The 1H-NMR assay has adequate sensitivity to measure real-time pyruvate-lactate exchange kinetics in vitro, offering a complementary and accessible assay of apparent LDH activity. PMID:23712817

Renal Cortical Pyruvate Depletion during AKI

PubMed Central

Johnson, Ali C.M.; Becker, Kirsten

2014-01-01

Pyruvate is a key intermediary in energy metabolism and can exert antioxidant and anti-inflammatory effects. However, the fate of pyruvate during AKI remains unknown. Here, we assessed renal cortical pyruvate and its major determinants (glycolysis, gluconeogenesis, pyruvate dehydrogenase [PDH], and H2O2 levels) in mice subjected to unilateral ischemia (15–60 minutes; 0–18 hours of vascular reflow) or glycerol-induced ARF. The fate of postischemic lactate, which can be converted back to pyruvate by lactate dehydrogenase, was also addressed. Ischemia and glycerol each induced persistent pyruvate depletion. During ischemia, decreasing pyruvate levels correlated with increasing lactate levels. During early reperfusion, pyruvate levels remained depressed, but lactate levels fell below control levels, likely as a result of rapid renal lactate efflux. During late reperfusion and glycerol-induced AKI, pyruvate depletion corresponded with increased gluconeogenesis (pyruvate consumption). This finding was underscored by observations that pyruvate injection increased renal cortical glucose content in AKI but not normal kidneys. AKI decreased PDH levels, potentially limiting pyruvate to acetyl CoA conversion. Notably, pyruvate therapy mitigated the severity of AKI. This renoprotection corresponded with increases in cytoprotective heme oxygenase 1 and IL-10 mRNAs, selective reductions in proinflammatory mRNAs (e.g., MCP-1 and TNF-α), and improved tissue ATP levels. Paradoxically, pyruvate increased cortical H2O2 levels. We conclude that AKI induces a profound and persistent depletion of renal cortical pyruvate, which may induce additional injury. PMID:24385590

(13)C MR spectroscopy study of lactate as substrate for rat brain.

PubMed

Qu, H; Håberg, A; Haraldseth, O; Unsgård, G; Sonnewald, U

2000-01-01

In order to address the question whether lactate in blood can serve as a precursor for cerebral metabolites, fully awake rats were injected intravenously with [U-(13)C]lactate or [U-(13)C]glucose followed 15 min later by decapitation. Incorporation of label from [U-(13)C]glucose was seen mainly in glutamate, GABA, glutamine, aspartate, alanine and lactate. More label was found in glutamate than glutamine, underscoring the predominantly neuronal metabolism of pyruvate from [U-(13)C]glucose. It was estimated that the neuronal metabolism of acetyl CoA from glucose accounts for at least 66% and the glial for no more than 34% of the total glucose consumption. When [U-(13)C]lactate was the precursor, label incorporation was similar to that observed from [U-(13)C]glucose, but much reduced. Plasma analysis revealed the presence of approximately equal amounts of [1,2,3-(13)C]- and [1,2-(13)C]glucose, showing gluconeogenesis from [U-(13)C]lactate. It was thus possible that the labeling seen in the cerebral amino acids originated from labeled glucose, not [U-(13)C]lactate. However, the presence of significantly more label in [U-(13)C]- than in [2,3-(13)C]alanine demonstrated that [U-(13)C]lactate did indeed cross the blood-brain barrier, and was metabolized further in the brain. Furthermore, contributions from pyruvate carboxylase (glial enzyme) were detectable in glutamine, glutamate and GABA, and were comparatively more pronounced in the glucose group. This indicated that relatively more pyruvate from lactate than glucose was metabolized in neurons. Surprisingly, the same amount of lactate was synthesized via the tricarboxylic acid cycle in both groups, indicating transfer of neurotransmitters from the neuronal to the astrocytic compartment, as previous studies have shown that this lactate is synthesized primarily in astrocytes. Taking into consideration that astrocytes take up glutamate more avidly than GABA, it is conceivable that neuronal lactate metabolism was more

Metabolic Imaging of Patients with Prostate Cancer Using Hyperpolarized [1-13C]Pyruvate

PubMed Central

Nelson, Sarah J.; Kurhanewicz, John; Vigneron, Daniel B.; Larson, Peder E. Z.; Harzstark, Andrea L.; Ferrone, Marcus; van Criekinge, Mark; Chang, Jose W.; Bok, Robert; Park, Ilwoo; Reed, Galen; Carvajal, Lucas; Small, Eric J.; Munster, Pamela; Weinberg, Vivian K.; Ardenkjaer-Larsen, Jan Henrik; Chen, Albert P.; Hurd, Ralph E.; Odegardstuen, Liv-Ingrid; Robb, Fraser J.; Tropp, James; Murray, Jonathan A.

2014-01-01

This first-in-man imaging study evaluated the safety and feasibility of hyperpolarized [1-13C]pyruvate as an agent for noninvasively characterizing alterations in tumor metabolism for patients with prostate cancer. Imaging living systems with hyperpolarized agents can result in more than 10,000-fold enhancement in signal relative to conventional magnetic resonance (MR) imaging. When combined with the rapid acquisition of in vivo 13C MR data, it is possible to evaluate the distribution of agents such as [1-13C]pyruvate and its metabolic products lactate, alanine, and bicarbonate in a matter of seconds. Preclinical studies in cancer models have detected elevated levels of hyperpolarized [1-13C]lactate in tumor, with the ratio of [1-13C]lactate/[1-13C]pyruvate being increased in high-grade tumors and decreased after successful treatment. Translation of this technology into humans was achieved by modifying the instrument that generates the hyperpolarized agent, constructing specialized radio frequency coils to detect 13C nuclei, and developing new pulse sequences to efficiently capture the signal. The study population comprised patients with biopsy-proven prostate cancer, with 31 subjects being injected with hyperpolarized [1-13C]pyruvate. The median time to deliver the agent was 66 s, and uptake was observed about 20 s after injection. No dose-limiting toxicities were observed, and the highest dose (0.43 ml/kg of 230 mM agent) gave the best signal-to-noise ratio for hyperpolarized [1-13C]pyruvate. The results were extremely promising in not only confirming the safety of the agent but also showing elevated [1-13C]lactate/[1-13C]pyruvate in regions of biopsy-proven cancer. These findings will be valuable for noninvasive cancer diagnosis and treatment monitoring in future clinical trials. PMID:23946197

Enzyme mechanisms for pyruvate-to-lactate flux attenuation: a study of Sherpas, Quechuas, and hummingbirds.

PubMed

Hochachka, P W; Stanley, C; McKenzie, D C; Villena, A; Monge, C

1992-10-01

During incremental exercise to fatigue under hypobaric hypoxia, Andean Quechua natives form and accumulate less plasma lactate than do lowlanders under similar conditions. This phenomenon of low lactate accumulation despite hypobaric hypoxia, first discovered some half century ago, is known in Quechuas to be largely unaffected by acute exposure to hypoxia or by acclimatization to sea level conditions. Earlier Nuclear Magnetic Resonance (NMR) spectroscopy and metabolic biochemistry studies suggest that closer coupling of energy demand and energy supply in Quechuas allows given changes in work rate with relatively modest changes in muscle adenylate and phosphagen concentrations, thus tempering the activation of glycolytic flux to pyruvate--a coarse control mechanism operating at the level of overall pathway flux. Later studies of enzyme activities in skeletal muscles of Quechuas and of Sherpas have identified a finely-tuned control mechanism which by adaptive modifications of a few key enzymes apparently serves to specifically attenuate pyruvate flux to lactate.

The progression from a lower to a higher invasive stage of bladder cancer is associated with severe alterations in glucose and pyruvate metabolism

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

Conde, Vanessa R.; Oliveira, Pedro F.; Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine, Abel Salazar Institute of Biomedical Sciences, University of Porto – UMIB/ICBAS/UP

Cancer cells present a particular metabolic behavior. We hypothesized that the progression of bladder cancer could be accompanied by changes in cells glycolytic profile. We studied two human bladder cancer cells, RT4 and TCCSUP, in which the latter represents a more invasive stage. The levels of glucose, pyruvate, alanine and lactate in the extracellular media were measured by Proton Nuclear Magnetic Resonance. The protein expression levels of glucose transporters 1 (GLUT1) and 3 (GLUT3), monocarboxylate transporter 4 (MCT4), phosphofructokinase-1 (PFK1), glutamic-pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) were determined. Our data showed that glucose consumption and GLUT3 levels were similarmore » in both cell lines, but TCCSUP cells displayed lower levels of GLUT1 and PFK expression. An increase in pyruvate consumption, concordant with the higher levels of lactate and alanine production, was also detected in TCCSUP cells. Moreover, TCCSUP cells presented lower protein expression levels of GPT and LDH. These results illustrate that bladder cancer progression is associated with alterations in cells glycolytic profile, namely the switch from glucose to pyruvate consumption in the more aggressive stage. This may be useful to develop new therapies and to identify biomarkers for cancer progression. - Highlights: • Metabolic phenotype of less and high invasive bladder cancer cells was studied. • Bladder cancer progression involves alterations in cells glycolytic profile. • More invasive bladder cancer cells switch from glucose to pyruvate consumption. • Our results may help to identify metabolic biomarkers of bladder cancer progression.« less

The kinetics of transport of lactate and pyruvate into rat hepatocytes. Evidence for the presence of a specific carrier similar to that in erythrocytes.

PubMed Central

Edlund, G L; Halestrap, A P

1988-01-01

Time courses of L-lactate and pyruvate uptake into isolated rat hepatocytes were measured in a citrate-based medium to generate a pH gradient (alkaline inside), by using the silicone-oil-filtration technique at 0 degrees C to minimize metabolism. At low concentrations of lactate and pyruvate (0.5 mM), transport was inhibited by over 95% by 5 mM-alpha-cyano-4-hydroxycinnamate, whereas at higher concentrations (greater than 10 mM) a significant proportion of transport could not be inhibited. The rate of this non-inhibitable transport was linearly related to the substrate concentration, was less with pyruvate than with L-lactate, and appeared to be due to diffusion of undissociated acid. Uptake of D-lactate was not inhibited by alpha-cyano-4-hydroxycinnamate and occurred only by diffusion. Kinetic parameters for the carrier-mediated transport process were obtained after correction of the initial rates of uptake of lactate and pyruvate in the absence of 5 mM-alpha-cyano-4-hydroxycinnamate by that in the presence of inhibitor. Under the conditions used, the Km values for L-lactate and pyruvate were 2.4 and 0.6 mM respectively and the Ki for alpha-cyano-4-hydroxycinnamate as a competitive inhibitor was 0.11 mM. Km values for the transport of L-lactate and pyruvate into rat erythrocytes under similar conditions were 3.0 and 0.96 mM. The Vmax. of lactate and pyruvate transport into hepatocytes at 0 degrees C was 3 nmol/min per mg of protein. Carrier-mediated transport of 0.5 mM-L-lactate was inhibited by 0.2 mM-p-chloromercuribenzenesulphonate (greater than 90%), 0.5 mM-quercetin (80%), 0.6 mM-isobutylcarbonyl-lactyl anhydride (70%) and 0.5 mM-4,4'-di-isothiocyanostilbene-2,2'-disulphonate (50%). A similar pattern of inhibition of lactate transport is seen in erythrocytes. It is suggested that the same or a similar carrier protein exists in both tissues. The results also show that L-lactate transport into rat hepatocytes is very rapid at physiological temperatures and is

l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria.

PubMed

Pizzuto, Roberto; Paventi, Gianluca; Porcile, Carola; Sarnataro, Daniela; Daniele, Aurora; Passarella, Salvatore

2012-09-01

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M. Copyright © 2012 Elsevier B.V. All rights reserved.

Cerebrospinal fluid acid-base status and lactate and pyruvate concentrations after convulsions of varied duration and aetiology in children.

PubMed Central

Simpson, H; Habel, A H; George, E L

1977-01-01

Twenty-two infants and children were studied after convulsions of varied cause and duration. Arterial and CSF acid-base variables, lactate and pyruvate concentrations, and lactate/pyruvate ratios were measured between 3 and 18 hours after convulsive episodes. Biochemical signs of cerebral hypoxia were found in 7 patients with prolonged (greater than 30 minutes) or recurrent short convulsions. These signs were absent in patients with single short convulsions. These findings indicate that cerebral hypoxia and possible brain damage is a hazard of prolonged or rapidly recurring short convulsions. PMID:23078

Cerebrospinal fluid acid-base status and lactate and pyruvate concentrations after short (less than 30 minutes) first febrile convulsions in children.

PubMed Central

Simpson, H; Habel, A H; George, E L

1977-01-01

Twenty-nine infants and children with short (less than 30 minutes) first febrile convulsions were studied between 3 and 22 hours after convulsive episodes. Arterial and CSF acid-base variables, lactate and pyruvate concentrations, and lactate/pyruvate ratios were measured. Biochemical signs of cerebral hypoxia were found in only 2 patients, one of whom had short, repeated convulsions. Our findings indicate that hypoxic damage is unlikely to result from a short-duration febrile convulsion. PMID:23077

Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae.

PubMed

Kim, Sujin; Bae, Sang-Jeong; Hahn, Ji-Sook

2016-04-07

Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging.

Quantification of in vivo metabolic kinetics of hyperpolarized pyruvate in rat kidneys using dynamic 13C MRSI.

PubMed

Xu, Tao; Mayer, Dirk; Gu, Meng; Yen, Yi-Fen; Josan, Sonal; Tropp, James; Pfefferbaum, Adolf; Hurd, Ralph; Spielman, Daniel

2011-10-01

With signal-to-noise ratio enhancements on the order of 10,000-fold, hyperpolarized MRSI of metabolically active substrates allows the study of both the injected substrate and downstream metabolic products in vivo. Although hyperpolarized [1-(13)C]pyruvate, in particular, has been used to demonstrate metabolic activities in various animal models, robust quantification and metabolic modeling remain important areas of investigation. Enzyme saturation effects are routinely seen with commonly used doses of hyperpolarized [1-(13)C]pyruvate; however, most metrics proposed to date, including metabolite ratios, time-to-peak of metabolic products and single exchange rate constants, fail to capture these saturation effects. In addition, the widely used small-flip-angle excitation approach does not correctly model the inflow of fresh downstream metabolites generated proximal to the target slice, which is often a significant factor in vivo. In this work, we developed an efficient quantification framework employing a spiral-based dynamic spectroscopic imaging approach. The approach overcomes the aforementioned limitations and demonstrates that the in vivo (13)C labeling of lactate and alanine after a bolus injection of [1-(13)C]pyruvate is well approximated by saturatable kinetics, which can be mathematically modeled using a Michaelis-Menten-like formulation, with the resulting estimated apparent maximal reaction velocity V(max) and apparent Michaelis constant K(M) being unbiased with respect to critical experimental parameters, including the substrate dose, bolus shape and duration. Although the proposed saturatable model has a similar mathematical formulation to the original Michaelis-Menten kinetics, it is conceptually different. In this study, we focus on the (13)C labeling of lactate and alanine and do not differentiate the labeling mechanism (net flux or isotopic exchange) or the respective contribution of various factors (organ perfusion rate, substrate transport

Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress.

PubMed

Delgado, María Graciela; Oliva, Carlos; López, Estefanía; Ibacache, Andrés; Galaz, Alex; Delgado, Ricardo; Barros, L Felipe; Sierralta, Jimena

2018-01-19

The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.

d-Alanine Oxidase from Escherichia coli: Localization and Induction by l-Alanine

PubMed Central

Raunio, R. P.; Jenkins, W. T.

1973-01-01

Dialyzed membranes of Escherichia coli prepared by an ethylenediaminetetraacetic acid-lysozyme method catalyze the oxidation of both l-alanine and d-alanine. The specific activities for the oxidations of both d-alanine and l-alanine are increased fivefold when the cells are grown in the presence of either l-alanine or dl-alanine, but are increased only slightly when grown in the presence of d-alanine. In the dl-alanine-induced system, the specific activities for the oxidations of some other d-amino acids are also raised. dl-alanine also induces two other alanine catabolizing enzymes, alanine dehydrogenase and alanine-glutamate aminotransferase which are found in the “soluble” fraction of lysozyme-treated cells. The oxidations of both l-alanine and d-alanine were associated with the membranes of induced cells. After the membranes were disintegrated by sonic treatment, both l-alanine and d-alanine oxidation catalysts sedimented in a sucrose density gradient together with d-lactate and l-lactate dehydrogenases, apparently as a single multienzyme complex. PMID:4146872

Lactate and Pyruvate Are Major Sources of Energy for Stallion Sperm with Dose Effects on Mitochondrial Function, Motility, and ROS Production.

PubMed

Darr, Christa R; Varner, Dickson D; Teague, Sheila; Cortopassi, Gino A; Datta, Sandipan; Meyers, Stuart A

2016-08-01

Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to direct utilization within the mitochondria. Measurements were performed after incubation in modified BWW media with varying concentrations of lactate, pyruvate, and glucose. The effects of media and duration of incubation on sperm motility, ROS production, and oxygen consumption were determined using a linear mixed-effects model. Duplicate ejaculates from four stallions were used in three separate experiments to determine the effects of substrate availability and concentration on sperm motility and mitochondrial function and the relationship of oxygen consumption with cellular ROS production. The present results indicate that lactate and pyruvate are the most important sources of energy for stallion sperm motility and velocity, and elicit a dose-dependent response. Additionally, lactate and pyruvate are ideal for maximal mitochondrial function, as sperm in these media operate at a very high level of their bioenergetic capability due to the high rate of energy metabolism. Moreover, we found that addition of glucose to the media is not necessary for short-term storage of equine sperm, and may even result in reduction of mitochondrial function. Finally, we have confirmed that ROS production can be the result of mitochondrial dysfunction as well as intense mitochondrial activity. © 2016 by the Society for the Study of Reproduction, Inc.

Role of L-alanine for redox self-sufficient amination of alcohols.

PubMed

Klatte, Stephanie; Wendisch, Volker F

2015-01-23

In white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578-5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed. The enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-ta Cv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily

Beneficial effect of pyruvate therapy on Leigh syndrome due to a novel mutation in PDH E1α gene.

PubMed

Koga, Yasutoshi; Povalko, Nataliya; Katayama, Koujyu; Kakimoto, Noriko; Matsuishi, Toyojiro; Naito, Etsuo; Tanaka, Masashi

2012-02-01

Leigh syndrome (LS) is a progressive untreatable degenerating mitochondrial disorder caused by either mitochondrial or nuclear DNA mutations. A patient was a second child of unconsanguineous parents. On the third day of birth, he was transferred to neonatal intensive care units because of severe lactic acidosis. Since he was showing continuous lactic acidosis, the oral supplementation of dichloroacetate (DCA) was introduced on 31st day of birth at initial dose of 50 mg/kg, followed by maintenance dose of 25 mg/kg/every 12 h. The patient was diagnosed with LS due to a point mutation of an A-C at nucleotide 599 in exon 6 in the pyruvate dehydrogenase E1α gene, resulting in the substitution of aspartate for threonine at position 200 (N200T). Although the concentrations of lactate and pyruvate in blood were slightly decreased, his clinical conditions were deteriorating progressively. In order to overcome the mitochondrial or cytosolic energy crisis indicated by lactic acidosis as well as clinical symptoms, we terminated the DCA and administered 0.5 g/kg/day TID of sodium pyruvate orally. We analyzed the therapeutic effects of DCA or sodium pyruvate in the patient, and found that pyruvate therapy significantly decreased lactate, pyruvate and alanine levels, showed no adverse effects such as severe neuropathy seen in DCA, and had better clinical response on development and epilepsy. Though the efficacy of pyruvate on LS will be evaluated by randomized double-blind placebo-controlled study design in future, pyruvate therapy is a possible candidate for therapeutic choice for currently incurable mitochondrial disorders such as LS. Copyright © 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Lactate shuttling and lactate use as fuel after traumatic brain injury: metabolic considerations

PubMed Central

Dienel, Gerald A

2014-01-01

Lactate is proposed to be generated by astrocytes during glutamatergic neurotransmission and shuttled to neurons as ‘preferred' oxidative fuel. However, a large body of evidence demonstrates that metabolic changes during activation of living brain disprove essential components of the astrocyte–neuron lactate shuttle model. For example, some glutamate is oxidized to generate ATP after its uptake into astrocytes and neuronal glucose phosphorylation rises during activation and provides pyruvate for oxidation. Extension of the notion that lactate is a preferential fuel into the traumatic brain injury (TBI) field has important clinical implications, and the concept must, therefore, be carefully evaluated before implementation into patient care. Microdialysis studies in TBI patients demonstrate that lactate and pyruvate levels and lactate/pyruvate ratios, along with other data, have important diagnostic value to distinguish between ischemia and mitochondrial dysfunction. Results show that lactate release from human brain to blood predominates over its uptake after TBI, and strong evidence for lactate metabolism is lacking; mitochondrial dysfunction may inhibit lactate oxidation. Claims that exogenous lactate infusion is energetically beneficial for TBI patients are not based on metabolic assays and data are incorrectly interpreted. PMID:25204393

Mitochondrial pyruvate transport in working guinea-pig heart. Work-related vs. carrier-mediated control of pyruvate oxidation.

PubMed

Bünger, R; Mallet, R T

1993-09-19

Myocardial pyruvate oxidation is work- or calcium-load-related, but control of pyruvate dehydrogenase (PDH) by the specific mitochondrial pyruvate transporter has also been proposed. To test the transport hypothesis distribution of pyruvate across the cell membrane as well as rates of mitochondrial pyruvate net transport plus oxidation were examined in isolated perfused but stable and physiologically working guinea-pig hearts. 150 microM-1.2 mM alpha-cyanohydroxycinnamate proved to specifically block mitochondrial pyruvate uptake in these hearts. When perfusate glucose as cytosolic pyruvate precursor was supplied in combination with octanoate (0.2 or 0.5 mM) as diffusible alternative fatty acid substrate, alpha-cyanohydroxycinnamate produced up to 20- and 3-fold increases in pyruvate and lactate efflux, respectively. Cinnamates did not alter myocardial hemodynamics nor sarcolemmal pyruvate and lactate export. In contrast the tested concentrations of cinnamate produced reversible, dose-dependent decreases in 14CO2 production from [1-14C]pyruvate or [U-14C]glucose by inhibiting mitochondrial pyruvate uptake. Linear least-squares estimates of available cinnamate-sensitive total pyruvate transport potential yielded rates close to 110 mumol/min per g dry mass at S0.5 approximately 120 microM, which compared reasonably well with literature values from isolated cardiac mitochondria. This transport potential was severalfold larger than total extractable myocardial PDH activity of approximately 32 mumol/min per g dry mass at 37 degrees C. Even when cytosolic pyruvate levels were in the lower physiologic range of about 90 microM, pyruvate oxidation readily kept pace with mitochondrial respiration over a wide range of workload and inotropism. Furthermore, dichloroacetate, a selective activator of PDH, stimulated pyruvate oxidation without affecting myocardial O2 consumption, regardless of the metabolic or inotropic state of the hearts. Consequently, little or no regulatory

Propionate stimulates pyruvate oxidation in the presence of acetate.

PubMed

Purmal, Colin; Kucejova, Blanka; Sherry, A Dean; Burgess, Shawn C; Malloy, Craig R; Merritt, Matthew E

2014-10-15

Flux through pyruvate dehydrogenase (PDH) in the heart may be reduced by various forms of injury to the myocardium, or by oxidation of alternative substrates in normal heart tissue. It is important to distinguish these two mechanisms because imaging of flux through PDH based on the appearance of hyperpolarized (HP) [(13)C]bicarbonate derived from HP [1-(13)C]pyruvate has been proposed as a method for identifying viable myocardium. The efficacy of propionate for increasing PDH flux in the setting of PDH inhibition by an alternative substrate was studied using isotopomer analysis paired with exams using HP [1-(13)C]pyruvate. Hearts from C57/bl6 mice were supplied with acetate (2 mM) and glucose (8.25 mM). (13)C NMR spectra were acquired in a cryogenically cooled probe at 14.1 Tesla. After addition of hyperpolarized [1-(13)C]pyruvate, (13)C NMR signals from lactate, alanine, malate, and aspartate were easily detected, in addition to small signals from bicarbonate and CO2. The addition of propionate (2 mM) increased appearance of HP [(13)C]bicarbonate >30-fold without change in O2 consumption. Isotopomer analysis of extracts from the freeze-clamped hearts indicated that acetate was the preferred substrate for energy production, glucose contribution to energy production was minimal, and anaplerosis was stimulated in the presence of propionate. Under conditions where production of acetyl-CoA is dominated by the availability of an alternative substrate, acetate, propionate markedly stimulated PDH flux as detected by the appearance of hyperpolarized [(13)C]bicarbonate from metabolism of hyperpolarized [1-(13)C]pyruvate. Copyright © 2014 the American Physiological Society.

Brain oxygen utilization is unchanged by hypoglycemia in normal humans: lactate, alanine, and leucine uptake are not sufficient to offset energy deficit.

PubMed

Lubow, Jeffrey M; Piñón, Ivan G; Avogaro, Angelo; Cobelli, Claudio; Treeson, David M; Mandeville, Katherine A; Toffolo, Gianna; Boyle, Patrick J

2006-01-01

During hypoglycemia, substrates other than glucose have been suggested to serve as alternate neural fuels. We evaluated brain uptake of endogenously produced lactate, alanine, and leucine at euglycemia and during insulin-induced hypoglycemia in 17 normal subjects. Cross-brain arteriovenous differences for plasma glucose, lactate, alanine, leucine, and oxygen content were quantitated. Cerebral blood flow (CBF) was measured by Fick methodology using N(2)O as the dilution indicator gas. Substrate uptake was measured as the product of CBF and the arteriovenous concentration difference. As arterial glucose concentration fell, cerebral oxygen utilization and CBF remained unchanged. Brain glucose uptake (BGU) decreased from 36.3+/-2.6 to 26.6+/-2.1 micromol.100 g of brain(-1).min(-1) (P<0.001), equivalent to a drop in ATP of 291 micromol.100 g(-1).min(-1). Arterial lactate rose (P<0.001), whereas arterial alanine and leucine fell (P<0.009 and P<0.001, respectively). Brain lactate uptake (BLU) increased from a net release of -1.8+/- 0.6 to a net uptake of 2.5+/-1.2 micromol.100 g(-1).min(-1) (P<0.001), equivalent to an increase in ATP of 74 micromol.100 g(-1).min(-1). Brain leucine uptake decreased from 7.1+/-1.2 to 2.5 +/- 0.5 micromol.100 g(-1).min(-1) (P<0.001), and brain alanine uptake trended downward (P<0.08). We conclude that the ATP generated from the physiological increase in BLU during hypoglycemia accounts for no more than 25% of the brain glucose energy deficit.

The rate of lactate production from glucose in hearts is not altered by per-deuteration of glucose

NASA Astrophysics Data System (ADS)

Funk, Alexander M.; Anderson, Brian L.; Wen, Xiaodong; Hever, Thomas; Khemtong, Chalermchai; Kovacs, Zoltan; Sherry, A. Dean; Malloy, Craig R.

2017-11-01

This study was designed to determine whether perdeuterated glucose experiences a kinetic isotope effect (KIE) as glucose passes through glycolysis and is further oxidized in the tricarboxylic acid (TCA) cycle. Metabolism of deuterated glucose was investigated in two groups of perfused rat hearts. The control group was supplied with a 1:1 mixture of [U-13C6]glucose and [1,6-13C2]glucose, while the experimental group received [U-13C6,U-2H7]glucose and [1,6-13C2]glucose. Tissue extracts were analyzed by 1H, 2H and proton-decoupled 13C NMR spectroscopy. Extensive 2H-13C scalar coupling plus chemical shift isotope effects were observed in the proton-decoupled 13C NMR spectra of lactate, alanine and glutamate. A small but measureable (∼8%) difference in the rate of conversion of [U-13C6]glucose vs. [1,6-13C2]glucose to lactate, likely reflecting rates of Csbnd C bond breakage in the aldolase reaction, but conversion of [U-13C6]glucose versus [U-13C6,U-2H7]glucose to lactate did not differ. This shows that the presence of deuterium in glucose does not alter glycolytic flux. However, there were two distinct effects of deuteration on metabolism of glucose to alanine and oxidation of glucose in the TCA. First, alanine undergoes extensive exchange of methyl deuterons with solvent protons in the alanine amino transferase reaction. Second, there is a substantial kinetic isotope effect in metabolism of [U-13C6,U-2H7]glucose to alanine and glutamate. In the presence of [U-13C6,U-2H7]glucose, alanine and lactate are not in rapid exchange with the same pool of pyruvate. These studies indicate that the appearance of hyperpolarized 13C-lactate from hyperpolarized [U-13C6,U-2H7]glucose is not substantially influenced by a deuterium kinetic isotope effect.

SU-E-QI-11: Measurement of Renal Pyruvate-To-Lactate Exchange with Hyperpolarized 13C MRI

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

Adamson, E; Johnson, K; Fain, S

Purpose: Previous work [1] modeling the metabolic flux between hyperpolarized [1-13C]pyruvate and [1-13C]lactate in magnetic resonance spectroscopic imaging (MRSI) experiments failed to account for vascular signal artifacts. Here, we investigate a method to minimize the vascular signal and its impact on the fidelity of metabolic modeling. Methods: MRSI was simulated for renal metabolism in MATLAB both with and without bipolar gradients. The resulting data were fit to a two-site exchange model [1], and the effects of vascular partial volume artifacts on kinetic modeling were assessed. Bipolar gradients were then incorporated into a gradient echo sequence to validate the simulations experimentally.more » The degree of diffusion weighting (b = 32 s/mm{sup 2}) was determined empirically from 1H imaging of murine renal vascular signal. The method was then tested in vivo using MRSI with bipolar gradients following injection of hyperpolarized [1-{sup 13}C]pyruvate (∼80 mM at 20% polarization). Results: In simulations, vascular signal contaminated the renal metabolic signal at resolutions as high as 2 × 2 mm{sup 2} due to partial volume effects. The apparent exchange rate from pyruvate to lactate (k{sub p}) was underestimated in the presence of these artifacts due to contaminating pyruvate signal. Incorporation of bipolar gradients suppressed vascular signal and improved the accuracy of kp estimation. Experimentally, the in vivo results supported the ability of bipolar gradients to suppress vascular signal. The in vivo exchange rate increased, as predicted in simulations, from k{sub p} = 0.012 s-{sup 1} to k{sub p} = 0.020-{sup 1} after vascular signal suppression. Conclusion: We have demonstrated the limited accuracy of the two-site exchange model in the presence of vascular partial volume artifacts. The addition of bipolar gradients suppressed vascular signal and improved model accuracy in simulations. Bipolar gradients largely affected kp estimation in vivo

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential.

PubMed

Pudakalakatti, Shivanand M; Uppangala, Shubhashree; D'Souza, Fiona; Kalthur, Guruprasad; Kumar, Pratap; Adiga, Satish Kumar; Atreya, Hanudatta S

2013-01-01

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5) ) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright © 2012 John Wiley & Sons, Ltd.

Alanine increases blood pressure during hypotension

NASA Technical Reports Server (NTRS)

Conlay, L. A.; Maher, T. J.; Wurtman, R. J.

1990-01-01

The effect of L-alanine administration on blood pressure (BP) during haemorrhagic shock was investigated using anesthetized rats whose left carotid arteries were cannulated for BP measurement, blood removal, and drug administration. It was found that L-alanine, in doses of 10, 25, 50, 100, and 200 mg/kg, increased the systolic BP of hypotensive rats by 38 to 80 percent (while 100 mg/kg pyruvate increased BP by only 9.4 mmhg, not significantly different from saline). The results suggest that L-alanine might influence cardiovascular function.

Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

DOE PAGES

Zhou, Jilai; Olson, Daniel G.; Lanahan, Anthony A.; ...

2015-09-15

We report that Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. We found that that pyruvate ferredoxin oxidoreductase enzymemore » (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.« less

Pyruvate cycling and implications for regulation of gluconeogenesis in the insect, Manduca sexta L.

PubMed

Thompson, S N

2000-08-11

Pyruvate cycling was examined in the insect Manduca sexta L. (2-(13)C)pyruvate was injected into 5th instar larvae maintained on a semisynthetic high sucrose, low sucrose, or sucrose-free diet. Pyruvate cycling and gluconeogenesis were determined from the distribution of (13)C in blood metabolites, including trehalose, the blood sugar of insects, and alanine. Pyruvate cycling was evident from the (13)C enrichment of alanine C3, synthesized by transamination of pyruvate following carboxylation to oxaloacetate and cycling through phosphoenolpyruvate. Based on the relative (13)C enrichments of alanine C2 and C3, insects maintained on the high sucrose diet displayed higher levels of cycling than insects on the other diets. Insects on all the diets, when subsequently starved, displayed low levels of cycling. Gluconeogenesis was evident in insects on sucrose-free or low sucrose diets from the selective (13)C enrichment in trehalose. The level of gluconeogenesis relative to glycolysis was indicated by the (13)C enrichment of trehalose C6 and alanine C3, both enrichments metabolically derived in the same manner. Insects starved after maintenance on the sucrose-free or low sucrose diets remained glucogenic. Insects on the high sucrose diet were not glucogenic, and subsequent starvation did not induce gluconeogenesis. The results indicate that pyruvate kinase plays a critical role in regulating the gluconeogenic/glycolytic balance, and that inhibition of pyruvate kinase is a principal regulatory event during induction of de novo trehalose synthesis. Gluconeogenesis failed to maintain homeostatic levels of blood trehalose, supporting the conclusion that blood sugar level may be important for mediating nutrient intake. Possible factors involved in the regulation of gluconeogenesis in insects are discussed. Copyright 2000 Academic Press.

Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes

PubMed Central

Bender, Tom; Pena, Gabrielle; Martinou, Jean-Claude

2015-01-01

At the pyruvate branch point, the fermentative and oxidative metabolic routes diverge. Pyruvate can be transformed either into lactate in mammalian cells or into ethanol in yeast, or transported into mitochondria to fuel ATP production by oxidative phosphorylation. The recently discovered mitochondrial pyruvate carrier (MPC), encoded by MPC1, MPC2, and MPC3 in yeast, is required for uptake of pyruvate into the organelle. Here, we show that while expression of Mpc1 is not dependent on the carbon source, expression of Mpc2 and Mpc3 is specific to fermentative or respiratory conditions, respectively. This gives rise to two alternative carrier complexes that we have termed MPCFERM and MPCOX. By constitutively expressing the two alternative complexes in yeast deleted for all three endogenous genes, we show that MPCOX has a higher transport activity than MPCFERM, which is dependent on the C-terminus of Mpc3. We propose that the alternative MPC subunit expression in yeast provides a way of adapting cellular metabolism to the nutrient availability. PMID:25672363

High brain lactate is a hallmark of aging and caused by a shift in the lactate dehydrogenase A/B ratio

PubMed Central

Ross, Jaime M.; Öberg, Johanna; Brené, Stefan; Coppotelli, Giuseppe; Terzioglu, Mügen; Pernold, Karin; Goiny, Michel; Sitnikov, Rouslan; Kehr, Jan; Trifunovic, Aleksandra; Larsson, Nils-Göran; Hoffer, Barry J.; Olson, Lars

2010-01-01

At present, there are few means to track symptomatic stages of CNS aging. Thus, although metabolic changes are implicated in mtDNA mutation-driven aging, the manifestations remain unclear. Here, we used normally aging and prematurely aging mtDNA mutator mice to establish a molecular link between mitochondrial dysfunction and abnormal metabolism in the aging process. Using proton magnetic resonance spectroscopy and HPLC, we found that brain lactate levels were increased twofold in both normally and prematurely aging mice during aging. To correlate the striking increase in lactate with tissue pathology, we investigated the respiratory chain enzymes and detected mitochondrial failure in key brain areas from both normally and prematurely aging mice. We used in situ hybridization to show that increased brain lactate levels were caused by a shift in transcriptional activities of the lactate dehydrogenases to promote pyruvate to lactate conversion. Separation of the five tetrameric lactate dehydrogenase (LDH) isoenzymes revealed an increase of those dominated by the Ldh-A product and a decrease of those rich in the Ldh-B product, which, in turn, increases pyruvate to lactate conversion. Spectrophotometric assays measuring LDH activity from the pyruvate and lactate sides of the reaction showed a higher pyruvate → lactate activity in the brain. We argue for the use of lactate proton magnetic resonance spectroscopy as a noninvasive strategy for monitoring this hallmark of the aging process. The mtDNA mutator mouse allows us to conclude that the increased LDH-A/LDH-B ratio causes high brain lactate levels, which, in turn, are predictive of aging phenotypes. PMID:21041631

High brain lactate is a hallmark of aging and caused by a shift in the lactate dehydrogenase A/B ratio.

PubMed

Ross, Jaime M; Öberg, Johanna; Brené, Stefan; Coppotelli, Giuseppe; Terzioglu, Mügen; Pernold, Karin; Goiny, Michel; Sitnikov, Rouslan; Kehr, Jan; Trifunovic, Aleksandra; Larsson, Nils-Göran; Hoffer, Barry J; Olson, Lars

2010-11-16

At present, there are few means to track symptomatic stages of CNS aging. Thus, although metabolic changes are implicated in mtDNA mutation-driven aging, the manifestations remain unclear. Here, we used normally aging and prematurely aging mtDNA mutator mice to establish a molecular link between mitochondrial dysfunction and abnormal metabolism in the aging process. Using proton magnetic resonance spectroscopy and HPLC, we found that brain lactate levels were increased twofold in both normally and prematurely aging mice during aging. To correlate the striking increase in lactate with tissue pathology, we investigated the respiratory chain enzymes and detected mitochondrial failure in key brain areas from both normally and prematurely aging mice. We used in situ hybridization to show that increased brain lactate levels were caused by a shift in transcriptional activities of the lactate dehydrogenases to promote pyruvate to lactate conversion. Separation of the five tetrameric lactate dehydrogenase (LDH) isoenzymes revealed an increase of those dominated by the Ldh-A product and a decrease of those rich in the Ldh-B product, which, in turn, increases pyruvate to lactate conversion. Spectrophotometric assays measuring LDH activity from the pyruvate and lactate sides of the reaction showed a higher pyruvate → lactate activity in the brain. We argue for the use of lactate proton magnetic resonance spectroscopy as a noninvasive strategy for monitoring this hallmark of the aging process. The mtDNA mutator mouse allows us to conclude that the increased LDH-A/LDH-B ratio causes high brain lactate levels, which, in turn, are predictive of aging phenotypes.

Lactate shuttles in nature.

PubMed

Brooks, G A

2002-04-01

Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously under fully aerobic conditions. "Cell-cell" and "intracellular lactate shuttle" concepts describe the roles of lactate in the delivery of oxidative and gluconeogenic substrates, as well as in cell signalling. Examples of cell-cell shuttles include lactate exchanges between white-glycolytic and red-oxidative fibres within a working muscle bed, between working skeletal muscle and heart, and between tissues of net lactate release and gluconeogenesis. Lactate exchange between astrocytes and neurons that is linked to glutamatergic signalling in the brain is an example of a lactate shuttle supporting cell-cell signalling. Lactate uptake by mitochondria and pyruvate-lactate exchange in peroxisomes are examples of intracellular lactate shuttles. Lactate exchange between sites of production and removal is facilitated by monocarboxylate transport proteins, of which there are several isoforms, and, probably, also by scaffolding proteins. The mitochondrial lactate-pyruvate transporter appears to work in conjunction with mitochondrial lactate dehydrogenase, which permits lactate to be oxidized within actively respiring cells. Hence mitochondria function to establish the concentration and proton gradients necessary for cells with high mitochondrial densities (e.g. cardiocytes) to take up and oxidize lactate. Arteriovenous difference measurements on working cardiac and skeletal muscle beds as well as NMR spectral analyses of these tissues show that lactate is formed and oxidized within the cells of formation in vivo. Glycolysis and lactate oxidation within cells permits high flux rates and the maintenance of redox balance in the cytosol and mitochondria. Other examples of intracellular lactate shuttles include lactate uptake and oxidation in sperm mitochondria and the facilitation of beta-oxidation in peroxisomes by pyruvate-lactate

Pyruvate transport in isolated cardiac mitochondria from two species of amphibian exhibiting dissimilar aerobic scope: Bufo marinus and Rana catesbeiana.

PubMed

Duerr, Jeffrey M; Tucker, Kristina

2007-08-01

Cardiac mitochondria were isolated from Bufo marinus and Rana catesbeiana, two species of amphibian whose cardiovascular systems are adapted to either predominantly aerobic or glycolytic modes of locomotion. Mitochondrial oxidative capacity was compared using VO2 max and respiratory control ratios in the presence of a variety of substrates including pyruvate, lactate, oxaloacetate, beta-hydroxybutyrate, and octanoyl-carnitine. B. marinus cardiac mitochondria exhibited VO2 max values twice that of R. catesbeiana cardiac mitochondria when oxidizing carbohydrate substrates. Pyruvate transport was measured via a radiolabeled-tracer assay in isolated B. marinus and R. catesbeiana cardiac mitochondria. Time-course experiments described both alpha-cyano-4-hydroxycinnamate-sensitive (MCT-like) and phenylsuccinate-sensitive pyruvate uptake mechanisms in both species. Pyruvate uptake by the MCT-like transporter was enhanced in the presence of a pH gradient, whereas the phenylsuccinate-sensitive transporter was inhibited. Notably, anuran cardiac mitochondria exhibited activities of lactate dehydrogenase and pyruvate carboxylase. The presence of both transporters on the inner mitochondrial membrane affords the net uptake of monocarboxylates including pyruvate, beta-hydroxybutyrate, and lactate; the latter potentially indicating the presence of a lactate/pyruvate shuttle allowing oxidation of extramitochondrial NADH. Intramitochondrial lactate dehydrogenase and pyruvate carboxylase enables lactate to be oxidized to pyruvate or converted to anaplerotic oxaloacetate. Kinetics of the MCT-like transporter differed significantly between the two species, suggesting differences in aerobic scope may be in part attributable to differences in mitochondrial carbohydrate utilization. (c) 2007 Wiley-Liss, Inc.

Pyruvate incubation enhances glycogen stores and sustains neuronal function during subsequent glucose deprivation.

PubMed

Shetty, Pavan K; Sadgrove, Matthew P; Galeffi, Francesca; Turner, Dennis A

2012-01-01

The use of energy substrates, such as lactate and pyruvate, has been shown to improve synaptic function when administered during glucose deprivation. In the present study, we investigated whether prolonged incubation with monocarboxylate (pyruvate or lactate) prior rather than during glucose deprivation can also sustain synaptic and metabolic function. Pyruvate pre-incubation(3-4h) significantly prolonged (>25 min) the tolerance of rat hippocampal slices to delayed glucose deprivation compared to control and lactate pre-incubated slices, as revealed by field excitatory post synaptic potentials (fEPSPs); pre-incubation with pyruvate also reduced the marked decrease in NAD(P)H fluorescence resulting from glucose deprivation. Moreover, pyruvate exposure led to the enhancement of glycogen stores with time, compared to glucose alone (12 μmol/g tissue at 4h vs. 3.5 μmol/g tissue). Prolonged resistance to glucose deprivation following exogenous pyruvate incubation was prevented by glycogenolysis inhibitors, suggesting that enhanced glycogen mediates the delay in synaptic activity failure. The application of an adenosine A1 receptor antagonist enhanced glycogen utilization and prolonged the time to synaptic failure, further confirming this hypothesis of the importance of glycogen. Moreover, tissue levels of ATP were also significantly maintained during glucose deprivation in pyruvate pretreated slices compared to control and lactate. In summary, these experiments indicate that pyruvate exposure prior to glucose deprivation significantly increased the energy buffering capacity of hippocampal slices, particularly by enhancing internal glycogen stores, delaying synaptic failure during glucose deprivation by maintaining ATP levels, and minimizing the decrease in the levels of NAD(P)H. Copyright © 2011 Elsevier Inc. All rights reserved.

Gluconeogenesis in Leishmania mexicana: contribution of glycerol kinase, phosphoenolpyruvate carboxykinase, and pyruvate phosphate dikinase.

PubMed

Rodriguez-Contreras, Dayana; Hamilton, Nicklas

2014-11-21

Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Microorganisms and methods for producing pyruvate, ethanol, and other compounds

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

Reed, Jennifer L.; Zhang, Xiaolin

Microorganisms comprising modifications for producing pyruvate, ethanol, and other compounds. The microorganisms comprise modifications that reduce or ablate activity of one or more of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, phosphate acetyltransferase, acetate kinase, pyruvate oxidase, lactate dehydrogenase, cytochrome terminal oxidase, succinate dehydrogenase, 6-phosphogluconate dehydrogenase, glutamate dehydrogenase, pyruvate formate lyase, pyruvate formate lyase activating enzyme, and isocitrate lyase. The microorganisms optionally comprise modifications that enhance expression or activity of pyruvate decarboxylase and alcohol dehydrogenase. The microorganisms are optionally evolved in defined media to enhance specific production of one or more compounds. Methods of producing compounds with the microorganisms are provided.

Requirement for the Mitochondrial Pyruvate Carrier in Mammalian Development Revealed by a Hypomorphic Allelic Series

PubMed Central

Bowman, Caitlyn E.; Hartung, Thomas

2016-01-01

Glucose and oxygen are two of the most important molecules transferred from mother to fetus during eutherian pregnancy, and the metabolic fates of these nutrients converge at the transport and metabolism of pyruvate in mitochondria. Pyruvate enters the mitochondrial matrix through the mitochondrial pyruvate carrier (MPC), a complex in the inner mitochondrial membrane that consists of two essential components, MPC1 and MPC2. Here, we define the requirement for mitochondrial pyruvate metabolism during development with a progressive allelic series of Mpc1 deficiency in mouse. Mpc1 deletion was homozygous lethal in midgestation, but Mpc1 hypomorphs and tissue-specific deletion of Mpc1 presented as early perinatal lethality. The allelic series demonstrated that graded suppression of MPC resulted in dose-dependent metabolic and transcriptional changes. Steady-state metabolomics analysis of brain and liver from Mpc1 hypomorphic embryos identified compensatory changes in amino acid and lipid metabolism. Flux assays in Mpc1-deficient embryonic fibroblasts also reflected these changes, including a dramatic increase in mitochondrial alanine utilization. The mitochondrial alanine transaminase GPT2 was found to be necessary and sufficient for increased alanine flux upon MPC inhibition. These data show that impaired mitochondrial pyruvate transport results in biosynthetic deficiencies that can be mitigated in part by alternative anaplerotic substrates in utero. PMID:27215380

Functional characteristics of pyruvate transport in Phycomyces blakesleeanus.

PubMed

Marcos, J A; de Arriaga, D; Busto, F; Soler, J

1998-12-01

A saturable and accumulative transport system for pyruvate has been detected in Phycomyces blakesleeanus NRRL 1555(-) mycelium. It was strongly inhibited by alpha-cyano-4-hydroxycinnamate. l-Lactate and acetate were competitive inhibitors of pyruvate transport. The initial pyruvate uptake velocity and accumulation ratio was dependent on the external pH. The Vmax of transport greatly decreased with increasing pH, whereas the affinity of the carrier for pyruvate was not affected. The pyruvate transport system mediated its homologous exchange, which was essentially pH independent, and efflux, which increased with increasing external pH. The uptake of pyruvate was energy dependent and was strongly inhibited by inhibitors of oxidative phosphorylation and of the formation of proton gradients. Glucose counteracted the inhibitory effect of the pyruvate transport produced by inhibitors of mitochondrial ATP synthesis. Our results are consistent with a pyruvate/proton cotransport in P. blakesleeanus probably driven by an electrochemical gradient of H+ generated by a plasma membrane H+-ATPase. Copyright 1998 Academic Press.

Improvement of Neuroenergetics by Hypertonic Lactate Therapy in Patients with Traumatic Brain Injury Is Dependent on Baseline Cerebral Lactate/Pyruvate Ratio

PubMed Central

Quintard, Hervé; Patet, Camille; Zerlauth, Jean-Baptiste; Suys, Tamarah; Bouzat, Pierre; Pellerin, Luc; Meuli, Reto; Magistretti, Pierre J.

2016-01-01

Abstract Energy dysfunction is associated with worse prognosis after traumatic brain injury (TBI). Recent data suggest that hypertonic sodium lactate infusion (HL) improves energy metabolism after TBI. Here, we specifically examined whether the efficacy of HL (3h infusion, 30–40 μmol/kg/min) in improving brain energetics (using cerebral microdialysis [CMD] glucose as a main therapeutic end-point) was dependent on baseline cerebral metabolic state (assessed by CMD lactate/pyruvate ratio [LPR]) and cerebral blood flow (CBF, measured with perfusion computed tomography [PCT]). Using a prospective cohort of 24 severe TBI patients, we found CMD glucose increase during HL was significant only in the subgroup of patients with elevated CMD LPR >25 (n = 13; +0.13 [95% confidence interval (CI) 0.08–0.19] mmol/L, p < 0.001; vs. +0.04 [–0.05–0.13] in those with normal LPR, p = 0.33, mixed-effects model). In contrast, CMD glucose increase was independent from baseline CBF (coefficient +0.13 [0.04–0.21] mmol/L when global CBF was <32.5 mL/100 g/min vs. +0.09 [0.04–0.14] mmol/L at normal CBF, both p < 0.005) and systemic glucose. Our data suggest that improvement of brain energetics upon HL seems predominantly dependent on baseline cerebral metabolic state and support the concept that CMD LPR – rather than CBF – could be used as a diagnostic indication for systemic lactate supplementation following TBI. PMID:26421521

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa

PubMed Central

Hereng, T.H.; Elgstøen, K.B.P.; Cederkvist, F.H.; Eide, L.; Jahnsen, T.; Skålhegg, B.S.; Rosendal, K.R.

2011-01-01

BACKGROUND There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. METHODS Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of 13C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. RESULTS The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed 13C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. CONCLUSIONS Human

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa.

PubMed

Hereng, T H; Elgstøen, K B P; Cederkvist, F H; Eide, L; Jahnsen, T; Skålhegg, B S; Rosendal, K R

2011-12-01

There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. Human spermatozoa seem to rely mainly, if

Prolonged continuous intravenous infusion of the dipeptide L-alanine- L-glutamine significantly increases plasma glutamine and alanine without elevating brain glutamate in patients with severe traumatic brain injury

PubMed Central

2014-01-01

Introduction Low plasma glutamine levels are associated with worse clinical outcome. Intravenous glutamine infusion dose- dependently increases plasma glutamine levels, thereby correcting hypoglutaminemia. Glutamine may be transformed to glutamate which might limit its application at a higher dose in patients with severe traumatic brain injury (TBI). To date, the optimal glutamine dose required to normalize plasma glutamine levels without increasing plasma and cerebral glutamate has not yet been defined. Methods Changes in plasma and cerebral glutamine, alanine, and glutamate as well as indirect signs of metabolic impairment reflected by increased intracranial pressure (ICP), lactate, lactate-to-pyruvate ratio, electroencephalogram (EEG) activity were determined before, during, and after continuous intravenous infusion of 0.75 g L-alanine-L-glutamine which was given either for 24 hours (group 1, n = 6) or 5 days (group 2, n = 6) in addition to regular enteral nutrition. Lab values including nitrogen balance, urea and ammonia were determined daily. Results Continuous L-alanine-L-glutamine infusion significantly increased plasma and cerebral glutamine as well as alanine levels, being mostly sustained during the 5 day infusion phase (plasma glutamine: from 295 ± 62 to 500 ± 145 μmol/ l; brain glutamine: from 183 ± 188 to 549 ± 120 μmol/ l; plasma alanine: from 327 ± 91 to 622 ± 182 μmol/ l; brain alanine: from 48 ± 55 to 89 ± 129 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test). Plasma glutamate remained unchanged and cerebral glutamate was decreased without any signs of cerebral impairment. Urea and ammonia were significantly increased within normal limits without signs of organ dysfunction (urea: from 2.7 ± 1.6 to 5.5 ± 1.5 mmol/ l; ammonia: from 12 ± 6.3 to 26 ± 8.3 μmol/ l; p < 0.05, ANOVA, post hoc Dunn’s test). Conclusions High dose L-alanine-L-glutamine infusion (0

Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure.

PubMed

Ferriero, Rosa; Nusco, Edoardo; De Cegli, Rossella; Carissimo, Annamaria; Manco, Giuseppe; Brunetti-Pierri, Nicola

2018-03-24

Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of

Volumetric spiral chemical shift imaging of hyperpolarized [2-(13) c]pyruvate in a rat c6 glioma model.

PubMed

Park, Jae Mo; Josan, Sonal; Jang, Taichang; Merchant, Milton; Watkins, Ron; Hurd, Ralph E; Recht, Lawrence D; Mayer, Dirk; Spielman, Daniel M

2016-03-01

MRS of hyperpolarized [2-(13)C]pyruvate can be used to assess multiple metabolic pathways within mitochondria as the (13)C label is not lost with the conversion of pyruvate to acetyl-CoA. This study presents the first MR spectroscopic imaging of hyperpolarized [2-(13)C]pyruvate in glioma-bearing brain. Spiral chemical shift imaging with spectrally undersampling scheme (1042 Hz) and a hard-pulse excitation was exploited to simultaneously image [2-(13)C]pyruvate, [2-(13)C]lactate, and [5-(13)C]glutamate, the metabolites known to be produced in brain after an injection of hyperpolarized [2-(13)C]pyruvate, without chemical shift displacement artifacts. A separate undersampling scheme (890 Hz) was also used to image [1-(13)C]acetyl-carnitine. Healthy and C6 glioma-implanted rat brains were imaged at baseline and after dichloroacetate administration, a drug that modulates pyruvate dehydrogenase kinase activity. The baseline metabolite maps showed higher lactate and lower glutamate in tumor as compared to normal-appearing brain. Dichloroacetate led to an increase in glutamate in both tumor and normal-appearing brain. Dichloroacetate-induced %-decrease of lactate/glutamate was comparable to the lactate/bicarbonate decrease from hyperpolarized [1-(13)C]pyruvate studies. Acetyl-carnitine was observed in the muscle/fat tissue surrounding the brain. Robust volumetric imaging with hyperpolarized [2-(13)C]pyruvate and downstream products was performed in glioma-bearing rat brains, demonstrating changes in mitochondrial metabolism with dichloroacetate. © 2015 Wiley Periodicals, Inc.

Lactate dehydrogenase activity is inhibited by methylmalonate in vitro.

PubMed

Saad, Laura O; Mirandola, Sandra R; Maciel, Evelise N; Castilho, Roger F

2006-04-01

Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K (i)=3.02+/-0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.

Changes in pyruvate metabolism detected by magnetic resonance imaging are linked to DNA damage and serve as a sensor of temozolomide response in glioblastoma cells

PubMed Central

Park, Ilwoo; Mukherjee, Joydeep; Ito, Motokazu; Chaumeil, Myriam M.; Jalbert, Llewellyn E.; Gaensler, Karin; Ronen, Sabrina M.; Nelson, Sarah J.; Pieper, Russell O.

2014-01-01

Recent findings show that exposure to temozolomide (TMZ), a DNA damaging drug used to treat glioblastoma, can suppress the conversion of pyruvate to lactate. To understand the mechanistic basis for this effect and its potential utility as a TMZ response biomarker, we compared the response of isogenic glioblastoma cell populations differing only in expression of the DNA repair protein MGMT, a TMZ-sensitivity determinant, after exposure to TMZ in vitro and in vivo. Hyperpolarized [1-(13)C]-pyruvate-based magnetic resonance imaging was used to monitor temporal effects on pyruvate metabolism in parallel with DNA damage responses and tumor cell growth. TMZ exposure decreased conversion of pyruvate to lactate only in MGMT-deficient cells. This effect coincided temporally with TMZ-induced increases in levels of the DNA damage response protein pChk1. Changes in pyruvate to lactate conversion triggered by TMZ preceded tumor growth suppression and were not associated with changes in levels of NADH or lactate dehydrogenase activity in tumors. Instead, they were associated with a TMZ-induced decrease in the expression and activity of pyruvate kinase PKM2, a glycolytic enzyme that indirectly controls pyruvate metabolism. PKM2 silencing decreased pyruvate kinase activity, intracellular lactate levels, and conversion of pyruvate to lactate in the same manner as TMZ, and Chk1 silencing blocked the TMZ-induced decrease in PKM2 expression. Overall, our findings showed how TMZ-induced DNA damage is linked through PKM2 to changes in pyruvate metabolism, and how these changes can be exploited by magnetic resonance imaging methods as an early sensor of TMZ therapeutic response. PMID:25320009

Pyruvate ingestion for 7 days does not improve aerobic performance in well-trained individuals

NASA Technical Reports Server (NTRS)

Morrison, M. A.; Spriet, L. L.; Dyck, D. J.

2000-01-01

The purposes of the present studies were to test the hypotheses that lower dosages of oral pyruvate ingestion would increase blood pyruvate concentration and that the ingestion of a commonly recommended dosage of pyruvate (7 g) for 7 days would enhance performance during intense aerobic exercise in well-trained individuals. Nine recreationally active subjects (8 women, 1 man) consumed 7, 15, and 25 g of pyruvate and were monitored for a 4-h period to determine whether blood metabolites were altered. Pyruvate consumption failed to significantly elevate blood pyruvate, and it had no effect on indexes of carbohydrate (blood glucose, lactate) or lipid metabolism (blood glycerol, plasma free fatty acids). As a follow-up, we administered 7 g/day of either placebo or pyruvate, for a 1-wk period to seven, well-trained male cyclists (maximal oxygen consumption, 62.3 +/- 3.0 ml. kg(-1). min(-1)) in a randomized, double-blind, crossover trial. Subjects cycled at 74-80% of their maximal oxygen consumption until exhaustion. There was no difference in performance times between the two trials (placebo, 91 +/- 9 min; pyruvate, 88 +/- 8 min). Measured blood parameters (insulin, peptide C, glucose, lactate, glycerol, free fatty acids) were also unaffected. Our results indicate that oral pyruvate supplementation does not increase blood pyruvate content and does not enhance performance during intense exercise in well-trained cyclists.

Function of the Pyruvate Oxidase-Lactate Oxidase Cascade in Interspecies Competition between Streptococcus oligofermentans and Streptococcus mutans

PubMed Central

Liu, Lei

2012-01-01

Complex interspecies interactions occur constantly between oral commensals and the opportunistic pathogen Streptococcus mutans in dental plaque. Previously, we showed that oral commensal Streptococcus oligofermentans possesses multiple enzymes for H2O2 production, especially lactate oxidase (Lox), allowing it to out-compete S. mutans. In this study, through extensive biochemical and genetic studies, we identified a pyruvate oxidase (pox) gene in S. oligofermentans. A pox deletion mutant completely lost Pox activity, while ectopically expressed pox restored activity. Pox was determined to produce most of the H2O2 in the earlier growth phase and log phase, while Lox mainly contributed to H2O2 production in stationary phase. Both pox and lox were expressed throughout the growth phase, while expression of the lox gene increased by about 2.5-fold when cells entered stationary phase. Since lactate accumulation occurred to a large degree in stationary phase, the differential Pox- and Lox-generated H2O2 can be attributed to differential gene expression and substrate availability. Interestingly, inactivation of pox causes a dramatic reduction in H2O2 production from lactate, suggesting a synergistic action of the two oxidases in converting lactate into H2O2. In an in vitro two-species biofilm experiment, the pox mutant of S. oligofermentans failed to inhibit S. mutans even though lox was active. In summary, S. oligofermentans develops a Pox-Lox synergy strategy to maximize its H2O2 formation so as to win the interspecies competition. PMID:22287002

Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by alpha-cyano-4-hydroxycinnamate.

PubMed

Halestrap, A P; Denton, R M

1974-02-01

alpha-Cyano-4-hydroxycinnamate greatly inhibits the transport of pyruvate but not that of acetate or butyrate in liver mitochondria and erythrocytes. In the latter, lactate uptake is also inhibited. It is concluded that a specific carrier is involved in membrane transport of pyruvate and that the plasma-membrane carrier may also be involved in lactate transport.

Lactate and the Lactate-to-Pyruvate Molar Ratio Cannot Be Used as Independent Biomarkers for Monitoring Brain Energetic Metabolism: A Microdialysis Study in Patients with Traumatic Brain Injuries

PubMed Central

Sahuquillo, Juan; Merino, Maria-Angels; Sánchez-Guerrero, Angela; Arikan, Fuat; Vidal-Jorge, Marian; Martínez-Valverde, Tamara; Rey, Anna; Riveiro, Marilyn; Poca, Maria-Antonia

2014-01-01

Background For decades, lactate has been considered an excellent biomarker for oxygen limitation and therefore of organ ischemia. The aim of the present study was to evaluate the frequency of increased brain lactate levels and the LP ratio (LPR) in a cohort of patients with severe or moderate traumatic brain injury (TBI) subjected to brain microdialysis monitoring to analyze the agreement between these two biomarkers and to indicate brain energy metabolism dysfunction. Methods Forty-six patients with an admission Glasgow coma scale score of ≤13 after resuscitation admitted to a dedicated 10-bed Neurotraumatology Intensive Care Unit were included, and 5305 verified samples of good microdialysis data were analyzed. Results Lactate levels were above 2.5 mmol/L in 56.9% of the samples. The relationships between lactate and the LPR could not be adequately modeled by any linear or non-linear model. Neither Cohen’s kappa nor Gwet’s statistic showed an acceptable agreement between both biomarkers to classify the samples in regard to normal or abnormal metabolism. The dataset was divided into four patterns defined by the lactate concentrations and the LPR. A potential interpretation for these patterns is suggested and discussed. Pattern 4 (low pyruvate levels) was found in 10.7% of the samples and was characterized by a significantly low concentration of brain glucose compared with the other groups. Conclusions Our study shows that metabolic abnormalities are frequent in the macroscopically normal brain in patients with traumatic brain injuries and a very poor agreement between lactate and the LPR when classifying metabolism. The concentration of lactate in the dialysates must be interpreted while taking into consideration the LPR to distinguish between anaerobic metabolism and aerobic hyperglycolysis. PMID:25025772

The BRAF inhibitor vemurafenib activates mitochondrial metabolism and inhibits hyperpolarized pyruvate-lactate exchange in BRAF mutant human melanoma cells

PubMed Central

Delgado-Goni, Teresa; Falck Miniotis, Maria; Wantuch, Slawomir; Parkes, Harold G.; Marais, Richard; Workman, Paul; Leach, Martin O.; Beloueche-Babari, Mounia

2016-01-01

Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity and potential as non-invasive imaging response biomarkers.1H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF mutant (WM266.4 and SKMEL28) but not BRAFWT (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. 13C NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of HKII, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase and monocarboxylate transporter (MCT) 1 and 4 in BRAF mutant but not BRAFWT cells and, interestingly, decreased BRAF mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized 13C-pyruvate-lactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as non-invasive imaging of response. PMID:27765851

A re-evaluation of the role of mitochondrial pyruvate transport in the hormonal control of rat liver mitochondrial pyruvate metabolism.

PubMed Central

Halestrap, A P; Armston, A E

1984-01-01

The inhibitor of mitochondrial pyruvate transport alpha-cyano-beta-(1-phenylindol-3-yl)-acrylate was used to inhibit progressively pyruvate carboxylation by liver mitochondria from control and glucagon-treated rats. The data showed that, contrary to our previous conclusions [Halestrap (1978) Biochem. J. 172, 389-398], pyruvate transport could not regulate metabolism under these conditions. This was confirmed by measuring the intramitochondrial pyruvate concentration, which almost equilibrated with the extramitochondrial pyruvate concentration in control mitochondria, but was significantly decreased in mitochondria from glucagon-treated rats, where rates of pyruvate metabolism were elevated. Computer-simulation studies explain how this is compatible with linear Dixon plots of the inhibition of pyruvate metabolism by alpha-cyano-4-hydroxycinnamate. Parallel measurements of the mitochondrial membrane potential by using [3H]triphenylmethylphosphonium ions showed that it was elevated by about 3 mV after pretreatment of rats with both glucagon and phenylephrine. There was no significant change in the transmembrane pH gradient. It is shown that the increase in pyruvate metabolism can be explained by a stimulation of the respiratory chain, producing an elevation in the protonmotive force and a consequent rise in the intramitochondrial ATP/ADP ratio, which in turn increases pyruvate carboxylase activity. Mild inhibition of the respiratory chain with Amytal reversed the effects of hormone treatment on mitochondrial pyruvate metabolism and ATP concentrations, but not on citrulline synthesis. The significance of these observations for the hormonal regulation of gluconeogenesis from L-lactate in vivo is discussed. PMID:6095807

Induction of triglyceride accumulation and mitochondrial maintenance in muscle cells by lactate

PubMed Central

Sun, Jingquan; Ye, Xin; Xie, Minhao; Ye, Jianping

2016-01-01

Muscle exercise induces intramuscular triglyceride (TG) accumulation and promotes mitochondrial maintenance in myotubes. However, the mechanism underlying exercise effects remains unknown. In this study, lactic acid was tested as a signaling molecule in C2C12 myotubes to understand the mechanism. Intracellular TG storage was induced in the cells by sodium lactate. The lactate activity was observed with an inhibition of the cAMP-PKA pathway as indicated by a reduction in the phosphorylation status of CREB (pCREB). Induction of pCREB signal by forskolin was blocked by pretreatment of cells with lactate. The impact of lactate on mitochondrial function was examined with a focus on the activities of two enzymes, MCAT (malonylCoA:ACP transferase) and PDH (pyruvate dehydrogenase). The enzyme activities were induced in the cells by lactate. Expression of the lactate receptor (GPR81) and lactate transporters (MCT1/4) were induced as well by lactate. The lactate activities were observed at concentrations between 4–64 mM, and were not dependent on the increase in intracellular pyruvate. Pyruvate treatment did not generate the same effects in the cells. Those results suggest that lactate may induce intramuscular TG storage and mitochondrial maintenance in myotubes through inhibition of the cAMP pathway by activation of GPR81 in a positive feedback manner. PMID:27645401

Selective spectroscopic imaging of hyperpolarized pyruvate and its metabolites using a single-echo variable phase advance method in balanced SSFP

PubMed Central

Varma, Gopal; Wang, Xiaoen; Vinogradov, Elena; Bhatt, Rupal S.; Sukhatme, Vikas; Seth, Pankaj; Lenkinski, Robert E.; Alsop, David C.; Grant, Aaron K.

2015-01-01

Purpose In balanced steady state free precession (bSSFP), the signal intensity has a well-known dependence on the off-resonance frequency, or, equivalently, the phase advance between successive radiofrequency (RF) pulses. The signal profile can be used to resolve the contributions from the spectrally separated metabolites. This work describes a method based on use of a variable RF phase advance to acquire spatial and spectral data in a time-efficient manner for hyperpolarized 13C MRI. Theory and Methods The technique relies on the frequency response from a bSSFP acquisition to acquire relatively rapid, high-resolution images that may be reconstructed to separate contributions from different metabolites. The ability to produce images from spectrally separated metabolites was demonstrated in-vitro, as well as in-vivo following administration of hyperpolarized 1-13C pyruvate in mice with xenograft tumors. Results In-vivo images of pyruvate, alanine, pyruvate hydrate and lactate were reconstructed from 4 images acquired in 2 seconds with an in-plane resolution of 1.25 × 1.25mm2 and 5mm slice thickness. Conclusions The phase advance method allowed acquisition of spectroscopically selective images with high spatial and temporal resolution. This method provides an alternative approach to hyperpolarized 13C spectroscopic MRI that can be combined with other techniques such as multi-echo or fluctuating equilibrium bSSFP. PMID:26507361

Two separate pathways for d-lactate oxidation by Saccharomyces cerevisiae mitochondria which differ in energy production and carrier involvement.

PubMed

Pallotta, Maria Luigia; Valenti, Daniela; Iacovino, Michelina; Passarella, Salvatore

2004-02-15

In this work we looked at whether and how mitochondria isolated from Saccharomyces cerevisiae (SCM) oxidize d-lactate. We found that: (1). externally added d-lactate causes oxygen uptake by SCM with P/O ratio equal to 1.5; in the presence of antimycin A (AA), P/O ratio was 1.8, differently in the presence of the non-penetrant alpha-cyanocinnamate (alpha-CCN-) no P/O ratio could be measured. Consistently, mitochondrial electrical membrane potential (deltapsi) generation was found, due to externally added d-lactate in the presence of antimycin A, but not of alpha-CCN-. (2). SCM oxidize d-lactate in two different manners: (i). via inner membrane d-lactate dehydrogenase which leads to d-lactate oxidation without driving deltapsi generation and ATP synthesis and (ii). via the matrix d-lactate dehydrogenase, which drives deltapsi generation and ATP synthesis by using taken up d-lactate. (3). Pyruvate newly synthesised in the mitochondrial matrix is exported via the novel d-lactate/pyruvate antiporter. d-Lactate/pyruvate antiport proved to regulate the rate of pyruvate efflux in vitro. (4). The existence of the d-lactate/H+ symporter is also proposed as shown by mitochondrial swelling. The d-lactate carriers and d-lactate dehydrogenases could account for the removal of the toxic methylglyoxal from cytosol, as well as for the d-lactate-dependent gluconeogenesis.

Transport of pyruvate into mitochondria is involved in methylmercury toxicity

PubMed Central

Lee, Jin-Yong; Ishida, Yosuke; Takahashi, Tsutomu; Naganuma, Akira; Hwang, Gi-Wook

2016-01-01

We have previously demonstrated that the overexpression of enzymes involved in the production of pyruvate, enolase 2 (Eno2) and D-lactate dehydrogenase (Dld3) renders yeast highly sensitive to methylmercury and that the promotion of intracellular pyruvate synthesis may be involved in intensifying the toxicity of methylmercury. In the present study, we showed that the addition of pyruvate to culture media in non-toxic concentrations significantly enhanced the sensitivity of yeast and human neuroblastoma cells to methylmercury. The results also suggested that methylmercury promoted the transport of pyruvate into mitochondria and that the increased pyruvate concentrations in mitochondria were involved in intensifying the toxicity of methylmercury without pyruvate being converted to acetyl-CoA. Furthermore, in human neuroblastoma cells, methylmercury treatment alone decreased the mitochondrial membrane potential, and the addition of pyruvate led to a further significant decrease. In addition, treatment with N-acetylcysteine (an antioxidant) significantly alleviated the toxicity of methylmercury and significantly inhibited the intensification of methylmercury toxicity by pyruvate. Based on these data, we hypothesize that methylmercury exerts its toxicity by raising the level of pyruvate in mitochondria and that mitochondrial dysfunction and increased levels of reactive oxygen species are involved in the action of pyruvate. PMID:26899208

Are arterial, muscle and working limb lactate exchange data obtained on men at altitude consistent with the hypothesis of an intracellular lactate shuttle?

PubMed

Brooks, G A

1999-01-01

The "Lactate Shuttle" Hypothesis posits that lactate removal requires exchange among producing and consuming cells. The "Intra-cellular Lactate Shuttle" hypothesis posits that lactate exchange occurs among compartments within cells, and that mitochondria are the major sites of cellular lactate disposal. Thus, cells with high mitochondrial densities (cardiocytes, myocytes, hepatocytes) are those which participate in lactate clearance. The model of an Intracellular Lactate Shuttle recognizes that the Keq for LDH is 3.6 x 10(4) M-1; thus, glycolysis results in cytosolic lactate production regardless of the intracellular PO2. The model also requires presence of a mitochondrial monocarboxylate transporter (MCT) that allows uptake of lactate as well as pyruvate, and intra-mitochondrial LDH whose function is linked to the ETC, and which permits lactate-->pyruvate conversion and oxidation. Recently, we have shown that liver, heart and muscle mitochondria readily oxidize lactate and contain LDH and MCT1. Accordingly, we have concluded that lactate is the predominant monocarboxylate oxidized by mitochondria in vivo. The model of an "Intra-cellular Lactate Shuttle" is consistent with many of the observations on men at sea level and altitude. The observations include: oxidation is the primary fate of lactate disposal during rest and exercise; lactate production and oxidation occur simultaneously within resting and working muscle; increasing [lactate]a increases muscle lactate extraction, and that by increasing SaO2 acclimatization reduces blood [lactate].

Properties of lactate dehydrogenase from the isopod, Saduria entomon.

PubMed

Mulkiewicz, E; Zietara, M S; Stachowiak, K; Skorkowski, E F

2000-07-01

Saduria entomon lactate dehydrogenase (LDH-A4*) from thorax muscle was purified about 89 fold to specific activity 510 micromol NADH/min/mg using Cibacron Blue 3GA Agarose and Oxamate-Agarose chromatographies. The enzyme is a tetramer, with molecular weight of 140 kDa for the native enzyme and 36 kDa for the subunit. The isoelectric point was at pH 5.7. The enzyme possesses high heat stability (T50 = 71.5 degrees C). The optimum pH for pyruvate reduction reaction was 6.5, while for lactate oxidation one, the maximum activity was at pH 9.1. The Km for pyruvate was minimal at 5 degrees C, the average environmental temperature of the isopod. The Km values determined at 30 degrees C and optimal pH for pyruvate reduction and lactate oxidation were 0.18 and 90.04 mM, respectively. Amino acid compositional analyses showed the strongest resemblance of the isopod isoenzyme to cod (Gadus morhua) LDH-C4.

Relative Carnitine Deficiency in Autism

ERIC Educational Resources Information Center

Filipek, Pauline A.; Juranek, Jenifer; Nguyen, Minh T.; Cummings, Christa; Gargus, J. Jay

2004-01-01

A random retrospective chart review was conducted to document serum carnitine levels on 100 children with autism. Concurrently drawn serum pyruvate, lactate, ammonia, and alanine levels were also available in many of these children. Values of free and total carnitine ([rho] less than 0.001), and pyruvate ([rho]=0.006) were significantly reduced…

Mitochondrial pyruvate transport: a historical perspective and future research directions

PubMed Central

McCommis, Kyle S.; Finck, Brian N.

2015-01-01

Pyruvate is the end-product of glycolysis, a major substrate for oxidative metabolism, and a branching point for glucose, lactate, fatty acid and amino acid synthesis. The mitochondrial enzymes that metabolize pyruvate are physically separated from cytosolic pyruvate pools and rely on a membrane transport system to shuttle pyruvate across the impermeable inner mitochondrial membrane (IMM). Despite long-standing acceptance that transport of pyruvate into the mitochondrial matrix by a carrier-mediated process is required for the bulk of its metabolism, it has taken almost 40 years to determine the molecular identity of an IMM pyruvate carrier. Our current understanding is that two proteins, mitochondrial pyruvate carriers MPC1 and MPC2, form a hetero-oligomeric complex in the IMM to facilitate pyruvate transport. This step is required for mitochondrial pyruvate oxidation and carboxylation – critical reactions in intermediary metabolism that are dysregulated in several common diseases. The identification of these transporter constituents opens the door to the identification of novel compounds that modulate MPC activity, with potential utility for treating diabetes, cardiovascular disease, cancer, neurodegenerative diseases, and other common causes of morbidity and mortality. The purpose of the present review is to detail the historical, current and future research investigations concerning mitochondrial pyruvate transport, and discuss the possible consequences of altered pyruvate transport in various metabolic tissues. PMID:25748677

Selective modification of the pyruvate dehydrogenase kinase isoform profile in skeletal muscle in hyperthyroidism: implications for the regulatory impact of glucose on fatty acid oxidation.

PubMed

Sugden, M C; Lall, H S; Harris, R A; Holness, M J

2000-11-01

The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Immunoblot analysis with antibodies raised against recombinant PDK isoforms demonstrated changes in PDK isoform expression in response to experimental hyperthyroidism (100 microg/100 g body weight; 3 days) that was selective for fast-twitch vs slow-twitch skeletal muscle in that PDK2 expression was increased in the fast-twitch skeletal muscle (the anterior tibialis) (by 1. 6-fold; P<0.05) but not in the slow-twitch muscle (the soleus). PDK4 protein expression was increased by experimental hyperthyroidism in both muscle types, there being a greater response in the anterior tibialis (4.2-fold increase; P<0.05) than in the soleus (3.2-fold increase; P<0.05). The hyperthyroidism-associated up-regulation of PDK4 expression was observed in conjunction with suppression of skeletal-muscle PDC activity, but not suppression of glucose uptake/phosphorylation, as measured in vivo in conscious unrestrained rats (using the 2-[(3)H]deoxyglucose technique). We propose that increased PDK isoform expression contributes to the pathology of hyperthyroidism and to PDC inactivation by facilitating the operation of the glucose --> lactate --> glucose (Cori) and glucose --> alanine --> glucose cycles. We also propose that enhanced relative expression of the pyruvate-insensitive PDK isoform (PDK4) in skeletal muscle in hyperthyroidism uncouples glycolytic flux from pyruvate oxidation, sparing pyruvate for non-oxidative entry into the tricarboxylic acid (TCA) cycle, and thereby supporting entry of acetyl-CoA (derived from fatty acid oxidation) into the TCA cycle.

[Effects of ß-alanine supplementation on wingate tests in university female footballers].

PubMed

Rodríguez Rodríguez, Fernando; Delgado Ormeño, Alex; Rivera Lobos, Patricio; Tapia Aranda, Víctor; Cristi-Montero, Carlos

2014-11-01

Football is a sport that develops actions intermittent high-intensity exercise using the anaerobic pathway, for that reason, the muscle fatigue would produce primarily by increasing acidosis. Carnosine, which is formed from L-histidine, ß-alanine, has proven to produce an effect "buffer" of acidosis. To determine the effect of ß-alanine supplementation, on three successive Wingate tests and compare the average power, maximum power and lactate blood in selected female college soccer. We evaluated 10 football players who were three Wingate, 5 min rest between each sprint, determining the average power, maximum and lactate at the end of each test, then consumed 2,4 gr/day of ß-alanine for 30 days and repeated the tests. The control group (n=8) performed the same tests, but without consuming the supplement. Monark cycle ergometer was used (Ergomedic 874E) and to measure lactate the Lactate Pro 2. The group with supplementation significantly improved mean power difference from the control group. The maximum power improved only in the first sprint unlike the control group and Lactate did not differ. The evidence shows that the ß-alanine improves performance on tests of more than 30 second long, but in our study improves average power and peak power even when performing consecutive sprint, being able to emulate the reality of the football game. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Lactate is oxidized outside of the mitochondrial matrix in rodent brain.

PubMed

Herbst, Eric A F; George, Mitchell A J; Brebner, Karen; Holloway, Graham P; Kane, Daniel A

2018-05-01

The nature and existence of mitochondrial lactate oxidation is debated in the literature. Obscuring the issue are disparate findings in isolated mitochondria, as well as relatively low rates of lactate oxidation observed in permeabilized muscle fibres. However, respiration with lactate has yet to be directly assessed in brain tissue with the mitochondrial reticulum intact. To determine if lactate is oxidized in the matrix of brain mitochondria, oxygen consumption was measured in saponin-permeabilized mouse brain cortex samples, and rat prefrontal cortex and hippocampus (dorsal) subregions. While respiration in the presence of ADP and malate increased with the addition of lactate, respiration was maximized following the addition of exogenous NAD + , suggesting maximal lactate metabolism involves extra-matrix lactate dehydrogenase. This was further supported when NAD + -dependent lactate oxidation was significantly decreased with the addition of either low-concentration α-cyano-4-hydroxycinnamate or UK-5099, inhibitors of mitochondrial pyruvate transport. Mitochondrial respiration was comparable between glutamate, pyruvate, and NAD + -dependent lactate oxidation. Results from the current study demonstrate that permeabilized brain is a feasible model for assessing lactate oxidation, and support the interpretation that lactate oxidation occurs outside the mitochondrial matrix in rodent brain.

Skeletal Muscle Pyruvate Dehydrogenase Phosphorylation and Lactate Accumulation During Sprint Exercise in Normoxia and Severe Acute Hypoxia: Effects of Antioxidants.

PubMed

Morales-Alamo, David; Guerra, Borja; Santana, Alfredo; Martin-Rincon, Marcos; Gelabert-Rebato, Miriam; Dorado, Cecilia; Calbet, José A L

2018-01-01

Compared to normoxia, during sprint exercise in severe acute hypoxia the glycolytic rate is increased leading to greater lactate accumulation, acidification, and oxidative stress. To determine the role played by pyruvate dehydrogenase (PDH) activation and reactive nitrogen and oxygen species (RNOS) in muscle lactate accumulation, nine volunteers performed a single 30-s sprint (Wingate test) on four occasions: two after the ingestion of placebo and another two following the intake of antioxidants, while breathing either hypoxic gas (P I O 2 = 75 mmHg) or room air (P I O 2 = 143 mmHg). Vastus lateralis muscle biopsies were obtained before, immediately after, 30 and 120 min post-sprint. Antioxidants reduced the glycolytic rate without altering performance or VO 2 . Immediately after the sprints, Ser 293 - and Ser 300 -PDH-E1α phosphorylations were reduced to similar levels in all conditions (~66 and 91%, respectively). However, 30 min into recovery Ser 293 -PDH-E1α phosphorylation reached pre-exercise values while Ser 300 -PDH-E1α was still reduced by 44%. Thirty minutes after the sprint Ser 293 -PDH-E1α phosphorylation was greater with antioxidants, resulting in 74% higher muscle lactate concentration. Changes in Ser 293 and Ser 300 -PDH-E1α phosphorylation from pre to immediately after the sprints were linearly related after placebo ( r = 0.74, P < 0.001; n = 18), but not after antioxidants ingestion ( r = 0.35, P = 0.15). In summary, lactate accumulation during sprint exercise in severe acute hypoxia is not caused by a reduced activation of the PDH. The ingestion of antioxidants is associated with increased PDH re-phosphorylation and slower elimination of muscle lactate during the recovery period. Ser 293 re-phosphorylates at a faster rate than Ser 300 -PDH-E1α during the recovery period, suggesting slightly different regulatory mechanisms.

Skeletal Muscle Pyruvate Dehydrogenase Phosphorylation and Lactate Accumulation During Sprint Exercise in Normoxia and Severe Acute Hypoxia: Effects of Antioxidants

PubMed Central

Morales-Alamo, David; Guerra, Borja; Santana, Alfredo; Martin-Rincon, Marcos; Gelabert-Rebato, Miriam; Dorado, Cecilia; Calbet, José A. L.

2018-01-01

Compared to normoxia, during sprint exercise in severe acute hypoxia the glycolytic rate is increased leading to greater lactate accumulation, acidification, and oxidative stress. To determine the role played by pyruvate dehydrogenase (PDH) activation and reactive nitrogen and oxygen species (RNOS) in muscle lactate accumulation, nine volunteers performed a single 30-s sprint (Wingate test) on four occasions: two after the ingestion of placebo and another two following the intake of antioxidants, while breathing either hypoxic gas (PIO2 = 75 mmHg) or room air (PIO2 = 143 mmHg). Vastus lateralis muscle biopsies were obtained before, immediately after, 30 and 120 min post-sprint. Antioxidants reduced the glycolytic rate without altering performance or VO2. Immediately after the sprints, Ser293- and Ser300-PDH-E1α phosphorylations were reduced to similar levels in all conditions (~66 and 91%, respectively). However, 30 min into recovery Ser293-PDH-E1α phosphorylation reached pre-exercise values while Ser300-PDH-E1α was still reduced by 44%. Thirty minutes after the sprint Ser293-PDH-E1α phosphorylation was greater with antioxidants, resulting in 74% higher muscle lactate concentration. Changes in Ser293 and Ser300-PDH-E1α phosphorylation from pre to immediately after the sprints were linearly related after placebo (r = 0.74, P < 0.001; n = 18), but not after antioxidants ingestion (r = 0.35, P = 0.15). In summary, lactate accumulation during sprint exercise in severe acute hypoxia is not caused by a reduced activation of the PDH. The ingestion of antioxidants is associated with increased PDH re-phosphorylation and slower elimination of muscle lactate during the recovery period. Ser293 re-phosphorylates at a faster rate than Ser300-PDH-E1α during the recovery period, suggesting slightly different regulatory mechanisms. PMID:29615918

PDK4 Inhibits Cardiac Pyruvate Oxidation in Late Pregnancy.

PubMed

Liu, Laura X; Rowe, Glenn C; Yang, Steven; Li, Jian; Damilano, Federico; Chan, Mun Chun; Lu, Wenyun; Jang, Cholsoon; Wada, Shogo; Morley, Michael; Hesse, Michael; Fleischmann, Bernd K; Rabinowitz, Joshua D; Das, Saumya; Rosenzweig, Anthony; Arany, Zoltan

2017-12-08

Pregnancy profoundly alters maternal physiology. The heart hypertrophies during pregnancy, but its metabolic adaptations, are not well understood. To determine the mechanisms underlying cardiac substrate use during pregnancy. We use here 13 C glucose, 13 C lactate, and 13 C fatty acid tracing analyses to show that hearts in late pregnant mice increase fatty acid uptake and oxidation into the tricarboxylic acid cycle, while reducing glucose and lactate oxidation. Mitochondrial quantity, morphology, and function do not seem altered. Insulin signaling seems intact, and the abundance and localization of the major fatty acid and glucose transporters, CD36 (cluster of differentiation 36) and GLUT4 (glucose transporter type 4), are also unchanged. Rather, we find that the pregnancy hormone progesterone induces PDK4 (pyruvate dehydrogenase kinase 4) in cardiomyocytes and that elevated PDK4 levels in late pregnancy lead to inhibition of PDH (pyruvate dehydrogenase) and pyruvate flux into the tricarboxylic acid cycle. Blocking PDK4 reverses the metabolic changes seen in hearts in late pregnancy. Taken together, these data indicate that the hormonal environment of late pregnancy promotes metabolic remodeling in the heart at the level of PDH, rather than at the level of insulin signaling. © 2017 American Heart Association, Inc.

Development of an enzymatic assay to measure lactate in perchloric acid-precipitated cerebrospinal fluid.

PubMed

Lu, Jun; Genzen, Jonathan R; Grenache, David G

2018-04-27

Individuals with inherited deficiencies of the pyruvate dehydrogenase complex or the respiratory chain complex can have increased concentrations of cerebrospinal fluid (CSF) lactate. Such measurements are clinical useful when measured in conjunction with pyruvate in order to calculate the lactate:pyruvate (L:P) ratio, a useful surrogate of cytosolic redox status. CSF pyruvate is measured in a protein-free supernatant prepared by the addition of CSF to perchloric acid while lactate is measured in untreated CSF. Utilizing the same sample for both lactate and pyruvate measurements is desirable. To develop a method to measure lactate in perchloric-acid precipitated CSF and validate the L:P ratio as calculated from the analysis of both analytes in the same sample. Samples were prepared by the addition of 1 mL CSF to 2 mL 8% (w/v) cold perchloric acid, incubated on ice for 10 min, then centrifuged to obtain a protein-free supernatant. Lactate was measured by its oxidation to pyruvate and hydrogen peroxide using lactate oxidase and the absorbance of the resulting chromogen determined at 540 nm on a Roche cobas c501 chemistry analyzer. Method accuracy, linearity, imprecision and sensitivity were determined and a reference interval was verified. To assess accuracy, this method was compared to lactate determined in unaltered CSF at another laboratory using 41 specimens with lactate concentrations from 0.6-11.9 mmol/L. Linear regression produced a slope of 1.09 and y-intercept of 0.26 (R 2  = 1.00). Recovery was performed by ad-mixes of a high lactate standard and a CSF pool in different ratios to create a set of 19 samples prior to preparing protein-free supernatants. Recovery was 94.6-100% (mean ± SD was 97.4 ± 1.4%) at lactate concentrations of 2.68 to 12.63 mmol/L. Linearity was determined by combining two supernatants with low and high lactate concentrations in different ratios to create a set of six samples (0.15-12.70 mmol/L) that were

A novel archaeal alanine dehydrogenase homologous to ornithine cyclodeaminase and mu-crystallin.

PubMed

Schröder, Imke; Vadas, Alexander; Johnson, Eric; Lim, Sierin; Monbouquette, Harold G

2004-11-01

A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of L-alanine to pyruvate and NH4+. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The Km values for L-alanine, NAD+, pyruvate, NADH, and NH4+ were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82 degrees C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25 degrees C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90 degrees C. At 25 degrees C in the presence of this salt solution, the enzyme was approximately 100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-mu-crystallin family of enzymes. Similar to the mu-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human mu-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.

Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons.

PubMed

Halim, Nader D; Mcfate, Thomas; Mohyeldin, Ahmed; Okagaki, Peter; Korotchkina, Lioubov G; Patel, Mulchand S; Jeoung, Nam Ho; Harris, Robert A; Schell, Michael J; Verma, Ajay

2010-08-01

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. (c) 2010 Wiley-Liss, Inc.

Alanine infusion during hypoglycaemia partly supports cognitive performance in healthy human subjects.

PubMed

Evans, M L; Hopkins, D; Macdonald, I A; Amiel, S A

2004-05-01

To investigate the potential for the non-glucose metabolic substrate alanine to support brain function during glucose deprivation in man. Seven healthy men were studied on two occasions using a hyperinsulinaemic glucose clamp to lower arterialized plasma glucose to 2.5 mmol/l, in the presence of either 2 mmol/kg/h alanine infusion or saline, measuring counter-regulatory hormonal responses, symptoms generated and cognitive function with a mini-battery of tests sensitive to hypoglycaemia. Alanine infusion elevated plasma alanine (peak value 1481 +/- 1260 vs. 138 +/- 32 micro mol/l, P = 0.02 alanine vs. saline) and lactate (peak value 3.09 +/- 0.14 vs. 2.05 +/- 0.12 mmol/l, P = 0.02). Cognitive function assessed by the Stroop word and colour subtests deteriorated less with alanine than saline (P < 0.01 for both). Other cognitive function tests deteriorated equally and counter-regulatory hormones rose equally during hypoglycaemia in both studies (P > 0.34) except for increased glucagon with alanine (peak 260 +/- 53 vs. 91 + 8 ng/l, P = 0.03). There was no significant effect of alanine on either autonomic or neuroglycopenic symptom scores. Some, but not all, aspects of cognitive performance may be supported by an alanine infusion during hypoglycaemia. It is not clear whether alanine supports brain function directly or via increased availability of lactate. These data contribute to the growing evidence that regional metabolic differences exist in the brain's ability to use non-glucose fuels during hypoglycaemia.

Reactivity landscape of pyruvate under simulated hydrothermal vent conditions

PubMed Central

Novikov, Yehor; Copley, Shelley D.

2013-01-01

Pyruvate is an important “hub” metabolite that is a precursor for amino acids, sugars, cofactors, and lipids in extant metabolic networks. Pyruvate has been produced under simulated hydrothermal vent conditions from alkyl thiols and carbon monoxide in the presence of transition metal sulfides at 250 °C [Cody GD et al. (2000) Science 289(5483):1337–1340], so it is plausible that pyruvate was formed in hydrothermal systems on the early earth. We report here that pyruvate reacts readily in the presence of transition metal sulfide minerals under simulated hydrothermal vent fluids at more moderate temperatures (25–110 °C) that are more conducive to survival of biogenic molecules. We found that pyruvate partitions among five reaction pathways at rates that depend upon the nature of the mineral present; the concentrations of H2S, H2, and NH4Cl; and the temperature. In most cases, high yields of one or two primary products are found due to preferential acceleration of certain pathways. Reactions observed include reduction of ketones to alcohols and aldol condensation, both reactions that are common in extant metabolic networks. We also observed reductive amination to form alanine and reduction to form propionic acid. Amino acids and fatty acids formed by analogous processes may have been important components of a protometabolic network that allowed the emergence of life. PMID:23872841

Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

PubMed

Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

2015-03-01

The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. Copyright © 2014 Elsevier B.V. All rights reserved.

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

PubMed Central

Fenton, Aron W.; Hutchinson, Myra

2009-01-01

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37°C. As a function of increasing pH, hL-PYK's affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and Ala slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to Ala is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for Ala inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response. PMID:19467627

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

PubMed

Halestrap, A P; Denton, R M

1975-04-01

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human

MCT1 modulates cancer cell pyruvate export and growth of tumors that co-express MCT1 and MCT4

PubMed Central

Hong, Candice Sun; Graham, Nicholas A.; Gu, Wen; Camacho, Carolina Espindola; Mah, Vei; Maresh, Erin L.; Alavi, Mohammed; Bagryanova, Lora; Krotee, Pascal A. L.; Gardner, Brian K.; Behbahan, Iman Saramipoor; Horvath, Steve; Chia, David; Mellinghoff, Ingo K.; Hurvitz, Sara A.; Dubinett, Steven M.; Critchlow, Susan E.; Kurdistani, Siavash K.; Goodglick, Lee; Braas, Daniel; Graeber, Thomas G.; Christofk, Heather R.

2016-01-01

SUMMARY Monocarboxylate Transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export, which when inhibited enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors further supporting their use as anti-cancer therapeutics. PMID:26876179

MCT1 Modulates Cancer Cell Pyruvate Export and Growth of Tumors that Co-express MCT1 and MCT4.

PubMed

Hong, Candice Sun; Graham, Nicholas A; Gu, Wen; Espindola Camacho, Carolina; Mah, Vei; Maresh, Erin L; Alavi, Mohammed; Bagryanova, Lora; Krotee, Pascal A L; Gardner, Brian K; Behbahan, Iman Saramipoor; Horvath, Steve; Chia, David; Mellinghoff, Ingo K; Hurvitz, Sara A; Dubinett, Steven M; Critchlow, Susan E; Kurdistani, Siavash K; Goodglick, Lee; Braas, Daniel; Graeber, Thomas G; Christofk, Heather R

2016-02-23

Monocarboxylate transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here, we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export that when inhibited, enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors, further supporting their use as anti-cancer therapeutics. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Glucose, Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals.

PubMed

Hohnholt, Michaela C; Andersen, Vibe H; Bak, Lasse K; Waagepetersen, Helle S

2017-01-01

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.

Depressed gluconeogenesis and ureogenesis in isolated hepatocytes after intermittent hypoxia in rats.

PubMed

Freminet, A; Megas, P; Puceat, M

1990-01-01

1. Rats were exposed to hypobaric hypoxia (equivalent altitude 4500 m), 2 x 2 hr per day, for 5 days. Isolated hepatocytes were prepared on day 6 after 18 hr of fast and also from control normoxic animals. The hepatocytes were incubated (120 min) with various substrates. 2. ATP contents were lower in hepatocytes from exposed as compared to control animals whether at the beginning (14%) or at the end (-6 to -33%) of incubation depending on the substrate. 3. Gluconeogenesis from all precursors (lactate, alanine, pyruvate, glutamine) was significantly reduced (40-50%) in exposed as compared to control animals. 4. Ureogenesis from alanine and from pyruvate + NH4Cl was also markedly depressed in exposed animals but no differences were noticed with glutamine or lactate + NH4Cl and alanine + NH4Cl. 5. Results are discussed in relation to known effects of acute and chronic hypoxia, interrelationship between gluconeogenesis and ureogenesis, taking into account the inhomogeneity of liver and the metabolic properties of periportal and perivenous hepatocytes.

Impaired hippocampal glucose metabolism during and after flurothyl-induced seizures in mice: Reduced phosphorylation coincides with reduced activity of pyruvate dehydrogenase.

PubMed

McDonald, Tanya S; Borges, Karin

2017-07-01

To determine changes in glucose metabolism and the enzymes involved in the hippocampus ictally and postictally in the acute mouse flurothyl seizure model. [U- 13 C]-Glucose was injected (i.p.) prior to, or following a 5 min flurothyl-induced seizure. Fifteen minutes later, mice were killed and the total metabolite levels and % 13 C enrichment were analyzed in the hippocampal formation using gas chromatography-mass spectrometry. Activities of key metabolic and antioxidant enzymes and the phosphorylation status of pyruvate dehydrogenase were measured, along with lipid peroxidation. During seizures, total lactate levels increased 1.7-fold; however, [M + 3] enrichment of both lactate and alanine were reduced by 30% and 43%, respectively, along with a 28% decrease in phosphofructokinase activity. Postictally the % 13 C enrichments of all measured tricarboxylic acid (TCA) cycle intermediates and the amino acids were reduced by 46-93%. At this time, pyruvate dehydrogenase (PDH) activity was 56% of that measured in controls, and there was a 1.9-fold increase in the phosphorylation of PDH at ser232. Phosphorylation of PDH is known to decrease its activity. Here, we show that the increase of lactate levels during flurothyl seizures is from a source other than [U- 13 C]-glucose, such as glycogen. Surprisingly, although we saw a reduction in phosphofructokinase activity during the seizure, metabolism of [U- 13 C]-glucose into the TCA cycle seemed unaffected. Similar to our recent findings in the chronic phase of the pilocarpine model, postictally the metabolism of glucose by glycolysis and the TCA cycle was impaired along with reduced PDH activity. Although this decrease in activity may be a protective mechanism to reduce oxidative stress, which is observed in the flurothyl model, ATP is critical to the recovery of ion and neurotransmitter balance and return to normal brain function. Thus we identified promising novel strategies to enhance energy metabolism and recovery from

Altered Kinetics Properties of Erythrocyte Lactate Dehydrogenase in Type II Diabetic Patients and Its Implications for Lactic Acidosis.

PubMed

Mali, Aniket V; Bhise, Sunita S; Katyare, Surendra S; Hegde, Mahabaleshwar V

2018-01-01

Recent studies have been noted that the erythrocytes from Type II diabetic patients show significantly altered structural and functional characteristics along with the changed intracellular concentrations of glycolytic intermediates. More recent studies from our laboratory have shown that the activities of enzymes of glycolytic pathway changed significantly in RBCs from Type II diabetic patients. In particular the levels of lactate dehydrogenase (LDH) increased significantly. Lactic acidosis is an established feature of diabetes and LDH plays a crucial role in conversion of pyruvate to lactate and reportedly, the levels of lactate are significantly high which is consistent with our observation on increased levels of LDH. Owing to this background, we examined the role of erythrocyte LDH in lactic acidosis by studying its kinetics properties in Type II diabetic patients. Km, Vmax and apparent catalytic efficiency were determined using pyruvate and NADH as the substrates. With pyruvate as the substrate the Km values were comparable but Vmax increased significantly in the diabetic group. With NADH as the substrate the enzyme activity of the diabetic group resolved in two components as against a single component in the controls. The Apparent Kcat and Kcat/Km values for pyruvate increased in the diabetic group. The Ki for pyruvate increased by two fold for the enzyme from diabetic group with a marginal decrease in Ki for NADH. The observed changes in catalytic attributes are conducive to enable the enzyme to carry the reaction in forward direction towards conversion of pyruvate to lactate leading to lactic acidosis.

Oxygen-Inducible Conversion of Lactate to Acetate in Heterofermentative Lactobacillus brevis ATCC 367.

PubMed

Guo, Tingting; Zhang, Li; Xin, Yongping; Xu, ZhenShang; He, Huiying; Kong, Jian

2017-11-01

Lactobacillus brevis is an obligatory heterofermentative lactic acid bacterium that produces high levels of acetate, which improve the aerobic stability of silages against deterioration caused by yeasts and molds. However, the mechanism involved in acetate accumulation has yet to be elucidated. Here, experimental evidence indicated that aerobiosis resulted in the conversion of lactate to acetate after glucose exhaustion in L. brevis ATCC 367 (GenBank accession number NC_008497). To elucidate the conversion pathway, in silico analysis showed that lactate was first converted to pyruvate by the reverse catalytic reaction of lactate dehydrogenase (LDH); subsequently, pyruvate conversion to acetate might be mediated by pyruvate dehydrogenase (PDH) or pyruvate oxidase (POX). Transcriptional analysis indicated that the pdh and pox genes of L. brevis ATCC 367 were upregulated 37.92- and 18.32-fold, respectively, by oxygen and glucose exhaustion, corresponding to 5.32- and 2.35-fold increases in the respective enzyme activities. Compared with the wild-type strain, the transcription and enzymatic activity of PDH remained stable in the Δ pox mutant, while those of POX increased significantly in the Δ pdh mutant. More lactate but less acetate was produced in the Δ pdh mutant than in the wild-type and Δ pox mutant strains, and more H 2 O 2 (a product of the POX pathway) was produced in the Δ pdh mutant. We speculated that the high levels of aerobic acetate accumulation in L. brevis ATCC 367 originated mainly from the reuse of lactate to produce pyruvate, which was further converted to acetate by the predominant and secondary functions of PDH and POX, respectively. IMPORTANCE PDH and POX are two possible key enzymes involved in aerobic acetate accumulation in lactic acid bacteria (LAB). It is currently thought that POX plays the major role in aerobic growth in homofermentative LAB and some heterofermentative LAB, while the impact of PDH remains unclear. In this study, we

Oxygen-Inducible Conversion of Lactate to Acetate in Heterofermentative Lactobacillus brevis ATCC 367

PubMed Central

Guo, Tingting; Zhang, Li; Xin, Yongping; Xu, ZhenShang; He, Huiying

2017-01-01

ABSTRACT Lactobacillus brevis is an obligatory heterofermentative lactic acid bacterium that produces high levels of acetate, which improve the aerobic stability of silages against deterioration caused by yeasts and molds. However, the mechanism involved in acetate accumulation has yet to be elucidated. Here, experimental evidence indicated that aerobiosis resulted in the conversion of lactate to acetate after glucose exhaustion in L. brevis ATCC 367 (GenBank accession number NC_008497). To elucidate the conversion pathway, in silico analysis showed that lactate was first converted to pyruvate by the reverse catalytic reaction of lactate dehydrogenase (LDH); subsequently, pyruvate conversion to acetate might be mediated by pyruvate dehydrogenase (PDH) or pyruvate oxidase (POX). Transcriptional analysis indicated that the pdh and pox genes of L. brevis ATCC 367 were upregulated 37.92- and 18.32-fold, respectively, by oxygen and glucose exhaustion, corresponding to 5.32- and 2.35-fold increases in the respective enzyme activities. Compared with the wild-type strain, the transcription and enzymatic activity of PDH remained stable in the Δpox mutant, while those of POX increased significantly in the Δpdh mutant. More lactate but less acetate was produced in the Δpdh mutant than in the wild-type and Δpox mutant strains, and more H2O2 (a product of the POX pathway) was produced in the Δpdh mutant. We speculated that the high levels of aerobic acetate accumulation in L. brevis ATCC 367 originated mainly from the reuse of lactate to produce pyruvate, which was further converted to acetate by the predominant and secondary functions of PDH and POX, respectively. IMPORTANCE PDH and POX are two possible key enzymes involved in aerobic acetate accumulation in lactic acid bacteria (LAB). It is currently thought that POX plays the major role in aerobic growth in homofermentative LAB and some heterofermentative LAB, while the impact of PDH remains unclear. In this study, we

Depolarizing actions of GABA in immature neurons depend neither on ketone bodies nor on pyruvate.

PubMed

Tyzio, Roman; Allene, Camille; Nardou, Romain; Picardo, Michel A; Yamamoto, Sumii; Sivakumaran, Sudhir; Caiati, Maddalena D; Rheims, Sylvain; Minlebaev, Marat; Milh, Mathieu; Ferré, Pascal; Khazipov, Rustem; Romette, Jean-Louis; Lorquin, Jean; Cossart, Rosa; Khalilov, Ilgam; Nehlig, Astrid; Cherubini, Enrico; Ben-Ari, Yehezkel

2011-01-05

GABA depolarizes immature neurons because of a high [Cl(-)](i) and orchestrates giant depolarizing potential (GDP) generation. Zilberter and coworkers (Rheims et al., 2009; Holmgren et al., 2010) showed recently that the ketone body metabolite DL-3-hydroxybutyrate (DL-BHB) (4 mM), lactate (4 mM), or pyruvate (5 mM) shifted GABA actions to hyperpolarizing, suggesting that the depolarizing effects of GABA are attributable to inadequate energy supply when glucose is the sole energy source. We now report that, in rat pups (postnatal days 4-7), plasma D-BHB, lactate, and pyruvate levels are 0.9, 1.5, and 0.12 mM, respectively. Then, we show that DL-BHB (4 mM) and pyruvate (200 μM) do not affect (i) the driving force for GABA(A) receptor-mediated currents (DF(GABA)) in cell-attached single-channel recordings, (2) the resting membrane potential and reversal potential of synaptic GABA(A) receptor-mediated responses in perforated patch recordings, (3) the action potentials triggered by focal GABA applications, or (4) the GDPs determined with electrophysiological recordings and dynamic two-photon calcium imaging. Only very high nonphysiological concentrations of pyruvate (5 mM) reduced DF(GABA) and blocked GDPs. Therefore, DL-BHB does not alter GABA signals even at the high concentrations used by Zilberter and colleagues, whereas pyruvate requires exceedingly high nonphysiological concentrations to exert an effect. There is no need to alter conventional glucose enriched artificial CSF to investigate GABA signals in the developing brain.

Impaired mitochondrial pyruvate importation in a patient and a fetus at risk.

PubMed

Brivet, M; Garcia-Cazorla, A; Lyonnet, S; Dumez, Y; Nassogne, M C; Slama, A; Boutron, A; Touati, G; Legrand, A; Saudubray, J M

2003-03-01

The patient was the first child of healthy consanguineous parents. She presented at birth with hypotonia, mild facial dysmorphism, periventricular cysts, marked metabolic acidosis, hyperlactacidemia with normal lactate/pyruvate molar ratios, normoglycemia, and normal ammonia. Hyperlactacidemia was severe (5-14 mmol/l) and not corrected with bicarbonate, thiamine (10 mg/d), 2-chloropropionate (100 mg/kg/d) and a ketogenic diet. Pyruvate dehydrogenase (PDHC) activity was normal in lymphocytes and fibroblasts. Functional assays were performed in digitonin-permeabilized fibroblasts to measure oxidation rates from radiolabeled pyruvate and malate. The production of [14C]acetylcarnitine or [14C]citric cycle intermediates derived from [2-14C]pyruvate as well as the release of 14CO(2) from [1-14C]pyruvate was severely impaired, whereas decarboxylation of [U-14C]malate was normal. With increasing concentrations of [1-14C]pyruvate, the patient's fibroblasts behave like control fibroblasts incubated in the presence of alpha-cyano-4-hydroxycinnamate, a specific inhibitor of mitochondrial pyruvate uptake: a progressive increase in 14CO(2) production was observed, likely due to passive diffusion of [1-14C]pyruvate through the mitochondrial membranes. Our results are consistent with a defect of mitochondrial pyruvate transport in the patient. Mutational analysis was precluded as the cDNA sequence of the pyruvate carrier has not been identified as yet in any organism. An affected fetus was recognized in a subsequent dichorionic twin pregnancy using the coupled assay measuring [2-14C]pyruvate oxidation rates on digitonin-permeabilized trophoblasts. After selective feticide, the pregnancy was uncomplicated with delivery at 37w of a healthy female, who is currently 2-month old. Copyright 2003 Elsevier Science (USA)

Effects of Systemic Metabolic Fuels on Glucose and Lactate Levels in the Brain Extracellular Compartment of the Mouse

PubMed Central

Béland-Millar, Alexandria; Larcher, Jeremy; Courtemanche, Justine; Yuan, Tina; Messier, Claude

2017-01-01

Classic neuroenergetic research has emphasized the role of glucose, its transport and its metabolism in sustaining normal neural function leading to the textbook statement that it is the necessary and sole metabolic fuel of the mammalian brain. New evidence, including the Astrocyte-to-Neuron Lactate Shuttle hypothesis, suggests that the brain can use other metabolic substrates. To further study that possibility, we examined the effect of intraperitoneally administered metabolic fuels (glucose, fructose, lactate, pyruvate, ß-hydroxybutyrate, and galactose), and insulin, on blood, and extracellular brain levels of glucose and lactate in the adult male CD1 mouse. Primary motor cortex extracellular levels of glucose and lactate were monitored in freely moving mice with the use of electrochemical electrodes. Blood concentration of these same metabolites were obtained by tail vein sampling and measured with glucose and lactate meters. Blood and extracellular fluctuations of glucose and lactate were monitored for a 2-h period. We found that the systemic injections of glucose, fructose, lactate, pyruvate, and ß-hydroxybutyrate increased blood lactate levels. Apart for a small transitory rise in brain extracellular lactate levels, the main effect of the systemic injection of glucose, fructose, lactate, pyruvate, and ß-hydroxybutyrate was an increase in brain extracellular glucose levels. Systemic galactose injections produced a small rise in blood glucose and lactate but almost no change in brain extracellular lactate and glucose. Systemic insulin injections led to a decrease in blood glucose and a small rise in blood lactate; however brain extracellular glucose and lactate monotonically decreased at the same rate. Our results support the concept that the brain is able to use alternative fuels and the current experiments suggest some of the mechanisms involved. PMID:28154523

Differences between magnesium-activated and manganese-activated pyruvate kinase from the muscle of Concholepas concholepas.

PubMed

González, R; Carvajal, N; Morán, A

1984-01-01

In contrast to the Mg2+-activated enzyme, in the presence of Mn2+ pyruvate kinase exhibits hyperbolic kinetics with respect to the substrate phosphoenolpyruvate and is insensitive to fructose 1,6-biphosphate, phenylalanine and alanine. However, with both metal activated species inhibition by excess ADP is observed. In contrast with Mg2+, which affords significant protection against inactivation caused by 5,5'-dithiobis (2-nitrobenzoic acid), the rate of inactivation by this reagent is increased in the presence of Mn2+. Differences in conformational changes induced by combination of pyruvate kinase with Mg2+ or Mn2+ were indicated by u.v. difference spectra.

Lactate/pyruvate transporter MCT-1 is a direct Wnt target that confers sensitivity to 3-bromopyruvate in colon cancer.

PubMed

Sprowl-Tanio, Stephanie; Habowski, Amber N; Pate, Kira T; McQuade, Miriam M; Wang, Kehui; Edwards, Robert A; Grun, Felix; Lyou, Yung; Waterman, Marian L

2016-01-01

There is increasing evidence that oncogenic Wnt signaling directs metabolic reprogramming of cancer cells to favor aerobic glycolysis or Warburg metabolism. In colon cancer, this reprogramming is due to direct regulation of pyruvate dehydrogenase kinase 1 ( PDK1 ) gene transcription. Additional metabolism genes are sensitive to Wnt signaling and exhibit correlative expression with PDK1. Whether these genes are also regulated at the transcriptional level, and therefore a part of a core metabolic gene program targeted by oncogenic WNT signaling, is not known. Here, we identify monocarboxylate transporter 1 (MCT-1; encoded by SLC16A1 ) as a direct target gene supporting Wnt-driven Warburg metabolism. We identify and validate Wnt response elements (WREs) in the proximal SLC16A1 promoter and show that they mediate sensitivity to Wnt inhibition via dominant-negative LEF-1 (dnLEF-1) expression and the small molecule Wnt inhibitor XAV939. We also show that WREs function in an independent and additive manner with c-Myc, the only other known oncogenic regulator of SLC16A1 transcription. MCT-1 can export lactate, the byproduct of Warburg metabolism, and it is the essential transporter of pyruvate as well as a glycolysis-targeting cancer drug, 3-bromopyruvate (3-BP). Using sulforhodamine B (SRB) assays to follow cell proliferation, we tested a panel of colon cancer cell lines for sensitivity to 3-BP. We observe that all cell lines are highly sensitive and that reduction of Wnt signaling by XAV939 treatment does not synergize with 3-BP, but instead is protective and promotes rapid recovery. We conclude that MCT-1 is part of a core Wnt signaling gene program for glycolysis in colon cancer and that modulation of this program could play an important role in shaping sensitivity to drugs that target cancer metabolism.

A comparison of quantitative methods for clinical imaging with hyperpolarized (13)C-pyruvate.

PubMed

Daniels, Charlie J; McLean, Mary A; Schulte, Rolf F; Robb, Fraser J; Gill, Andrew B; McGlashan, Nicholas; Graves, Martin J; Schwaiger, Markus; Lomas, David J; Brindle, Kevin M; Gallagher, Ferdia A

2016-04-01

Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized (13)C-labelled molecules, such as the conversion of [1-(13)C]pyruvate to [1-(13)C]lactate, to be dynamically and non-invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model-free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two-way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time-to-peak and the lactate-to-pyruvate area under the curve ratio were simple model-free approaches that accurately represented the full reaction, with the time-to-peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized (13)C data. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

Protein Homeostasis Defects of Alanine-Glyoxylate Aminotransferase: New Therapeutic Strategies in Primary Hyperoxaluria Type I

PubMed Central

Pey, Angel L.; Albert, Armando; Salido, Eduardo

2013-01-01

Alanine-glyoxylate aminotransferase catalyzes the transamination between L-alanine and glyoxylate to produce pyruvate and glycine using pyridoxal 5′-phosphate (PLP) as cofactor. Human alanine-glyoxylate aminotransferase is a peroxisomal enzyme expressed in the hepatocytes, the main site of glyoxylate detoxification. Its deficit causes primary hyperoxaluria type I, a rare but severe inborn error of metabolism. Single amino acid changes are the main type of mutation causing this disease, and considerable effort has been dedicated to the understanding of the molecular consequences of such missense mutations. In this review, we summarize the role of protein homeostasis in the basic mechanisms of primary hyperoxaluria. Intrinsic physicochemical properties of polypeptide chains such as thermodynamic stability, folding, unfolding, and misfolding rates as well as the interaction of different folding states with protein homeostasis networks are essential to understand this disease. The view presented has important implications for the development of new therapeutic strategies based on targeting specific elements of alanine-glyoxylate aminotransferase homeostasis. PMID:23956997

The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane monocarboxylate transporters.

PubMed

Nancolas, Bethany; Guo, Lili; Zhou, Rong; Nath, Kavindra; Nelson, David S; Leeper, Dennis B; Blair, Ian A; Glickson, Jerry D; Halestrap, Andrew P

2016-04-01

Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitizing tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, whereas indirect evidence suggests it may also inhibit L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). In the present study, we test these possibilities directly. We demonstrate that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki2.5 μM) and co-operatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevisoocytes with K0.5 and Hill coefficient values of 36-40 μM and 1.65-1.85 respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50~ 7 μM) than other substrates including glutamate (IC50~ 20 μM). In isolated DB-1 melanoma cells 1-10 μM LND increased L-lactate output, consistent with MPC inhibition, but higher concentrations (150 μM) decreased L-lactate output whereas increasing intracellular [L-lactate] > 5-fold, consistent with MCT inhibition. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux and glutamine/glutamate oxidation. Together these actions can account for published data on the selective tumour effects of LND onL-lactate, intracellular pH (pHi) and ATP levels that can be partially mimicked by the established MPC and MCT inhibitor α-cyano-4-hydroxycinnamate (CHC). © 2016 Authors; published by Portland Press Limited.

The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane monocarboxylate transporters

PubMed Central

Nancolas, Bethany; Guo, Lili; Zhou, Rong; Nath, Kavindra; Nelson, David S.; Leeper, Dennis B.; Blair, Ian A.; Glickson, Jerry D.; Halestrap, Andrew P.

2016-01-01

Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitising tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, while indirect evidence suggests it may also inhibit L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). Here we test these possibilities directly. We demonstrate that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki 2.5 μM) and cooperatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K0.5 and Hill Coefficient values of 36–40 μM and 1.65–1.85. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50 ~7 μM) than other substrates including glutamate (IC50 ~20 μM). In isolated DB-1 melanoma cells 1–10 μM LND increased L-lactate output, consistent with MPC inhibition, but higher concentrations (150 μM) decreased L-lactate output while increasing intracellular [L-lactate] > five-fold, consistent with MCT inhibition. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux and glutamine/glutamate oxidation. Together these actions can account for published data on the selective tumour effects of LND on L-lactate, intracellular pH (pHi) and ATP levels that can be partially mimicked by the established MPC and MCT inhibitor α-cyano-4-hydroxycinnamate. PMID:26831515

Metabolic fate of glucose in rats with traumatic brain injury and pyruvate or glucose treatments: A NMR spectroscopy study.

PubMed

Shijo, Katsunori; Sutton, Richard L; Ghavim, Sima S; Harris, Neil G; Bartnik-Olson, Brenda L

2017-01-01

Administration of sodium pyruvate (SP; 9.08 μmol/kg, i.p.), ethyl pyruvate (EP; 0.34 μmol/kg, i.p.) or glucose (GLC; 11.1 μmol/kg, i.p.) to rats after unilateral controlled cortical impact (CCI) injury has been reported to reduce neuronal loss and improve cerebral metabolism. In the present study these doses of each fuel or 8% saline (SAL; 5.47 nmoles/kg) were administered immediately and at 1, 3, 6 and 23 h post-CCI. At 24 h all CCI groups and non-treated Sham injury controls were infused with [1,2 13 C] glucose for 68 min 13 C nuclear magnetic resonance (NMR) spectra were obtained from cortex + hippocampus tissues from left (injured) and right (contralateral) hemispheres. All three fuels increased lactate labeling to a similar degree in the injured hemisphere. The amount of lactate labeled via the pentose phosphate and pyruvate recycling (PPP + PR) pathway increased in CCI-SAL and was not improved by SP, EP, and GLC treatments. Oxidative metabolism, as assessed by glutamate labeling, was reduced in CCI-SAL animals. The greatest improvement in oxidative metabolism was observed in animals treated with SP and fewer improvements after EP or GLC treatments. Compared to SAL, all three fuels restored glutamate and glutamine labeling via pyruvate carboxylase (PC), suggesting improved astrocyte metabolism following fuel treatment. Only SP treatments restored the amount of [4 13 C] glutamate labeled by the PPP + PR pathway to sham levels. Milder injury effects in the contralateral hemisphere appear normalized by either SP or EP treatments, as increases in the total pool of 13 C lactate and labeling of lactate in glycolysis, or decreases in the ratio of PC/PDH labeling of glutamine, were found only for CCI-SAL and CCI-GLC groups compared to Sham. The doses of SP, EP and GLC examined in this study all enhanced lactate labeling and restored astrocyte-specific PC activity but differentially affected neuronal metabolism after CCI injury. The restoration of

Metabolic fate of glucose in rats with traumatic brain injury and pyruvate or glucose treatments: A NMR spectroscopy study

PubMed Central

Shijo, Katsunori; Sutton, Richard L.; Ghavim, Sima S.; Harris, Neil G.; Bartnik-Olson, Brenda L.

2016-01-01

Administration of sodium pyruvate (SP; 9.08 μmol/kg, i.p.), ethyl pyruvate (EP; 0.34 μmol/kg, i.p.) or glucose (GLC; 11.1 μmol/kg, i.p.) to rats after unilateral controlled cortical impact (CCI) injury has been reported to reduce neuronal loss and improve cerebral metabolism. In the present study these doses of each fuel or 8% saline (SAL; 5.47 nmoles/kg) were administered immediately and at 1, 3, 6 and 23 h post-CCI. At 24 h all CCI groups and non-treated Sham injury controls were infused with [1,2 13C] glucose for 68 min 13C nuclear magnetic resonance (NMR) spectra were obtained from cortex + hippocampus tissues from left (injured) and right (contralateral) hemispheres. All three fuels increased lactate labeling to a similar degree in the injured hemisphere. The amount of lactate labeled via the pentose phosphate and pyruvate recycling (PPP + PR) pathway increased in CCI-SAL and was not improved by SP, EP, and GLC treatments. Oxidative metabolism, as assessed by glutamate labeling, was reduced in CCI-SAL animals. The greatest improvement in oxidative metabolism was observed in animals treated with SP and fewer improvements after EP or GLC treatments. Compared to SAL, all three fuels restored glutamate and glutamine labeling via pyruvate carboxylase (PC), suggesting improved astrocyte metabolism following fuel treatment. Only SP treatments restored the amount of [4 13C] glutamate labeled by the PPP + PR pathway to sham levels. Milder injury effects in the contralateral hemisphere appear normalized by either SP or EP treatments, as increases in the total pool of 13C lactate and labeling of lactate in glycolysis, or decreases in the ratio of PC/PDH labeling of glutamine, were found only for CCI-SAL and CCI-GLC groups compared to Sham. The doses of SP, EP and GLC examined in this study all enhanced lactate labeling and restored astrocyte-specific PC activity but differentially affected neuronal metabolism after CCI injury. The restoration of astrocyte metabolism

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.

Neuroenergetic Response to Prolonged Cerebral Glucose Depletion after Severe Brain Injury and the Role of Lactate.

PubMed

Patet, Camille; Quintard, Hervé; Suys, Tamarah; Bloch, Jocelyne; Daniel, Roy T; Pellerin, Luc; Magistretti, Pierre J; Oddo, Mauro

2015-10-15

Lactate may represent a supplemental fuel for the brain. We examined cerebral lactate metabolism during prolonged brain glucose depletion (GD) in acute brain injury (ABI) patients monitored with cerebral microdialysis (CMD). Sixty episodes of GD (defined as spontaneous decreases of CMD glucose from normal to low [<1.0 mmol/L] for at least 2 h) were identified among 26 patients. During GD, we found a significant increase of CMD lactate (from 4 ± 2.3 to 5.4 ± 2.9 mmol/L), pyruvate (126.9 ± 65.1 to 172.3 ± 74.1 μmol/L), and lactate/pyruvate ratio (LPR; 27 ± 6 to 35 ± 9; all, p < 0.005), while brain oxygen and blood lactate remained normal. Dynamics of lactate and glucose supply during GD were further studied by analyzing the relationships between blood and CMD samples. There was a strong correlation between blood and brain lactate when LPR was normal (r = 0.56; p < 0.0001), while an inverse correlation (r = -0.11; p = 0.04) was observed at elevated LPR >25. The correlation between blood and brain glucose also decreased from r = 0.62 to r = 0.45. These findings in ABI patients suggest increased cerebral lactate delivery in the absence of brain hypoxia when glucose availability is limited and support the concept that lactate acts as alternative fuel.

Purification and functional characterisation of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae).

PubMed

Nałecz, M J; Nałecz, K A; Azzi, A

1991-08-09

Isolated yeast mitochondria were subjected to solubilization by Triton X-114 and the detergent extract was subsequently chromatrographed on dry hydroxyapatite. Purification of the yeast monocarboxylate (pyruvate) carrier was achieved by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate, as described previously for bovine heart mitochondria (Bolli, R., Nałecz K.A. and Azzi, A. (1989) J. Biol. Chem. 264 18024-18030). The final preparation contained two polypeptides of apparent molecular mass 26 and 50 kDa. The yeast carrier appeared to be less abundant, but more active, than the analogous protein from higher eukaryotes. The carrier was able to catalyse the pyruvate / pyruvate and pyruvate / acetoacetate exchange reactions, both reactions being sensitive to cyanocinnamate and its derivatives, to phenylpyruvate and to mersalyl and p-chloromercuribenzoate. In the pyruvate / acetoacetate exchange reaction (200 mM internal acetoacetate, enzymatic assay), the Km value for external pyruvate was found to be 0.8 mM and the Vmax 135 mumol/min per mg protein. Among other substrates of the yeast carrier, all transported with similar affinity and identical maximal velocity against acetoacetate, we identified 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate. Lactate was not translocated by this carrier with a measurable rate, neither were di- or tricarboxylates.

White shrimp Litopenaeus vannamei recombinant lactate dehydrogenase: Biochemical and kinetic characterization.

PubMed

Fregoso-Peñuñuri, Ambar A; Valenzuela-Soto, Elisa M; Figueroa-Soto, Ciria G; Peregrino-Uriarte, Alma B; Ochoa-Valdez, Manuel; Leyva-Carrillo, Lilia; Yepiz-Plascencia, Gloria

2017-09-01

Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2. For this, the cDNA from muscle was cloned and overexpressed in E. coli as a fusion protein containing an intein and a chitin binding protein domain (CBD). The recombinant protein was purified by chitin affinity chromatography column that retained the CBD and released solely the full and active LDH. The active protein appears to be a tetramer with molecular mass of approximately 140 kDa and can use pyruvate or lactate as substrates, but has higher specific activity with pyruvate. The enzyme is stable between pH 7.0 to 8.5, and between 20 and 50 °C with an optimal temperature of 50 °C. Two pK a of 9.3 and 6.6, and activation energy of 44.8 kJ/mol°K were found. The kinetic constants K m for NADH was 23.4 ± 1.8 μM, and for pyruvate was 203 ± 25 μM, while V max was 7.45 μmol/min/mg protein. The shrimp LDH that is mainly expressed in shrimp muscle preferentially converts pyruvate to lactate and is an important enzyme for the response to hypoxia. Copyright © 2017 Elsevier Inc. All rights reserved.

Determination of ammonium ion using a reagentless amperometric biosensor based on immobilized alanine dehydrogenase.

PubMed

Tan, Ling Ling; Musa, Ahmad; Lee, Yook Heng

2011-01-01

The use of the enzyme alanine dehydrogenase (AlaDH) for the determination of ammonium ion (NH(4)(+)) usually requires the addition of pyruvate substrate and reduced nicotinamide adenine dinucleotide (NADH) simultaneously to effect the reaction. This addition of reagents is inconvenient when an enzyme biosensor based on AlaDH is used. To resolve the problem, a novel reagentless amperometric biosensor using a stacked methacrylic membrane system coated onto a screen-printed carbon paste electrode (SPE) for NH(4)(+) ion determination is described. A mixture of pyruvate and NADH was immobilized in low molecular weight poly(2-hydroxyethyl methacrylate) (pHEMA) membrane, which was then deposited over a photocured pHEMA membrane (photoHEMA) containing alanine dehydrogenase (AlaDH) enzyme. Due to the enzymatic reaction of AlaDH and the pyruvate substrate, NH(4)(+) was consumed in the process and thus the signal from the electrocatalytic oxidation of NADH at an applied potential of +0.55 V was proportional to the NH(4)(+) ion concentration under optimal conditions. The stacked methacrylate membranes responded rapidly and linearly to changes in NH(4)(+) ion concentrations between 10-100 mM, with a detection limit of 0.18 mM NH(4)(+) ion. The reproducibility of the amperometrical NH(4)(+) biosensor yielded low relative standard deviations between 1.4-4.9%. The stacked membrane biosensor has been successfully applied to the determination of NH(4)(+) ion in spiked river water samples without pretreatment. A good correlation was found between the analytical results for NH(4)(+) obtained from the biosensor and the Nessler spectrophotometric method.

A metabolic switch in brain: glucose and lactate metabolism modulation by ascorbic acid.

PubMed

Castro, Maite A; Beltrán, Felipe A; Brauchi, Sebastián; Concha, Ilona I

2009-07-01

In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K(+)]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD(+)/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.

Mechanisms driving the lactate switch in Chinese hamster ovary cells.

PubMed

Hartley, Fiona; Walker, Tracy; Chung, Vicky; Morten, Karl

2018-03-31

The metabolism of Chinese Hamster Ovary (CHO) cells in a production environment has been extensively investigated. However, a key metabolic transition, the switch from lactate production to lactate consumption, remains enigmatic. Though commonly observed in CHO cultures, the mechanism(s) by which this metabolic shift is triggered is unknown. Despite this, efforts to control the switch have emerged due to the association of lactate consumption with improved cell growth and productivity. This review aims to consolidate current theories surrounding the lactate switch. The influence of pH, NAD + /NADH, pyruvate availability and mitochondrial function on lactate consumption are explored. A hypothesis based on the cellular redox state is put forward to explain the onset of lactate consumption. Various techniques implemented to control the lactate switch, including manipulation of the culture environment, genetic engineering, and cell line selection are also discussed. © 2018 Wiley Periodicals, Inc.

In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons.

PubMed

Mächler, Philipp; Wyss, Matthias T; Elsayed, Maha; Stobart, Jillian; Gutierrez, Robin; von Faber-Castell, Alexandra; Kaelin, Vincens; Zuend, Marc; San Martín, Alejandro; Romero-Gómez, Ignacio; Baeza-Lehnert, Felipe; Lengacher, Sylvain; Schneider, Bernard L; Aebischer, Patrick; Magistretti, Pierre J; Barros, L Felipe; Weber, Bruno

2016-01-12

Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons. Copyright © 2016 Elsevier Inc. All rights reserved.

A Comparison of Oxidative Lactate Metabolism in Traumatically Injured Brain and Control Brain.

PubMed

Jalloh, Ibrahim; Helmy, Adel; Howe, Duncan J; Shannon, Richard J; Grice, Peter; Mason, Andrew; Gallagher, Clare N; Murphy, Michael P; Pickard, John D; Menon, David K; Carpenter, T Adrian; Hutchinson, Peter J; Carpenter, Keri L H

2018-05-18

Metabolic abnormalities occur after traumatic brain injury (TBI). Glucose is conventionally regarded as the major energy substrate, although lactate can also be an energy source. We compared 3- 13 C lactate metabolism in TBI with "normal" control brain and muscle, measuring 13 C-glutamine enrichment to assess tricarboxylic acid (TCA) cycle metabolism. Microdialysis catheters in brains of nine patients with severe TBI, five non-TBI brain surgical patients, and five resting muscle (non-TBI) patients were perfused (24 h in brain, 8 h in muscle) with 8 mmol/L sodium 3- 13 C lactate. Microdialysate analysis employed ISCUS and nuclear magnetic resonance. In TBI, with 3- 13 C lactate perfusion, microdialysate glucose concentration increased nonsignificantly (mean +11.9%, p = 0.463), with significant increases (p = 0.028) for lactate (+174%), pyruvate (+35.8%), and lactate/pyruvate ratio (+101.8%). Microdialysate 13 C-glutamine fractional enrichments (median, interquartile range) were: for C4 5.1 (0-11.1) % in TBI and 5.7 (4.6-6.8) % in control brain, for C3 0 (0-5.0) % in TBI and 0 (0-0) % in control brain, and for C2 2.9 (0-5.7) % in TBI and 1.8 (0-3.4) % in control brain. 13 C-enrichments were not statistically different between TBI and control brain, showing both metabolize 3- 13 C lactate via TCA cycle, in contrast to muscle. Several patients with TBI exhibited 13 C-glutamine enrichment above the non-TBI control range, suggesting lactate oxidative metabolism as a TBI "emergency option."

Hyperpolarized 13C pyruvate mouse brain metabolism with absorptive-mode EPSI at 1 T

NASA Astrophysics Data System (ADS)

Miloushev, Vesselin Z.; Di Gialleonardo, Valentina; Salamanca-Cardona, Lucia; Correa, Fabian; Granlund, Kristin L.; Keshari, Kayvan R.

2017-02-01

The expected signal in echo-planar spectroscopic imaging experiments was explicitly modeled jointly in spatial and spectral dimensions. Using this as a basis, absorptive-mode type detection can be achieved by appropriate choice of spectral delays and post-processing techniques. We discuss the effects of gradient imperfections and demonstrate the implementation of this sequence at low field (1.05 T), with application to hyperpolarized [1-13C] pyruvate imaging of the mouse brain. The sequence achieves sufficient signal-to-noise to monitor the conversion of hyperpolarized [1-13C] pyruvate to lactate in the mouse brain. Hyperpolarized pyruvate imaging of mouse brain metabolism using an absorptive-mode EPSI sequence can be applied to more sophisticated murine disease and treatment models. The simple modifications presented in this work, which permit absorptive-mode detection, are directly translatable to human clinical imaging and generate improved absorptive-mode spectra without the need for refocusing pulses.

Role of the mitochondrial metabolism of pyruvate on the regulation of ketogenesis in rat hepatocytes.

PubMed

Demaugre, F; Buc, H; Girard, J; Leroux, J P

1983-01-01

In hepatocytes isolated from fed rats the inhibition of lipogenesis (-80%) by 5-tetradecyloxy-2-furoate (an inhibitor of acetylCoA carboxylase) and alpha-cyano-3-hydroxycinnamate (an inhibitor of pyruvate entry into mitochondria) increases the oxidation of 0.35 mM oleate respectively by 70% and 90%. 5-tetradecyloxy-2-furoate increases ketone body production from oleate only by 30% and has no effect on ketogenesis from octanoate, whereas alpha-cyano-3-hydroxycinnamate mimics the effects of fasting on ketone body production: It increases ketogenesis from 0.35 mM oleate by 90%, from 0.78 mM oleate by 25% and from 1.57 mM butyrate by 37%. alpha-cyano-3-hydroxycinnamate also decreases the activity of tricarboxylic acid cycle and the production of malate and citrate. In hepatocytes from fasted rats, alpha-cyano-3-hydroxycinnamate does not modify ketogenesis from oleate, unless cells are incubated with a mixture of lactate and pyruvate. A lactate and pyruvate mixture decreases ketogenesis from oleate and octanoate and increases citrate and malate production without modifying the uptake of fatty acids. This effect is potentiated by 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase. The results cannot be interpreted only by the effects of malonylCoA on carnitine acyltransferase. They are discussed with respect to the possible involvement of mitochondrial oxaloacetate concentration in the regulation of ketogenesis.

The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.

PubMed

Halestrap, A P

1975-04-01

1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of

[Characterization of D-lactate dehydrogenase isozymes from a D-lactic acid producing bacterium Sporolactobacillus inulinus].

PubMed

Zhang, Danru; Zheng, Lu; Wu, Bin; He, Bingfang

2016-11-04

Sporolactobacillus inulinus, a typical homofermentative lactic acid bacterium, is an efficient D-lactic acid producer. Various environment factors affect the productivity of S. inulinus. Glucokinase, phosphofructokinase, pyruvate kinase and lactic dehydrogenase are the key enzymes of D-lactic acid production from glucose by S. inulinus. The characteristics of these enzymes are important in controlling and regulating the fermentation process. According to the genome bioinformatics analysis of S. inulinus CASD, three putative D-lactate dehydrogenases were identified, among which the bifunctional protein had been reported. In this study, we provided insights into the characteristics of the other two D-lactate dehydrogenase isozymes. S. inulinus Y2-8 genome was used as the template to amplify D-lactate dehydrogenase gene (dldh) and D-isomer specific 2-hydroxyacid dehydrogenase gene (dhdh). The two recombinant strains E-pET-28a/dldh and E-pET-28a/dhdh were constructed for enzyme expression. Both recombinants DLDH and DHDH could convert pyruvic acid into D-lactic acid. Enzymes expressed by recombinant strains were purified by Ni-NTA chromatography. The apparent molecular mass of DLDH was approximately 37 kDa by SDS-PAGE analysis, and DLDH showed a high affinity to pyruvate with the Km value of (0.58±0.04) mmol/L. The optimal reaction temperature and pH for DLDH was 35℃ and 6.5, respectively. The apparent molecular mass of DHDH was approximately 39 kDa, and the Km of DHDH toward pyruvate was (1.70±0.08) mmol/L. The optimum catalysis temperature and pH of DHDH were 30℃ and 7.5, respectively. According to the Km and optimal reaction pH, DLDH was suggested as the main catalyst in formation D-lactic acid from pyruvate during the fermentation. The enzymatic properties would contribute to the regulation of the fermentation of S. inulinus.

Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

PubMed

Wojtkowiak, Jonathan W; Cornnell, Heather C; Matsumoto, Shingo; Saito, Keita; Takakusagi, Yoichi; Dutta, Prasanta; Kim, Munju; Zhang, Xiaomeng; Leos, Rafael; Bailey, Kate M; Martinez, Gary; Lloyd, Mark C; Weber, Craig; Mitchell, James B; Lynch, Ronald M; Baker, Amanda F; Gatenby, Robert A; Rejniak, Katarzyna A; Hart, Charles; Krishna, Murali C; Gillies, Robert J

2015-01-01

Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized (13)C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to

Pyruvate Formate-Lyase Is Essential for Fumarate-Independent Anaerobic Glycerol Utilization in the Enterococcus faecalis Strain W11

PubMed Central

Ikegami, Yuki

2014-01-01

Although anaerobic glycerol metabolism in Enterococcus faecalis requires exogenous fumarate for NADH oxidation, E. faecalis strain W11 can metabolize glycerol in the absence of oxygen without exogenous fumarate. In this study, metabolic end product analyses and reporter assays probing the expression of enzymes involved in pyruvate metabolism were performed to investigate this fumarate-independent anaerobic metabolism of glycerol in W11. Under aerobic conditions, the metabolic end products of W11 cultured with glycerol were similar to those of W11 cultured with glucose. However, when W11 was cultured anaerobically, most of the glucose was converted to l-lactate, but glycerol was converted to ethanol and formate. During anaerobic culture with glycerol, the expression of the l-lactate dehydrogenase and pyruvate dehydrogenase E1αβ genes in W11 was downregulated, whereas the expression of the pyruvate formate-lyase (Pfl) and aldehyde/alcohol dehydrogenase genes was upregulated. These changes in the expression levels caused the change in the composition of end products. A pflB gene disruptant (Δpfl mutant) of W11 could barely utilize glycerol under anaerobic conditions, but the growth of the Δpfl mutant cultured with either glucose or dihydroxyacetone (DHA) under anaerobic conditions was the same as that of W11. Glucose metabolism and DHA generates one NADH molecule per pyruvate molecule, whereas glycerol metabolism in the dehydrogenation pathway generates two NADH molecules per pyruvate molecule. These findings demonstrate that NADH generated from anaerobic glycerol metabolism in the absence of fumarate is oxidized through the Pfl-ethanol fermentation pathway. Thus, Pfl is essential to avoid the accumulation of excess NADH during fumarate-independent anaerobic glycerol metabolism. PMID:24769696

Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model

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

Ledee, Dolena R.; Kajimoto, Masaki; O'Kelly-Priddy, Colleen M.

Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO, although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we closely examined the role of prolonged systemic pyruvate supplementation in modifying metabolic parameters during the unique conditions of ventricular unloading provided by ECMO. Twelve male mixed breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (Group C) or pyruvate (Group P) during ECMO for 8 hours. Over themore » final hour piglets received [2-13C] pyruvate, and [13C6]-L-leucine, as an indicator for oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of all measured CAC intermediates. Group P showed greater anaplerotic flux through pyruvate carboxylation although pyruvate oxidation relative to citrate synthase flux was similar to Group C. The groups demonstrated similar leucine fractional contributions to acetyl-CoA and fractional protein synthesis rates. Pyruvate also promoted an increase in the phosphorylation state of several nutrient sensitive enzymes, such as AMPK and ACC, and promoted O-GlcNAcylation through the hexosamine biosynthetic pathway (HBP). In conclusion, prolonged pyruvate supplementation during ECMO modified anaplerotic pyruvate flux and elicited changes in important nutrient and energy sensitive pathways, while preserving protein synthesis. Therefore, the observed results support the further study of nutritional supplementation and its downstream effects on cardiac adaptation during ventricular unloading.« less

Regulation of Blood Glucose by Hypothalamic Pyruvate Metabolism

NASA Astrophysics Data System (ADS)

Lam, Tony K. T.; Gutierrez-Juarez, Roger; Pocai, Alessandro; Rossetti, Luciano

2005-08-01

The brain keenly depends on glucose for energy, and mammalians have redundant systems to control glucose production. An increase in circulating glucose inhibits glucose production in the liver, but this negative feedback is impaired in type 2 diabetes. Here we report that a primary increase in hypothalamic glucose levels lowers blood glucose through inhibition of glucose production in rats. The effect of glucose requires its conversion to lactate followed by stimulation of pyruvate metabolism, which leads to activation of adenosine triphosphate (ATP)-sensitive potassium channels. Thus, interventions designed to enhance the hypothalamic sensing of glucose may improve glucose homeostasis in diabetes.

Technique development of 3D dynamic CS-EPSI for hyperpolarized 13 C pyruvate MR molecular imaging of human prostate cancer.

PubMed

Chen, Hsin-Yu; Larson, Peder E Z; Gordon, Jeremy W; Bok, Robert A; Ferrone, Marcus; van Criekinge, Mark; Carvajal, Lucas; Cao, Peng; Pauly, John M; Kerr, Adam B; Park, Ilwoo; Slater, James B; Nelson, Sarah J; Munster, Pamela N; Aggarwal, Rahul; Kurhanewicz, John; Vigneron, Daniel B

2018-03-25

The purpose of this study was to develop a new 3D dynamic carbon-13 compressed sensing echoplanar spectroscopic imaging (EPSI) MR sequence and test it in phantoms, animal models, and then in prostate cancer patients to image the metabolic conversion of hyperpolarized [1- 13 C]pyruvate to [1- 13 C]lactate with whole gland coverage at high spatial and temporal resolution. A 3D dynamic compressed sensing (CS)-EPSI sequence with spectral-spatial excitation was designed to meet the required spatial coverage, time and spatial resolution, and RF limitations of the 3T MR scanner for its clinical translation for prostate cancer patient imaging. After phantom testing, animal studies were performed in rats and transgenic mice with prostate cancers. For patient studies, a GE SPINlab polarizer (GE Healthcare, Waukesha, WI) was used to produce hyperpolarized sterile GMP [1- 13 C]pyruvate. 3D dynamic 13 C CS-EPSI data were acquired starting 5 s after injection throughout the gland with a spatial resolution of 0.5 cm 3 , 18 time frames, 2-s temporal resolution, and 36 s total acquisition time. Through preclinical testing, the 3D CS-EPSI sequence developed in this project was shown to provide the desired spectral, temporal, and spatial 5D HP 13 C MR data. In human studies, the 3D dynamic HP CS-EPSI approach provided first-ever simultaneously volumetric and dynamic images of the LDH-catalyzed conversion of [1- 13 C]pyruvate to [1- 13 C]lactate in a biopsy-proven prostate cancer patient with full gland coverage. The results demonstrate the feasibility to characterize prostate cancer metabolism in animals, and now patients using this new 3D dynamic HP MR technique to measure k PL , the kinetic rate constant of [1- 13 C]pyruvate to [1- 13 C]lactate conversion. © 2018 International Society for Magnetic Resonance in Medicine.

Metabolic and histologic effects of sodium pyruvate treatment in the rat after cortical contusion injury.

PubMed

Fukushima, Masamichi; Lee, Stefan M; Moro, Nobuhiro; Hovda, David A; Sutton, Richard L

2009-07-01

This study determined the effects of intraperitoneal sodium pyruvate (SP) treatment on the levels of circulating fuels and on cerebral microdialysis levels of glucose (MD(glc)), lactate (MD(lac)), and pyruvate (MD(pyr)), and the effects of SP treatment on neuropathology after left cortical contusion injury (CCI) in rats. SP injection (1000 mg/kg) 5 min after sham injury (Sham-SP) or CCI (CCI-SP) significantly increased arterial pyruvate (p < 0.005) and lactate (p < 0.001) compared to that of saline-treated rats with CCI (CCI-Sal). Serum glucose also increased significantly in CCI-SP compared to that in CCI-Sal rats (p < 0.05), but not in Sham-SP rats. MD(pyr) was not altered after CCI-Sal, whereas MD(lac) levels within the cerebral cortex significantly increased bilaterally (p < 0.05) and those for MD(glc) decreased bilaterally (p < 0.05). MD(pyr) levels increased significantly in both Sham-SP and CCI-SP rats (p < 0.05 vs. CCI-Sal) and were higher in left/injured cortex of the CCI-SP group (p < 0.05 vs. sham-SP). In CCI-SP rats the contralateral MD(lac) decreased below CCI-Sal levels (p < 0.05) and the ipsilateral MD(glc) levels exceeded those of CCI-Sal rats (p < 0.05). Rats with a single low (500 mg/kg) or high dose (1000 mg/kg) SP treatment had fewer damaged cortical cells 6 h post-CCI than did saline-treated rats (p < 0.05), but three hourly injections of SP (1000 mg/kg) were needed to significantly reduce contusion volume 2 weeks after CCI. Thus, a single intraperitoneal SP treatment increases circulating levels of three potential brain fuels, attenuates a CCI-induced reduction in extracellular glucose while increasing extracellular levels of pyruvate, but not lactate, and can attenuate cortical cell damage occurring within 6 h of injury. Enduring (2 week) neuronal protection was achieved only with multiple SP treatments within the first 2 h post-CCI, perhaps reflecting the need for additional fuel throughout the acute period of increased metabolic demands

Metabolic and Histologic Effects of Sodium Pyruvate Treatment in the Rat after Cortical Contusion Injury

PubMed Central

Fukushima, Masamichi; Lee, Stefan M.; Moro, Nobuhiro; Hovda, David A.

2009-01-01

Abstract This study determined the effects of intraperitoneal sodium pyruvate (SP) treatment on the levels of circulating fuels and on cerebral microdialysis levels of glucose (MDglc), lactate (MDlac), and pyruvate (MDpyr), and the effects of SP treatment on neuropathology after left cortical contusion injury (CCI) in rats. SP injection (1000 mg/kg) 5 min after sham injury (Sham-SP) or CCI (CCI-SP) significantly increased arterial pyruvate (p < 0.005) and lactate (p < 0.001) compared to that of saline-treated rats with CCI (CCI-Sal). Serum glucose also increased significantly in CCI-SP compared to that in CCI-Sal rats (p < 0.05), but not in Sham-SP rats. MDpyr was not altered after CCI-Sal, whereas MDlac levels within the cerebral cortex significantly increased bilaterally (p < 0.05) and those for MDglc decreased bilaterally (p < 0.05). MDpyr levels increased significantly in both Sham-SP and CCI-SP rats (p < 0.05 vs. CCI-Sal) and were higher in left/injured cortex of the CCI-SP group (p < 0.05 vs. sham-SP). In CCI-SP rats the contralateral MDlac decreased below CCI-Sal levels (p < 0.05) and the ipsilateral MDglc levels exceeded those of CCI-Sal rats (p < 0.05). Rats with a single low (500 mg/kg) or high dose (1000 mg/kg) SP treatment had fewer damaged cortical cells 6 h post-CCI than did saline-treated rats (p < 0.05), but three hourly injections of SP (1000 mg/kg) were needed to significantly reduce contusion volume 2 weeks after CCI. Thus, a single intraperitoneal SP treatment increases circulating levels of three potential brain fuels, attenuates a CCI-induced reduction in extracellular glucose while increasing extracellular levels of pyruvate, but not lactate, and can attenuate cortical cell damage occurring within 6 h of injury. Enduring (2 week) neuronal protection was achieved only with multiple SP treatments within the first 2 h post-CCI, perhaps reflecting the need for additional fuel throughout the

Carnitine status and lactate increase in patients with type I juvenile diabetes.

PubMed

Evangeliou, A; Gourgiotis, D; Karagianni, C; Markouri, M; Anogianaki, N; Mamoulakis, D; Maropoulos, G; Tsakalidis, C; Frentzayias, A; Nicolaidou, P

2010-12-01

In 32 juvenile patients suffering from insulin dependent diabetes we observed a carnitine imbalance (increase in acylcarnitine and reduction of free carnitine), which was higher in patients with the highest levels of glycosylated hemoglobin. Parallel to that, in patients with the most prominent carnitine imbalance, there was the highest increase in the postprandial lactic acid level and the highest increase in the lactate/pyruvate ratio, without relating to ketosis. In addition, we observed a decrease in free carnitine related to the length of time after appearance of diabetes. This was a prospective study of a cohort of 32 children and young adolescents with insulin dependent diabetes mellitus. All patients were on insulin treatment. Plasma concentrations of total, free and acyl-Carnitine were evaluated in 12 hours fasting blood samples and before the morning administration of insulin. Blood glucose, cholesterol, triglycerides, and lactate, pyruvate, beta-hydroxybutyrate and free fatty acid levels were measured. The postprandial highest increase of the lactate and lactate/pyruvate ratio observed in patients with the highest degree of carnitine imbalance, namely with poorliest regulated diabetes, raises the question of a coincidental mitochondrial dysfunction. On the ground of our own data, such a claim cannot be substantiated for our patients. In contrast we suggest that the role of other factors like increased gluconeogenesis, degree of ketosis need to be sought. In order to clarify the role of carnitine in the pathophysiology of disease we need also data from other tissues. Carnitine in the peripheral blood reflects only the 1% of the total body carnitine ; furthermore, patients with diabetes exhibit changes in carnitine status not only in the peripheral blood but also in other body tissues, mainly in muscles.

Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae)

PubMed Central

Wang, Yang; Wei, Lian; Wei, Dengbang; Li, Xiao; Xu, Lina; Wei, Linna

2016-01-01

Testis-specific lactate dehydrogenase (LDH-C4) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4), Lactate Dehydrogenase B4 (LDH-B4), and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L), LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4. PMID:26751442

Lactate dehydrogenase activity drives hair follicle stem cell activation

PubMed Central

Aimee, Flores; John, Schell; Abby, Krall; David, Jelinek; Matilde, Miranda; Melina, Grigorian; Daniel, Braas; White Andrew, C; Jessica, Zhou; Nick, Graham; Thomas, Graeber; Pankaj, Seth; Denis, Evseenko; Hilary, Coller; Jared, Rutter; Heather, Christofk; Lowry William, E

2017-01-01

Summary While normally dormant, Hair Follicle Stem Cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allow them to remain dormant and yet quickly respond to appropriate proliferative stimuli. PMID:28812580

Lactate oxidation coupled to energy production in mitochondria like particles from Setaria digitata, a filarial parasite.

PubMed

Sivan, V M; Raj, R K

1994-10-14

In the filarial parasite, Setaria digitata, the mitochondria like particles (MLP) show NAD reduction with sodium lactate. The MLP also reduces dye and ferricyanide with lactate. The ferricyanide reduction by lactate is found to be sensitive to the cytochrome o inhibitor orthohydroxy diphenyl (OHD) and complex I inhibitor rotenone, modulated by ADP (+) and ATP (-) and inhibited by pyruvate and oxaloacetate. MLP shows lactate oxidation sensitive to OHD, rotenone and sodium malonate. Thus, the lactate utilizing complex system, consisting of an NADH generating MLP bound lactate dehydrogenase and a lactate flavocytochrome reductase tightly linked to complex I and cytochrome o, produces ATP in functional association with fumarate reductase complex and other enzyme systems. Hence, this study provides new dimensions to the study of metabolism in filarial parasites.

Blood concentrations of amino acids, glucose and lactate during experimental swine dysentery.

PubMed

Jonasson, R; Essén-Gustavsson, B; Jensen-Waern, M

2007-06-01

The aim of this study was to examine blood concentrations of amino acids, glucose and lactate in association with experimental swine dysentery. Ten pigs (approximately 23kg) were orally inoculated with Brachyspira hyodysenteriae. Eight animals developed muco-haemorrhagic diarrhoea with impaired general appearance, changes in white blood cell counts and increased levels of the acute phase protein Serum Amyolid A. Blood samples were taken before inoculation, during the incubation period, during clinical signs of dysentery and during recovery. Neither plasma glucose nor lactate concentrations changed during the course of swine dysentery, but the serum concentrations of gluconeogenic non-essential amino acids decreased during dysentery. This was mainly due to decreases in alanine, glutamine, serine and tyrosine. Lysine increased during dysentery and at the beginning of the recovery period, and leucine increased during recovery. Glutamine, alanine and tyrosine levels show negative correlations with the numbers of neutrophils and monocytes. In conclusion, swine dysentery altered the blood concentrations of amino acids, but not of glucose or lactate.

Dynamic metabolic imaging of hyperpolarized [2-(13) C]pyruvate using spiral chemical shift imaging with alternating spectral band excitation.

PubMed

Josan, Sonal; Hurd, Ralph; Park, Jae Mo; Yen, Yi-Fen; Watkins, Ron; Pfefferbaum, Adolf; Spielman, Daniel; Mayer, Dirk

2014-06-01

In contrast to [1-(13) C]pyruvate, hyperpolarized [2-(13) C]pyruvate permits the ability to follow the (13) C label beyond flux through pyruvate dehydrogenase complex and investigate the incorporation of acetyl-coenzyme A into different metabolic pathways. However, chemical shift imaging (CSI) with [2-(13) C]pyruvate is challenging owing to the large spectral dispersion of the resonances, which also leads to severe chemical shift displacement artifacts for slice-selective acquisitions. This study introduces a sequence for three-dimensional CSI of [2-(13) C]pyruvate using spectrally selective excitation of limited frequency bands containing a subset of metabolites. Dynamic CSI data were acquired alternately from multiple frequency bands in phantoms for sequence testing and in vivo in rat heart. Phantom experiments verified the radiofrequency pulse design and demonstrated that the signal behavior of each group of resonances was unaffected by excitation of the other frequency bands. Dynamic three-dimensional (13) C CSI data demonstrated the sequence capability to image pyruvate, lactate, acetylcarnitine, glutamate, and acetoacetate, enabling the analysis of organ-specific spectra and metabolite time courses. The presented method allows CSI of widely separated resonances without chemical shift displacement artifact, acquiring multiple frequency bands alternately to obtain dynamic time-course information. This approach enables robust imaging of downstream metabolic products of acetyl-coenzyme A with hyperpolarized [2-(13) C]pyruvate. Copyright © 2013 Wiley Periodicals, Inc.

A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila, and Humans

PubMed Central

Bricker, Daniel K.; Taylor, Eric B.; Schell, John C.; Orsak, Thomas; Boutron, Audrey; Chen, Yu-Chan; Cox, James E.; Cardon, Caleb M.; Van Vranken, Jonathan G.; Dephoure, Noah; Redin, Claire; Boudina, Sihem; Gygi, Steven P.; Brivet, Michèle; Thummel, Carl S.; Rutter, Jared

2013-01-01

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, and silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier. PMID:22628558

Trehalose Mediated Inhibition of Lactate Dehydrogenase from Rabbit Muscle. The Application of Kramers' Theory in Enzyme Catalysis.

PubMed

Hernández-Meza, Juan M; Sampedro, José G

2018-04-19

Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to lactate by using NADH. LDH kinetics has been proposed to be dependent on the dynamics of a loop over the active site. Kramers' theory has been useful in the study of enzyme catalysis dependent on large structural dynamics. In this work, LDH kinetics was studied in the presence of trehalose and at different temperatures. In the absence of trehalose, temperature increase raised exponentially the LDH V max and revealed a sigmoid transition of K m toward a low-affinity state similar to protein unfolding. Notably, LDH V max diminished when in the presence of trehalose, while pyruvate affinity increased and the temperature-mediated binding site transition was hindered. The effect of trehalose on k cat was viscosity dependent as described by Kramers' theory since V max correlated inversely with the viscosity of the medium. As a result, activation energy ( E a ) for pyruvate reduction was dramatically increased by trehalose presence. This work provides experimental evidence that the dynamics of a structural component in LDH is essential for catalysis, i.e., the closing of the loop on the active site. While the trehalose mediated-increased of pyruvate affinity is proposed to be due to the compaction and/or increase of structural order at the binding site.

Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

PubMed

Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

2014-03-01

Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production

PubMed Central

Dave, Khyati K.; Punekar, Narayan S.

2015-01-01

Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production. PMID:26683313

Ergogenic Effects of β-Alanine and Carnosine: Proposed Future Research to Quantify Their Efficacy

PubMed Central

Caruso, John; Charles, Jessica; Unruh, Kayla; Giebel, Rachel; Learmonth, Lexis; Potter, William

2012-01-01

β-alanine is an amino acid that, when combined with histidine, forms the dipeptide carnosine within skeletal muscle. Carnosine and β-alanine each have multiple purposes within the human body; this review focuses on their roles as ergogenic aids to exercise performance and suggests how to best quantify the former’s merits as a buffer. Carnosine normally makes a small contribution to a cell’s total buffer capacity; yet β-alanine supplementation raises intracellular carnosine concentrations that in turn improve a muscle’s ability to buffer protons. Numerous studies assessed the impact of oral β-alanine intake on muscle carnosine levels and exercise performance. β-alanine may best act as an ergogenic aid when metabolic acidosis is the primary factor for compromised exercise performance. Blood lactate kinetics, whereby the concentration of the metabolite is measured as it enters and leaves the vasculature over time, affords the best opportunity to assess the merits of β-alanine supplementation’s ergogenic effect. Optimal β-alanine dosages have not been determined for persons of different ages, genders and nutritional/health conditions. Doses as high as 6.4 g day−1, for ten weeks have been administered to healthy subjects. Paraesthesia is to date the only side effect from oral β-alanine ingestion. The severity and duration of paraesthesia episodes are dose-dependent. It may be unwise for persons with a history of paraesthesia to ingest β-alanine. As for any supplement, caution should be exercised with β-alanine supplementation. PMID:22852051

Partial reconstruction of in vitro gluconeogenesis arising from mitochondrial l-lactate uptake/metabolism and oxaloacetate export via novel L-lactate translocators.

PubMed

De Bari, Lidia; Atlante, Anna; Valenti, Daniela; Passarella, Salvatore

2004-05-15

In the light of the occurrence of L-lactate dehydrogenase inside the mitochondrial matrix, we looked at whether isolated rat liver mitochondria can take up and metabolize L-lactate, and provide oxaloacetate outside mitochondria, thus contributing to a partial reconstruction of gluconeogenesis in vitro. We found that: (1) L-lactate (10 mM), added to mitochondria in the presence of a cocktail of glycolysis/gluconeogenesis enzymes and cofactors, can lead to synthesis of glyceraldehyde-3-phosphate at a rate of about 7 nmol/min per mg mitochondrial protein. (2) Three novel translocators exist to mediate L-lactate traffic across the inner mitochondrial membrane. An L-lactate/H+ symporter was identified by measuring fluorimetrically the rate of endogenous pyridine nucleotide reduction. Consistently, L-lactate oxidation was found to occur with P/O ratio=3 (where P/O ratio is the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) and with generation of membrane potential. Proton uptake, which occurred as a result of addition of L-lactate to RLM together with electron flow inhibitors, and mitochondrial swelling in ammonium L-lactate solutions were also monitored. L-Lactate/oxaloacetate and L-lactate/pyruvate anti-porters were identified by monitoring photometrically the appearance of L-lactate counter-anions outside mitochondria. These L-lactate translocators, which are distinct from the monocarboxylate carrier, were found to differ from each other in V(max) values and in inhibition and pH profiles, and proved to regulate mitochondrial L-lactate metabolism in vitro. The role of lactate/mitochondria interactions in gluconeogenesis is discussed.

Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae.

PubMed

Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki

2015-01-01

Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit, Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

PubMed Central

Sailasree, Purnima; Singh, Durgesh K.; Kameshwari, Duvurri B.; Shivaji, Sisinthy

2014-01-01

Background/Aims The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. Methodology and Principal Findings Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. Conclusions This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the

Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barkeri and Methanobacterium thermoautotrophicum.

PubMed Central

Kenealy, W R; Thompson, T E; Schubert, K R; Zeikus, J G

1982-01-01

The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting. PMID:6122678

D-Lactate transport and metabolism in rat liver mitochondria.

PubMed

de Bari, Lidia; Atlante, Anna; Guaragnella, Nicoletta; Principato, Giovanni; Passarella, Salvatore

2002-07-15

In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize D-lactate. We found the following: (1) externally added D-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation)=2] and membrane potential (Delta(psi)) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by alpha-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (D-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate D-lactate traffic across the mitochondrial inner membrane: the D-lactate/H(+) symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the D-lactate/oxoacid antiporter (which mediates both the D-lactate/pyruvate and D-lactate/oxaloacetate exchanges) and D-lactate/malate antiporter studied by monitoring photometrically the appearance of the D-lactate counteranions outside mitochondria. The D-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the V(max) values and in the inhibition and pH profiles and were shown to regulate mitochondrial D-lactate metabolism in vitro. The D-lactate translocators and the D-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for D-lactate-dependent gluconeogenesis.

Molecular mechanisms of citrus flavanones on hepatic gluconeogenesis.

PubMed

Constantin, Rodrigo Polimeni; Constantin, Renato Polimeni; Bracht, Adelar; Yamamoto, Nair Seiko; Ishii-Iwamoto, Emy Luiza; Constantin, Jorgete

2014-01-01

It is well known that hyperglycaemia is the initiating cause of tissue damage associated with type 2 diabetes mellitus and that enhanced hepatic gluconeogenesis may account for the increase in blood glucose levels. The purpose of this work was to investigate the possible actions and mechanisms of three related citrus flavanones, namely hesperidin, hesperetin and naringenin, on hepatic gluconeogenesis and related parameters using isolated perfused rat liver. Hesperetin and naringenin (but not hesperidin) inhibited gluconeogenesis from lactate plus pyruvate, alanine and dihydroxyacetone. The inhibitory effects of these flavanones on gluconeogenesis from lactate and pyruvate (hesperetin IC50 75.6 μM; naringenin IC50 85.5 μM) as well as from alanine were considerably more pronounced than those from dihydroxyacetone. The main cause of gluconeogenesis inhibition is the reduction of pyruvate carboxylation by hesperetin (IC50 134.2 μM) and naringenin (IC50 143.5 μM) via inhibition of pyruvate transport into the mitochondria. Secondary causes are likely inhibition of energy metabolism, diversion of glucose 6-phosphate for glucuronidation reactions and oxidation of NADH by flavanone phenoxyl radicals. The influence of the structural differences between hesperetin and naringenin on their metabolic effects was negligible. Analytical evidence indicated that the presence of a rutinoside moiety in hesperidin noticeably decreases its metabolic effects, confirming that hesperetin and naringenin interact with intracellular enzymes and mitochondrial or cellular membranes better than hesperidin. Thus, the inhibition of the gluconeogenic pathway by citrus flavanones, which was similar to that of the drug metformin, may represent an attractive novel treatment strategy for type 2 diabetes. Copyright © 2013 Elsevier B.V. All rights reserved.

Disruption of BCAA metabolism in mice impairs exercise metabolism and endurance.

PubMed

She, Pengxiang; Zhou, Yingsheng; Zhang, Zhiyou; Griffin, Kathleen; Gowda, Kavitha; Lynch, Christopher J

2010-04-01

Exercise enhances branched-chain amino acid (BCAA) catabolism, and BCAA supplementation influences exercise metabolism. However, it remains controversial whether BCAA supplementation improves exercise endurance, and unknown whether the exercise endurance effect of BCAA supplementation requires catabolism of these amino acids. Therefore, we examined exercise capacity and intermediary metabolism in skeletal muscle of knockout (KO) mice of mitochondrial branched-chain aminotransferase (BCATm), which catalyzes the first step of BCAA catabolism. We found that BCATm KO mice were exercise intolerant with markedly decreased endurance to exhaustion. Their plasma lactate and lactate-to-pyruvate ratio in skeletal muscle during exercise and lactate release from hindlimb perfused with high concentrations of insulin and glucose were significantly higher in KO than wild-type (WT) mice. Plasma and muscle ammonia concentrations were also markedly higher in KO than WT mice during a brief bout of exercise. BCATm KO mice exhibited 43-79% declines in the muscle concentration of alanine, glutamine, aspartate, and glutamate at rest and during exercise. In response to exercise, the increments in muscle malate and alpha-ketoglutarate were greater in KO than WT mice. While muscle ATP concentration tended to be lower, muscle IMP concentration was sevenfold higher in KO compared with WT mice after a brief bout of exercise, suggesting elevated ammonia in KO is derived from the purine nucleotide cycle. These data suggest that disruption of BCAA transamination causes impaired malate/aspartate shuttle, thereby resulting in decreased alanine and glutamine formation, as well as increases in lactate-to-pyruvate ratio and ammonia in skeletal muscle. Thus BCAA metabolism may regulate exercise capacity in mice.

Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae

PubMed Central

Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki

2015-01-01

Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved. PMID:26588105

Detection of intracellular lactate with localized diffusion { 1H- 13C}-spectroscopy in rat glioma in vivo

NASA Astrophysics Data System (ADS)

Pfeuffer, Josef; Lin, Joseph C.; DelaBarre, Lance; Ugurbil, Kamil; Garwood, Michael

2005-11-01

The aim of this study was to compare the diffusion characteristic of lactate and alanine in a brain tumor model to that of normal brain metabolites known to be mainly intracellular such as N-acetylaspartate or creatine. The diffusion of 13C-labeled metabolites was measured in vivo with localized NMR spectroscopy at 9.4 T (400 MHz) using a previously described localization and editing pulse sequence known as ACED-STEAM ('adiabatic carbon editing and decoupling'). 13C-labeled glucose was administered and the apparent diffusion coefficients of the glycolytic products, { 1H- 13C}-lactate and { 1H- 13C}-alanine, were determined in rat intracerebral 9L glioma. To obtain insights into { 1H- 13C}-lactate compartmentation (intra- versus extracellular), the pulse sequence used very large diffusion weighting (50 ms/μm 2). Multi-exponential diffusion attenuation of the lactate metabolite signals was observed. The persistence of a lactate signal at very large diffusion weighting provided direct experimental evidence of significant intracellular lactate concentration. To investigate the spatial distribution of lactate and other metabolites, 1H spectroscopic images were also acquired. Lactate and choline-containing compounds were consistently elevated in tumor tissue, but not in necrotic regions and surrounding normal-appearing brain. Overall, these findings suggest that lactate is mainly associated with tumor tissue and that within the time-frame of these experiments at least some of the glycolytic product ([ 13C] lactate) originates from an intracellular compartment.

Inhibition, by 2-oxo acids that accumulate in maple-syrup-urine disease, of lactate, pyruvate, and 3-hydroxybutyrate transport across the blood-brain barrier.

PubMed

Cremer, J E; Teal, H M; Cunningham, V J

1982-09-01

Data are presented in support of the transport of (-)-D-3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were Vmax 2.0 mumol.g-1.min-1, Km 29 mM, and KD 0.024 ml.g-1.min-1. The value for Vmax was the same as that for L-lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-oxo-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The Ki values for the 2-oxo acids were severalfold lower than the respective Km values. 2-Oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.

Pyruvate and ketone-body transport across the mitochondrial membrane. Exchange properties, pH-dependence and mechanism of the carrier.

PubMed

Halestrap, A P

1978-06-15

The effects of exchangeable ions and pH on the efflux of pyruvate from preloaded mitochondria are reported. Efflux obeys first-order kinetics, and the stimulation of efflux by exchangeable ions such as acetoacetate and lactate obeys Michaelis--Menten kinetics. The apparent Km value +/- S.E. for acetoacetate was 0.56 +/- 0.14 mM (n = 5) and that for lactate 12.3 +/- 2.3 mM (n = 6). The Vmax. values +/- S.E. at 0 degrees C were 16.2 +/- 2.0 and 21.9 +/- 2.7 nmol/min per mg of protein. The exchange of a variety of other substituted monocarboxylates was also studied. Efflux was also stimulated by increasing the external pH. The data gave a pK for the transport process of 8.35 and a Vmax. of 3.31 +/- 0.14 nmol/min per mg. The similarity of the Vmax. values for various exchangeable ions but the difference of this from the Vmax. in the absence of exchangeable ions may indicate that transport of pyruvate occurs with H+ and not in exchange for an OH- ion. The inhibition of transport by alpha-cyano-4-hydroxycinnamate took several seconds to reach completion at 0 degrees C. It is proposed that inhibition occurs by binding to the substrate site and subsequent reaction with an -SH group on the inside of the membrane. The inhibitor can be displaced by substrates that can also enter the mitochondria independently of the carrier and so compete with the inhibitor for the substrate-binding site on the inside of the membrane. A mechanism for transport is proposed that invokes a transition state of pyruvate involving addition of an -SH group to the 2-carbon of pyruvate. Evidence is presented that suggests that ketone bodies may cross the mitochondrial membrane either on the carrier or by free diffusion. The physiological involvement of the carrier in ketone-body metabolism is discussed. The role of ketone bodies and pH in the physiological regulation of pyruvate transport is considered.

Dichloroacetate effects on glucose and lactate oxidation by neurons and astroglia in vitro and on glucose utilization by brain in vivo.

PubMed

Itoh, Yoshiaki; Esaki, Takanori; Shimoji, Kazuaki; Cook, Michelle; Law, Mona J; Kaufman, Elaine; Sokoloff, Louis

2003-04-15

Neuronal cultures in vitro readily oxidized both D-[(14)C]glucose and l-[(14)C]lactate to (14)CO(2), whereas astroglial cultures oxidized both substrates sparingly and metabolized glucose predominantly to lactate and released it into the medium. [(14)C]Glucose oxidation to (14)CO(2) varied inversely with unlabeled lactate concentration in the medium, particularly in neurons, and increased progressively with decreasing lactate concentration. Adding unlabeled glucose to the medium inhibited [(14)C]lactate oxidation to (14)CO(2) only in astroglia but not in neurons, indicating a kinetic preference in neurons for oxidation of extracellular lactate over intracellular pyruvatelactate produced by glycolysis. Protein kinase-catalyzed phosphorylation inactivates pyruvate dehydrogenase (PDH), which regulates pyruvate entry into the tricarboxylic acid cycle. Dichloroacetate inhibits this kinase, thus enhancing PDH activity. In vitro dichloroacetate stimulated glucose and lactate oxidation to CO(2) and reduced lactate release mainly in astroglia, indicating that limitations in glucose and lactate oxidation by astroglia may be due to a greater balance of PDH toward the inactive form. To assess the significance of astroglial export of lactate to neurons in vivo, we attempted to diminish this traffic in rats by administering dichloroacetate (50 mgkg) intravenously to stimulate astroglial lactate oxidation and then examined the effects on baseline and functionally activated local cerebral glucose utilization (lCMR(glc)). Dichloroacetate raised baseline lCMR(glc) throughout the brain and decreased the percent increases in lCMR(glc) evoked by functional activation. These studies provide evidence in support of the compartmentalization of glucose metabolism between astroglia and neurons but indicate that the compartmentalization may be neither complete nor entirely obligatory.

Regulation of pyruvate metabolism and human disease.

PubMed

Gray, Lawrence R; Tompkins, Sean C; Taylor, Eric B

2014-07-01

Pyruvate is a keystone molecule critical for numerous aspects of eukaryotic and human metabolism. Pyruvate is the end-product of glycolysis, is derived from additional sources in the cellular cytoplasm, and is ultimately destined for transport into mitochondria as a master fuel input undergirding citric acid cycle carbon flux. In mitochondria, pyruvate drives ATP production by oxidative phosphorylation and multiple biosynthetic pathways intersecting the citric acid cycle. Mitochondrial pyruvate metabolism is regulated by many enzymes, including the recently discovered mitochondria pyruvate carrier, pyruvate dehydrogenase, and pyruvate carboxylase, to modulate overall pyruvate carbon flux. Mutations in any of the genes encoding for proteins regulating pyruvate metabolism may lead to disease. Numerous cases have been described. Aberrant pyruvate metabolism plays an especially prominent role in cancer, heart failure, and neurodegeneration. Because most major diseases involve aberrant metabolism, understanding and exploiting pyruvate carbon flux may yield novel treatments that enhance human health.

Macromolecular crowding effect upon in vitro enzyme kinetics: mixed activation-diffusion control of the oxidation of NADH by pyruvate catalyzed by lactate dehydrogenase.

PubMed

Balcells, Cristina; Pastor, Isabel; Vilaseca, Eudald; Madurga, Sergio; Cascante, Marta; Mas, Francesc

2014-04-17

Enzyme kinetics studies have been usually designed as dilute solution experiments, which differ substantially from in vivo conditions. However, cell cytosol is crowded with a high concentration of molecules having different shapes and sizes. The consequences of such crowding in enzymatic reactions remain unclear. The aim of the present study is to understand the effect of macromolecular crowding produced by dextran of different sizes and at diverse concentrations in the well-known reaction of oxidation of NADH by pyruvate catalyzed by L-lactate dehydrogenase (LDH). Our results indicate that the reaction rate is determined by both the occupied volume and the relative size of dextran obstacles with respect to the enzyme present in the reaction. Moreover, we analyzed the influence of macromolecular crowding on the Michaelis-Menten constants, vmax and Km. The obtained results show that only high concentrations and large sizes of dextran reduce both constants suggesting a mixed activation-diffusion control of this enzymatic reaction due to the dextran crowding action. From our knowledge, this is the first experimental study that depicts mixed activation-diffusion control in an enzymatic reaction due to the effect of crowding.

A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization

PubMed Central

Jiang, Tianyi; Guo, Xiaoting; Yan, Jinxin; Zhang, Yingxin; Wang, Yujiao; Zhang, Manman; Sheng, Binbin; Ma, Cuiqing; Xu, Ping

2017-01-01

ABSTRACT Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10. Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH. IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been

Effect of ornithine and lactate on urea synthesis in isolated hepatocytes.

PubMed Central

Briggs, S; Freedland, R A

1976-01-01

1. In hepatocytes isolated from 24 h-starved rats, urea production from ammonia was stimulated by addition of lactate, in both the presence and the absence of ornithine. The relationship of lactate concentration to the rate of urea synthesis was hyperbolic. 2. Other glucose precursors also stimulated urea production to varying degrees, but none more than lactate. Added oleate and butyrate did not stimulate urea synthesis. 3. Citrulline accumulation was largely dependent on ornithine concentration. As ornithine was increased from 0 to 40 mM, the rate of citrulline accumulation increased hyperbolically, and was half-maximal when ornithine was 8-12 mM. 4. The rate of citrulline accumulation was independent of the presence of lactate, but with pyruvate the rate increased. 5. The rate of urea production continued to increase as ornithine was varied from 0 to 40 mM. 6. It was concluded that intermediates provided by both ornithine and lactate are limiting for urea production from ammonia in isolated liver cells. It was suggested that the stimulatory effect of lactate lies in increased availability of cytosolic aspartate for condensation with citrulline. PMID:1008850

Peroxisomal Alanine: Glyoxylate Aminotransferase AGT1 Is Indispensable for Appressorium Function of the Rice Blast Pathogen, Magnaporthe oryzae

PubMed Central

Bhadauria, Vijai; Banniza, Sabine; Vandenberg, Albert; Selvaraj, Gopalan; Wei, Yangdou

2012-01-01

The role of β-oxidation and the glyoxylate cycle in fungal pathogenesis is well documented. However, an ambiguity still remains over their interaction in peroxisomes to facilitate fungal pathogenicity and virulence. In this report, we characterize a gene encoding an alanine, glyoxylate aminotransferase 1 (AGT1) in Magnaporthe oryzae, the causative agent of rice blast disease, and demonstrate that AGT1 is required for pathogenicity of M. oryzae. Targeted deletion of AGT1 resulted in the failure of penetration via appressoria; therefore, mutants lacking the gene were unable to induce blast symptoms on the hosts rice and barley. This penetration failure may be associated with a disruption in lipid mobilization during conidial germination as turgor generation in the appressorium requires mobilization of lipid reserves from the conidium. Analysis of enhanced green fluorescent protein expression using the transcriptional and translational fusion with the AGT1 promoter and open reading frame, respectively, revealed that AGT1 expressed constitutively in all in vitro grown cell types and during in planta colonization, and localized in peroxisomes. Peroxisomal localization was further confirmed by colocalization with red fluorescent protein fused with the peroxisomal targeting signal 1. Surprisingly, conidia produced by the Δagt1 mutant were unable to form appressoria on artificial inductive surfaces, even after prolonged incubation. When supplemented with nicotinamide adenine dinucleotide (NAD+)+pyruvate, appressorium formation was restored on an artificial inductive surface. Taken together, our data indicate that AGT1-dependent pyruvate formation by transferring an amino group of alanine to glyoxylate, an intermediate of the glyoxylate cycle is required for lipid mobilization and utilization. This pyruvate can be converted to non-fermentable carbon sources, which may require reoxidation of NADH generated by the β-oxidation of fatty acids to NAD+ in peroxisomes

Hyperpolarized 13C lactate-to-bicarbonate ratio as a biomarker for monitoring acute response of anti-VEGF treatment

PubMed Central

Park, Jae Mo; Spielman, Daniel M.; Josan, Sonal; Jang, Taichang; Merchant, Milton; Hurd, Ralph E.; Mayer, Dirk; Recht, Lawrence D.

2016-01-01

Hyperpolarized [1-13C]pyruvate MRS provides a unique imaging opportunity to study reaction kinetics and enzyme activities of in vivo metabolism both because of its favorable imaging characteristics as well as its critical position in the cellular metabolic pathway where it can either be reduced to lactate (reflecting glycolysis) or converted to acetyl-CoA and bicarbonate (reflecting oxidative phosphorylation). Cancer tissue metabolism is altered in such a way as to result in a relative preponderance of glycolysis as compared to oxidative phosphorylation (i.e., Warburg effect). While there is a strong theoretical basis for presuming that readjusting the metabolic balance towards normal could alter tumor growth, a robust noninvasive in vivo tool with which to measure the balance between these two metabolic processes has yet to be developed. Until recently, hyperpolarized 13C-pyruvate imaging studies have focused solely on [1-13C]lactate production because of its strong signal. However, without a concomitant measure of pyruvate entry into the mitochondria, the lactate signal provides no information on the balance between the glycolytic and oxidative metabolic pathways. Consistent measurement of 13C-bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report reliable measurement of 13C-bicarbonate production both in healthy brain and a highly glycolytic experimental glioblastoma model using an optimized 13C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of 13C-lactate-to-bicarbonate provides a more robust metric than does 13C-lactate for assessing the metabolic effects of an anti-angiogenic therapy. Our data suggest a potential application of this ratio as an early biomarker to assess therapeutic effectiveness. Furthermore, although further study is needed, the results suggest that anti-angiogenic treatment results in a rapid

Fast Dynamic 3D MRSI with Compressed Sensing and Multiband Excitation Pulses for Hyperpolarized 13C Studies

PubMed Central

Larson, Peder E. Z.; Hu, Simon; Lustig, Michael; Kerr, Adam B.; Nelson, Sarah J.; Kurhanewicz, John; Pauly, John M.; Vigneron, Daniel B.

2010-01-01

Hyperpolarized 13C MRSI can detect not only the uptake of the pre-polarized molecule but also its metabolic products in vivo, thus providing a powerful new method to study cellular metabolism. Imaging the dynamic perfusion and conversion of these metabolites provides additional tissue information but requires methods for efficient hyperpolarization usage and rapid acquisitions. In this work, we have developed a time-resolved 3D MRSI method for acquiring hyperpolarized 13C data by combining compressed sensing methods for acceleration and multiband excitation pulses to efficiently use the magnetization. This method achieved a 2 sec temporal resolution with full volumetric coverage of a mouse, and metabolites were observed for up to 60 sec following injection of hyperpolarized [1-13C]-pyruvate. The compressed sensing acquisition used random phase encode gradient blips to create a novel random undersampling pattern tailored to dynamic MRSI with sampling incoherency in four (time, frequency and two spatial) dimensions. The reconstruction was also tailored to dynamic MRSI by applying a temporal wavelet sparsifying transform in order to exploit the inherent temporal sparsity. Customized multiband excitation pulses were designed with a lower flip angle for the [1-13C]-pyruvate substrate given its higher concentration than its metabolic products ([1-13C]-lactate and [1-13C]-alanine), thus using less hyperpolarization per excitation. This approach has enabled the monitoring of perfusion and uptake of the pyruvate, and the conversion dynamics to lactate and alanine throughout a volume with high spatial and temporal resolution. PMID:20939089

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine.

PubMed

Borodina, Irina; Kildegaard, Kanchana R; Jensen, Niels B; Blicher, Thomas H; Maury, Jérôme; Sherstyk, Svetlana; Schneider, Konstantin; Lamosa, Pedro; Herrgård, Markus J; Rosenstand, Inger; Öberg, Fredrik; Forster, Jochen; Nielsen, Jens

2015-01-01

Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the β-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of β-alanine and its subsequent conversion into 3HP using a novel β-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Lethal neonatal case and review of primary short-chain enoyl-CoA hydratase (SCEH) deficiency associated with secondary lymphocyte pyruvate dehydrogenase complex (PDC) deficiency.

PubMed

Bedoyan, Jirair K; Yang, Samuel P; Ferdinandusse, Sacha; Jack, Rhona M; Miron, Alexander; Grahame, George; DeBrosse, Suzanne D; Hoppel, Charles L; Kerr, Douglas S; Wanders, Ronald J A

2017-04-01

Mutations in ECHS1 result in short-chain enoyl-CoA hydratase (SCEH) deficiency which mainly affects the catabolism of various amino acids, particularly valine. We describe a case compound heterozygous for ECHS1 mutations c.836T>C (novel) and c.8C>A identified by whole exome sequencing of proband and parents. SCEH deficiency was confirmed with very low SCEH activity in fibroblasts and nearly absent immunoreactivity of SCEH. The patient had a severe neonatal course with elevated blood and cerebrospinal fluid lactate and pyruvate concentrations, high plasma alanine and slightly low plasma cystine. 2-Methyl-2,3-dihydroxybutyric acid was markedly elevated as were metabolites of the three branched-chain α-ketoacids on urine organic acids analysis. These urine metabolites notably decreased when lactic acidosis decreased in blood. Lymphocyte pyruvate dehydrogenase complex (PDC) activity was deficient, but PDC and α-ketoglutarate dehydrogenase complex activities in cultured fibroblasts were normal. Oxidative phosphorylation analysis on intact digitonin-permeabilized fibroblasts was suggestive of slightly reduced PDC activity relative to control range in mitochondria. We reviewed 16 other cases with mutations in ECHS1 where PDC activity was also assayed in order to determine how common and generalized secondary PDC deficiency is associated with primary SCEH deficiency. For reasons that remain unexplained, we find that about half of cases with primary SCEH deficiency also exhibit secondary PDC deficiency. The patient died on day-of-life 39, prior to establishing his diagnosis, highlighting the importance of early and rapid neonatal diagnosis because of possible adverse effects of certain therapeutic interventions, such as administration of ketogenic diet, in this disorder. There is a need for better understanding of the pathogenic mechanisms and phenotypic variability in this relatively recently discovered disorder. Copyright © 2017 Elsevier Inc. All rights reserved.

Multi-timescale analysis of a metabolic network in synthetic biology: a kinetic model for 3-hydroxypropionic acid production via beta-alanine.

PubMed

Dalwadi, Mohit P; King, John R; Minton, Nigel P

2018-07-01

A biosustainable production route for 3-hydroxypropionic acid (3HP), an important platform chemical, would allow 3HP to be produced without using fossil fuels. We are interested in investigating a potential biochemical route to 3HP from pyruvate through [Formula: see text]-alanine and, in this paper, we develop and solve a mathematical model for the reaction kinetics of the metabolites involved in this pathway. We consider two limiting cases, one where the levels of pyruvate are never replenished, the other where the levels of pyruvate are continuously replenished and thus kept constant. We exploit the natural separation of both the time scales and the metabolite concentrations to make significant asymptotic progress in understanding the system without resorting to computationally expensive parameter sweeps. Using our asymptotic results, we are able to predict the most important reactions to maximize the production of 3HP in this system while reducing the maximum amount of the toxic intermediate compound malonic semi-aldehyde present at any one time, and thus we are able to recommend which enzymes experimentalists should focus on manipulating.

Warburg-like Glycolysis and Lactate Shuttle in Mouse Decidua during Early Pregnancy*

PubMed Central

Zuo, Ru-Juan; Gu, Xiao-Wei; Qi, Qian-Rong; Wang, Tong-Song; Zhao, Xu-Yu; Liu, Ji-Long; Yang, Zeng-Ming

2015-01-01

Decidualization is an essential process of maternal endometrial stromal cells to support pregnancy. Although it is known that enhanced glucose influx is critical for decidualization, the underlying mechanism in regulating glucose metabolism in decidua remains insufficiently understood. Here, we demonstrate that aerobic glycolysis-related genes and factors are all substantially induced during decidualization, indicating the existence of Warburg-like glycolysis in decidua. In vitro, progesterone activates hypoxia-inducible factor 1α (Hif1α) and c-Myc through Pi3k-Akt signaling pathway to maintain aerobic glycolysis in decidualizing cells. Knocking down of pyruvate kinase M2 (Pkm2) attenuates the induction of decidual marker gene. Decidual formation in vivo is also impaired by glycolysis inhibitor 3-bromopyruvate. Besides, lactate exporter monocarboxylate transporter 4 (Mct4) is induced in newly formed decidual cells, whereas lactate importer Mct1 and proliferation marker Ki-67 are complementarily located in the surrounding undifferentiated cells, which are supposed to consume lactate for proliferation. Hif1α activation is required for lactate-dependent proliferation of the undifferentiated cells. Inhibition of lactate flux leads to compromised decidualization and decelerated lactate-dependent proliferation. In summary, we reveal that Warburg-like glycolysis and local lactate shuttle are activated in decidua and play important roles for supporting early pregnancy. PMID:26178372

Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart: ROLE OF MITOCHONDRIAL PYRUVATE CARRIER 2 (MPC2) ACETYLATION.

PubMed

Vadvalkar, Shraddha S; Matsuzaki, Satoshi; Eyster, Craig A; Giorgione, Jennifer R; Bockus, Lee B; Kinter, Caroline S; Kinter, Michael; Humphries, Kenneth M

2017-03-17

Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Non-thermal atmospheric pressure plasma activates lactate in Ringer’s solution for anti-tumor effects

NASA Astrophysics Data System (ADS)

Tanaka, Hiromasa; Nakamura, Kae; Mizuno, Masaaki; Ishikawa, Kenji; Takeda, Keigo; Kajiyama, Hiroaki; Utsumi, Fumi; Kikkawa, Fumitaka; Hori, Masaru

2016-11-01

Non-thermal atmospheric pressure plasma is a novel approach for wound healing, blood coagulation, and cancer therapy. A recent discovery in the field of plasma medicine is that non-thermal atmospheric pressure plasma not only directly but also indirectly affects cells via plasma-treated liquids. This discovery has led to the use of non-thermal atmospheric pressure plasma as a novel chemotherapy. We refer to these plasma-treated liquids as plasma-activated liquids. We chose Ringer’s solutions to produce plasma-activated liquids for clinical applications. In vitro and in vivo experiments demonstrated that plasma-activated Ringer’s lactate solution has anti-tumor effects, but of the four components in Ringer’s lactate solution, only lactate exhibited anti-tumor effects through activation by non-thermal plasma. Nuclear magnetic resonance analyses indicate that plasma irradiation generates acetyl and pyruvic acid-like groups in Ringer’s lactate solution. Overall, these results suggest that plasma-activated Ringer’s lactate solution is promising for chemotherapy.

Pyruvate transport by thermogenic-tissue mitochondria.

PubMed

Proudlove, M O; Beechey, R B; Moore, A L

1987-10-15

1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.

Characterization of free and alginate-polylysine-alginate microencapsulated Erwinia herbicola for the conversion of ammonia, pyruvate, and phenol into L-tyrosine

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

Lloyd-George, I.; Chang, T.M.S.

1995-12-20

The whole cell tyrosine phenol-lyase activity of Erwinia herbicola was microencapsulated. The authors studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22 C to 32 C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37 C the cells grow rapidly, but they also enter themore » death phase rapidly. There is only limited growth of E. herbicola at 42 C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 {+-} 100 {micro}m, mean {+-} std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenol-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent K{sub M} of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent K{sub M} for phenol is 0.5 mM. The apparent K{sub M} values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme.« less

Toxicovigilance: new biochemical tool used in sulfonylurea herbicides toxicology studies.

PubMed

Belhadj-Tahar, Hafid; Adamczewski, Nicolas; Nassar, Bertrand; Coulais, Yvon

2003-06-01

In vitro toxic effects of sulfonylurea herbicides (thifensulfuron-methyl and metsulfuron-methyl) were evaluated according to a new protocol. Physiological conditions were reproduced in order to boost toxicovigilance. Sulfonylureas and their hydrolysis products were added to biological substrates such as urea, alanine, aspartic acid, alpha-ketoglutarate, oxaloacetate, pyruvate and then incubated with some specific enzymes. Addition of these sulfonylureas and their degradation products did not significantly change the enzymatic activity of the urease, aspartate-aminotransferase, glutamate dehydrogenase, malate dehydrogenase and lactate dehydrogenase. However, the acid hydrolysis products inhibited up to 95% of the activity of the alanine-aminotransferase at low concentrations (0.27 micromol L(-1)). Inhibition did not affect the mitochondrial aspartate-aminotransferase.

Alterations in carbohydrates and the protein metabolism of the harmful freshwater vector snail Lymnaea acuminata induced by the Euphorbia tirucalli latex extract.

PubMed

Tiwari, Sudhanshu; Singh, A

2005-11-01

To know the short- as well as long-term effect of aqueous latex extracts of Euphorbia tirucalli on carbohydrate and protein metabolism, the snail Lymnaea acuminata was exposed to sublethal doses of 0.37 and 0.55 mg/L for a 24-h and 0.20 and 0.31 mg/L for a 96-h exposure period. Significant (P<0.05) alterations in the glycogen, pyruvate, lactate, total protein, and free amino acid level, as well as in the activity of enzyme lactic dehydrogenase, succinic dehydrogenase, cytochrome oxidase, protease, aspartate aminotransaminase, and alanine aminotransaminase were observed in the nervous, hepatopancreatic, and ovotestis tissues of the freshwater vector snail L. acuminata exposed to sublethal doses of E. tirucalli latex extract. The alterations in all biochemical parameters were significantly (P<0.05) time and dose dependent. After the 7th day of the withdrawal of treatment, there was significant (P<0.05) recovery in glycogen, pyruvate, lactate, total protein, and the free amino acid level and in the activity of the lactic dehydrogenase, succinic dehydrogenase, cytochrome oxidase, protease, aspartate aminotransaminase and alanine aminotransaminase enzymes in all three of the studied tissues of the snail, which supports the view that the plant product is safe for use as a molluscicide for the control of harmful freshwater vector snails in the aquatic environment.

Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress.

PubMed

Rocha, Marcio; Sodek, Ladaslav; Licausi, Francesco; Hameed, Muhammad Waqar; Dornelas, Marcelo Carnier; van Dongen, Joost T

2010-10-01

Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again.

Kinetics of lactate metabolism during acellular normothermic ex vivo lung perfusion.

PubMed

Koike, Terumoto; Yeung, Jonathan C; Cypel, Marcelo; Rubacha, Matthew; Matsuda, Yasushi; Sato, Masaaki; Waddell, Thomas K; Liu, Mingyao; Keshavjee, Shaf

2011-12-01

Plasma lactate has been used as a marker of poor prognosis in clinical conditions. However, the relationship between lactate production and lung function during acellular normothermic ex vivo lung perfusion (EVLP) is unclear. We investigated the kinetics of lactate metabolism during EVLP and the correlation of this marker with outcomes after transplant. Human donor lungs in our clinical EVLP trial (CLs; n = 28) and rejected donor lungs for experimental use (Els; n = 8) were perfused ex vivo using the Toronto technique. Lactate level, lactate/pyruvate (L/P) ratio, and glucose level in the perfusate were measured. In CLs, we examined the relationship between lactate metabolism during EVLP and early post-transplant outcomes. The hypoxia-inducible factor 1 sub-unit 1α (HIF-1α) level in lung tissue was examined in ELs. We performed double-lung EVLP in CLs and single-lung EVLP in ELs. In CLs, the lactate and L/P ratios at the end of EVLP had no correlation with early post-transplant outcomes despite lactate elevation during EVLP. Although lactate elevation was also present in all ELs, we were able to identify 2 groups based on L/P ratio at the end of EVLP. The group with the high L/P ratio had higher airway pressure during EVLP and higher HIF-1α in lung tissue at the end of EVLP. Lactate increases seen in the EVLP perfusate most often represent physiologic lactate production by the lung in a setting with reduced lactate clearance. Thus, patients who underwent transplantation after EVLP had good outcomes despite lactate elevation during EVLP. Copyright © 2011 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

Conformational heterogeneity within the Michaelis complex of lactate dehydrogenase†

PubMed Central

Deng, Hua; Vu, Dung V.; Clinch, Keith; Desamero, Ruel; Dyer, R. Brian; Callender, Robert

2011-01-01

A series of isotope edited IR measurements, both static as well as temperature jump relaxation spectroscopy, are performed on lactate dehydrogenase (LDH) to determine the ensemble of structures available to its Michaelis complex. There clearly has been a substantial reduction in the number of states available to the pyruvate substrate (as modeled by the substrate mimic, oxamate) and NADH when bound to protein compared to dissolved in solution, as determined by the bandwidths and positions of the critical C2=O band of bound substrate mimic and the C4-H stretch of NADH reduced nicotinamide group. Moreover, it is found that a strong ionic bond (characterized by a signature IR band discovered in this study) is formed between the carboxyl group of bound pyruvate with (presumably) Arg171, forming a strong ‘anchor’ within the protein matrix. However, conformational heterogeneity within the Michaelis complex is found that has an impact on both catalytic efficiency and thermodynamics of the enzyme. PMID:21568287

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

PubMed

Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2013-02-14

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Lactate as a Metabolite and a Regulator in the Central Nervous System

PubMed Central

Proia, Patrizia; Di Liegro, Carlo Maria; Schiera, Gabriella; Fricano, Anna; Di Liegro, Italia

2016-01-01

More than two hundred years after its discovery, lactate still remains an intriguing molecule. Considered for a long time as a waste product of metabolism and the culprit behind muscular fatigue, it was then recognized as an important fuel for many cells. In particular, in the nervous system, it has been proposed that lactate, released by astrocytes in response to neuronal activation, is taken up by neurons, oxidized to pyruvate and used for synthesizing acetyl-CoA to be used for the tricarboxylic acid cycle. More recently, in addition to this metabolic role, the discovery of a specific receptor prompted a reconsideration of its role, and lactate is now seen as a sort of hormone, even involved in processes as complex as memory formation and neuroprotection. As a matter of fact, exercise offers many benefits for our organisms, and seems to delay brain aging and neurodegeneration. Now, exercise induces the production and release of lactate into the blood which can reach the liver, the heart, and also the brain. Can lactate be a beneficial molecule produced during exercise, and offer neuroprotection? In this review, we summarize what we have known on lactate, discussing the roles that have been attributed to this molecule over time. PMID:27598136

Pyruvate metabolism in castor-bean mitochondria.

PubMed Central

Brailsford, M A; Thompson, A G; Kaderbhai, N; Beechey, R B

1986-01-01

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation. PMID:3814077

Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

PubMed

Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

2011-11-22

Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.

Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose

PubMed Central

Wang, Qingzhao; Ingram, Lonnie O.; Shanmugam, K. T.

2011-01-01

Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(−)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L-1 of optically pure D(−)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min-1 (mg protein)-1. By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(−) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase*

PubMed Central

Ikehara, Yoko; Arai, Kazuhito; Furukawa, Nayuta; Ohno, Tadashi; Miyake, Tatsuya; Fushinobu, Shinya; Nakajima, Masahiro; Miyanaga, Akimasa; Taguchi, Hayao

2014-01-01

For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S0.5 value 103-fold and increased the Vmax value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172–185) and MR2 (positions 211–221), form a compact core for allosteric motion, and His179 of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased Vmax 4-fold but reduced pyruvate S0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase Vmax, but 102-reduced pyruvate S0.5, and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule. PMID:25258319

Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

PubMed Central

Reed, L J; Pettit, F H; Eley, M H; Hamilton, L; Collins, J H; Oliver, R M

1975-01-01

The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube. Images PMID:1103138

Inhibitory effects of ethyl pyruvate on platelet aggregation and phosphatidylserine exposure.

PubMed

Li, Wenjin; Yang, Xinyu; Peng, Minyuan; Li, Can; Mu, Guangfu; Chen, Fangping

2017-06-03

Ethyl pyruvate (EP) is a stable lipophilic pyruvate derivative. Studies demonstrated that EP shows potent anti-oxidation, anti-inflammatory and anti-coagulant effects. Inflammation and coagulation are closely interacted with platelet activation. However, it is unclear whether EP has anti-platelet effects. Therefore, we investigated the anti-platelet effect of EP in this study in vitro. We found that EP inhibited agonists induced platelets aggregation, ATP release and adhesion to collagen. Flow cytometric analysis revealed that EP inhibited agonist induced platelets PAC-1 binding, as well as P-selectin and CD40L expression. The underlying mechanism of action may involve the inhibition of platelet PI3K/Akt and Protein Kinase C (PKC) signaling pathways. Additionally, EP dose dependently inhibited platelet PS exposure induced by high concentration thrombin. Lactate dehydrogenase (LDH) activity assay and mice platelet count implied that EP may have no toxic effect on platelets. Therefore, we are the first to report that EP has potent anti-platelet activity and attenuates platelet PS exposure in vitro, suggesting that the inhibitory effects of EP on platelets may also play important roles in improvement of inflammation and coagulation disorder in related animal models. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel mode of lactate metabolism in strictly anaerobic bacteria.

PubMed

Weghoff, Marie Charlotte; Bertsch, Johannes; Müller, Volker

2015-03-01

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E0 ' = -190 mV excludes direct NAD(+) reduction (E0 ' = -320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron-transferring flavoprotein (Etf) that exhibited NAD(+) reduction only when reduced ferredoxin (Fd(2-) ) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin-based electron confurcation to drive endergonic lactate oxidation with NAD(+) as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E0 ' ≈ -500 mV) to NAD(+) according to: lactate + Fd(2-)  + 2 NAD(+)  → pyruvate + Fd + 2 NADH. The reduced Fd(2-) is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical ( Δ μ ˜ Na + ) and finally redox energy (Fd(2-) from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Increased Interstitial Concentrations of Glutamate and Pyruvate in Vastus Lateralis of Women with Fibromyalgia Syndrome Are Normalized after an Exercise Intervention – A Case-Control Study

PubMed Central

Gerdle, Björn; Ernberg, Malin; Mannerkorpi, Kaisa; Larsson, Britt; Kosek, Eva; Christidis, Nikolaos; Ghafouri, Bijar

2016-01-01

Background Fibromyalgia syndrome (FMS) is associated with central alterations, but controversies exist regarding the presence and role of peripheral factors. Microdialysis (MD) can be used in vivo to study muscle alterations in FMS. Furthermore for chronic pain conditions such as FMS, the mechanisms for the positive effects of exercise are unclear. This study investigates the interstitial concentrations of algesics and metabolites in the vastus lateralis muscle of 29 women with FMS and 28 healthy women before and after an exercise intervention. Methods All the participants went through a clinical examination and completed a questionnaire. In addition, their pressure pain thresholds (PPTs) in their upper and lower extremities were determined. For both groups, MD was conducted in the vastus lateralis muscle before and after a 15-week exercise intervention of mainly resistance training of the lower limbs. Muscle blood flow and interstitial muscle concentrations of lactate, pyruvate, glutamate, glucose, and glycerol were determined. Results FMS was associated with significantly increased interstitial concentrations of glutamate, pyruvate, and lactate. After the exercise intervention, the FMS group exhibited significant decreases in pain intensity and in mean interstitial concentrations of glutamate, pyruvate, and glucose. The decrease in pain intensity in FMS correlated significantly with the decreases in pyruvate and glucose. In addition, the FMS group increased their strength and endurance. Conclusion This study supports the suggestion that peripheral metabolic and algesic muscle alterations are present in FMS patients and that these alterations contribute to pain. After an exercise intervention, alterations normalized, pain intensity decreased (but not abolished), and strength and endurance improved, all findings that suggest the effects of exercise are partially peripheral. PMID:27695113

Lactate Chemical Exchange Saturation Transfer (LATEST) Imaging in vivo A Biomarker for LDH Activity.

PubMed

DeBrosse, Catherine; Nanga, Ravi Prakash Reddy; Bagga, Puneet; Nath, Kavindra; Haris, Mohammad; Marincola, Francesco; Schnall, Mitchell D; Hariharan, Hari; Reddy, Ravinder

2016-01-22

Non-invasive imaging of lactate is of enormous significance in cancer and metabolic disorders where glycolysis dominates. Here, for the first time, we describe a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) method (LATEST), based on the exchange between lactate hydroxyl proton and bulk water protons to image lactate with high spatial resolution. We demonstrate the feasibility of imaging lactate with LATEST in lactate phantoms under physiological conditions, in a mouse model of lymphoma tumors, and in skeletal muscle of healthy human subjects pre- and post-exercise. The method is validated by measuring LATEST changes in lymphoma tumors pre- and post-infusion of pyruvate and correlating them with lactate determined from multiple quantum filtered proton magnetic resonance spectroscopy (SEL-MQC (1)H-MRS). Similarly, dynamic LATEST changes in exercising human skeletal muscle are correlated with lactate determined from SEL-MQC (1)H-MRS. The LATEST method does not involve injection of radioactive isotopes or labeled metabolites. It has over two orders of magnitude higher sensitivity compared to conventional (1)H-MRS. It is anticipated that this technique will have a wide range of applications including diagnosis and evaluation of therapeutic response of cancer, diabetes, cardiac, and musculoskeletal diseases. The advantages of LATEST over existing methods and its potential challenges are discussed.

Lactate Chemical Exchange Saturation Transfer (LATEST) Imaging in vivo A Biomarker for LDH Activity

PubMed Central

DeBrosse, Catherine; Nanga, Ravi Prakash Reddy; Bagga, Puneet; Nath, Kavindra; Haris, Mohammad; Marincola, Francesco; Schnall, Mitchell D.; Hariharan, Hari; Reddy, Ravinder

2016-01-01

Non-invasive imaging of lactate is of enormous significance in cancer and metabolic disorders where glycolysis dominates. Here, for the first time, we describe a chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) method (LATEST), based on the exchange between lactate hydroxyl proton and bulk water protons to image lactate with high spatial resolution. We demonstrate the feasibility of imaging lactate with LATEST in lactate phantoms under physiological conditions, in a mouse model of lymphoma tumors, and in skeletal muscle of healthy human subjects pre- and post-exercise. The method is validated by measuring LATEST changes in lymphoma tumors pre- and post-infusion of pyruvate and correlating them with lactate determined from multiple quantum filtered proton magnetic resonance spectroscopy (SEL-MQC 1H-MRS). Similarly, dynamic LATEST changes in exercising human skeletal muscle are correlated with lactate determined from SEL-MQC 1H-MRS. The LATEST method does not involve injection of radioactive isotopes or labeled metabolites. It has over two orders of magnitude higher sensitivity compared to conventional 1H-MRS. It is anticipated that this technique will have a wide range of applications including diagnosis and evaluation of therapeutic response of cancer, diabetes, cardiac, and musculoskeletal diseases. The advantages of LATEST over existing methods and its potential challenges are discussed. PMID:26794265

Enhanced pyruvate production in Candida glabrata by carrier engineering.

PubMed

Luo, Zhengshan; Liu, Song; Du, Guocheng; Xu, Sha; Zhou, Jingwen; Chen, Jian

2018-02-01

Pyruvate is an important organic acid that plays a key role in the central metabolic pathway. Manipulating transporters is an efficient strategy to enhance production of target organic acids and a means to understand the effects of altered intracellular pyruvate content on global metabolic networks. Efforts have been made to manipulate mitochondrial pyruvate carrier (MPC) to transport pyruvate into different subcellular compartments in Candida glabrata to demonstrate the effects of the subcellular distribution of pyruvate on central carbon metabolism. By increasing the mitochondrial pyruvate content through enhancing the rate of pyruvate transport into mitochondria, a high central carbon metabolism rate, specific growth rate and specific pyruvate production rate were obtained. Comparing the intracellular pyruvate content of engineered and control strains showed that higher intracellular pyruvate levels were not conducive to improving pyruvate productivity or central carbon metabolism. Plasma membrane expression of MPCs significantly increased the expression levels of key rate-limiting glycolytic enzymes. Moreover, pyruvate production of CGΔura3-Sp-MPC1, CGΔura3-Sp-MPC2, and CGΔura3-Sp-MPC1-Sp-MPC2 increased 134.4%, 120.3%, and 30.0%, respectively. In conclusion, lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms. © 2017 Wiley Periodicals, Inc.

Insulin-induced inhibition of gluconeogenesis genes, including glutamic pyruvic transaminase 2, is associated with reduced histone acetylation in a human liver cell line.

PubMed

Honma, Kazue; Kamikubo, Michiko; Mochizuki, Kazuki; Goda, Toshinao

2017-06-01

Hepatic glutamic pyruvic transaminase (GPT; also known as alanine aminotransferase) is a gluconeogenesis enzyme that catalyzes conversions between alanine and pyruvic acid. It is also used as a blood biomarker for hepatic damage. In this study, we investigated whether insulin regulates GPT expression, as it does for other gluconeogenesis genes, and if this involves the epigenetic modification of histone acetylation. Human liver-derived HepG2 cells were cultured with 0.5-100nM insulin for 8h, and the mRNA expression of GPT, glutamic-oxaloacetic transaminase (GOT), γ-glutamyltransferase (GGT), PCK1, G6PC and FBP1 was measured. We also investigated the extent of histone acetylation around these genes. Insulin suppressed the mRNA expression of gluconeogenesis genes (GPT2, GOT1, GOT2, GGT1, GGT2, G6PC, and PCK1) in HepG2 cells in a dose-dependent manner. mRNA levels of GPT2, but not GPT1, were decreased by insulin. Histone acetylation was also reduced around GPT2, G6PC, and PCK1 in response to insulin. The expression of GPT2 and other gluconeogenesis genes such as G6PC and PCK1 was suppressed by insulin, in association with decreases in histone H3 and H4 acetylation surrounding these genes. Copyright © 2017 Elsevier Inc. All rights reserved.

Atmospheric measurements of pyruvic and formic acid

NASA Technical Reports Server (NTRS)

Andreae, Meinrat O.; Li, Shao-Meng; Talbot, Robert W.

1987-01-01

Pyruvic acid, a product of the atmospheric oxidation of cresols and probably of isoprene, has been determined together with formic acid in atmospheric aerosols and rain as well as in the vapor phase. Both acids are present predominantly as vapor; only about 10-20 percent of the total atmospheric pyruvate and 1-2 percent of the total formate are in the particulate phase. The concentrations of pyruvic and formic acid are highly correlated, with typical formic-to-pyruvic ratios of 10-30 in the gas phase, 20-30 in rain, and 2-10 in aerosols. The gas-phase and rain ratios are comparable to those predicted to result from isoprene oxidation. Pyruvic acid levels were similar in the eastern United States (during summer) and the Amazon Basin, suggesting that natural processes, particularly the photochemical oxidation of isoprene, could account for most of the pyruvic acid present in the atmosphere.

An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor.

PubMed

Yuan, Meng; McNae, Iain W; Chen, Yiyuan; Blackburn, Elizabeth A; Wear, Martin A; Michels, Paul A M; Fothergill-Gilmore, Linda A; Hupp, Ted; Walkinshaw, Malcolm D

2018-05-10

We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer-monomer dissociation K d is estimated to be ~0.9 µM with a slow dissociation rate (t 1/2 ~ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R-states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such 'allostatic' regulation may be important in metabolic reprogramming and influencing cell fate. ©2018 The Author(s).

An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor

PubMed Central

McNae, Iain W.; Chen, Yiyuan; Blackburn, Elizabeth A.; Wear, Martin A.; Hupp, Ted

2018-01-01

We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that, within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors, while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer–monomer dissociation Kd is estimated to be ∼0.9 µM with a slow dissociation rate (t1/2 ∼ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such ‘allostatic’ regulation may be important in metabolic reprogramming and influencing cell fate. PMID:29748232

Adiposity and Fat Metabolism in Lactating and Fasting Northern Elephant Seals12

PubMed Central

Crocker, Daniel E.; Champagne, Cory D.; Fowler, Melinda A.; Houser, Dorian S.

2014-01-01

Several taxa of animals fast completely from food and water during energy-intensive periods such as lactation, breeding, and development. In elephant seals, these behaviors are sustained by high adiposity, high rates of fat mobilization, and reduced oxidation of carbohydrates and proteins. Adiposity and the regulation of lipolysis directly affect lactation energetics, milk composition, and mating success. Long-term fasting induces changes in regulation of lipolysis and lipid metabolism that influence fatty acid (FA) availability and the onset of insulin resistance. Hypoinsulinemia and elevated circulating FAs are also associated with several unique features of carbohydrate metabolism, including elevated plasma glucose, gluconeogenesis, and Cori cycle activity as well as high rates of pyruvate and tricarboxylic acid cycling. Glucose-lactate pools and triacylglycerol-FA cycles may be linked via glyceroneogenesis and this may be an important pathway influencing both fat and carbohydrate metabolism. Together, these features allow a sustained, high intensity, fat-based metabolism without substantial accumulation of ketoacids. PMID:24425723

Lactate production as representative of the fermentation potential of Corynebacterium glutamicum 2262 in a one-step process.

PubMed

Khuat, Hoang Bao Truc; Kaboré, Abdoul Karim; Olmos, Eric; Fick, Michel; Boudrant, Joseph; Goergen, Jean-Louis; Delaunay, Stéphane; Guedon, Emmanuel

2014-01-01

The fermentative properties of thermo-sensitive strain Corynebacterium glutamicum 2262 were investigated in processes coupling aerobic cell growth and the anaerobic fermentation phase. In particular, the influence of two modes of fermentation on the production of lactate, the fermentation product model, was studied. In both processes, lactate was produced in significant amount, 27 g/L in batch culture, and up to 55.8 g/L in fed-batch culture, but the specific production rate in the fed-batch culture was four times lower than that in the batch culture. Compared to other investigated fermentation processes, our strategy resulted in the highest yield of lactic acid from biomass. Lactate production by C. glutamicum 2262 thus revealed the capability of the strain to produce various fermentation products from pyruvate.

Protective effects of ethyl pyruvate in cisplatin-induced nephrotoxicity

PubMed Central

Kelle, Ilker; Akkoc, Hasan; Tunik, Selcuk; Nergiz, Yusuf; Erdinc, Meral; Erdinc, Levent

2014-01-01

This study was performed to investigate the effect of ethyl pyruvate on changes in renal functions and oxidative stress related renal injury caused by cisplatin (cis-dichlorodiammine platinum-II; CDDP). Male Wistar albino rats were divided into four groups (n = 8): (1) control group (1 ml Ringer's lactate solution i.p.); (2) ethyl pyruvate (EP) group (50 mg/kg Ringer's EP solution (REPS) i.p.); (3) cisplatin group (a single dose of cisplatin (5 mg/kg, i.p.); and (4) cisplatin + EP group (a single dose of cisplatin (5 mg/kg, i.p.) + REPS 50 mg/kg/day, i.p.) for five days. At the sixth day, kidneys of rats were mounted to a Langendorff apparatus. Renal perfusion pressures were recorded. Blood samples were taken for serum urea, creatinine, total oxidant status (TOS), total antioxidant status (TAS) and oxidative stres index (OSI) evaluations. Kidney tissues were obtained for malondialdehyde (MDA) analyses and histopathological examination. Perfusion pressures, serum urea, creatinine, TOS, OSI and tissue MDA levels were found significantly higher, whereas TAS was notably lower in cisplatin group. Histopathological examination showed apparent renal paranchymal injury in cisplatin group. In cisplatin + REPS group, perfusion pressures, serum urea, creatinine and tissue MDA levels were decreased. Moreover, EP co-administration provided less inflammatory cell infiltration, tubular dilatation, whereas TOS, TAS and OSI improved significantly versus cisplatin group. These findings show that EP has protective effects against cisplatin nephrotoxicity. PMID:26019553

Stimulation of pyruvate transport in metabolizing mitochondria through changes in the transmembrane pH gradient induced by glucagon treatment of rats.

PubMed

Halestrap, A P

1978-06-15

Glucagon treatment of rats allowed the isolation of liver mitochondria with enhanced rates of pyruvate metabolism measured in either sucrose or KCl media. No change in the activity of the pyruvate carrier itself was apparent, but under metabolizing conditions, use of the inhibitor of pyruvate transport, alpha-cyano-4-hydroxycinnamate, demonstrated that pyruvate transport limited the rate of pyruvate metabolism. The maximum rate of transport under metabolizing conditions was enhanced by glucagon treatment. Problems involved in measuring the transmembrane pH gradient under metabolizing conditions are discussed and a variety of techniques are used to estimate the matrix pH. From the distribution of methylamine, ammonia and D-lactate and the Ki for inhibition by alpha-cyano-4-hydroxycinnamate it is concluded that the matrix is more acid than the medium and that the pH of the matrix rises after glucagon treatment. The increase in matrix pH stimulates pyruvate transport. The membrane potential, ATP concentration and O2 uptake were also increased under metabolizing conditions in glucagon-treated mitochondria. These changes were correlated with a stimulation of the respiratory chain which can be observed in uncoupled mitochondria [Yamazaki (1975) J. Biol. Chem. 250, 7924--7930]. The mitochondrial Mg2+ content (mean +/- S.E.M.) was increased from 38.8 +/- 1.2 (n = 26) to 47.5 +/- 2.0 (n = 26) ng-atoms/mg by glucagon and the K+ content from 126.7 +/- 10.3 (n = 19) ng-atoms/mg. This may represent a change in membrane potential induced by glucagon in vivo. The physiological significance of these results in the control of gluconeogenesis is discussed.

The effect of extracellular alkalinization on lactate metabolism of breast cancer stem cells: Overview of LDH-A, LDH-B, MCT1 and MCT4 gene expression

NASA Astrophysics Data System (ADS)

Neolaka, G. M. G.; Yustisia, I.; Sadikin, M.; Wanandi, S. I.

2017-08-01

Changes in the metabolic status of cancer cells are presumed to be correlated with the adjustment of these cells to extracellular changes. Cell glycolysis increases the production of intracellular lactate catalyzed by the lactate dehydrogenases, both LDH-A and LDH-B. An increase in intracellular lactate can affect extracellular pH balance through monocarboxylate transporters, particularly MCT1 and MCT4. This study aimed to analyze the effects of extracellular alkalinization on the lactate metabolism of human breast cancer stem cells (BCSCs). In this study, human primary BCSCs (CD24-/CD44+ cells) were treated with 100 mM sodium bicarbonate for 0.5, 24, and 48 h in DMEM F12/HEPES. After incubation, extracellular pH was measured and cells were harvested to extract the total RNA and protein. The expression of LDH-A, LDH-B, MCT1, and MCT4 mRNA genes were analyzed using qRT-PCR method. Our study shows that administration of sodium bicarbonate in the BCSC culture medium could increase extracellular pH. To balance the increase of extracellular pH, BCSCs regulated the expression of LDH-A, LDH-B, MCT1, and MCT4 genes. As the extracellular pH increases, the expression of LDH-A that converts pyruvate to lactate increased along with the increase of MCT 4 and MCT 1 expression, which act as lactate transporters. As the incubation time increases, the pH decreases, leading to the suppression of LDH-A and increase of LDH-B expression that converts lactate into pyruvate. Therefore, we suggest that the extracellular alkalinization by sodium bicarbonate in BCSCs affected the genes that regulate lactate metabolism.

Identification of regions of rabbit muscle pyruvate kinase important for allosteric regulation by phenylalanine, detected by H/D exchange mass spectrometry†

PubMed Central

Prasannan, Charulata B.; Villar, Maria T.; Artigues, Antonio; Fenton, Aron W.

2013-01-01

Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a non-allosteric analogue of the inhibitor. Alanine is used as the non-allosteric analogue since it binds competitively with phenylalanine, but elicits a negligible allosteric inhibition, i.e. a negligible reduction of the affinity of rM1-PYK for the substrate, phosphoenolpyruvate (PEP). This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e. those changes common upon the addition of alanine vs. phenylalanine) from changes associated with allosteric regulation (i.e. those elicited by the addition of phenylalanine binding, but not alanine binding). High quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique include residues previously identified by mutagenesis to have roles in the allosteric regulation by phenylalanine. PMID:23418858

Functional Characterization of Alanine Racemase from Schizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase

PubMed Central

Uo, Takuma; Yoshimura, Tohru; Tanaka, Naotaka; Takegawa, Kaoru; Esaki, Nobuyoshi

2001-01-01

Schizosaccharomyces pombe has an open reading frame, which we named alr1+, encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1+ gene in Escherichia coli and purified the gene product (Alr1p), with an Mr of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent Km and Vmax values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of l-alanine, respectively. S. pombe uses d-alanine as a sole nitrogen source, but deletion of the alr1+ gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for l-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but not d-alanine as a sole nitrogen source. Moreover, d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1+ gene enabled S. cerevisiae to grow efficiently on d-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine. PMID:11244061

Genetics Home Reference: pyruvate dehydrogenase deficiency

MedlinePlus

... form that cells can use. The pyruvate dehydrogenase complex converts a molecule called pyruvate, which is formed from the breakdown of carbohydrates, into another molecule called acetyl-CoA. This conversion ...

Lactate production by swine adipocytes: effects of age, nutritional status, glucose concentration, and insulin.

PubMed

Heckler, B K; Carey, G B

1997-06-01

To develop an alternative model in which to study the relationship between adipose tissue lactate production, obesity, and non-insulin-dependent diabetes mellitus (NIDDM), we investigated lactate production by swine adipocytes. Subcutaneous adipocytes from fasted 3-wk-old, fasted 7-mo-old, and fed 7-mo-old Yucatan minIature swine were isolated and incubated with 0.2, 1, 5, 10, or 25 mM glucose +/- 1 mU/ml insulin. Total glucose metabolism (TGM) was estimated by product summation. Results showed that 1) TGM was threefold greater in cells from fasted 7-mo- vs. 3-wk-old swine (P < 0.05), 2) TGM was 2.7-fold greater in cells from fed 7-mo-old vs. fasted 7-mo-old swine (P < 0.05), 3) insulin failed to stimulate TGM in adipocytes from swine of either age and either nutritional status, and 4) lactate and pyruvate accounted for 34 and 30% of TGM, respectively, in adipocytes from swine of both ages. Similarities in glucose metabolism and lactate production in adipocytes from swine and obese NIDDM humans make the swine a potentially valuable model for studying lactate production associated with obesity and NIDDM.

Metabolic Response to Heat Stress in Late-Pregnant and Early Lactation Dairy Cows: Implications to Liver-Muscle Crosstalk.

PubMed

Koch, Franziska; Lamp, Ole; Eslamizad, Mehdi; Weitzel, Joachim; Kuhla, Björn

2016-01-01

Climate changes lead to rising temperatures during summer periods and dramatic economic losses in dairy production. Modern high-yielding dairy cows experience severe metabolic stress during the transition period between late gestation and early lactation to meet the high energy and nutrient requirements of the fetus or the mammary gland, and additional thermal stress during this time has adverse implications on metabolism and welfare. The mechanisms enabling metabolic adaptation to heat apart from the decline in feed intake and milk yield are not fully elucidated yet. To distinguish between feed intake and heat stress related effects, German Holstein dairy cows were first kept at thermoneutral conditions at 15°C followed by exposure to heat-stressed (HS) at 28°C or pair-feeding (PF) at 15°C for 6 days; in late-pregnancy and again in early lactation. Liver and muscle biopsies and plasma samples were taken to assess major metabolic pathway regulation using real-time PCR and Western Blot. The results indicate that during heat stress, late pregnant cows activate Cahill but reduce Cori cycling, prevent increase in skeletal muscle fatty acid oxidation, and utilize increased amounts of pyruvate for gluconeogenesis, without altering ureagenesis despite reduced plane of nutrition. These homeorhetic adaptations are employed to reduce endogenous heat production while diverting amino acids to the growing fetus. Metabolic adaptation to heat stress in early lactation involves increased long-chain fatty acid degradation in muscle peroxisomes, allowance for muscle glucose utilization but diminished hepatic use of amino acid-derived pyruvate for gluconeogenesis and reduced peroxisomal fatty acid oxidation and ATP production in liver of HS compared to PF cows in early lactation. Consequently, metabolic adaptation to heat stress and reduced feed intake differ between late pregnancy and early lactation of dairy cows to maintain energy supply for fetus development or milk production

Metabolic Response to Heat Stress in Late-Pregnant and Early Lactation Dairy Cows: Implications to Liver-Muscle Crosstalk

PubMed Central

Eslamizad, Mehdi; Weitzel, Joachim; Kuhla, Björn

2016-01-01

Climate changes lead to rising temperatures during summer periods and dramatic economic losses in dairy production. Modern high-yielding dairy cows experience severe metabolic stress during the transition period between late gestation and early lactation to meet the high energy and nutrient requirements of the fetus or the mammary gland, and additional thermal stress during this time has adverse implications on metabolism and welfare. The mechanisms enabling metabolic adaptation to heat apart from the decline in feed intake and milk yield are not fully elucidated yet. To distinguish between feed intake and heat stress related effects, German Holstein dairy cows were first kept at thermoneutral conditions at 15°C followed by exposure to heat-stressed (HS) at 28°C or pair-feeding (PF) at 15°C for 6 days; in late-pregnancy and again in early lactation. Liver and muscle biopsies and plasma samples were taken to assess major metabolic pathway regulation using real-time PCR and Western Blot. The results indicate that during heat stress, late pregnant cows activate Cahill but reduce Cori cycling, prevent increase in skeletal muscle fatty acid oxidation, and utilize increased amounts of pyruvate for gluconeogenesis, without altering ureagenesis despite reduced plane of nutrition. These homeorhetic adaptations are employed to reduce endogenous heat production while diverting amino acids to the growing fetus. Metabolic adaptation to heat stress in early lactation involves increased long-chain fatty acid degradation in muscle peroxisomes, allowance for muscle glucose utilization but diminished hepatic use of amino acid-derived pyruvate for gluconeogenesis and reduced peroxisomal fatty acid oxidation and ATP production in liver of HS compared to PF cows in early lactation. Consequently, metabolic adaptation to heat stress and reduced feed intake differ between late pregnancy and early lactation of dairy cows to maintain energy supply for fetus development or milk production

Identification and characterisation of a new class of highly specific and potent inhibitors of the mitochondrial pyruvate carrier.

PubMed

Hildyard, John C W; Ammälä, Carina; Dukes, Iain D; Thomson, Stephen A; Halestrap, Andrew P

2005-01-01

Two novel thiazolidine compounds, GW604714X and GW450863X, were found to be potent inhibitors of mitochondrial respiration supported by pyruvate but not other substrates. Direct measurement of pyruvate transport into rat liver and yeast mitochondria confirmed that these agents inhibited the mitochondrial pyruvate carrier (MPC) with K(i) values <0.1 muM. Inhibitor titrations of pyruvate-dependent respiration by heart mitochondria gave values (+/-S.E.) for the concentration of inhibitor binding sites (pmol per mg protein) and their K(i) (nM) of 56.0+/-0.9 and 0.057+/-0.010 nM for the more hydrophobic GW604714X; for GW450863X the values were 59.9+/-4.6 and 0.60+/-0.12 nM. [(3)H]-methoxy-GW450863X binding was also used to determine the MPC content of the heart, kidney, liver and brain mitochondria giving values of 56, 40, 26 and 20 pmol per mg protein respectively. Binding to yeast mitochondria was <10% of that in rat liver mitochondria, consistent with the slow rate of pyruvate transport into yeast mitochondria. [(3)H]-methoxy-GW450863X binding was inhibited by GW604714X and by the established MPC inhibitor, UK5099. The absorbance spectra of GW450863X and GW604714X were markedly changed by the addition of beta-mercaptoethanol suggesting that the novel inhibitors, like alpha-cyanocinnamate, possess an activated double bond that attacks a critical cysteine residue on the MPC. However, no labelled protein was detected following SDS-PAGE suggesting that the covalent modification is reversible. GW604714X and GW450863X inhibited l-lactate transport by the plasma membrane monocarboxylate transporter MCT1, but at concentrations more than four orders of magnitude greater than the MPC.

Alanine synthesis from glyceraldehyde and ammonium ion in aqueous solution

NASA Technical Reports Server (NTRS)

Weber, A. L.

1985-01-01

The formation of alanine (ala) form C(14)-glyceraldehyde and ammonium phosphate in the presence or absence of a thiol is reported. At ambient temperature, ala synthesis was six times more rapid in the presence of 3-mercaptopropionic acid than in its absence (0.6 and 0.1 percent, respectively, after 60 days). Similarly, the presence of another thiol, N-acetylcysteinate, increased the production of ala, as well as of lactate. The reaction pathway of thiol-catalyzed synthesis of ala, with the lactic acid formed in a bypath, is suggested. In this, dehydration of glyceraldehyde is followed by the formation of hemithioacetal. In the presence of ammonia, an imine is formed, which eventually yields ala. This pathway is consistent with the observation that the rate ratio of ala/lactate remains constant throughout the process. The fact that the reaction takes place under anaerobic conditions in the presence of H2O and with the low concentrations of simple substrates and catalysts makes it an attractive model prebiotic reaction in the process of molecular evolution.

Elevated CSF-lactate is a reliable marker of mitochondrial disorders in children even after brief seizures.

PubMed

Magner, Martin; Szentiványi, Karol; Svandová, Ivana; Ješina, Pavel; Tesařová, Markéta; Honzík, Tomáš; Zeman, Jiří

2011-03-01

Increased lactate is an important biochemical marker in diagnosis of children with suspicion of mitochondrial disorders. A diagnostic dilemma may originate if analyses are performed after seizures, when the increased lactate levels may be considered to result from the seizures. To address this problem, we ascertained the diagnostic value of lactate and alanine in blood (B) and cerebrospinal fluid (CSF) in children with mitochondrial disorders (n = 24), epilepsy (n = 32), psychomotor retardation (n = 23), meningitis (n = 12) and meningism (n = 16). Lactate concentration was measured using a spectrophotometric method. Amino acids in serum and CSF were analyzed by ion exchange chromatography with ninhydrin detection. Average blood and CSF-lactate levels were significantly higher in children with mitochondrial disorders (3.87 ± 0.48 and 4.43 ± 0.55 mmol/l) and meningitis (2.77 ± 0.45 and 8.58 ± 1.08 mmol/l) than in children with epilepsy (1.72 ± 0.13 and 1.62 ± 0.04 mmol/l), psychomotor retardation (1.79 ± 1.40 and 1.68 ± 0.06 mmol/l) or meningism (1.70 ± 0.13 and 1.64 ± 0.07 mmol/l). Blood and CSF-alanine levels were also higher in children with mitochondrial disorders (558 ± 44 and 51 ± 8 μmol/l) than in children with epilepsy (327 ± 23 and 27 ± 3 μmol/l) or psychomotor retardation (323 ± 27 and 26 ± 3 μmol/l). The CSF-lactate levels of children with epilepsy were similar whether the samples were obtained 3 ± 0.6 h after an attack of brief seizures or from children without history of recent seizures. Elevated cerebrospinal fluid lactate level is a reliable marker pointing to mitochondrial origin of disease, even in children who have recently suffered short-lasting seizures. Some children with mitochondrial disorders manifest only mild or intermittent elevation of lactate levels. Copyright © 2010 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Enzyme-coated microelectrodes to monitor lactate production in a nanoliter microfluidic cell culture device

PubMed Central

Ges, Igor A.; Baudenbacher, Franz

2015-01-01

Monitoring the degree of anaerobic respiration of cells in high density microscale culture systems is an enabling key technology and essential for cell-based biosensors. We have fabricated and incorporated miniature amperometric lactate sensing electrodes with working areas from 3 to 5×10−2 mm2 into a microfluidic-based microscale cell culture system to measure the lactate production rate of fibroblasts in nanoliter volumes. Planar thin film platinum electrode arrays on glass substrates were spin coated with lactate oxidase and a protective Nafion layer. The lactate electrodes had a high enzymatic activity described by a Michaelis-Menten constant of 2.6±0.1 mM, a linear response in the range 0.01÷2.5mM and a sensitivity of 7.3×10−2mA/mM·cm2. A replica-molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. We trapped fibroblasts in the cell culture volume and measured the lactate production rate using a stop and flow protocol. The average lactate production rate was 0.011±0.0049mM/min. The lactate production was suppressed with the addition of 2-deoxy-D-glucose, which binds to hexokinase. The blocking of hexokinase prevents the generation of pyruvate, the intermittent substrate required for lactate production even in the presence of glucose. PMID:20566279

Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart

PubMed Central

Vadvalkar, Shraddha S.; Matsuzaki, Satoshi; Eyster, Craig A.; Giorgione, Jennifer R.; Bockus, Lee B.; Kinter, Caroline S.; Kinter, Michael

2017-01-01

Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart. PMID:28154187

Production and Recovery of Pyruvic Acid: Recent Advances

NASA Astrophysics Data System (ADS)

Pal, Dharm; Keshav, Amit; Mazumdar, Bidyut; Kumar, Awanish; Uslu, Hasan

2017-12-01

Pyruvic acid is an important keto-carboxylic acid and can be manufactured by both chemical synthesis and biotechnological routes. In the present paper an overview of recent developments and challenges in various existing technique for the production and recovery of pyruvic acid from fermentation broth or from waste streams has been presented. The main obstacle in biotechnological production of pyruvic acid is development of suitable microorganism which can provide high yield and selectivity. On the other hand, technical limitation in recovery of pyruvic acid from fermentation broth is that, it could not be separated as other carboxylic acid in the form of salts by addition of alkali. Besides, pyruvic acid cannot be crystallized. Commercial separation by distillation is very expensive because pyruvic acid decomposes at higher temperature. It is also chemically reactive due to its peculiar molecular structure and has tendency to polymerize. Thus, at high concentration the various type of reaction leads to lower yield of the product, and hence, conventional methods are not favorable. Alternate separation technologies viable to both synthetic and biological routes are the current research areas. Latest techniques such as reactive extraction is new to the field of recovery of pyruvic acid. Recent development and future prospects in downstream processing of biochemically produced pyruvic acids has been discussed in this review article.

21 CFR 862.1650 - Pyruvate kinase test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

....1650 Pyruvate kinase test system. (a) Identification. A pyruvate kinase test system is a device intended to measure the activity of the enzyme pyruvate kinase in erythrocytes (red blood cells...). The device is exempt from the premarket notification procedures in subpart E of part 807 of this...

21 CFR 862.1650 - Pyruvate kinase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

....1650 Pyruvate kinase test system. (a) Identification. A pyruvate kinase test system is a device intended to measure the activity of the enzyme pyruvate kinase in erythrocytes (red blood cells...). The device is exempt from the premarket notification procedures in subpart E of part 807 of this...

21 CFR 862.1650 - Pyruvate kinase test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

....1650 Pyruvate kinase test system. (a) Identification. A pyruvate kinase test system is a device intended to measure the activity of the enzyme pyruvate kinase in erythrocytes (red blood cells...). The device is exempt from the premarket notification procedures in subpart E of part 807 of this...

21 CFR 862.1650 - Pyruvate kinase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

....1650 Pyruvate kinase test system. (a) Identification. A pyruvate kinase test system is a device intended to measure the activity of the enzyme pyruvate kinase in erythrocytes (red blood cells...). The device is exempt from the premarket notification procedures in subpart E of part 807 of this...

21 CFR 862.1650 - Pyruvate kinase test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

....1650 Pyruvate kinase test system. (a) Identification. A pyruvate kinase test system is a device intended to measure the activity of the enzyme pyruvate kinase in erythrocytes (red blood cells...). The device is exempt from the premarket notification procedures in subpart E of part 807 of this...

A replaceable dual-enzyme capillary microreactor using magnetic beads and its application for simultaneous detection of acetaldehyde and pyruvate.

PubMed

Shi, Jing; Zhao, Wenwen; Chen, Yuanfang; Guo, Liping; Yang, Li

2012-07-01

A novel replaceable dual-enzyme capillary microreactor was developed and evaluated using magnetic fields to immobilize the alcohol dehydrogenase (ADH)- and lactate dehydrogenase (LDH)-coated magnetic beads at desired positions in the capillary. The dual-enzyme assay was achieved by measuring the two consumption peaks of the coenzyme β-nicotinamide adenine dinucleotide (NADH), which were related to the ADH reaction and LDH reaction. The dual-enzyme capillary microreactor was constructed using magnetic beads without any modification of the inner surface of the capillary, and showed great stability and reproducibility. The electrophoretic resolution for different analytes can be easily controlled by altering the relative distance of different enzyme-coated magnetic beads. The apparent K(m) values for acetaldehyde with ADH-catalyzed reaction and for pyruvate with LDH-catalyzed reaction were determined. The detection limits for acetaldehyde and pyruvate determination are 0.01 and 0.016 mM (S/N = 3), respectively. The proposed method was successfully applied to simultaneously determine the acetaldehyde and pyruvate contents in beer samples. The results indicated that combing magnetic beads with CE is of great value to perform replaceable and controllable multienzyme capillary microreactor for investigation of a series of enzyme reactions and determination of multisubstrates. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of anoxia on the extra- and intracellular acid-base status in the land snail helix lucorum (L.): lack of evidence for a relationship between pyruvate kinase down-regulation and acid-base status

PubMed

Michaelidis; Pallidou; Vakouftsi

1999-06-01

The aims of the present study were to describe a possible correlation between the regulation of the key glycolytic enzyme pyruvate kinase and the acid-base status in the haemolymph and in several other tissues of land snails during anoxia. To illustrate whether such a relationship exists, we determined (i) the acid-base variables in the haemolymph and tissues of the land snail Helix lucorum, (ii) the kinetic properties of pyruvate kinase from several tissues and (iii) the levels of the anaerobic end-products d-lactate and succinate in the haemolymph and tissues of aerobic and anoxic Helix lucorum. The results showed that the pH of haemolymph (pHe) decreased significantly over the first 20 h of anoxia and then recovered slowly towards control values. A similar pattern was observed for intracellular pH (pHi), which decreased significantly over the first 16 h of anoxia and slowly returned towards control levels. The reduction and recovery of pHi and pHe seem to reflect the rate of anaerobic metabolism. The main anaerobic end-products, d-lactate and succinate, accumulated rapidly during the initial stages of anoxia and more slowly as anoxia progressed. The decrease in the rate of accumulation of anaerobic end-products during prolonged anoxia was due to the conversion of tissue pyruvate kinase to a less active form. The results demonstrate a correlation between pyruvate kinase down-regulation and the recovery of acid-base status in the haemolymph and the tissues of land snails during anoxia.

Mechanism of d-Cycloserine Action: Transport Systems for d-Alanine, d-Cycloserine, l-Alanine, and Glycine1

PubMed Central

Wargel, Robert J.; Shadur, Craig A.; Neuhaus, Francis C.

1970-01-01

The accumulation of d-alanine, l-alanine, glycine, and d-cycloserine in Escherichia coli was found to be mediated by at least two transport systems. The systems for d-alanine and glycine are related, and are separate from that involved in the accumulation of l-alanine. d-Cycloserine appears to be primarily transported by the d-alanine-glycine system. The accumulation of d-alanine, glycine, and d-cycloserine was characterized by two line segments in the Lineweaver-Burk analysis, whereas the accumulation of l-alanine was characterized by a single line segment. d-Cycloserine was an effective inhibitor of glycine and d-alanine accumulation, and l-cycloserine was an effective inhibitor of l-alanine transport. The systems were further differentiated by effects of azide, enhancement under various growth conditions, and additional inhibitor studies. Since the primary access of d-cycloserine in E. coli is via the d-alanine-glycine system, glycine might be expected to be a better antagonist of d-cycloserine inhibition than l-alanine. Glycine and d-alanine at 10−5m antagonized the effect of d-cycloserine in E. coli, whereas this concentration of l-alanine had no effect. PMID:4919992

Microdialysate concentration changes do not provide sufficient information to evaluate metabolic effects of lactate supplementation in brain-injured patients

PubMed Central

Rothman, Douglas L; Nordström, Carl-Henrik

2016-01-01

Cerebral microdialysis is a widely used clinical tool for monitoring extracellular concentrations of selected metabolites after brain injury and to guide neurocritical care. Extracellular glucose levels and lactate/pyruvate ratios have high diagnostic value because they can detect hypoglycemia and deficits in oxidative metabolism, respectively. In addition, patterns of metabolite concentrations can distinguish between ischemia and mitochondrial dysfunction, and are helpful to choose and evaluate therapy. Increased intracranial pressure can be life-threatening after brain injury, and hypertonic solutions are commonly used for pressure reduction. Recent reports have advocated use of hypertonic sodium lactate, based on claims that it is glucose sparing and provides an oxidative fuel for injured brain. However, changes in extracellular concentrations in microdialysate are not evidence that a rise in extracellular glucose level is beneficial or that lactate is metabolized and improves neuroenergetics. The increase in glucose concentration may reflect inhibition of glycolysis, glycogenolysis, and pentose phosphate shunt pathway fluxes by lactate flooding in patients with mitochondrial dysfunction. In such cases, lactate will not be metabolizable and lactate flooding may be harmful. More rigorous approaches are required to evaluate metabolic and physiological effects of administration of hypertonic sodium lactate to brain-injured patients. PMID:27604313

Hypertonic 15% sodium pyruvate offers no initial resuscitation advantage compared with 8% hypertonic NACl in sheep with multiple hemorrhages.

PubMed

do Nascimento, Paulo; Vaid, Sumreen U; Hoskins, Stephen L; Espana, Jonathan M; Kinsky, Michael P; Kramer, George C

2007-05-01

Initial fluid resuscitation of hemorrhagic shock might be enhanced by the infusion of monocarboxylate-energy substrates. We evaluated hemodynamics, metabolism, and fluid dynamics for initial resuscitation of hemorrhage using small volume 15% sodium pyruvate solution (HPY) compared with osmotically matched 8% hypertonic saline (HS). Instrumented conscious sheep were hemorrhaged 25 mL/kg at time zero through 15 min (T0-T15) and 5 mL/kg for 5 min at T50 to T55 and T70 to T75. Fluid resuscitation from T30 to T180 was performed by a computer-controlled closed-loop system, which titrated infusion rate to a mean arterial pressure of 90 mmHg. Initial infusion was 4 mL/kg of either HPY or HS, followed by the administration of lactated Ringer. Both HPY and HS restored cardiac index similarly. The lactate/pyruvate ratio was used to assess metabolic debt and was significantly higher (T180), whereas oxygen delivery was significantly lower (T120) with HPY versus HS. Total fluid administered was similar, with 43.7 +/- 6.2 mL/kg for HPY and 39.4 +/- 6.8 mL/kg for HS. Plasma volume was similarly increased and approached baseline values for both groups. Initial resuscitation with small volume HPY offered no hemodynamic or metabolic advantage compared with small volume HS when the fluids were infused to an end point pressure.

Gluconeogenesis in the amphibian retina. Lactate is preferred to glutamate as the gluconeogenic precursor.

PubMed Central

Goldman, S S

1988-01-01

The capacity for gluconeogenesis in the isolated amphibian retina was found to be approx. 70-fold greater with lactate than with glutamate as the gluconeogenic precursor, 1426 versus 21 pmol of glucose incorporated into glycogen/h per mg of protein. It was also found that 11-15% of the glucosyl units in glycogen are derived from C3 metabolites of the glycolytic pathway, suggesting that lactate is recycled within the retina. In concert with these metabolic observations, a full complement of the gluconeogenic enzymes was detected in retinal homogenates. These included: glucose-6-phosphatase, fructose-1,6-bisphosphatase, acetyl-CoA-dependent pyruvate carboxylase and phosphoenolpyruvate carboxykinase. Agents that regulate the rate of gluconeogenesis in hepatic tissue were tested on the retina. At concentrations of glutamate and lactate that are presumed to be relevant physiologically, it was found that vasoactive intestinal peptide, ionophore A23187 and elevated [K+] each enhanced the rate of gluconeogenesis in Ringer containing 50 microM-glutamate, whereas in Ringer containing 8.5 mM-lactate these agents inhibited the rate of gluconeogenesis. Further, it was found that the classic gluconeogenic hormone glucagon inhibited gluconeogenesis in both glutamate- and lactate-containing Ringer. Retinal energy metabolism was found to be altered in lactate-containing Ringer, in that lactate production was suppressed completely. In addition, glycogen metabolism appeared to be dependent on increased cytosolic Ca2+ and was insensitive to increased retinal cyclic AMP. PMID:2902849

21 CFR 862.1655 - Pyruvic acid test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... treatment of acid-base and electrolyte disturbances or anoxia (the reduction of oxygen in body tissues). (b... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Pyruvic acid test system. 862.1655 Section 862....1655 Pyruvic acid test system. (a) Identification. A pyruvic acid test system is a device intended to...

21 CFR 862.1655 - Pyruvic acid test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... treatment of acid-base and electrolyte disturbances or anoxia (the reduction of oxygen in body tissues). (b... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Pyruvic acid test system. 862.1655 Section 862....1655 Pyruvic acid test system. (a) Identification. A pyruvic acid test system is a device intended to...

21 CFR 862.1655 - Pyruvic acid test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... treatment of acid-base and electrolyte disturbances or anoxia (the reduction of oxygen in body tissues). (b... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Pyruvic acid test system. 862.1655 Section 862....1655 Pyruvic acid test system. (a) Identification. A pyruvic acid test system is a device intended to...

21 CFR 862.1655 - Pyruvic acid test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... treatment of acid-base and electrolyte disturbances or anoxia (the reduction of oxygen in body tissues). (b... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Pyruvic acid test system. 862.1655 Section 862....1655 Pyruvic acid test system. (a) Identification. A pyruvic acid test system is a device intended to...

21 CFR 862.1655 - Pyruvic acid test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... treatment of acid-base and electrolyte disturbances or anoxia (the reduction of oxygen in body tissues). (b... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Pyruvic acid test system. 862.1655 Section 862....1655 Pyruvic acid test system. (a) Identification. A pyruvic acid test system is a device intended to...

Glycolysis without pyruvate kinase in Clostridium thermocellum

DOE PAGES

Olson, Daniel G.; Horl, Manuel; Fuhrer, Tobias; ...

2016-12-01

The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay.more » Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic 13C labeling. In the wild-type strain, the malate shunt accounts for about 33 ± 2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. Lastly, this provides the first direct evidence of the in-vivo function of the malate shunt.« less

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.

Changes in mRNA expression for gluconeogenic enzymes in liver of dairy cattle during the transition to lactation.

PubMed

Greenfield, R B; Cecava, M J; Donkin, S S

2000-06-01

The objective of this study was to profile phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) mRNA expression in the liver of dairy cattle during the peripartum transition and determine changes in abundance of these mRNA in response to protein fed during the prepartum period. Thirty-eight multiparous Holstein cows were fed diets containing either 12% crude protein (CP) and 26% rumen undegradable protein (RUP), 16% CP and 26% RUP, 16% CP and 33% RUP, or 16% CP and 40% RUP on a dry-matter basis beginning 28 d before expected calving. After calving, all cows were fed a common diet through 56 d in milk (DIM). Northern analysis of RNA from liver biopsy samples obtained on days -28, -14, +1, +28, and +56 relative to calving indicated that PC and PEPCK mRNA expression were responsive to onset of lactation but not to prepartum protein or RUP concentration. Abundance of PEPCK mRNA was similar at -28, -14, and +1 DIM but was elevated by +28 and +56 DIM relative to precalving levels. Liver PC mRNA abundance was elevated on +1 DIM, remained elevated through 28 DIM, and declined to precalving levels by 56 DIM. The activity of PC enzyme was correlated (r2 = 0.89) with PC mRNA abundance. The data demonstrate increased abundance of PC mRNA during the early transition period followed by increased abundance of PEPCK mRNA during the postpartum period and suggest increased potential metabolism of lactate, pyruvate, and amino acids that contribute to the liver pyruvate pool.

Mitochondrial pyruvate import and its effects on homeostasis.

PubMed

Vanderperre, Benoît; Bender, Tom; Kunji, Edmund R S; Martinou, Jean-Claude

2015-04-01

Pyruvate metabolism plays a pivotal role in cell homeostasis and energy production. Pyruvate, the end product of glycolysis, is either catabolized in the cytosol, or enters into mitochondria to promote oxidative phosphorylation. The import of pyruvate into mitochondria requires a specific carrier in the inner mitochondrial membrane, the mitochondrial pyruvate carrier (MPC), whose identity was only recently discovered. Here we report our current knowledge of the structure and function of the MPC and we describe how dysfunction of the MPC could participate in various pathologies, including type 2 diabetes and cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

Branched Chain Amino Acid Oxidation in Cultured Rat Skeletal Muscle Cells

PubMed Central

Pardridge, William M.; Casanello-Ertl, Delia; Duducgian-Vartavarian, Luiza

1980-01-01

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-14C]leucine to 14CO2 or to the [1-14C]keto-acid of leucine (α-keto-isocaproate) was linear for at least 2 h of incubation; the production of 14CO2 from [1-14C]leucine was saturable with a Km = 6.3 mM and a maximum oxidation rate (Vmax) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-14C]leucine (Ki = 0.85 mM) and [U-14C]isoleucine, but had no effect on the oxidation of [U-14C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-14C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of 14CO2 and had relatively little effect on the production of [1-14C]keto-acid of leucine. A physiological concentration—3.0 g/100 ml—of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD+ ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug

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

Kajimoto, Masaki; O'Kelly-Priddy, Colleen M.; Ledee, Dolena R.

Extracorporeal membrane oxygenation (ECMO) supports infants and children with severe cardiopulmonary compromise. Nutritional support for these children includes provision of medium- and long-chain fatty acids (FAs). However, ECMO induces a stress response, which could limit the capacity for FA oxidation. Metabolic impairment could induce new or exacerbate existing myocardial dysfunction. Using a clinically relevant piglet model, we tested the hypothesis that ECMO maintains the myocardial capacity for FA oxidation and preserves myocardial energy state. Provision of 13-Carbon labeled medium-chain FA (octanoate), longchain free FAs (LCFAs), and lactate into systemic circulation showed that ECMO promoted relative increases in myocardial LCFA oxidationmore » while inhibiting lactate oxidation. Loading of these labeled substrates at high dose into the left coronary artery demonstrated metabolic flexibility as the heart preferentially oxidized octanoate. ECMO preserved this octanoate metabolic response, but also promoted LCFA oxidation and inhibited lactate utilization. Rapid upregulation of pyruvate dehydrogenase kinase-4 (PDK4) protein appeared to participate in this metabolic shift during ECMO. ECMO also increased relative flux from lactate to alanine further supporting the role for pyruvate dehydrogenase inhibition by PDK4. High dose substrate loading during ECMO also elevated the myocardial energy state indexed by phosphocreatine to ATP ratio. ECMO promotes LCFA oxidation in immature hearts, while maintaining myocardial energy state. These data support the appropriateness of FA provision during ECMO support for the immature heart.« less

Metabolism of pyruvate and malate by isolated fat-cell mitochondria.

PubMed

Martin, B R; Denton, R M

1971-11-01

1. Metabolism of pyruvate and malate by isolated fat-cell mitochondria incubated in the presence of ADP and phosphate has been studied by measuring rates of pyruvate uptake, malate utilization or production, citrate production and oxygen consumption. From these measurements calculations of the flow rates through pyruvate carboxylase, pyruvate dehydrogenase and citrate cycle have been made under various conditions. 2. In the presence of bicarbonate, pyruvate was largely converted into citrate and malate and only about 10% was oxidized by the citrate cycle; citrate and malate outputs were linear after lag periods of 6-9min and 3min respectively, and no other end products of pyruvate metabolism were detected. On the further addition of malate or hydroxymalonate, the lag in the rate of citrate output was less marked but no net malate disappearance was detected. If, however, bicarbonate was omitted then net malate uptake was observed. Addition of butyl malonate was found to greatly inhibit the metabolism of pyruvate to citrate and malate in the presence of bicarbonate. 3. These results are in agreement with earlier conclusions that in adipose tissue acetyl units for fatty acid synthesis are transferred to the cytoplasm as citrate and that this transfer requires malate presumably for counter transport. They also support the view that oxaloacetate for citrate synthesis is preferentially formed from pyruvate through pyruvate carboxylase rather than malate through malate dehydrogenase and that the mitochondrial metabolism of citrate in fat-cells is restricted. The possible consequences of these conclusions are discussed. 4. Studies on the effects of additions of adenine nucleotides to pyruvate metabolism by isolated fat-cell mitochondria are consistent with inhibition of pyruvate carboxylase in the presence of ADP and pyruvate dehydrogenase in the presence of ATP.

Experimental and computational thermochemical study of α-alanine (DL) and β-alanine.

PubMed

da Silva, Manuel A V Ribeiro; da Silva, Maria das Dores M C Ribeiro; Santos, Ana Filipa L O M; Roux, Maria Victoria; Foces-Foces, Concepción; Notario, Rafael; Guzmán-Mejía, Ramón; Juaristi, Eusebio

2010-12-16

This paper reports an experimental and theoretical study of the gas phase standard (p° = 0.1 MPa) molar enthalpies of formation, at T = 298.15 K, of α-alanine (DL) and β-alanine. The standard (p° = 0.1 MPa) molar enthalpies of formation of crystalline α-alanine (DL) and β-alanine were calculated from the standard molar energies of combustion, in oxygen, to yield CO2(g), N2(g), and H2O(l), measured by static-bomb combustion calorimetry at T = 298.15 K. The vapor pressures of both amino acids were measured as function of temperature by the Knudsen effusion mass-loss technique. The standard molar enthalpies of sublimation at T = 298.15 K was derived from the Clausius−Clapeyron equation. The experimental values were used to calculate the standard (p° = 0.1 MPa) enthalpy of formation of α-alanine (DL) and β-alanine in the gaseous phase, Δ(f)H(m)°(g), as −426.3 ± 2.9 and −421.2 ± 1.9 kJ·mol(−1), respectively. Standard ab initio molecular orbital calculations at the G3 level were performed. Enthalpies of formation, using atomization reactions, were calculated and compared with experimental data. Detailed inspections of the molecular and electronic structures of the compounds studied were carried out.

-Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression.

PubMed

Mattice, Amanda M S; MacLean, Isabelle A; Childers, Christine L; Storey, Kenneth B

2018-01-01

Pyruvate kinase (PK) is responsible for the final reaction in glycolysis. As PK is a glycolytic control point, the analysis of PK posttranslational modifications (PTM) and kinetic changes reveals a key piece of the reorganization of energy metabolism in an anoxia tolerant vertebrate. To explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles ( Trachemys scripta elegans ). Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia. Both muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. I 50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I 50 L-alanine was only reduced in the liver. Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver) and lysine methylation (by 43% in muscle and 70% in liver) during anoxia which have been linked to suppression of PK activity in other organisms. Liver PK also showed a 26% decrease in tyrosine phosphorylation under anoxia. Anoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state. This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia.

Resistance to hypoxia-induced necroptosis is conferred by glycolytic pyruvate scavenging of mitochondrial superoxide in colorectal cancer cells.

PubMed

Huang, C-Y; Kuo, W-T; Huang, Y-C; Lee, T-C; Yu, L C H

2013-05-02

Cancer cells may survive under oxygen and nutrient deprivation by metabolic reprogramming for high levels of anaerobic glycolysis, which contributes to tumor growth and drug resistance. Abnormally expressed glucose transporters (GLUTs) are colocalized with hypoxia (Hx) inducible factor (HIF)1α in peri-necrotic regions in human colorectal carcinoma. However, the underlying mechanisms of anti-necrotic resistance conferred by glucose metabolism in hypoxic cancer cells remain poorly understood. Our aim was to investigate signaling pathways of Hx-induced necroptosis and explore the role of glucose pyruvate metabolite in mechanisms of death resistance. Human colorectal carcinoma cells were Hx exposed with or without glucose, and cell necroptosis was examined by receptor-interacting protein (RIP)1/3 kinase immunoprecipitation and (32)P kinase assays. Our results showed increased RIP1/3 complex formation and phosphorylation in hypoxic, but not normoxic cells in glucose-free media. Blocking RIP1 signaling, by necrostatin-1 or gene silencing, decreased lactodehydrogenase (LDH) leakage and plasma membrane disintegration. Generation of mitochondrial superoxide was noted after hypoxic challenge; its reduction by antioxidants inhibited RIP signaling and cell necrosis. Supplementation of glucose diminished the RIP-dependent LDH leakage and morphological damage in hypoxic cells, whereas non-metabolizable sugar analogs did not. Hypoxic cells given glucose showed nuclear translocation of HIF1α associated with upregulation of GLUT-1 and GLUT-4 expression, as well as increase of intracellular ATP, pyruvate and lactate levels. The glucose-mediated death resistance was ablated by iodoacetate (an inhibitor to glyceraldehyde-3-phosphate dehydrogenase), but not by UK5099 (an inhibitor to mitochondrial pyruvate carrier), suggesting that glycolytic pathway was involved in anti-necrotic mechanism. Lastly, replacing glucose with cell-permeable pyruvate derivative also led to decrease of Hx

Pyruvate production and excretion by the luminous marine bacteria.

PubMed Central

Ruby, E G; Nealson, K H

1977-01-01

During aerobic growth on glucose, several species of luminous marine bacteria exhibited an imcomplete oxidative catabolism of substrate. Pyruvate, one of the products of glucose metabolism, was excreted into the medium during exponential growth and accounted for up to 50% of the substrate carbon metabolized. When glucose was depleted from the medium, the excreted pyruvate was promptly utilized, demonstrating that the cells are capable of pyruvate catabolism. Pyruvate excretion is not a general phenomenon of carbohydrate metabolism since it does not occur during the utilization of glycerol or maltose. When cells pregrown on glycerol were exposed to glucose, they began to excrete pyruvate, even if protein synthesis was blocked with chloramphenicol. Glucose thus appears to have an effect on the activity of preexisting catabolic enzymes. PMID:303077

The role of the mitochondrial pyruvate carrier in substrate regulation

PubMed Central

Vacanti, Nathaniel M.; Divakaruni, Ajit S.; Green, Courtney R.; Parker, Seth J.; Henry, Robert R.; Ciaraldi, Theodore P.; Murphy, Anne N.; Metallo, Christian M.

2014-01-01

SUMMARY Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied 13C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with use of lipids and amino acids as catabolic and anabolic fuels. PMID:25458843

Pioglitazone inhibits mitochondrial pyruvate metabolism and glucose production in hepatocytes

PubMed Central

Shannon, Christopher E.; Daniele, Giuseppe; Galindo, Cynthia; Abdul-Ghani, Muhammad A.; DeFronzo, Ralph A.; Norton, Luke

2017-01-01

Pioglitazone is used globally for the treatment of type 2 diabetes mellitus (T2DM) and is one of the most effective therapies for improving glucose homeostasis and insulin resistance in T2DM patients. However, its mechanism of action in the tissues and pathways that regulate glucose metabolism are incompletely defined. Here we investigated the direct effects of pioglitazone on hepatocellular pyruvate metabolism and the dependency of these observations on the purported regulators of mitochondrial pyruvate transport, MPC1 and MPC2. In cultured H4IIE hepatocytes, pioglitazone inhibited [2-14C]-pyruvate oxidation and pyruvate-driven oxygen consumption and, in mitochondria isolated from both hepatocytes and human skeletal muscle, pioglitazone selectively and dose-dependently inhibited pyruvate-driven ATP synthesis. Pioglitazone also suppressed hepatocellular glucose production (HGP), without influencing the mRNA expression of key HGP regulatory genes. Targeted siRNA silencing of MPC1 and 2 caused a modest inhibition of pyruvate oxidation and pyruvate-driven ATP synthesis, but did not alter pyruvate-driven HGP and, importantly, it did not influence the actions of pioglitazone on either pathway. In summary, these findings outline a novel mode of action of pioglitazone relevant to the pathogenesis of T2DM and suggest that targeting pyruvate metabolism may lead to the development of effective new T2DM therapies. PMID:27987376

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

PubMed Central

Yamamoto, Yoko; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Usuda, Yoshihiro; Sode, Koji

2014-01-01

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production. PMID:25416770

Real-time measurement of hyperpolarized lactate production and efflux as a biomarker of tumor aggressiveness in an MR compatible 3D cell culture bioreactor.

PubMed

Sriram, Renuka; Van Criekinge, Mark; Hansen, Ailin; Wang, Zhen J; Vigneron, Daniel B; Wilson, David M; Keshari, Kayvan R; Kurhanewicz, John

2015-09-01

We have developed a 3D cell/tissue culture bioreactor compatible with hyperpolarized (HP) (13)C MR and interrogated HP [1-(13)C]lactate production and efflux in human renal cell carcinoma (RCC) cells. This platform is capable of resolving intracellular and extracellular HP lactate pools, allowing the kinetic measurement of lactate production and efflux in the context of cancer aggressiveness and response to therapy. HP (13)C MR studies were performed on three immortalized human renal cell lines: HK2, a normal renal proximal tubule cell line from which a majority of RCCs arise, UMRC6, a cell line derived from a localized RCC, and UOK262, an aggressive and metastatic RCC. The intra- (Lacin ) and extracellular (Lacex ) HP lactate signals were robustly resolved in dynamic (13)C spectra of the cell lines due to a very small but reproducible chemical shift difference (0.031 ± 0.0005 ppm). Following HP [1-(13)C]pyruvate delivery, the ratio of HP Lacin /Lacex was significantly lower for UOK262 cells compared with both UMRC6 and HK2 cells due to a significant (p < 0.05) increase in the Lacex pool size. Lacin /Lacex correlated with the MCT4 mRNA expression of the cell lines, and inhibition of MCT4 transport using DIDS resulted in a significant reduction in the HP Lacex pool size. The extension of these studies to living patient-derived RCC tissue slices using HP [1,2-(13)C2]pyruvate demonstrated a similarly split lactate doublet with a high Lacex pool fraction; in contrast, only a single NMR resonance is noted for HP [5-(13)C]glutamate, consistent with intracellular localization. These studies support the importance of lactate efflux as a biomarker of cancer aggressiveness and metastatic potential, and the utility of the MR compatible 3D cell/tissue culture bioreactor to study not only cellular metabolism but also transport. Additionally, this platform offers a sophisticated way to follow therapeutic interventions and screen novel therapies that target lactate export

Stereocomplexes Formed From Select Oligomers of Polymer d-lactic Acid (PDLA) and l-lactate May Inhibit Growth of Cancer Cells and Help Diagnose Aggressive Cancers-Applications of the Warburg Effect.

PubMed

Goldberg, Joel S

2011-02-15

It is proposed that select oligomers of polymer d-lactic acid (PDLA) will form a stereocomplex with l-lactate in vivo, producing lactate deficiency in tumor cells. Those cancer cells that utilize transport of lactate to maintain electrical neutrality may cease to multiply or die because of lactate trapping, and those cancer cells that benefit from utilization of extracellular lactate may be impaired. Intracellular trapping of lactate produces a different physiology than inhibition of LDH because the cell loses the option of shuttling pyruvate to an alternative pathway to produce an anion. Conjugated with stains or fluorescent probes, PDLA oligomers may be an agent for the diagnosis of tissue lactate and possibly cell differentiation in biopsy specimens. Preliminary experimental evidence is presented confirming that PDLA in high concentrations is cytotoxic and that l-lactate forms a presumed stereocomplex with PDLA. Future work should be directed at isolation of biologically active oligomers of PDLA.

Stereocomplexes Formed From Select Oligomers of Polymer d-lactic Acid (PDLA) and l-lactate May Inhibit Growth of Cancer Cells and Help Diagnose Aggressive Cancers—Applications of the Warburg Effect

PubMed Central

Goldberg, Joel S.

2011-01-01

It is proposed that select oligomers of polymer d-lactic acid (PDLA) will form a stereocomplex with l-lactate in vivo, producing lactate deficiency in tumor cells. Those cancer cells that utilize transport of lactate to maintain electrical neutrality may cease to multiply or die because of lactate trapping, and those cancer cells that benefit from utilization of extracellular lactate may be impaired. Intracellular trapping of lactate produces a different physiology than inhibition of LDH because the cell loses the option of shuttling pyruvate to an alternative pathway to produce an anion. Conjugated with stains or fluorescent probes, PDLA oligomers may be an agent for the diagnosis of tissue lactate and possibly cell differentiation in biopsy specimens. Preliminary experimental evidence is presented confirming that PDLA in high concentrations is cytotoxic and that l-lactate forms a presumed stereocomplex with PDLA. Future work should be directed at isolation of biologically active oligomers of PDLA. PMID:21487535

Metabolomic and proteomic analysis of D-lactate-producing Lactobacillus delbrueckii under various fermentation conditions.

PubMed

Liang, Shaoxiong; Gao, Dacheng; Liu, Huanhuan; Wang, Cheng; Wen, Jianping

2018-05-28

As an important feedstock monomer for the production of biodegradable stereo-complex poly-lactic acid polymer, D-lactate has attracted much attention. To improve D-lactate production by microorganisms such as Lactobacillus delbrueckii, various fermentation conditions were performed, such as the employment of anaerobic fermentation, the utilization of more suitable neutralizing agents, and exploitation of alternative nitrogen sources. The highest D-lactate titer could reach 133 g/L under the optimally combined fermentation condition, increased by 70.5% compared with the control. To decipher the potential mechanisms of D-lactate overproduction, the time-series response of intracellular metabolism to different fermentation conditions was investigated by GC-MS and LC-MS/MS-based metabolomic analysis. Then the metabolomic datasets were subjected to weighted correlation network analysis (WGCNA), and nine distinct metabolic modules and eight hub metabolites were identified to be specifically associated with D-lactate production. Moreover, a quantitative iTRAQ-LC-MS/MS proteomic approach was employed to further analyze the change of intracellular metabolism under the combined fermentation condition, identifying 97 up-regulated and 42 down-regulated proteins compared with the control. The in-depth analysis elucidated how the key factors exerted influence on D-lactate biosynthesis. The results revealed that glycolysis and pentose phosphate pathways, transport of glucose, amino acids and peptides, amino acid metabolism, peptide hydrolysis, synthesis of nucleotides and proteins, and cell division were all strengthened, while ATP consumption for exporting proton, cell damage, metabolic burden caused by stress response, and bypass of pyruvate were decreased under the combined condition. These might be the main reasons for significantly improved D-lactate production. These findings provide the first omics view of cell growth and D-lactate overproduction in L. delbrueckii, which

Metabolic profiling indicates impaired pyruvate dehydrogenase function in myalgic encephalopathy/chronic fatigue syndrome

PubMed Central

Mella, Olav; Bruland, Ove; Risa, Kristin; Dyrstad, Sissel E.; Alme, Kine; Rekeland, Ingrid G.; Sapkota, Dipak; Røsland, Gro V.; Fosså, Alexander; Ktoridou-Valen, Irini; Lunde, Sigrid; Sørland, Kari; Lien, Katarina; Herder, Ingrid; Thürmer, Hanne; Gotaas, Merete E.; Baranowska, Katarzyna A.; Bohnen, Louis M.L.J.; Schäfer, Christoph; McCann, Adrian; Sommerfelt, Kristian; Helgeland, Lars; Ueland, Per M.; Dahl, Olav

2016-01-01

Myalgic encephalopathy/chronic fatigue syndrome (ME/CFS) is a debilitating disease of unknown etiology, with hallmark symptoms including postexertional malaise and poor recovery. Metabolic dysfunction is a plausible contributing factor. We hypothesized that changes in serum amino acids may disclose specific defects in energy metabolism in ME/CFS. Analysis in 200 ME/CFS patients and 102 healthy individuals showed a specific reduction of amino acids that fuel oxidative metabolism via the TCA cycle, mainly in female ME/CFS patients. Serum 3-methylhistidine, a marker of endogenous protein catabolism, was significantly increased in male patients. The amino acid pattern suggested functional impairment of pyruvate dehydrogenase (PDH), supported by increased mRNA expression of the inhibitory PDH kinases 1, 2, and 4; sirtuin 4; and PPARδ in peripheral blood mononuclear cells from both sexes. Myoblasts grown in presence of serum from patients with severe ME/CFS showed metabolic adaptations, including increased mitochondrial respiration and excessive lactate secretion. The amino acid changes could not be explained by symptom severity, disease duration, age, BMI, or physical activity level among patients. These findings are in agreement with the clinical disease presentation of ME/CFS, with inadequate ATP generation by oxidative phosphorylation and excessive lactate generation upon exertion. PMID:28018972

Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview.

PubMed

Oppici, Elisa; Montioli, Riccardo; Cellini, Barbara

2015-09-01

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT) (EC 2.6.1.44) catalyses the conversion of l-alanine and glyoxylate to pyruvate and glycine, a reaction that allows glyoxylate detoxification. Inherited mutations on the AGXT gene encoding AGT lead to Primary Hyperoxaluria Type I (PH1), a rare disorder characterized by the deposition of calcium oxalate crystals primarily in the urinary tract. Here we describe the results obtained on the biochemical features of AGT as well as on the molecular and cellular effects of polymorphic and pathogenic mutations. A complex scenario on the molecular pathogenesis of PH1 emerges in which the co-inheritance of polymorphic changes and the condition of homozygosis or compound heterozygosis are two important factors that determine the enzymatic phenotype of PH1 patients. All the reported data represent relevant steps toward the understanding of genotype/phenotype correlations, the prediction of the response of the patients to the available therapies, and the development of new therapeutic approaches. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Metabolism of 13C-enriched D-fructose in hepatocytes from Goto-Kakizaki rats.

PubMed

Malaisse, Willy J; Ladriere, Laurence; Verbruggen, Ingrid; Willem, Rudolph

2004-05-01

This study aims at assessing the conversion of exogenous D-[1-13C]fructose, D-[2-13C]fructose or D-[6-13C]-fructose (10 mM) to 13C-enriched and either hydrogenated or deuterated D-glucose, L-lactate and L-alanine released by rat liver cells prepared from Goto-Kakizaki rats and incubated for 120 min in the presence of unlabelled D-glucose (also 10 mM) and D2O. The results of this study are relevant to the relative contribution of fructokinase and hexokinase isoenzyme to the phosphorylation of D-fructose, the capacity of D-glucose to confer to glucokinase positive cooperativity towards D-fructose, the circulation of D-fructose 6-phosphate in the pentose phosphate pathway, the regulation of the cytosolic NADD/NADH ratio, the respective fate of D-fructose-derived D-glyceraldehyde and dihydroxyacetone phosphate, the deuteration of fructose-derived glycolytic intermediates at the phosphoglucoisomerase, phosphomannoisomerase, enolase, pyruvate kinase and glutamate-alanine transaminase levels, and the unequal generation of L-[1-13C]lactate by cells exposed to D-[1-13C]fructose or D-[6-13C]fructose versus D-[2-13C]-fructose.

Gluconeogenesis is associated with high rates of tricarboxylic acid and pyruvate cycling in fasting northern elephant seals.

PubMed

Champagne, Cory D; Houser, Dorian S; Fowler, Melinda A; Costa, Daniel P; Crocker, Daniel E

2012-08-01

Animals that endure prolonged periods of food deprivation preserve vital organ function by sparing protein from catabolism. Much of this protein sparing is achieved by reducing metabolic rate and suppressing gluconeogenesis while fasting. Northern elephant seals (Mirounga angustirostris) endure prolonged fasts of up to 3 mo at multiple life stages. During these fasts, elephant seals maintain high levels of activity and energy expenditure associated with breeding, reproduction, lactation, and development while maintaining rates of glucose production typical of a postabsorptive mammal. Therefore, we investigated how fasting elephant seals meet the requirements of glucose-dependent tissues while suppressing protein catabolism by measuring the contribution of glycogenolysis, glycerol, and phosphoenolpyruvate (PEP) to endogenous glucose production (EGP) during their natural 2-mo postweaning fast. Additionally, pathway flux rates associated with the tricarboxylic acid (TCA) cycle were measured specifically, flux through phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate cycling. The rate of glucose production decreased during the fast (F(1,13) = 5.7, P = 0.04) but remained similar to that of postabsorptive mammals. The fractional contributions of glycogen, glycerol, and PEP did not change with fasting; PEP was the primary gluconeogenic precursor and accounted for ∼95% of EGP. This large contribution of PEP to glucose production occurred without substantial protein loss. Fluxes through the TCA cycle, PEPCK, and pyruvate cycling were higher than reported in other species and were the most energetically costly component of hepatic carbohydrate metabolism. The active pyruvate recycling fluxes detected in elephant seals may serve to rectify gluconeogeneic PEP production during restricted anaplerotic inflow in these fasting-adapted animals.

Cloning of D-lactate dehydrogenase genes of Lactobacillus delbrueckii subsp. bulgaricus and their roles in D-lactic acid production.

PubMed

Huang, Yanna; You, Chunping; Liu, Zhenmin

2017-07-01

Lactobacillus delbrueckii subsp. bulgaricus is a heterogenous lactic acid bacterium that converts pyruvate mainly to D-lactic acid using D-lactate dehydrogenases (D-LDHs), whose functional properties remain poorly characterized. Here, the D-LDHs genes (ldb0101, ldb0813, ldb1010, ldb1147 and ldb2021) were cloned and overexpressed in Escherichia coli JM109 from an inducible pUC18 vector, respectively, and the resulting strains were compared in terms of D-lactic acid production. The strain expressing ldb0101 and ldb1010 gene individually produced more D-lactate than other three strains. Further study revealed that Ldb0101 activity was down-regulated by the oxygen and, therefore, achieved a highest titer of D-lactate (1.94 g/L) under anaerobic condition, and introduction of ldb1010 gene enhanced D-lactate formation (0.94 and 0.85 g/L, respectively) both in aerobic and anaerobic conditions due to a relatively stable q d-lactate . Our results suggested that the enzyme Ldb0101 and Ldb1010 played a role of more importance in D-lactate formation. To the best of our knowledge, we demonstrate for the first time the roles of different D-LDH homologs from L. bulgaricus in D-lactic acid production.

Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier

PubMed Central

Divakaruni, Ajit S.; Wiley, Sandra E.; Rogers, George W.; Andreyev, Alexander Y.; Petrosyan, Susanna; Loviscach, Mattias; Wall, Estelle A.; Yadava, Nagendra; Heuck, Alejandro P.; Ferrick, David A.; Henry, Robert R.; McDonald, William G.; Colca, Jerry R.; Simon, Melvin I.; Ciaraldi, Theodore P.; Murphy, Anne N.

2013-01-01

Facilitated pyruvate transport across the mitochondrial inner membrane is a critical step in carbohydrate, amino acid, and lipid metabolism. We report that clinically relevant concentrations of thiazolidinediones (TZDs), a widely used class of insulin sensitizers, acutely and specifically inhibit mitochondrial pyruvate carrier (MPC) activity in a variety of cell types. Respiratory inhibition was overcome with methyl pyruvate, localizing the effect to facilitated pyruvate transport, and knockdown of either paralog, MPC1 or MPC2, decreased the EC50 for respiratory inhibition by TZDs. Acute MPC inhibition significantly enhanced glucose uptake in human skeletal muscle myocytes after 2 h. These data (i) report that clinically used TZDs inhibit the MPC, (ii) validate that MPC1 and MPC2 are obligatory components of facilitated pyruvate transport in mammalian cells, (iii) indicate that the acute effect of TZDs may be related to insulin sensitization, and (iv) establish mitochondrial pyruvate uptake as a potential therapeutic target for diseases rooted in metabolic dysfunction. PMID:23513224

Ethyl pyruvate ameliorates hepatic injury following blunt chest trauma and hemorrhagic shock by reducing local inflammation, NF-kappaB activation and HMGB1 release.

PubMed

Wagner, Nils; Dieteren, Scott; Franz, Niklas; Köhler, Kernt; Mörs, Katharina; Nicin, Luka; Schmidt, Julia; Perl, Mario; Marzi, Ingo; Relja, Borna

2018-01-01

The treatment of patients with multiple trauma including blunt chest/thoracic trauma (TxT) and hemorrhagic shock (H) is still challenging. Numerous studies show detrimental consequences of TxT and HS resulting in strong inflammatory changes, organ injury and mortality. Additionally, the reperfusion (R) phase plays a key role in triggering inflammation and worsening outcome. Ethyl pyruvate (EP), a stable lipophilic ester, has anti-inflammatory properties. Here, the influence of EP on the inflammatory reaction and liver injury in a double hit model of TxT and H/R in rats was explored. Female Lewis rats were subjected to TxT followed by hemorrhage/H (60 min, 35±3 mm Hg) and resuscitation/R (TxT+H/R). Reperfusion was performed by either Ringer`s lactated solution (RL) alone or RL supplemented with EP (50 mg/kg). Sham animals underwent all surgical procedures without TxT+H/R. After 2h, blood and liver tissue were collected for analyses, and survival was assessed after 24h. Resuscitation with EP significantly improved haemoglobin levels and base excess recovery compared with controls after TxT+H/R, respectively (p<0.05). TxT+H/R-induced significant increase in alanine aminotransferase levels and liver injury were attenuated by EP compared with controls (p<0.05). Local inflammation as shown by increased gene expression of IL-6 and ICAM-1, enhanced ICAM-1 and HMGB1 protein expression and infiltration of the liver with neutrophils were also significantly attenuated by EP compared with controls after TxT+H/R (p<0.05). EP significantly reduced TxT+H/R-induced p65 activation in liver tissue. Survival rates improved by EP from 50% to 70% after TxT+H/R. These data support the concept that the pronounced local pro-inflammatory response in the liver after blunt chest trauma and hemorrhagic shock is associated with NF-κB. In particular, the beneficial anti-inflammatory effects of ethyl pyruvate seem to be regulated by the HMGB1/NF-κB axis in the liver, thereby, restraining

Misconceptions regarding basic thermodynamics and enzyme kinetics have led to erroneous conclusions regarding the metabolic importance of lactate dehydrogenase isoenzyme expression.

PubMed

Bak, Lasse K; Schousboe, Arne

2017-11-01

Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate involving the coenzyme NAD + . Part of the foundation for the proposed shuttling of lactate from astrocytes to neurons during brain activation is the differential distribution of LDH isoenzymes between the two cell types. In this short review, we outline the basic kinetic properties of the LDH isoenzymes expressed in neurons and astrocytes, and argue that the distribution of LDH isoenzymes does not in any way govern directional flow of lactate between the two cellular compartments. The two main points are as follows. First, in line with the general concept of chemical catalysis, enzymes do not influence the thermodynamic equilibrium of a chemical reaction but merely the speed at which equilibrium is obtained. Thus, differential distribution of LDH isoenzymes with different kinetic parameters does not predict which cells are producing and which are consuming lactate. Second, the thermodynamic equilibrium of the reaction is toward the reduced substrate (i.e., lactate), which is reflected in the concentrations measured in brain tissue, suggesting that the reaction is at near-equilibrium at steady state. To conclude, the cellular distribution of LDH isoenzymes is of little if any consequence in determining any directional flow of lactate between neurons and astrocytes. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Pea Fiber and Wheat Bran Fiber Show Distinct Metabolic Profiles in Rats as Investigated by a 1H NMR-Based Metabolomic Approach

PubMed Central

Liu, Guangmang; Xiao, Liang; Fang, Tingting; Cai, Yimin; Jia, Gang; Zhao, Hua; Wang, Jing; Chen, Xiaoling; Wu, Caimei

2014-01-01

This study aimed to examine the effect of pea fiber (PF) and wheat bran fiber (WF) supplementation in rat metabolism. Rats were assigned randomly to one of three dietary groups and were given a basal diet containing 15% PF, 15% WF, or no supplemental fiber. Urine and plasma samples were analyzed by NMR-based metabolomics. PF significantly increased the plasma levels of 3-hydroxybutyrate, and myo-inositol as well as the urine levels of alanine, hydroxyphenylacetate, phenylacetyglycine, and α-ketoglutarate. However, PF significantly decreased the plasma levels of isoleucine, leucine, lactate, and pyruvate as well as the urine levels of allantoin, bile acids, and trigonelline. WF significantly increased the plasma levels of acetone, isobutyrate, lactate, myo-inositol, and lipids as well as the urine levels of alanine, lactate, dimethylglycine, N-methylniconamide, and α-ketoglutarate. However, WF significantly decreased the plasma levels of amino acids, and glucose as well as the urine levels of acetate, allantoin, citrate, creatine, hippurate, hydroxyphenylacetate, and trigonelline. Results suggest that PF and WF exposure can promote antioxidant activity and can exhibit common systemic metabolic changes, including lipid metabolism, energy metabolism, glycogenolysis and glycolysis metabolism, protein biosynthesis, and gut microbiota metabolism. PF can also decrease bile acid metabolism. These findings indicate that different fiber diet may cause differences in the biofluid profile in rats. PMID:25541729

Association of brain metabolites with blood lactate and glucose levels with respect to neurological outcomes after out-of-hospital cardiac arrest: A preliminary microdialysis study.

PubMed

Hifumi, Toru; Kawakita, Kenya; Yoda, Takeshi; Okazaki, Tomoya; Kuroda, Yasuhiro

2017-01-01

Out-of-hospital cardiac arrest (OHCA) is associated with poor prognosis. Cerebral microdialysis (CMD) is an efficient sampling technique to detect neurochemical changes in brain interstitial tissue. In this retrospective study, we hypothesised that there are different CMD levels between patients with favourable and unfavourable neurological outcomes. Data of patients with OHCA admitted to Kagawa University Hospital and administered therapeutic hypothermia (TH) were collected. Using a CMD probe, extracellular glucose, lactate and pyruvate levels were measured hourly along with intracranial perfusion pressure (ICP) and cerebral perfusion pressure (CPP) for the initial 72h during TH. The lactate/pyruvate (LP) ratio was calculated. Patients were divided into favourable [Glasgow-Pittsburgh cerebral performance category 1-2 at 30days after cardiac arrest] or unfavourable neurological outcome groups. CMD biochemical markers and blood lactate and glucose levels were compared between two groups. Ten patients were included. ICP was significantly higher in the unfavourable than in the favourable neurological outcome group; there were no significant differences with respect to CPP. The CMD LP ratio in the unfavourable outcome group progressively increased; significant differences were observed on days 2, 3 and 4 (p<0.01). Significant differences in blood lactate levels were observed between the groups only on day 3.5. CMD and blood glucose levels were higher in the unfavourable than in the favourable outcome group during TH. The association of CMD levels with long-term outcomes would be better defined in a large randomised prospective study. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Mitochondrial Pyruvate Carrier Function and Cancer Metabolism

PubMed Central

Rauckhorst, Adam J.

2016-01-01

Metabolic reprograming in cancer supports the increased biosynthesis required for unchecked proliferation. Increased glucose utilization is a defining feature of many cancers that is accompanied by altered pyruvate partitioning and mitochondrial metabolism. Cancer cells also require mitochondrial tricarboxylic acid cycle activity and electron transport chain function for biosynthetic competency and proliferation. Recent evidence demonstrates that mitochondrial pyruvate carrier (MPC) function is abnormal in some cancers and that increasing MPC activity may decrease cancer proliferation. Here we examine recent findings on MPC function and cancer metabolism. Special emphasis is placed on the compartmentalization of pyruvate metabolism and the alternative routes of metabolism that maintain the cellular biosynthetic pools required for unrestrained proliferation in cancer. PMID:27269731

Re-evaluation of amino-oxyacetate as an inhibitor.

PubMed Central

Smith, S B; Briggs, S; Triebwasser, K C; Freedland, R A

1977-01-01

Data are provided which indicate that pyruvate and/or acetaldehyde can reverse the inhibition of alanine aminotransferase and aspartate aminotransferase by amino-oxyacetate. It was shown that acetaldehyde could reverse the inhibition of gluconeogenesis from alanine and that pyruvate could reverse the inhibition of urea synthesis by amino-oxyacetate. PMID:849292

Determining In Vivo Regulation of Cardiac Pyruvate Dehydrogenase Based on Label Flux from Hyperpolarized [1-13C]Pyruvate

PubMed Central

Heather, Lisa C.; Griffin, Julian L.; Clarke, Kieran; Radda, George K.; Tyler, Damian J.

2015-01-01

Background Pyruvate dehydrogenase (PDH) is a key regulator of cardiac substrate selection and is regulated by both pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation and feedback inhibition. The extent to which chronic upregulation of PDK protein levels, acutely increased PDK activity and acute feedback inhibition limit PDH flux remains unclear because existing in vitro assessment methods inherently disrupt the enzyme complex. We have previously demonstrated that hyperpolarized 13C-labelled metabolic tracers with magnetic resonance spectroscopy (MRS) can monitor flux through PDH in vivo. The aim of this study was to determine the relative contributions of acute and chronic changes in PDK and PDH activities to in vivo myocardial PDH flux. Methodology/Principal Findings We examined both fed and fasted rats with either hyperpolarized [1-13C]pyruvate alone or hyperpolarized [1-13C]pyruvate co-infused with malate (to modulate mitochondrial NADH/NAD+ and acetyl-CoA/CoA ratios, which alter both PDH activity and flux). To confirm the metabolic fate of infused malate, we performed in vitro 1H NMR spectroscopy on cardiac tissue extracts. We observed that in fed rats, where PDH activity was high, the presence of malate increased PDH flux by 27%, whereas in the fasted state, malate infusion had no effect on PDH flux. Conclusions/Significance These observations suggest that pyruvate oxidation is limited by feedback inhibition from acetyl-CoA only when PDH activity is high. Therefore, in the case of PDH, and potentially other enzymes, hyperpolarized 13C MR can be used to non-invasively assess enzymatic regulation. PMID:21387444

Pyruvate metabolism: A therapeutic opportunity in radiation-induced skin injury

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

Yoo, Hyun; Kang, Jeong Wook; Lee, Dong Won

Ionizing radiation is used to treat a range of cancers. Despite recent technological progress, radiation therapy can damage the skin at the administration site. The specific molecular mechanisms involved in this effect have not been fully characterized. In this study, the effects of pyruvate, on radiation-induced skin injury were investigated, including the role of the pyruvate dehydrogenase kinase 2 (PDK2) signaling pathway. Next generation sequencing (NGS) identified a wide range of gene expression differences between the control and irradiated mice, including reduced expression of PDK2. This was confirmed using Q-PCR. Cell culture studies demonstrated that PDK2 overexpression and a highmore » cellular pyruvate concentration inhibited radiation-induced cytokine expression. Immunohistochemical studies demonstrated radiation-induced skin thickening and gene expression changes. Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness and inflammatory cytokine expression. These findings indicated that regulation of the pyruvate metabolic pathway could provide an effective approach to the control of radiation-induced skin damage. - Highlights: • The effects of radiation on skin thickness in mice. • Next generation sequencing revealed that radiation inhibited pyruvate dehydrogenase kinase 2 expression. • PDK2 inhibited irradiation-induced cytokine gene expression. • Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness.« less

Effects of eliminating pyruvate node pathways and of coexpression of heterogeneous carboxylation enzymes on succinate production by Enterobacter aerogenes.

PubMed

Tajima, Yoshinori; Yamamoto, Yoko; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Usuda, Yoshihiro; Sode, Koji

2015-02-01

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans

PubMed Central

Dawson, Neal J.; Bell, Ryan A. V.; Storey, Kenneth B.

2013-01-01

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) K m for L-lactate and a higher V max value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the K m of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the K m of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions. PMID:23533717

Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans.

PubMed

Dawson, Neal J; Bell, Ryan A V; Storey, Kenneth B

2013-01-01

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD(+). The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) K m for L-lactate and a higher V max value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the K m of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the K m of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

The lactate paradox revisited in lowlanders during acclimatization to 4100 m and in high-altitude natives

PubMed Central

van Hall, G; Lundby, C; Araoz, M; Calbet, J A L; Sander, M; Saltin, B

2009-01-01

Chronic hypoxia has been proposed to induce a closer coupling in human skeletal muscle between ATP utilization and production in both lowlanders (LN) acclimatizing to high altitude and high-altitude natives (HAN), linked with an improved match between pyruvate availability and its use in mitochondrial respiration. This should result in less lactate being formed during exercise in spite of the hypoxaemia. To test this hypothesis six LN (22–31 years old) were studied during 15 min warm up followed by an incremental bicycle exercise to exhaustion at sea level, during acute hypoxia and after 2 and 8 weeks at 4100 m above sea level (El Alto, Bolivia). In addition, eight HAN (26–37 years old) were studied with a similar exercise protocol at altitude. The leg net lactate release, and the arterial and muscle lactate concentrations were elevated during the exercise in LN in acute hypoxia and remained at this higher level during the acclimatization period. HAN had similar high values; however, at the moment of exhaustion their muscle lactate, ADP and IMP content and Cr/PCr ratio were higher than in LN. In conclusion, sea-level residents in the course of acclimatization to high altitude did not exhibit a reduced capacity for the active muscle to produce lactate. Thus, the lactate paradox concept could not be demonstrated. High-altitude natives from the Andes actually exhibit a higher anaerobic energy production than lowlanders after 8 weeks of acclimatization reflected by an increased muscle lactate accumulation and enhanced adenine nucleotide breakdown. PMID:19139048

The effect of additives on red cell 2,3 diphosphoglycerate levels in CPDA preservatives.

PubMed

Vora, S; West, C; Beutler, E

1989-01-01

Forty-two chemical substances, chosen because they might influence red cell metabolism, were screened for effect on red cell adenosine triphosphate and 2,3 diphosphoglycerate (2,3 DPG) levels during storage in CPD or CPDA-1 at 4 degrees C. Of these substances, six appeared on initial screening to increase 2,3 DPG levels during storage; on repeated examination, four compounds, i.e., oxalate, glyoxalate, ethyl oxaloacetate, and L-phenylalanyl-L-alanine, consistently increased 2,3 DPG levels during storage. It was shown that glyoxalate was converted rapidly to oxalate in blood, presumably through the lactate dehydrogenase reaction. Ethyl oxaloacetate is known to hydrolyze, giving rise to oxalate. Thus, the effect of both glyoxalate and ethyl oxaloacetate can be explained by the formation of oxalate, a compound already known to increase 2,3 DPG levels. The effect of L-phenylalanyl-L-alanine remains to be explained, but it may be hydrolyzed to L-alanine and L-phenylalanine, both of which are thought to have the capacity to increase red cell 2,3 DPG levels by inhibiting pyruvate kinase activity.

Anti-apoptotic and myocardial protective effects of ethyl pyruvate after regional ischaemia/reperfusion myocardial damage in an in vivo rat model.

PubMed

Shim, Haeng Seon; Lee, Wang Gyu; Kim, Yeon A; Han, Jeong Yeol; Park, Miyeong; Song, Yun Gyu; Kim, Joon Soo; Shin, Il-Woo

2017-09-01

The integration of reactive oxygen species is strongly associated with important pathophysiological mechanisms that mediate myocardial ischaemia/reperfusion (I/R) damage. Pyruvate is an efficacious scavenger of reactive oxygen species and a previous study has shown that ethyl pyruvate (EP) has a myocardial protective effect against regional I/R damage in an in vivo rat model. The purpose of this study was to determine whether the myocardial protective effect of EP is associated with anti-apoptosis. Rats were allocated to receive EP dissolved in lactated Ringer's solution or lactated Ringer's solution alone, via intraperitoneal infusion one hour before ischaemia. They were exposed to 30 minutes of ischaemia followed by reperfusion of the left coronary artery territory over two hours. Anti-apoptotic effects were checked using several biochemical parameters after two hours of reperfusion. Apoptosis was analysed using measured caspase-3 activity, Western blotting of B-cell lymphoma 2 (Bcl-2) family protein cleaved by caspase-3, and assessment of DNA laddering patterns and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining test. In ischaemic myocardium, EP increased Bcl-2 expression, but reduced Bcl-2-associated X protein and cleaved caspase-3 expressions. EP reduced the expression of DNA laddering and the number of myocardial I/R-damaged TUNEL-positive cells. This study demonstrated that EP has an anti-apoptotic effect after regional I/R damage in an in vivo rat heart model. The myocardial protective effect of EP may be related to its anti-apoptotic effect. Copyright: © Singapore Medical Association

Circulating metabolite predictors of glycemia in middle-aged men and women.

PubMed

Würtz, Peter; Tiainen, Mika; Mäkinen, Ville-Petteri; Kangas, Antti J; Soininen, Pasi; Saltevo, Juha; Keinänen-Kiukaanniemi, Sirkka; Mäntyselkä, Pekka; Lehtimäki, Terho; Laakso, Markku; Jula, Antti; Kähönen, Mika; Vanhala, Mauno; Ala-Korpela, Mika

2012-08-01

Metabolite predictors of deteriorating glucose tolerance may elucidate the pathogenesis of type 2 diabetes. We investigated associations of circulating metabolites from high-throughput profiling with fasting and postload glycemia cross-sectionally and prospectively on the population level. Oral glucose tolerance was assessed in two Finnish, population-based studies consisting of 1,873 individuals (mean age 52 years, 58% women) and reexamined after 6.5 years for 618 individuals in one of the cohorts. Metabolites were quantified by nuclear magnetic resonance spectroscopy from fasting serum samples. Associations were studied by linear regression models adjusted for established risk factors. Nineteen circulating metabolites, including amino acids, gluconeogenic substrates, and fatty acid measures, were cross-sectionally associated with fasting and/or postload glucose (P < 0.001). Among these metabolic intermediates, branched-chain amino acids, phenylalanine, and α1-acid glycoprotein were predictors of both fasting and 2-h glucose at 6.5-year follow-up (P < 0.05), whereas alanine, lactate, pyruvate, and tyrosine were uniquely associated with 6.5-year postload glucose (P = 0.003-0.04). None of the fatty acid measures were prospectively associated with glycemia. Changes in fatty acid concentrations were associated with changes in fasting and postload glycemia during follow-up; however, changes in branched-chain amino acids did not follow glucose dynamics, and gluconeogenic substrates only paralleled changes in fasting glucose. Alterations in branched-chain and aromatic amino acid metabolism precede hyperglycemia in the general population. Further, alanine, lactate, and pyruvate were predictive of postchallenge glucose exclusively. These gluconeogenic precursors are potential markers of long-term impaired insulin sensitivity that may relate to attenuated glucose tolerance later in life.

A pyruvate-proton symport and an H+-ATPase regulate the intracellular pH of Trypanosoma brucei at different stages of its life cycle.

PubMed

Vanderheyden, N; Wong, J; Docampo, R

2000-02-15

, oxaloacetate, 3-oxoisovalerate and 3-oxoisohexanoate significantly decreased pH(i), as well as proton and pyruvate efflux, whereas lactate had only a small effect, and no effect was observed with citrate or fumarate. The inhibition by pyruvate analogues of pyruvate efflux, proton efflux and acidification of pH(i) supports the hypothesis that pyruvate efflux is accompanied by proton efflux and that this is the major pH(i) control mechanism in bloodstream forms. Inhibition by H(+)-ATPase inhibitors of residual H(+) efflux in the absence of glucose or in the presence of high extracellular pyruvate indicates a minor role for H(+)-ATPase(s) in control of pH(i) in bloodstream forms.

Pyruvate dehydrogenase deficiency and epilepsy.

PubMed

Prasad, Chitra; Rupar, Tony; Prasad, Asuri N

2011-11-01

The pyruvate dehydrogenase complex (PDHc) is a mitochondrial matrix multienzyme complex that provides the link between glycolysis and the tricarboxylic acid (TCA) cycle by catalyzing the conversion of pyruvate into acetyl-CoA. PDHc deficiency is one of the commoner metabolic disorders of lactic acidosis presenting with neurological phenotypes that vary with age and gender. In this mini-review, we postulate mechanisms of epilepsy in the setting of PDHc deficiency using two illustrative cases (one with pyruvate dehydrogenase complex E1-alpha polypeptide (PDHA1) deficiency and the second one with pyruvate dehydrogenase complex E1-beta subunit (PDHB) deficiency (a rare subtype of PDHc deficiency)) and a selected review of published case series. PDHc plays a critical role in the pathway of carbohydrate metabolism and energy production. In severe deficiency states the resulting energy deficit impacts on brain development in utero resulting in structural brain anomalies and epilepsy. Milder deficiency states present with variable manifestations that include cognitive delay, ataxia, and seizures. Epileptogenesis in PDHc deficiency is linked to energy failure, development of structural brain anomalies and abnormal neurotransmitter metabolism. The use of the ketogenic diet bypasses the metabolic block, by providing a direct source of acetyl-CoA, leading to amelioration of some symptoms. Genetic counseling is essential as PDHA1 deficiency (commonest defect) is X-linked although females can be affected due to unfavorable lyonization, while PDHB and PDH phosphatase (PDP) deficiencies (much rarer defects) are of autosomal recessive inheritance. Research is in progress for looking into animal models to better understand pathogenesis and management of this challenging disorder. Copyright © 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Effect of metformin therapy on circulating amino acids in a randomized trial: the CAMERA study.

PubMed

Preiss, D; Rankin, N; Welsh, P; Holman, R R; Kangas, A J; Soininen, P; Würtz, P; Ala-Korpela, M; Sattar, N

2016-11-01

To investigate whether metformin therapy alters circulating aromatic and branched-chain amino acid concentrations, increased levels amino acid concentrations, increased levels of which have been found to predict Type 2 diabetes. In the Carotid Atherosclerosis: Metformin for Insulin Resistance (CAMERA) study (NCT00723307), 173 individuals without Type 2 diabetes, but with coronary disease, were randomized to metformin (n=86) or placebo (n=87) for 18 months. Plasma samples, taken every 6 months, were analysed using quantitative nuclear magnetic resonance spectroscopy. Ten metabolites consisting of eight amino acids [three branched-chain (isoleucine, leucine, valine), three aromatic (tyrosine, phenylalanine, histidine) and two other amino acids (alanine, glutamine)], lactate and pyruvate were quantified and analysed using repeated-measures models. On-treatment analyses were conducted to investigate whether amino acid changes were dependent on changes in weight, fat mass or insulin resistance estimated using homeostasis model assessment (HOMA-IR). Tyrosine decreased [-6.1 μmol/l (95% CI -8.5, -3.7); P<0.0001], while alanine [42 umol/l (95% CI 25, 59); P<0.0001] increased in the metformin-treated group compared with the placebo-treated group. Decreases in phenylalanine [-2.0 μmol/l (95% CI -3.6, -0.3); P=0.018] and increases in histidine [2.3 μmol/l (95% CI 0.1, 4.6); P=0.045] were also observed in the metformin group, although these changes were less statistically robust. Changes in these four amino acids were not accounted for by changes in weight, fat mass or HOMA-IR values. Levels of branched-chain amino acids, glutamine, pyruvate and lactate were not altered by metformin therapy. Metformin therapy results in a sustained and specific pattern of changes in aromatic amino acid and alanine concentrations. These changes are independent of any effects on weight and insulin sensitivity. Any causal link to metformin's unexplained cardiometabolic benefit requires further

Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Korotchkina, Lioubov; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand

2003-01-01

The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

Monocarboxylate transporters, blood lactate removal after supramaximal exercise, and fatigue indexes in humans.

PubMed

Thomas, C; Perrey, S; Lambert, K; Hugon, G; Mornet, D; Mercier, J

2005-03-01

The present study investigated whether muscular monocarboxylate transporter (MCT) 1 and 4 contents are related to the blood lactate removal after supramaximal exercise, fatigue indexes measured during different supramaximal exercises, and muscle oxidative parameters in 15 humans with different training status. Lactate recovery curves were obtained after a 1-min all-out exercise. A biexponential time function was then used to determine the velocity constant of the slow phase (gamma(2)), which denoted the blood lactate removal ability. Fatigue indexes were calculated during 1-min all-out (FI(AO)) and repeated 10-s (FI(Sprint)) cycling sprints. Biopsies were taken from the vastus lateralis muscle. MCT1 and MCT4 contents were quantified by Western blots, and maximal muscle oxidative capacity (V(max)) was evaluated with pyruvate + malate and glutamate + malate as substrates. The results showed that the blood lactate removal ability (i.e., gamma(2)) after a 1-min all-out test was significantly related to MCT1 content (r = 0.70, P < 0.01) but not to MCT4 (r = 0.50, P > 0.05). However, greater MCT1 and MCT4 contents were negatively related with a reduction of blood lactate concentration at the end of 1-min all-out exercise (r = -0.56, and r = -0.61, P < 0.05, respectively). Among skeletal muscle oxidative indexes, we only found a relationship between MCT1 and glutamate + malate V(max) (r = 0.63, P < 0.05). Furthermore, MCT1 content, but not MCT4, was inversely related to FI(AO) (r = -0.54, P < 0.05) and FI(Sprint) (r = -0.58, P < 0.05). We concluded that skeletal muscle MCT1 expression was associated with the velocity constant of net blood lactate removal after a 1-min all-out test and with the fatigue indexes. It is proposed that MCT1 expression may be important for blood lactate removal after supramaximal exercise based on the existence of lactate shuttles and, in turn, in favor of a better tolerance to muscle fatigue.

­Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression

PubMed Central

2018-01-01

Background Pyruvate kinase (PK) is responsible for the final reaction in glycolysis. As PK is a glycolytic control point, the analysis of PK posttranslational modifications (PTM) and kinetic changes reveals a key piece of the reorganization of energy metabolism in an anoxia tolerant vertebrate. Methods To explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles (Trachemys scripta elegans). Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia. Results Both muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. I50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I50 L-alanine was only reduced in the liver. Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver) and lysine methylation (by 43% in muscle and 70% in liver) during anoxia which have been linked to suppression of PK activity in other organisms. Liver PK also showed a 26% decrease in tyrosine phosphorylation under anoxia. Discussion Anoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state. This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia. PMID:29900073

Variability in spectrophotometric pyruvate analyses for predicting onion pungency and nutraceutical value.

PubMed

Beretta, Vanesa H; Bannoud, Florencia; Insani, Marina; Galmarini, Claudio R; Cavagnaro, Pablo F

2017-06-01

Onion pyruvate concentration is used as a predictor of flavor intensity and nutraceutical value. The protocol of Schwimmer and Weston (SW) (1961) is the most widespread methodology for estimating onion pyruvate. Anthon and Barret (AB) (2003) proposed modifications to this procedure. Here, we compared these spectrophotometry-based procedures for pyruvate analysis using a diverse collection of onion cultivars. The SW method always led to over-estimation of pyruvate levels in colored, but not in white onions, by up to 65%. Identification of light-absorbance interfering compounds was performed by spectrophotometry and HPLC analysis. Interference by quercetin and anthocyanins, jointly, accounted for more than 90% of the over-estimation of pyruvate. Pyruvate determinations according to AB significantly reduced absorbance interference from compounds other than pyruvate. This study provides evidence about the mechanistic basis underlying differences between the SW and AB methods for indirect assessment of onion flavor and nutraceutical value. Copyright © 2016 Elsevier Ltd. All rights reserved.

21 CFR 172.540 - DL-Alanine.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Agents and Related Substances § 172.540 DL-Alanine. DL-Alanine (a racemic mixture of D- and L-alanine; CAS Reg. No. 302-72-7) may be safely used as a flavor enhancer for sweeteners in pickling mixtures at a level not to exceed 1 percent of the pickling spice that is added to the pickling brine. [56 FR...

Differential effects of safflower oil versus fish oil feeding on insulin-stimulated glycogen synthesis, glycolysis, and pyruvate dehydrogenase flux in skeletal muscle: a 13C nuclear magnetic resonance study.

PubMed

Jucker, B M; Cline, G W; Barucci, N; Shulman, G I

1999-01-01

To examine the effects of safflower oil versus fish oil feeding on in vivo intramuscular glucose metabolism and relative pyruvate dehydrogenase (PDH) versus tricarboxylic acid (TCA) cycle flux, rats were pair-fed on diets consisting of 1) 59% safflower oil, 2) 59% menhaden fish oil, or 3) 59% carbohydrate (control) in calories. Rates of glycolysis and glycogen synthesis were assessed by monitoring [1-(13)C]glucose label incorporation into [1-(13)C]glycogen, [3-(13)C]lactate, and [3-(13)C]alanine in the hindlimb of awake rats via 13C nuclear magnetic resonance (NMR) spectroscopy during a euglycemic (approximately 6 mmol/l) hyperinsulinemic (approximately 180 microU/ml) clamp. A steady-state isotopic analysis of lactate, alanine, and glutamate was used to determine the relative PDH versus TCA cycle flux present in muscle under these conditions. The safflower oil-fed rats were insulin resistant compared with control and fish oil-fed rats, as reflected by a markedly reduced glucose infusion rate (Ginf) during the clamp (21.4 +/- 2.3 vs. 31.6 +/- 2.8 and 31.7 +/- 1.9 mg x kg(-1) x min(-1) in safflower oil versus control and fish oil groups, respectively, P < 0.006). This decrease in insulin-stimulated glucose disposal in the safflower oil group was associated with a lower rate of glycolysis (21.7 +/- 2.2 nmol x g(-1) x min(-1)) versus control (62.1 +/- 10.3 nmol x g(-1) x min(-1), P < 0.001) and versus fish oil (45.7 +/- 6.7 nmol x g(-1) x min(-1), P < 0.04), as no change in glycogen synthesis (103 +/- 15, 133 +/- 19, and 125 +/- 14 nmol x g(-1) x min(-1) in safflower oil, fish oil, and control, respectively) was detected. The intramuscular triglyceride (TG) content was increased in the safflower oil group (7.3 +/- 0.8 micromol/g) compared with the control group (5.2 +/- 0.8 micromol/g, P < 0.05) and the fish oil group (3.6 +/- 1.1 micromol/g, P < 0.01). Conversely, the percent PDH versus TCA cycle flux was decreased in the safflower oil (43 +/- 8%) versus the control

Effect of various alanine analogues on the L-alanine-adding enzyme from Escherichia coli.

PubMed

Liger, D; Blanot, D; van Heijenoort, J

1991-05-01

An extract from Escherichia coli containing the L-alanine-adding enzyme with a high specific activity was prepared. Several compounds structurally related to L-alanine were tested as inhibitors of this activity. Intact amino and carboxyl groups were necessary for an interaction with the enzyme. Certain halogenated (haloalanines) or unsaturated (L-vinylglycine, L-propargylglycine, 3-cyano-L-alanine) amino acids were good inhibitors. Radioactive glycine, serine and 1-aminoethylphosphonic acid were tested as substrates. Whereas glycine or L-serine gave rise to the formation of the corresponding nucleotide product, no synthesis of UDP-N-acetylmuramyl-L-1-aminoethylphosphonic acid could be detected.

Pyruvate in reduced osmolarity oral rehydration salt corrected lactic acidosis in sever scald rats.

PubMed

Liu, Rui; Wang, Shu-Ming; Li, Zong-Yu; Yu, Wen; Zhang, Hui-Ping; Zhou, Fang-Qiang

2018-06-01

A novel pyruvate-based oral rehydration salt (Pyr-ORS) was demonstrated of superiority over bicarbonate- or citrate-based one to preserve organ function and correct lactic acidosis in rehydration of lethal shock in animals. This study further compared these effects between low-osmolar Pyr-ORS and equimolar citrate-based counterpart. Eighty rats, using a fatal burn shock model, were randomized into four groups (two subgroups per group: n = 10): the sham group (group SR), Pyr-ORS group (group PR), WHO-ORS III group (group CR), and no rehydration group. ORS was delivered by manual gavage during 24 h following burns. Oral administration consisted of half of counted volume in the initial 8 h plus the rest in the later 16 h. Systemic hemodynamics, visceral organ surface blood flow, organ function, and metabolic acidosis were determined at 8 h and 24 h after burn. Another set of rats with identical surgical procedures without tests was observed for survival. Survival was markedly improved in the groups PR and CR; the former showed a higher survival rate than the latter at 24 h (40% versus 20%, P < 0.05). Systemic hemodynamics, visceral blood flow, and function of heart, liver, and kidney were greatly restored in group PR, compared with group CR (all P < 0.05). Hypoxic lactic acidosis was efficiently reversed in group PR, instead of group CR, (pH 7.36 versus 7.11, base excess 2.1 versus -9.1 mmol/L, lactate 4.28 versus 8.18 mmol/L; all P < 0.05) at 24 h after injury. Pyruvate was advantageous over citrate in low-osmolar ORS for protection of organs and survival; pyruvate, but not citrate, in the ORS corrected hypoxic lactic acidosis in rats subjected to lethal burn shock in 24 h. Copyright © 2018 Elsevier Inc. All rights reserved.

Metabolic effects of p-coumaric acid in the perfused rat liver.

PubMed

Lima, Leonardo C N; Buss, Gisele D; Ishii-Iwamoto, Emy L; Salgueiro-Pagadigorria, Clairce; Comar, Jurandir Fernando; Bracht, Adelar; Constantin, Jorgete

2006-01-01

The p-coumaric acid, a phenolic acid, occurs in several plant species and, consequently, in many foods and beverages of vegetable origin. Its antioxidant activity is well documented, but there is also a single report about an inhibitory action on the monocarboxylate carrier, which operates in the plasma and mitochondrial membranes. The latter observation suggests that p-coumaric acid could be able to inhibit gluconeogenesis and related parameters. The present investigation was planned to test this hypothesis in the isolated and hemoglobin-free perfused rat liver. Transformation of lactate and alanine into glucose (gluconeogenesis) in the liver was inhibited by p-coumaric acid (IC50 values of 92.5 and 75.6 microM, respectively). Transformation of fructose into glucose was inhibited to a considerably lower degree (maximally 28%). The oxygen uptake increase accompanying gluconeogenesis from lactate was also inhibited. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC50 = 160.1 microM); no such effect was observed in freeze-thawing disrupted mitochondria. Glucose 6-phosphatase and fructose 1,6-bisphosphatase were not inhibited. In isolated intact mitochondria, p-coumaric acid inhibited respiration dependent on pyruvate oxidation but was ineffective on respiration driven by succinate and beta-hydroxybutyrate. It can be concluded that inhibition of pyruvate transport into the mitochondria is the most prominent primary effect of p-coumaric acid and also the main cause for gluconeogenesis inhibition. The existence of additional actions of p-coumaric acid, such as enzyme inhibitions and interference with regulatory mechanisms, cannot be excluded. 2006 Wiley Periodicals, Inc.

Imaging mitochondrial flux in single cells with a FRET sensor for pyruvate.

PubMed

San Martín, Alejandro; Ceballo, Sebastián; Baeza-Lehnert, Felipe; Lerchundi, Rodrigo; Valdebenito, Rocío; Contreras-Baeza, Yasna; Alegría, Karin; Barros, L Felipe

2014-01-01

Mitochondrial flux is currently accessible at low resolution. Here we introduce a genetically-encoded FRET sensor for pyruvate, and methods for quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution. In HEK293 cells, neurons and astrocytes, mitochondrial pyruvate uptake was saturated at physiological levels, showing that the metabolic rate is determined by intrinsic properties of the organelle and not by substrate availability. The potential of the sensor was further demonstrated in neurons, where mitochondrial flux was found to rise by 300% within seconds of a calcium transient triggered by a short theta burst, while glucose levels remained unaltered. In contrast, astrocytic mitochondria were insensitive to a similar calcium transient elicited by extracellular ATP. We expect the improved resolution provided by the pyruvate sensor will be of practical interest for basic and applied researchers interested in mitochondrial function.

Imaging Mitochondrial Flux in Single Cells with a FRET Sensor for Pyruvate

PubMed Central

Baeza-Lehnert, Felipe; Lerchundi, Rodrigo; Valdebenito, Rocío; Contreras-Baeza, Yasna; Alegría, Karin; Barros, L. Felipe

2014-01-01

Mitochondrial flux is currently accessible at low resolution. Here we introduce a genetically-encoded FRET sensor for pyruvate, and methods for quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution. In HEK293 cells, neurons and astrocytes, mitochondrial pyruvate uptake was saturated at physiological levels, showing that the metabolic rate is determined by intrinsic properties of the organelle and not by substrate availability. The potential of the sensor was further demonstrated in neurons, where mitochondrial flux was found to rise by 300% within seconds of a calcium transient triggered by a short theta burst, while glucose levels remained unaltered. In contrast, astrocytic mitochondria were insensitive to a similar calcium transient elicited by extracellular ATP. We expect the improved resolution provided by the pyruvate sensor will be of practical interest for basic and applied researchers interested in mitochondrial function. PMID:24465702

Effect of beta-alanine supplementation on the onset of blood lactate accumulation (OBLA) during treadmill running: Pre/post 2 treatment experimental design.

PubMed

Jordan, Thomas; Lukaszuk, Judith; Misic, Mark; Umoren, Josephine

2010-05-19

beta-Alanine (betaA) has been shown to improve performance during cycling. This study was the first to examine the effects of betaA supplementation on the onset of blood lactate accumulation (OBLA) during incremental treadmill running. Seventeen recreationally-active men (mean +/- SE 24.9 +/- 4.7 yrs, 180.6 +/- 8.9 cm, 79.25 +/- 9.0 kg) participated in this randomized, double-blind, placebo-controlled pre/post test 2-treatment experimental design. Subjects participated in two incremental treadmill tests before and after 28 days of supplementation with either betaA (6.0 g.d-1)(betaA, n = 8) or an equivalent dose of Maltodextrin as the Placebo (PL, n = 9). Heart rate, percent heart rate maximum (%HRmax), %VO2max@OBLA (4.0 mmol.L-1 blood lactate concentration) and VO2max (L.min-1) were determined for each treadmill test. Friedman test was used to determine within group differences; and Mann-Whitney was used to determine between group differences for pre and post values (p < 0.05). The betaA group experienced a significant rightward shift in HR@OBLA beats.min-1 (p < 0.01) pre/post (161.6 +/- 19.2 to 173.6 +/- 9.9) but remained unchanged in the PL group (166.8 +/- 15.8 to 169.6 +/- 16.1). The %HRmax@OBLA increased (p < 0.05) pre/post in the betaA group (83.0% +/- 9.7 to 88.6% +/- 3.7) versus no change in the PL group (86.3 +/- % 4.8 to 87.9% +/- 7.2). The %VO2max@OBLA increased (p < 0.05) in the betaA group pre/post (69.1 +/- 11.0 to 75.6 +/- 10.7) but remained unchanged in the PL group (73.3 +/- 7.3 to 74.3 +/- 7.3). VO2max (L.min-1) decreased (p < 0.01) in the betaA group pre/post (4.57 +/- 0.8 to 4.31 +/- 0.8) versus no change in the PL group (4.04 +/- 0.7 to 4.18 +/- 0.8). Body mass kg increased (p < 0.05) in the betaA group pre/post (77.9 +/- 9.0 to 78.3 +/- 9.3) while the PL group was unchanged (80.6 +/- 9.1 to 80.4 +/- 9.0). betaA supplementation for 28 days enhanced sub-maximal endurance performance by delaying OBLA. However, betaA supplemented individuals had a

Combined use of l-alanine tert butyl ester lactate and trimethyl-β-cyclodextrin for the enantiomeric separations of 2-arylpropionic acids nonsteroidal anti-inflammatory drugs.

PubMed

Mavroudi, Maria C; Kapnissi-Christodoulou, Constantina P

2015-10-01

In this study, a new CE method, employing a binary system of trimethyl-β-CD (TM-β-CD) and a chiral amino acid ester-based ionic liquid (AAIL), was developed for the chiral separation of seven 2-arylpropionic acid nonsteroidal anti-inflammatory drugs (NSAIDs). In particular, the enantioseparation of ibuprofen, ketoprofen, carprofen, indoprofen, flurbiprofen, naproxen, and fenoprofen was improved significantly by supporting the BGE with the chiral AAIL l-alanine tert butyl ester lactate (l-AlaC4 Lac). Parameters, such as concentrations of TM-β-CD and l-AlaC4 Lac, and buffer pH, were systematically examined in order to optimize the chiral separation of each NSAID. It was observed that the addition of the AAIL into the BGE improved both resolution and efficiency significantly. After optimization of separation conditions, baseline separation (Rs >1.5) of five of the analytes was achieved in less than 11 min, while the resolution of ibuprofen and flurbiprofen was approximately 1.2. The optimized enantioseparation conditions for all analytes involve a BGE of 5 mM sodium acetate/acetic acid (pH 5.0), an applied voltage of 30 kV, and a temperature of 20°C. In addition, the results obtained by computing the %-RSD values of the EOF and the two enantiomer peaks, demonstrated excellent run-to-run, batch-to-batch, and day-to-day reproducibilities. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

STUDIES ON MAMMALIAN AND HUMAN PYRUVATE AND ALPHA-KETOGLUTARATE DEHYDROGENATION COMPLEXES.

DTIC Science & Technology

The pig heart pyruvate and alpha - ketoglutarate dehydrogenase complex were isolated in highly purified state as multienzyme units with molecular...weights of approximately 9 million and 2.8 million, respectively. The aims were to resolve the pig heart pyruvate and alpha - ketoglutarate dehydrogenase...complexes was isolated from three sources; (1) pyruvate dehydrogenase complex, (2) alpha - ketoglutarate dehydrogenase, and (3) amber-color extract free

A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity*

PubMed Central

Gray, Lawrence R.; Rauckhorst, Adam J.; Taylor, Eric B.

2016-01-01

The discovery that the MPC1 and MPC2 genes encode the protein components of the mitochondrial pyruvate carrier (MPC) has invigorated studies of mitochondrial pyruvate transport and its regulation in normal and disease states. Indeed, recent reports have demonstrated MPC involvement in the control of cell fate in cancer and gluconeogenesis in models of type 2 diabetes. Biochemical measurements of MPC activity are foundational for understanding the role of pyruvate transport in health and disease. We developed a 96-well scaled method of [14C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPC Km (28.0 ± 3.9 μm) and Vmax (1.08 ± 0.05 nmol/min/mg), which have not previously been reported. Km and Vmax of the rat liver MPC were found to be 71.2 ± 17 μm and 1.42 ± 0.14 nmol/min/mg, respectively. Additionally, we performed parallel pyruvate uptake and oxidation experiments with the same biological samples and show differential results in response to fasting, demonstrating the continued importance of a direct MPC activity assay. We expect this method will be of value for understanding the contribution of the MPC activity to health and disease states where pyruvate metabolism is expected to play a prominent role. PMID:26823462

[The role of lactate acidosis in the development and treatment of various neurologic syndromes in children and adolescents].

PubMed

Arveladze, G A; Geladze, N M; Sanikidze, T B; Khachapuridze, N S; Bakhtadze, S Z

2015-02-01

The aim of the study was to detect the role of lactate acidosis, also to find the share of mitochondrial insufficiency in development of various neurologic syndromes in children and adolescents. The detection of cellular energetic metabolism and acid based imbalance is also important for finding the specific method of management. We have studied 200 patients with various degree of neurodevelopment delay with epilepsy and epileptic syndromes, headache, vertigo, early strokes, floppy infant syndrome, atrophy of ophthalmic nerve, cataracta, neurosensory deafness, systemic myopathy, cerebral palsy. In 27% of cases with various ages we have detected lactate acidosis and increase level of pyruvate. Mitochondrial insufficiency was seen in 8% of cases which gives us opportunity to find the specific method of treatment in this group of patients. Each patient with neurological symptoms requires correction of parameters of energetic and oxidative metabolism.

Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr(215) in Aerococcus viridans lactate oxidase.

PubMed

Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K; Nidetzky, Bernd

2016-06-15

L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr(215) in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr(215), effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr(215) can thus lead to a kinetic bottleneck in product release.

Prebiotic synthesis of phosphoenol pyruvate by α-phosphorylation-controlled triose glycolysis

NASA Astrophysics Data System (ADS)

Coggins, Adam J.; Powner, Matthew W.

2017-04-01

Phosphoenol pyruvate is the highest-energy phosphate found in living organisms and is one of the most versatile molecules in metabolism. Consequently, it is an essential intermediate in a wide variety of biochemical pathways, including carbon fixation, the shikimate pathway, substrate-level phosphorylation, gluconeogenesis and glycolysis. Triose glycolysis (generation of ATP from glyceraldehyde 3-phosphate via phosphoenol pyruvate) is among the most central and highly conserved pathways in metabolism. Here, we demonstrate the efficient and robust synthesis of phosphoenol pyruvate from prebiotic nucleotide precursors, glycolaldehyde and glyceraldehyde. Furthermore, phosphoenol pyruvate is derived within an α-phosphorylation controlled reaction network that gives access to glyceric acid 2-phosphate, glyceric acid 3-phosphate, phosphoserine and pyruvate. Our results demonstrate that the key components of a core metabolic pathway central to energy transduction and amino acid, sugar, nucleotide and lipid biosyntheses can be reconstituted in high yield under mild, prebiotically plausible conditions.

Gluconeogenesis in Leishmania mexicana

PubMed Central

Rodriguez-Contreras, Dayana; Hamilton, Nicklas

2014-01-01

Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. PMID:25288791

Elevated levels of liver methylglyoxal and d-lactate in early-stage hepatitis in rats.

PubMed

Wang, Wen-Chuang; Chou, Chu-Kuang; Chuang, Ming-Cheng; Li, Yi-Chieh; Lee, Jen-Ai

2018-02-01

Methylglyoxal (MGO) is highly cytotoxic and its levels are elevated in diabetes, nephropathy and atherosclerosis. However, it has never been studied in liver disease. For this reason, we aimed to assess the levels of MGO and its metabolite d-lactate in an early hepatitis model. Wistar rats were administered CCl 4 (0.75 mL/kg, i.p.) to induce hepatitis. In either CCl 4 -treated or untreated rats, alanine transaminase and aspartate transaminase levels did not change over the course of the study, indicating that significant liver damage did not occur following CCl 4 treatment. However, the levels of MGO and d-lactate were higher in the livers of CCl 4 -treated animals than in untreated animals (MGO: 128.2 ± 18.8 and 248.1 ± 64.9 μg/g protein, p < 0.01; d-lactate: 0.860 ± 0.040 and 1.293 ± 0.078 μmol/g protein, respectively p < 0.01). Furthermore, in untreated and treated animals, serum d-lactate levels were 57.65 ± 2.59 and 92.16 ± 16.69 μm and urine d-lactate levels were 1.060 ± 0.007 and 1.555 ± 0.366 μmol/mg UCr, respectively (p < 0.01). These data show that in this model of early-stage liver damage, the levels of MGO and its metabolite d-lactate are elevated and that d-lactate could be useful as a reference marker for the early stage of hepatitis. Copyright © 2017 John Wiley & Sons, Ltd.

Effects of insulin combined with ethyl pyruvate on inflammatory response and oxidative stress in multiple-organ dysfunction syndrome rats with severe burns.

PubMed

Wang, Zhanke; Chen, Rongjian; Zhu, Zhongzhen; Zhang, Xiaoyun; Wang, Shiliang

2016-11-01

Inflammation response and oxidative stress promote the occurrence and development of multiple-organ dysfunction syndrome (MODS). Eighty MODS rats with third-degree burns were divided randomly into 4 groups: insulin, ethyl pyruvate (EP), insulin combined with EP, and control. Blood levels of glucose, alanine aminotransferase (ALT), creatine (CRE), creatine kinase (CK), tumor necrosis factor α (TNF-α), high-mobility group box 1 (HMGB-1), malondialdehyde (MDA), and total antioxidant capacity (TAC) before as well as 1, 3, 5, and 7 days after burns were measured. Blood levels of ALT, CRE, CK, TNF-α, HMGB-1, and MDA in INS, EP, and INS+EP groups at different time points were significantly lower, and TAC was significantly higher than that in the control group (C) (P<.01). These parameters in the INS+EP group were significantly lower, and TAC was significantly higher than that in INS and EP groups (P<.01). Blood levels of TNF-α, HMGB-1, and TAC in the INS group at different time points after burns were significantly lower, and MDA was significantly higher than that in the EP group (P<.01). Insulin combined with EP can effectively reduce the inflammatory response, oxidative stress, and main organ dysfunctions in MODS rats after severe burns. The therapeutic effect of insulin combined with EP is superior to single-agent treatment. The insulin anti-inflammatory effect is better than that of pyruvic acid ethyl ester, and the ethyl pyruvate antioxidation effect is better than that of insulin. The insulin can treat inflammation, whereas EP can reduce oxidative stress in MODS rats. Copyright © 2016 Elsevier Inc. All rights reserved.

Therapeutic Targeting of the Pyruvate Dehydrogenase Complex/Pyruvate Dehydrogenase Kinase (PDC/PDK) Axis in Cancer.

PubMed

Stacpoole, Peter W

2017-11-01

The mitochondrial pyruvate dehydrogenase complex (PDC) irreversibly decarboxylates pyruvate to acetyl coenzyme A, thereby linking glycolysis to the tricarboxylic acid cycle and defining a critical step in cellular bioenergetics. Inhibition of PDC activity by pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation has been associated with the pathobiology of many disorders of metabolic integration, including cancer. Consequently, the PDC/PDK axis has long been a therapeutic target. The most common underlying mechanism accounting for PDC inhibition in these conditions is post-transcriptional upregulation of one or more PDK isoforms, leading to phosphorylation of the E1α subunit of PDC. Such perturbations of the PDC/PDK axis induce a "glycolytic shift," whereby affected cells favor adenosine triphosphate production by glycolysis over mitochondrial oxidative phosphorylation and cellular proliferation over cellular quiescence. Dichloroacetate is the prototypic xenobiotic inhibitor of PDK, thereby maintaining PDC in its unphosphorylated, catalytically active form. However, recent interest in the therapeutic targeting of the PDC/PDK axis for the treatment of cancer has yielded a new generation of small molecule PDK inhibitors. Ongoing investigations of the central role of PDC in cellular energy metabolism and its regulation by pharmacological effectors of PDKs promise to open multiple exciting vistas into the biochemical understanding and treatment of cancer and other diseases. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Dietary nutrient levels regulate protein and carbohydrate intake, gluconeogenic/glycolytic flux and blood trehalose level in the insect Manduca sexta L.

PubMed

Thompson, S N; Borchardt, D B; Wang, L-W

2003-03-01

This study examined the effects of dietary casein and sucrose levels on nutrient intake, and distinguished the effects of carbohydrate and protein consumption on growth, fat content, pyruvate metabolism and blood trehalose level of 5th instar Manduca sexta larvae. Growth increased with increasing casein consumption but was unaffected by carbohydrate intake. Fat content also increased with carbohydrate consumption, but on carbohydrate-free diets fat content increased with increased protein consumption. Blood trehalose level and pyruvate metabolism were examined by nuclear magnetic resonance spectroscopy analysis of blood following administration of (3-(13)C)pyruvate. On diets containing sucrose, blood trehalose increased with increasing carbohydrate intake, and on most diets trehalose was synthesized entirely from dietary sucrose. Pyruvate cycling, indicated by the alanine C2/C3 (13)C enrichment ratio, increased with carbohydrate consumption reflecting increased glycolysis, and pyruvate decarboxylation exceeded carboxylation on all sucrose diets. Larvae that consumed <75 mg/day sucrose were gluconeogenic, based on the [2 (trehalose C6)(Glx C3/C2)]/alanine C2] (13)C enrichment ratio. On carbohydrate-free diets, blood trehalose levels were low and maintained entirely by gluconeogenesis. Blood trehalose level increased with increasing protein intake. Pyruvate cycling was very low, although many insects displayed higher levels of pyruvate decarboxylation than carboxylation. All gluconeogenic larvae displayed alanine (13)C enrichment ratios <0.35 and had blood trehalose levels <50 mM.

Improved sake metabolic profile during fermentation due to increased mitochondrial pyruvate dissimilation.

PubMed

Agrimi, Gennaro; Mena, Maria C; Izumi, Kazuki; Pisano, Isabella; Germinario, Lucrezia; Fukuzaki, Hisashi; Palmieri, Luigi; Blank, Lars M; Kitagaki, Hiroshi

2014-03-01

Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

β-Alanine supplementation and military performance.

PubMed

Hoffman, Jay R; Stout, Jeffrey R; Harris, Roger C; Moran, Daniel S

2015-12-01

During sustained high-intensity military training or simulated combat exercises, significant decreases in physical performance measures are often seen. The use of dietary supplements is becoming increasingly popular among military personnel, with more than half of the US soldiers deployed or garrisoned reported to using dietary supplements. β-Alanine is a popular supplement used primarily by strength and power athletes to enhance performance, as well as training aimed at improving muscle growth, strength and power. However, there is limited research examining the efficacy of β-alanine in soldiers conducting operationally relevant tasks. The gains brought about by β-alanine use by selected competitive athletes appears to be relevant also for certain physiological demands common to military personnel during part of their training program. Medical and health personnel within the military are expected to extrapolate and implement relevant knowledge and doctrine from research performed on other population groups. The evidence supporting the use of β-alanine in competitive and recreational athletic populations suggests that similar benefits would also be observed among tactical athletes. However, recent studies in military personnel have provided direct evidence supporting the use of β-alanine supplementation for enhancing combat-specific performance. This appears to be most relevant for high-intensity activities lasting 60-300 s. Further, limited evidence has recently been presented suggesting that β-alanine supplementation may enhance cognitive function and promote resiliency during highly stressful situations.

Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans

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

Lu, Yong-Zhi; Sheng, Yu; Li, Lan-Fen

2007-09-01

A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU-599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous proteinmore » was obtained using a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.« less

Alteration of pyruvate metabolism in African trypanosomes during differentiation from bloodstream into insect forms.

PubMed

Barnard, J P; Pedersen, P L

1994-08-15

In the presence of glucose and ample oxygen, insect form African trypanosomes release pyruvate more than 100-fold more slowly than do bloodstream forms. This rate decrease could not be accounted for simply by an increased mitochondrial pyruvate oxidation rate as inhibiting mitochondrial respiration increases pyruvate efflux to rates only 2-3% of that observed for bloodstream form trypanosomes. Alternatively, decreased pyruvate efflux from insect form trypanosomes could not be accounted for by decreased pyruvate transporter activity, which, surprisingly, was nearly as high in insect form trypanosomes as reported by us earlier for bloodstream forms (J.P. Barnard, B. Reynafarje, and P.L. Pedersen (1993) J. Biol. Chem. 268, 3654-3661). Rather, the low pyruvate efflux rate appears to be due primarily to reduced levels of the enzyme pyruvate kinase, which, in contrast to conclusions of an earlier study, is readily detected in insect form trypanosomes in the absence of added activators at an activity level about 4% of that found in bloodstream forms. Insect form pyruvate kinase seems to be located in the cytosol and exhibits kinetic profiles and constants nearly identical to those reported by us earlier for the bloodstream form enzyme (J.P. Barnard, and P.L. Pedersen (1988) Mol. Biochem. Parasitol. 31, 141-148). It is suggested that the reduced levels of pyruvate kinase, and hence the reduced pyruvate efflux rates, in insect form trypanosomes result from down regulation of the gene encoding the cytosolic enzyme.

A catalyzing phantom for reproducible dynamic conversion of hyperpolarized [1-¹³C]-pyruvate.

PubMed

Walker, Christopher M; Lee, Jaehyuk; Ramirez, Marc S; Schellingerhout, Dawid; Millward, Steven; Bankson, James A

2013-01-01

In vivo real time spectroscopic imaging of hyperpolarized ¹³C labeled metabolites shows substantial promise for the assessment of physiological processes that were previously inaccessible. However, reliable and reproducible methods of measurement are necessary to maximize the effectiveness of imaging biomarkers that may one day guide personalized care for diseases such as cancer. Animal models of human disease serve as poor reference standards due to the complexity, heterogeneity, and transient nature of advancing disease. In this study, we describe the reproducible conversion of hyperpolarized [1-¹³C]-pyruvate to [1-¹³C]-lactate using a novel synthetic enzyme phantom system. The rate of reaction can be controlled and tuned to mimic normal or pathologic conditions of varying degree. Variations observed in the use of this phantom compare favorably against within-group variations observed in recent animal studies. This novel phantom system provides crucial capabilities as a reference standard for the optimization, comparison, and certification of quantitative imaging strategies for hyperpolarized tracers.

Complete Oxidation of Propionate, Valerate, Succinate, and Other Organic Compounds by Newly Isolated Types of Marine, Anaerobic, Mesophilic, Gram-Negative, Sulfur-Reducing Eubacteria

PubMed Central

Finster, Kai; Bak, Friedhelm

1993-01-01

Anaerobic enrichment cultures with either propionate, succinate, lactate, or valerate and elemental sulfur and inocula from shallow marine or deep-sea sediments were dominated by rod-shaped motile bacteria after three transfers. By application of deep-agar dilutions, five eubacterial strains were obtained in pure culture and designated Kyprop, Gyprop, Kysw2, Gylac, and Kyval. All strains were gram negative and grew by complete oxidation of the electron donors and concomitant stoichiometric reduction of elemental sulfur to hydrogen sulfide. The isolates used acetate, propionate, succinate, lactate, pyruvate, oxaloacetate, maleate, glutamate, alanine, aspartate, and yeast extract. All isolates, except strain Gylac, used citrate as an electron donor but valerate was oxidized only by strain Kyval. Fumarate and malate were degraded by all strains without an additional electron donor or acceptor. Kyprop, Gyprop, and Gylac utilized elemental sulfur as the sole inorganic electron acceptor, while Kysw2 and Kyval also utilized nitrate, dimethyl sulfoxide, or Fe(III)-citrate as an electron acceptor. Images PMID:16348934

[Bone histomorphometry of lactating and no lactating hyperthyroid rats].

PubMed

Serakides, Rogéria; Ocarino, Natália de Melo; Magalhães, Fernanda do Carmo; Souza, Cíntia de Almeida; Leite, Eveline Dias; Freitas, Edmilson Santos de

2008-06-01

The objective of this study was to verify if hyperthyroidism potentiates the osteopenia lactational. 24 adult female rats were distributed in four groups: euthyroid no lactating (control), euthyroid lactating, hyperthyroid no lactating and hyperthyroid lactating. 20 days after gestation, all the animals were necropsied. The thoracic and lumbar vertebrae, the femur and tibia were decalcified and processed for histomorphometric analysis. The euthyroid lactating group presented intense osteopenia in the studied bones. In the hyperthyroid no lactating group, there was not any change in trabecular bone percentage in none of the analyzed bone. In the hyperthyroid lactating group, there was osteopenia in the tibia and femur, similar to the one in the euthyroid lactating group. But the trabecular bone percentage in all the vertebral bodies was significantly larger in comparison with the euthyroid lactating group. It was concluded that the hyperthyroidism does not potentiate the osteopenia lactational in female rats, but it minimizes the vertebral osteopenia once it stimulates the osteoblastic activity.

Effect of hyperthyroidism on the transport of pyruvate in rat-heart mitochondria.

PubMed

Paradies, G; Ruggiero, F M

1988-08-17

A comparative study of the transport of pyruvate in heart mitochondria from normal and triiodothyronine-treated rats has been carried out. It has been found that the rate of carrier-mediated (alpha-cyanocinnamate-sensitive) pyruvate uptake is significantly enhanced in mitochondria from triiodothyronine-treated rats as compared with mitochondria from control rats. The kinetic parameters of the pyruvate uptake indicate that only the Vmax of this process is enhanced whilst there is no change in the Km value. The enhanced rate of pyruvate uptake is not dependent on the increase of the transmembrane delta pH value (both mitochondria from normal and triiodothyronine-treated rats exhibit the same delta pH value) neither does it depend on the increase of the pyruvate carrier molecules (titration of these last with alpha-cyanocinnamate gives the same total number of binding sites). the pyruvate-dependent oxygen uptake is stimulated by 35-40% in mitochondria from hyperthyroid rats when compared with mitochondria from control rats. There is, however, no difference in either the respiratory control ratios or in the ADP/O ratios between these two types of mitochondria. The heart mitochondrial phospholipid composition is altered significantly in hyperthyroid rats; in particular, negatively charged phospholipid such as cardiolipin and phosphatidylserine were found to increase by more than 50%. Minor alterations were found in the pattern of fatty acids with an increase of 20:4/18:2 ratio. It is suggested that the changes in the kinetic parameters of pyruvate transport in mitochondria from hyperthyroid rats involve hormone-mediated changes in the lipid composition of the mitochondrial membranes which in turn modulate the activity of the pyruvate carrier.

Effect of Pyruvate Decarboxylase Knockout on Product Distribution Using Pichia pastoris (Komagataella phaffii) Engineered for Lactic Acid Production.

PubMed

Melo, Nadiele T M; Mulder, Kelly C L; Nicola, André Moraes; Carvalho, Lucas S; Menino, Gisele S; Mulinari, Eduardo; Parachin, Nádia S

2018-02-16

Lactic acid is the monomer unit of the bioplastic poly-lactic acid (PLA). One candidate organism for lactic acid production is Pichia pastoris , a yeast widely used for heterologous protein production. Nevertheless, this yeast has a poor fermentative capability that can be modulated by controlling oxygen levels. In a previous study, lactate dehydrogenase (LDH) activity was introduced into P. pastoris, enabling this yeast to produce lactic acid. The present study aimed to increase the flow of pyruvate towards the production of lactic acid in P. pastoris . To this end, a strain designated GLp was constructed by inserting the bovine lactic acid dehydrogenase gene (LDHb) concomitantly with the interruption of the gene encoding pyruvate decarboxylase (PDC). Aerobic fermentation, followed by micro-aerophilic culture two-phase fermentations, showed that the GLp strain achieved a lactic acid yield of 0.65 g/g. The distribution of fermentation products demonstrated that the acetate titer was reduced by 20% in the GLp strain with a concomitant increase in arabitol production: arabitol increased from 0.025 g/g to 0.174 g/g when compared to the GS115 strain. Taken together, the results show a significant potential for P. pastoris in producing lactic acid. Moreover, for the first time, physiological data regarding co-product formation have indicated the redox balance limitations of this yeast.

Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.

PubMed

Rodrigues, A F; Guerreiro, M R; Formas-Oliveira, A S; Fernandes, P; Blechert, A-K; Genzel, Y; Alves, P M; Hu, W S; Coroadinha, A S

2016-01-01

Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype. © 2015 Wiley Periodicals, Inc.

Metabolic networks to generate pyruvate, PEP and ATP from glycerol in Pseudomonas fluorescens.

PubMed

Alhasawi, Azhar; Thomas, Sean C; Appanna, Vasu D

2016-04-01

Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H2O2) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O2-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H2O2-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit. Copyright © 2016 Elsevier Inc. All rights reserved.

Beneficial effect of feeding a ketogenic diet to mothers on brain development in their progeny with a murine model of pyruvate dehydrogenase complex deficiency.

PubMed

Pliss, Lioudmila; Jatania, Urvi; Patel, Mulchand S

2016-06-01

Pyruvate dehydrogenase complex (PDC) deficiency is a major inborn error of oxidative metabolism of pyruvate in the mitochondria causing congenital lactic acidosis and primarily structural and functional abnormalities of the central nervous system. To provide an alternate source of acetyl-CoA derived from ketone bodies to the developing brain, a formula high in fat content is widely employed as a treatment. In the present study we investigated efficacy of a high-fat diet given to mothers during pregnancy and lactation on lessening of the impact of PDC deficiency on brain development in PDC-deficient female progeny. A murine model of systemic PDC deficiency by interrupting the X-linked Pdha1 gene was employed in this study. Maternal consumption of a high-fat diet during pregnancy and lactation had no effect on number of live-birth, body growth, tissue PDC activity levels, as well as the in vitro rates of glucose oxidation and fatty acid biosynthesis by the developing brain of PDC-deficient female offspring during the postnatal age 35 days, as compared to the PDC-deficient progeny born to dams on a chow diet. Interestingly, brain weight was normalized in PDC-deficient progeny of high fat-fed mothers with improvement in impairment in brain structure deficit whereas brain weight was significantly decreased and was associated with greater cerebral structural defects in progeny of chow-fed mothers as compared to control progeny of mothers fed either a chow or high fat diet. The findings provide for the first time experimental support for beneficial effects of a ketogenic diet during the prenatal and early postnatal periods on the brain development of PDC-deficient mammalian progeny.

Value of acid metabolic products in identification of certain corynebacteria.

PubMed Central

Reddy, C A; Kao, M

1978-01-01

Acid metabolic products of 23 strains of human and animal pathogenic corynebacteria, representing eight different species, were determined by gas chromatography. The results showed that the species examined were metabolically heterogeneous and could be presumptively identified based on the acid products produced. Corynebacterium equi did not produce any acids; C. renale produced lactate; and C. pyogenes produced major amounts of lactate, variable amounts of acetate, and minor amounts of succinate and pyruvate. C. kutscheri produced propionate and lactate as major products and pyruvate and oxalacetate as minor products. C. diphtheriae and C. pseudotuberculosis produced major amounts of propionate, acetate, and formate. In addition, C. pseudotuberculosis produced major amounts of pyruvate and minor amounts of succinate, lactate, and oxalacetate, whereas C. diphtheriae strains produced minor but variable amounts of lactate, succinate, fumarate, pyruvate, and oxalacetate. C. bovis produced aicd products similar to those of C. pyogenes but was readily distinguishable from the latter by the lack of hemolysis on blood agar, colony morphology, catalase reaction, and biochemicals. C. suis characteristically produced major amounts of ethanol, acetate, and formate and minor amounts of lactate and succinate but no propionate. PMID:96126

Anaplerotic roles of pyruvate carboxylase in mammalian tissues.

PubMed

Jitrapakdee, S; Vidal-Puig, A; Wallace, J C

2006-04-01

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC serves an anaplerotic role for the tricarboxylic acid cycle, when intermediates are removed for different biosynthetic purposes. In liver and kidney, PC provides oxaloacetate for gluconeogenesis. In adipocytes PC is involved in de novo fatty acid synthesis and glyceroneogenesis, and is regulated by the peroxisome proliferator-activated receptor-gamma, suggesting that PC is involved in the metabolic switch controlling fuel partitioning toward lipogenesis. In islets, PC is necessary for glucose-induced insulin secretion by providing oxaloacetate to form malate that participates in the 'pyruvate/malate cycle' to shuttle 3C or 4C between mitochondria and cytoplasm. Hyperglycemia and hyperlipidemia impair this cycle and affect glucose-stimulated insulin release. In astrocytes, PC is important for de novo synthesis of glutamate, an important excitatory neurotransmitter supplied to neurons. Transcriptional studies of the PC gene pinpoint some transcription factors that determine tissue-specific expression.

Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles

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

Faraci, W.S.; Walsh, C.T.

1988-05-03

Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L ..-->.. D and D..-->.. L directions for all three enzymes to assess the degree to which abstraction of the ..cap alpha..-proton or protonation of substratemore » PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of ..cap alpha..-/sup 3/H from substrate to product and solvent exchange/substrate conversion experiments in /sup 3/H/sub 2/O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.« less

Inhibition of monocarboxylate transporter 2 in the retrotrapezoid nucleus in rats – a test of the astrocyte-neuron lactate-shuttle hypothesis

PubMed Central

Erlichman, J.S.; Hewitt, Amy; Damon, Tracey L.; Hart, Michael; Kurascz, Jennifer; Li, A.; Leiter, J.C.

2009-01-01

The astrocyte-neuronal lactate shuttle hypothesis (ANLSH) posits that lactate released from astrocytes into the extracellular space is metabolized by neurons. The lactate released should alter extracellular pH (pHe), and changes in pH in central chemosensory regions of the brainstem stimulate ventilation. Therefore, we assessed the impact of disrupting the lactate shuttle by administering 100 microM α-cyano-4-hydroxy-cinnamate (4-CIN), a dose that blocks the neuronal monocarboxylate transporter (MCT2), but not the astrocytic MCTs (MCT1 and MCT4). Administration of 4-CIN focally in the retrotrapezoid nucleus (RTN), a medullary central chemosensory nucleus, increased ventilation and decreased pHe in intact animals. In medullary brain slices, 4-CIN reduced astrocytic intracellular pH (pHi) slightly, but alkalinized neuronal pHi. Nonetheless, pHi fell significantly in both cell types when they were treated with exogenous lactate, although 100 microM 4-CIN significantly reduced the magnitude of the acidosis in neurons, but not astrocytes. Finally, 4-CIN treatment increased the uptake of a fluorescent 2-deoxy-d-glucose analogue in neurons, but did not alter the uptake rate of this 2-deoxy-d-glucose analogue in astrocytes. These data confirm the existence of an astrocyte to neuron lactate shuttle in intact animals in the RTN, and lactate derived from astrocytes forms part of the central chemosensory stimulus for ventilation in this nucleus. When the lactate shuttle was disrupted by treatment with 4-CIN, neurons increased the uptake of glucose. Thus, neurons seem to metabolize a combination of glucose and lactate (and other substances such as pyruvate) depending, in part, on the availability of each of these particular substrates. PMID:18463242

Ethyl pyruvate inhibits hypoxic pulmonary vasoconstriction and attenuates pulmonary artery cytokine expression

PubMed Central

Tsai, Ben M.; Lahm, Tim; Morrell, Eric D.; Crisostomo, Paul R.; Markel, Troy; Wang, Meijing; Meldrum, Daniel R.

2009-01-01

Hypoxic pulmonary vasoconstriction is a common consequence of acute lung injury and may be mediated by increased local production of proinflammatory cytokines. Ethyl pyruvate is a novel anti-inflammatory agent that has been shown to downregulate proinflammatory genes following hemorrhagic shock; however, its effects on hypoxic pulmonary vasoconstriction are unknown. We hypothesized that ethyl pyruvate would inhibit hypoxic pulmonary vasoconstriction and downregulate pulmonary artery cytokine expression during hypoxia. To study this, isometric force displacement was measured in isolated rat pulmonary artery rings (n=8/group) during hypoxia (95% N2/5% CO2) with or without prior ethyl pyruvate (10 mM) treatment. Following 60 minutes of hypoxia, pulmonary artery rings were analyzed for TNF-α and IL-1 mRNA via RT-PCR. Ethyl pyruvate inhibited hypoxic pulmonary artery contraction (4.49±2.32% vs. 88.80±5.68% hypoxia alone) and attenuated the hypoxic upregulation of pulmonary artery TNF and IL-1 mRNA (p<0.05). These data indicate that: 1) hypoxia increases pulmonary artery vasoconstriction and proinflammatory cytokine gene expression; 2) ethyl pyruvate decreases hypoxic pulmonary vasoconstriction and downregulates hypoxia-induced pulmonary artery proinflammatory cytokine gene expression; and 3) ethyl pyruvate may represent a novel therapeutic adjunct in the treatment of acute lung injury. PMID:17574585

Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism.

PubMed

Rankin, Joel A; Mauban, Robert C; Fellner, Matthias; Desguin, Benoît; McCracken, John; Hu, Jian; Varganov, Sergey A; Hausinger, Robert P

2018-03-09

Lactate racemase (LarA) of Lactobacillus plantarum contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect ( k H / k D of 3.11 ± 0.17) was established using 2-α- 2 H-lactate, further supporting a PCHT mechanism. UV-visible spectroscopy revealed a lactate-induced perturbation of the cofactor spectrum, notably increasing the absorbance at 340 nm, and demonstrated an interaction of the cofactor with the inhibitor sulfite. A crystal structure of LarA provided greater resolution (2.4 Å) than previously reported and revealed sulfite binding to the pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride reduction during a PCHT catalytic cycle. Finally, computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand. In aggregate, these studies provide compelling evidence that the nickel-pincer cofactor acts by a PCHT mechanism.

Sequential enzymatic derivatization coupled with online microdialysis sampling for simultaneous profiling of mouse tumor extracellular hydrogen peroxide, lactate, and glucose.

PubMed

Su, Cheng-Kuan; Tseng, Po-Jen; Chiu, Hsien-Ting; Del Vall, Andrea; Huang, Yu-Fen; Sun, Yuh-Chang

2017-03-01

Probing tumor extracellular metabolites is a vitally important issue in current cancer biology. In this study an analytical system was constructed for the in vivo monitoring of mouse tumor extracellular hydrogen peroxide (H 2 O 2 ), lactate, and glucose by means of microdialysis (MD) sampling and fluorescence determination in conjunction with a smart sequential enzymatic derivatization scheme-involving a loading sequence of fluorogenic reagent/horseradish peroxidase, microdialysate, lactate oxidase, pyruvate, and glucose oxidase-for step-by-step determination of sampled H 2 O 2 , lactate, and glucose in mouse tumor microdialysate. After optimization of the overall experimental parameters, the system's detection limit reached as low as 0.002 mM for H 2 O 2 , 0.058 mM for lactate, and 0.055 mM for glucose, based on 3 μL of microdialysate, suggesting great potential for determining tumor extracellular concentrations of lactate and glucose. Spike analyses of offline-collected mouse tumor microdialysate and monitoring of the basal concentrations of mouse tumor extracellular H 2 O 2 , lactate, and glucose, as well as those after imparting metabolic disturbance through intra-tumor administration of a glucose solution through a prior-implanted cannula, were conducted to demonstrate the system's applicability. Our results evidently indicate that hyphenation of an MD sampling device with an optimized sequential enzymatic derivatization scheme and a fluorescence spectrometer can be used successfully for multi-analyte monitoring of tumor extracellular metabolites in living animals. Copyright © 2016 Elsevier B.V. All rights reserved.

Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells

PubMed Central

2013-01-01

Background Most normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA. Methods High throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment. Results 3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu

Physiological function of hydrogen metabolism during growth of sulfidogenic bacteria on organic substrates.

PubMed Central

Lupton, F S; Conrad, R; Zeikus, J G

1984-01-01

Desulfovibrio vulgaris Madison and Thermodesulfobacterium commune contained functionally distinct hydrogenase activities, one which exchanged 3H2 into 3H2O and was inhibited by carbon monoxide and a second activity which produced H2 in the presence of CO. Cell suspensions of D. vulgaris used either lactate, pyruvate, or CO as the electron donor for H2 production in the absence of sulfate. Both sulfidogenic species produced and consumed hydrogen as a trace gas during growth on lactate or pyruvate as electron donors and on thiosulfate or sulfate as electron acceptors. Higher initial levels of hydrogen were detected during growth on lactate-sulfate than on pyruvate-sulfate. D. vulgaris but not T. commune also produced and then consumed CO during growth on organic electron donors and sulfate or thiosulfate. High partial pressures of exogenous H2 inhibited growth and substrate consumption when D. vulgaris was cultured on pyruvate alone but not when it was metabolizing pyruvate plus sulfate or lactate plus sulfate. The data are discussed in relation to supporting two different models for the physiological function of H2 metabolism during growth of sulfidogenic bacteria on organic electron donors plus sulfate. A trace H2 transformation model is proposed for control of redox processes during growth on either pyruvate or lactate plus sulfate, and an obligate H2 cycling model is proposed for chemiosmotic energy coupling during growth on CO plus sulfate. PMID:6480553

Genetics Home Reference: pyruvate kinase deficiency

MedlinePlus

... glucose is broken down to produce adenosine triphosphate (ATP), the cell's main energy source. PKLR gene mutations ... pyruvate kinase enzyme function, causing a shortage of ATP in red blood cells and increased levels of ...

Carrier detection of pyruvate carboxylase deficiency in fibroblasts and lymphocytes.

PubMed

Atkin, B M

1979-10-01

Pyruvate carboxylase (E.C. 6.4.1.1) activity was determined in the circulating peripheral lymphocytes and cultured skin fibroblasts from the family of a patient with hepatic, cerebral, renal cortical, leukocyte, and fibroblast pyruvate carboxylase deficiency (PC Portland deficiency). Lymphocyte activities were: mother, 33--39%; father, 11--29%; brother, 82--103%; and sister, 38--48% of the lowest normal. Fibroblasts from the patient's mother and father had 42 and 34%, respectively, of the activity of the lowest normal. These data demonstrate that the disease is inherited in an autosomal recessive manner and that lymphocytes and fibroblasts can be used to detect carriers. Neither pyruvate carboxylase nor mitochondrial PEPCK activity in lymphocytes was increased by a 21-hr fast.

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

USDA-ARS?s Scientific Manuscript database

Pyruvate phosphate dikinase reversibly converts AMP, pyrophosphate and phosphoenolpyruvate (PEP) to ATP, orthophosphate and pyruvate. Maize PPDK functions in mesophyll in C4 photosynthesis, yet also is highly abundant in starchy endosperm during grain fill where its function is unknown. To investiga...

Ketone bodies effectively compete with glucose for neuronal acetyl-CoA generation in rat hippocampal slices.

PubMed

Valente-Silva, Paula; Lemos, Cristina; Köfalvi, Attila; Cunha, Rodrigo A; Jones, John G

2015-09-01

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1-(13)C]glucose and 1.0 mM [2-(13)C]lactate + 0.1 mM pyruvate (combined [2-(13)C]lactate + [2-(13)C]pyruvate) to acetyl-CoA production were measured both without and with 5.0 mM [U-(13)C]3-hydroxybutyrate in superfused rat hippocampal slices by (13)C NMR non-steady-state isotopomer analysis of tissue glutamate and GABA. Without [U-(13)C]3-hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl-CoA, respectively. With [U-(13)C]3-hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U-(13)C]3-hydroxybutyrate became the major acetyl-CoA contributor (68 ± 3%)--about three-times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U-(13)C]3-hydroxybutyrate contributed approximately the same acetyl-CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1-(13)C]glucose did not decrease significantly in the presence of 3-hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3-hydroxybutyrate. This indicates that, under these conditions, 3-hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase. Copyright © 2015 John Wiley & Sons, Ltd.

Peroxisome proliferator-activated receptor γ agonism attenuates endotoxaemia-induced muscle protein loss and lactate accumulation in rats.

PubMed

Crossland, Hannah; Constantin-Teodosiu, Dumitru; Gardiner, Sheila M; Greenhaff, Paul L

2017-07-01

The peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (Rosi) appears to provide protection against organ dysfunction during endotoxaemia. We examined the potential benefits of Rosi on skeletal muscle protein maintenance and carbohydrate metabolism during lipopolysaccharide (LPS)-induced endotoxaemia. Sprague-Dawley rats were fed either standard chow (control) or standard chow containing Rosi (8.5 ± 0.1 mg·kg -1 ·day -1 ) for 2 weeks before and during 24 h continuous intravenous infusion of LPS (15 μg·kg -1 ·h -1 ) or saline. Rosi blunted LPS-induced increases in muscle tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA by 70% ( P <0.05) and 64% ( P <0.01) respectively. Furthermore, Rosi suppressed the LPS-induced reduction in phosphorylated AKT and phosphorylated Forkhead box O (FOXO) 1 protein, as well as the up-regulation of muscle RING finger 1 (MuRF1; P <0.01) mRNA and the LPS-induced increase in 20S proteasome activity ( P <0.05). Accordingly, LPS reduced the muscle protein:DNA ratio (∼30%, P <0.001), which Rosi offset. Increased muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA ( P <0.001) and muscle lactate accumulation ( P <0.001) during endotoxaemia were suppressed by Rosi. Thus, pre-treatment with Rosi reduced muscle cytokine accumulation and blunted muscle protein loss and lactate accumulation during endotoxaemia, and at least in part by reducing activation of molecular events known to increase muscle protein breakdown and mitochondrial pyruvate use. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Pig liver pyruvate carboxylase. The reaction pathway for the decarboxylation of oxaloacetate

PubMed Central

Warren, Graham B.; Tipton, Keith F.

1974-01-01

1. The reaction pathway for the decarboxylation of oxaloacetate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K+ and acetyl-CoA. 2. Free Mg2+ binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgADP− and Pi bind randomly, at equilibrium, followed by the binding of oxaloacetate. Pyruvate is released before the ordered steay-state release of HCO3− and MgATP2−. 3. These results are entirely consistent with studies on the carboxylation of pyruvate presented in the preceding paper (Warren & Tipton, 1974b) and together they allow a quantitative description of the reaction mechanism of pig liver pyruvate carboxylase. 4. In the absence of other substrates of the back reaction pig liver pyruvate carboxylase will decarboxylate oxaloacetate in a manner that is not inhibited by avidin. 5. Reciprocal plots involving oxaloacetate are non-linear curves, which suggest a negatively co-operative interaction between this substrate and the enzyme. PMID:4447613

High-Throughput Nuclear Magnetic Resonance Metabolomic Footprinting for Tissue Engineering

PubMed Central

Seagle, Christopher; Christie, Megan A.; Winnike, Jason H.; McClelland, Randall E.; Ludlow, John W.; O'Connell, Thomas M.; Gamcsik, Michael P.

2008-01-01

Abstract We report a high-throughput (HTP) nuclear magnetic resonance (NMR) method for analysis of media components and a metabolic schematic to help easily interpret the data. Spin-lattice relaxation values and concentrations were measured for 19 components and 2 internal referencing agents in pure and 2-day conditioned, hormonally defined media from a 3-dimensional (3D) multicoaxial human bioartificial liver (BAL). The 1H NMR spectral signal-to-noise ratio is 21 for 0.16 mM alanine in medium and is obtained in 12 min using a 400 MHz NMR spectrometer. For comparison, 2D gel cultures and 3D multicoaxial BALs were batch cultured, with medium changed every day for 15 days after inoculation with human liver cells in Matrigel–collagen type 1 gels. Glutamine consumption was higher by day 8 in the BAL than in 2D culture; lactate production was lower through the 15-day culture period. Alanine was the primary amino acid produced and tracked with lactate or urea production. Glucose and pyruvate consumption were similar in the BAL and 2D cultures. NMR analysis permits quality assurance of the bioreactor by identifying contaminants. Ethanol was observed because of a bioreactor membrane “wetting” procedure. A biochemical scheme is presented illustrating bioreactor metabolomic footprint results and demonstrating how this can be translated to modify bioreactor operational parameters or quality assurance issues. PMID:18544027

Mitochondrial uncoupling protein may participate in futile cycling of pyruvate and other monocarboxylates.

PubMed

Jezek, P; Borecký, J

1998-08-01

The physiological role of monocarboxylate transport in brown adipose tissue mitochondria has been reevaluated. We studied pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and phenylpyruvate uniport via the uncoupling protein (UCP1) as a GDP-sensitive swelling in K+ salts induced by valinomycin or by monensin and carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone in Na+ salts. We have demonstrated that this uniport is inhibited by fatty acids. GDP inhibition in K+ salts was not abolished by an uncoupler, indicating a negligible monocarboxylic acid penetration via the lipid bilayer. In contrast, the electroneutral pyruvate uptake (swelling in ammonium pyruvate or potassium pyruvate induced by change in pH) mediated by the pyruvate carrier was inhibited by its specific inhibitor alpha-cyano-4-hydroxycinnamate but not by fatty acids. Moreover, alpha-cyano-4-hydroxycinnamate enhanced the energization of brown adipose tissue mitochondria, which was monitored fluorometrically by 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide and safranin O. Consequently, we suggest that UCP1 might participate in futile cycling of unipolar ketocarboxylates under certain physiological conditions while expelling these anions from the matrix. The cycle is completed on their return via the pyruvate carrier in an H+ symport mode.

The activity of pyruvate carrier in a reconstituted system: substrate specificity and inhibitor sensitivity.

PubMed

Nałecz, K A; Kamińska, J; Nałecz, M J; Azzi, A

1992-08-15

The pyruvate carrier, of molecular mass 34 kDa, was purified from mitochondria isolated from rat liver, rat brain, and bovine heart, by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate. Its activity after reconstitution in phosphatidylcholine vesicles was measured either as uptake of [1-14C]pyruvate or as exchange with different 2-oxoacids. All preparations exhibited similar apparent Km values for pyruvate, but somewhat different V(max) values. The ability to exchange different anions of physiological significance, including branched-chain 2-oxoacids, confirmed the known substrate specificity described for the pyruvate carrier in mitochondria. The sensitivity of pyruvate transport toward phenylglyoxal suggested an important role of arginyl residues in the transport activity, while a role of lysyl and histidyl residues was not confirmed.

Pyruvate stabilizes electrocardiographic and hemodynamic function in pigs recovering from cardiac arrest

PubMed Central

Cherry, Brandon H; Nguyen, Anh Q; Hollrah, Roger A; Williams, Arthur G; Hoxha, Besim; Olivencia-Yurvati, Albert H

2015-01-01

Cardiac electromechanical dysfunction may compromise recovery of patients who are initially resuscitated from cardiac arrest, and effective treatments remain elusive. Pyruvate, a natural intermediary metabolite, energy substrate, and antioxidant, has been found to protect the heart from ischemia-reperfusion injury. This study tested the hypothesis that pyruvate-enriched resuscitation restores hemodynamic, metabolic, and electrolyte homeostasis following cardiac arrest. Forty-two Yorkshire swine underwent pacing-induced ventricular fibrillation and, after 6 min pre-intervention arrest, 4 min precordial compressions followed by transthoracic countershocks. After defibrillation and recovery of spontaneous circulation, the pigs were monitored for another 4 h. Sodium pyruvate or NaCl were infused i.v. (0.1 mmol·kg−1·min−1) throughout precordial compressions and the first 60 min recovery. In 8 of the 24 NaCl-infused swine, the first countershock converted ventricular fibrillation to pulseless electrical activity unresponsive to subsequent countershocks, but only 1 of 18 pyruvate-treated swine developed pulseless electrical activity (relative risk 0.17; 95% confidence interval 0.13–0.22). Pyruvate treatment also lowered the dosage of vasoconstrictor phenylephrine required to maintain systemic arterial pressure at 15–60 min recovery, hastened clearance of excess glucose, elevated arterial bicarbonate, and raised arterial pH; these statistically significant effects persisted up to 3 h after sodium pyruvate infusion, while infusion-induced hypernatremia subsided. These results demonstrate that pyruvate-enriched resuscitation achieves electrocardiographic and hemodynamic stability in swine during the initial recovery from cardiac arrest. Such metabolically based treatment may offer an effective strategy to support cardiac electromechanical recovery immediately after cardiac arrest. PMID:26088865

Free energy landscape of the Michaelis complex of lactate dehydrogenase: A network analysis of atomistic simulations

NASA Astrophysics Data System (ADS)

Pan, Xiaoliang; Schwartz, Steven

2015-03-01

It has long been recognized that the structure of a protein is a hierarchy of conformations interconverting on multiple time scales. However, the conformational heterogeneity is rarely considered in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they are catalytic competent at different reaction rates. In this study, millisecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network the Michaelis complex and the structures of the substates at atomistic scale. It also shed some light on understanding the complete picture of the catalytic mechanism of LDH.

Free energy surface of the Michaelis complex of lactate dehydrogenase: a network analysis of microsecond simulations.

PubMed

Pan, Xiaoliang; Schwartz, Steven D

2015-04-30

It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD(+)). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.

Supplementation of pyruvate prevents palmitate-induced impairment of glucose uptake in C2 myotubes.

PubMed

Jung, Jong Gab; Choi, Sung-E; Hwang, Yoon-Jung; Lee, Sang-A; Kim, Eun Kyoung; Lee, Min-Seok; Han, Seung Jin; Kim, Hae Jin; Kim, Dae Jung; Kang, Yup; Lee, Kwan-Woo

2011-10-15

Elevated fatty acid levels have been thought to contribute to insulin resistance. Repression of the glucose transporter 4 (GLUT4) gene as well as impaired GLUT4 translocation may be a mediator for fatty acid-induced insulin resistance. This study was initiated to determine whether palmitate treatment repressed GLUT4 expression, whether glucose/fatty acid metabolism influenced palmitate-induced GLUT4 gene repression (PIGR), and whether attempts to prevent PIGR restored palmitate-induced impairment of glucose uptake (PIIGU) in C2 myotubes. Not only stimulators of fatty acid oxidation, such as bezafibrate, AICAR, and TOFA, but also TCA cycle substrates, such as pyruvate, leucine/glutamine, and α-ketoisocaproate/monomethyl succinate, significantly prevented PIGR. In particular, supplementing with pyruvate through methyl pyruvate resulted in nearly complete prevention of PIIGU, whereas palmitate treatment reduced the intracellular pyruvate level. These results suggest that pyruvate depletion plays a critical role in PIGR and PIIGU; thus, pyruvate supplementation may help prevent obesity-induced insulin resistance in muscle cells. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

Neuroprotective effect of pyruvate and oxaloacetate during pilocarpine induced status epilepticus in rats.

PubMed

Carvalho, Andrezza Sossai Rodrigues; Torres, Laila Brito; Persike, Daniele Suzete; Fernandes, Maria José Silva; Amado, Debora; Naffah-Mazzacoratti, Maria da Graça; Cavalheiro, Esper Abrão; da Silva, Alexandre Valotta

2011-02-01

Recent research data have shown that systemic administration of pyruvate and oxaloacetate causes an increased brain-to-blood glutamate efflux. Since increased release of glutamate during epileptic seizures can lead to excitotoxicity and neuronal cell death, we tested the hypothesis that glutamate scavenging mediated by pyruvate and oxaloacetate systemic administration could have a neuroprotective effect in rats subjected to status epilepticus (SE). SE was induced by a single dose of pilocarpine (350mg/kgi.p.). Thirty minutes after SE onset, a single dose of pyruvate (250mg/kgi.p.), oxaloacetate (1.4mg/kgi.p.), or both substances was administrated. Acute neuronal loss in hippocampal regions CA1 and hilus was quantitatively determined five hours after SE onset, using the optical fractionator method for stereological cell counting. Apoptotic cascade in the hippocampus was also investigated seven days after SE using caspase-1 and -3 activity assays. SE-induced neuronal loss in CA1 was completely prevented in rats treated with pyruvate plus oxaloacetate. The SE-induced caspase-1 activation was significantly reduced when rats were treated with oxaloacetate or pyruvate plus oxaloacetate. The treatment with pyruvate and oxaloacetate caused a neuroprotective effect in rats subjected to pilocarpine-induced SE. Copyright © 2010 Elsevier Ltd. All rights reserved.

Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

PubMed

Vogel, O; Hoehn, B; Henning, U

1972-06-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex.

Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

PubMed Central

Vogel, Otto; Hoehn, Barbara; Henning, Ulf

1972-01-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 × 106. All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This “excess” component is bound differently than are the eight dimers in the core complex. Images PMID:4556465

Triiodothyronine activates lactate oxidation without impairing fatty acid oxidation and improves weaning from extracorporeal membrane oxygenation.

PubMed

Kajimoto, Masaki; Ledee, Dolena R; Xu, Chun; Kajimoto, Hidemi; Isern, Nancy G; Portman, Michael A

2014-01-01

Extracorporeal membrane oxygenation (ECMO) provides a rescue for children with severe cardiac failure. It has previously been shown that triiodothyronine (T3) improves cardiac function by modulating pyruvate oxidation during weaning. This study focused on fatty acid (FA) metabolism modulated by T3 for weaning from ECMO after cardiac injury. METHODS AND RESULTS: Nineteen immature piglets (9.1-15.3 kg) were separated into 3 groups with ECMO (6.5 h) and wean: normal circulation (Group-C); transient coronary occlusion (10 min) for ischemia-reperfusion (IR) followed by ECMO (Group-IR); and IR with T3 supplementation (Group-IR-T3). 13-Carbon ((13)C)-labeled lactate, medium-chain and long-chain FAs, was infused as oxidative substrates. Substrate fractional contribution (FC) to the citric acid cycle was analyzed by(13)C-nuclear magnetic resonance. ECMO depressed circulating T3 levels to 40% of the baseline at 4 h and were restored in Group-IR-T3. Group-IR decreased cardiac power, which was not fully restorable and 2 pigs were lost because of weaning failure. Group-IR also depressed FC-lactate, while the excellent contractile function and energy efficiency in Group-IR-T3 occurred along with a marked FC-lactate increase and [adenosine triphosphate]/[adenosine diphosphate] without either decreasing FC-FAs or elevating myocardial oxygen consumption over Group-C or -IR. T3 releases inhibition of lactate oxidation following IR injury without impairing FA oxidation. These findings indicate that T3 depression during ECMO is maladaptive, and that restoring levels improves metabolic flux and enhances contractile function during weaning.

Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle

PubMed Central

Patel, Anant B.; Lai, James C. K.; Chowdhury, Golam M. I.; Hyder, Fahmeed; Rothman, Douglas L.; Shulman, Robert G.; Behar, Kevin L.

2014-01-01

Previous 13C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-d-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG6P), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions. PMID:24706914

Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle.

PubMed

Patel, Anant B; Lai, James C K; Chowdhury, Golam M I; Hyder, Fahmeed; Rothman, Douglas L; Shulman, Robert G; Behar, Kevin L

2014-04-08

Previous (13)C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-D-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG6P), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions.

Use of 5-deazaFAD to study hydrogen transfer in the D-amino acid oxidase reaction.

PubMed

Hersh, L B; Jorns, M S

1975-11-25

The apoprotein of hog kidney D-amino acid oxidase was reconstituted with 5-deazaflavin adenine dinucleotide (5-deazaFAD) to yield a protein which contains 1.5 mol of 5-deazaFAD/mol of enzyme. The deazaFAD-containing enzyme forms complexes with benzoate, 2-amino benzoate, and 4-aminobenzoate which are both qualitatively and quantitatively similar to those observed with native enzyme. The complex with 2-aminobenzoate exhibits a new long wavelength absorption band characteristic of a flavin charge-transfer complex. The reconstituted enzyme exhibits no activity when assayed by D-alanine oxidation. However, the bound chromophore can be reduced by alanine, phenylalanine, proline, methionine, and valine, but not by glutamate or aspartate, indicating the deazaFAD enzyme retains the substrate specificity of the native enzyme. Reduction of the enzyme by D-alanine exhibits a 1.6-fold deuterium isotope effect. Reoxidation of the reduced enzyme occurred in the presence of pyruvate plus ammonia, but not with pyruvate alone or ammonia alone. beta-Phenylpyruvate and alpha-ketobutyrate, but not alpha-ketoglutarate could replace pyruvate. Reduced enzyme isolated following reaction with [alpha-3H]alanine was found to contain 0.5 mol of tritium/mol of deazaFADH2. After denaturation of the tritium-labeled enzyme, the radioactivity was identified as deazaFADH2. Reaction of the reduced tritium-labeled enzyme with pyruvate plus ammonia prior to denaturation yields [alpha-3H]alanine and unlabeled deazaFAD. These results suggest that reduction and reoxidation of enzyme-bound deazaFAD involves the stereo-specific transfer of alpha-hydrogen from substrate to deazaFAD.

Evidence for compromised metabolic function and limited glucose uptake in spermatozoa from the teratospermic domestic cat (Felis catus) and cheetah (Acinonyx jubatus).

PubMed

Terrell, Kimberly A; Wildt, David E; Anthony, Nicola M; Bavister, Barry D; Leibo, Stanley P; Penfold, Linda M; Marker, Laurie L; Crosier, Adrienne E

2010-11-01

Cheetahs and certain other felids consistently ejaculate high proportions (≥ 60%) of malformed spermatozoa, a condition known as teratospermia, which is prevalent in humans. Even seemingly normal spermatozoa from domestic cat teratospermic ejaculates have reduced fertilizing capacity. To understand the role of sperm metabolism in this phenomenon, we conducted a comparative study in the normospermic domestic cat versus the teratospermic cat and cheetah with the general hypothesis that sperm metabolic function is impaired in males producing predominantly pleiomorphic spermatozoa. Washed ejaculates were incubated in chemically defined medium containing glucose and pyruvate. Uptake of glucose and pyruvate and production of lactate were assessed using enzyme-linked fluorescence assays. Spermatozoa from domestic cats and cheetahs exhibited similar metabolic profiles, with minimal glucose metabolism and approximately equimolar rates of pyruvate uptake and lactate production. Compared to normospermic counterparts, pyruvate and lactate metabolism were reduced in teratospermic cat and cheetah ejaculates, even when controlling for sperm motility. Rates of pyruvate and lactate (but not glucose) metabolism were correlated positively with sperm motility, acrosomal integrity, and normal morphology. Collectively, our findings reveal that pyruvate uptake and lactate production are reliable, quantitative indicators of sperm quality in these two felid species and that metabolic function is impaired in teratospermic ejaculates. Furthermore, patterns of substrate utilization are conserved between these species, including the unexpected lack of exogenous glucose metabolism. Because glycolysis is required to support sperm motility and capacitation in certain other mammals (including dogs), the activity of this pathway in felid spermatozoa is a target for future investigation.

Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons.

PubMed

Bak, Lasse K; Obel, Linea F; Walls, Anne B; Schousboe, Arne; Faek, Sevan A A; Jajo, Farah S; Waagepetersen, Helle S

2012-04-05

We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca2+ during activation cause influx of Ca2+ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate-aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca2+ (i.e. independent of synaptic activity) on neuronal energy metabolism employing 13C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca2+ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca2+ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca2+-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca2+ signalling.

Adaptation of skeletal muscle energy metabolism to repeated hypoxic-normoxic exposures and drug treatment.

PubMed

Pastoris, O; Dossena, M; Gorini, A; Vercesi, L; Benzi, G

1985-03-01

Muscular glycolytic fuels, intermediates and end-products (glycogen, glucose, glucose-6-phosphate, pyruvate, lactate), Krebs cycle intermediates (citrate, alpha-ketoglutarate, succinate, malate), related free amino acids (glutamate, alanine), ammonia, energy store (creatine phosphate), energy mediators (ATP, ADP, AMP) and energy charge potential were evaluated. Furthermore the maximum rate (Vmax) of the following muscular enzyme activities was evaluated in the crude extract and/or mitochondrial fraction: for the anaerobic glycolytic pathway: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; for the tricarboxylic acid cycle: citrate synthase, malate dehydrogenase; for the electron transfer chain: total NADH cytochrome c reductase, cytochrome oxidase. The rat gastrocnemius muscles were analyzed in normoxia and after repeated, alternate hypoxic and normoxic exposures (12 hours of hypoxia daily; for 5 days). Naftidrofuryl was administered daily at three different doses: 10, 15 and 22.5 mg/kg i.m., 30 min before the beginning of the experimental hypoxia. The biochemical adaptation to intermittent normobaric hypoxic-normoxic exposures was characterized by the decrease of the muscular contents of creatine phosphate, citrate, alpha-ketoglutarate and glutamate. This adaptation occurred in absence of significant changes in the Vmax of the muscle enzymes tested. By naftidrofuryl treatment, in gastrocnemius muscle from hypoxic rats both alpha-ketoglutarate and creatine phosphate contents maintained normal values, while glutamate concentration remained reduced to subnormal values. With the exception of hexokinase, naftidrofuryl treatment did not modify the Vmax of marker enzymes related to energy transduction.

Energy metabolism and biotransformation as endpoints to pre-screen hepatotoxicity using a liver spheroid model

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

Xu Jinsheng; Purcell, Wendy M.

2006-10-15

The current study investigated liver spheroid culture as an in vitro model to evaluate the endpoints relevant to the status of energy metabolism and biotransformation after exposure to test toxicants. Mature rat liver spheroids were exposed to diclofenac, galactosamine, isoniazid, paracetamol, m-dinitrobenzene (m-DNB) and 3-nitroaniline (3-NA) for 24 h. Pyruvate uptake, galactose biotransformation, lactate release and glucose secretion were evaluated after exposure. The results showed that pyruvate uptake and lactate release by mature liver spheroids in culture were maintained at a relatively stable level. These endpoints, together with glucose secretion and galactose biotransformation, were related to and could reflect themore » status of energy metabolism and biotransformation in hepatocytes. After exposure, all of the test agents significantly reduced glucose secretion, which was shown to be the most sensitive endpoint of those evaluated. Diclofenac, isoniazid, paracetamol and galactosamine reduced lactate release (P < 0.01), but m-DNB increased lactate release (P < 0.01). Diclofenac, isoniazid and paracetamol also reduced pyruvate uptake (P < 0.01), while galactosamine had little discernible effect. Diclofenac, galactosamine, paracetamol and m-DNB also reduced galactose biotransformation (P < 0.01), by contrast, isoniazid did not. The metabolite of m-DNB, 3-NA, which served as a negative control, did not cause significant changes in lactate release, pyruvate uptake or galactose biotransformation. It is concluded that pyruvate uptake, galactose biotransformation, lactate release and glucose secretion can be used as endpoints for evaluating the status of energy metabolism and biotransformation after exposure to test agents using the liver spheroid model to pre-screen hepatotoxicity.« less

The Positive Inotropic Effect of Pyruvate Involves an Increase in Myofilament Calcium Sensitivity

PubMed Central

Torres, Carlos A. A.; Varian, Kenneth D.; Canan, Cynthia H.; Davis, Jonathan P.; Janssen, Paul M. L.

2013-01-01

Pyruvate is a metabolic fuel that is a potent inotropic agent. Despite its unique inotropic and antioxidant properties, the molecular mechanism of its inotropic mechanism is still largely unknown. To examine the inotropic effect of pyruvate in parallel with intracellular calcium handling under near physiological conditions, we measured pH, myofilament calcium sensitivity, developed force, and calcium transients in ultra thin rabbit heart trabeculae at 37 °C loaded iontophoretically with the calcium indicator bis-fura-2. By contrasting conditions of control versus sarcoplasmic reticulum block (with either cyclopiazonic acid and ryanodine or with thapsigargin) we were able to characterize and isolate the effects of pyruvate on sarcoplasmic reticulum calcium handling and developed force. A potassium contracture technique was subsequently utilized to assess the force-calcium relationship and thus the myofilament calcium sensitivity. Pyruvate consistently increased developed force whether or not the sarcoplasmic reticulum was blocked (16.8±3.5 to 24.5±5.1 vs. 6.9±2.6 to 12.5±4.4 mN/mm2, non-blocked vs. blocked sarcoplasmic reticulum respectively, p<0.001, n = 9). Furthermore, the sensitizing effect of pyruvate on the myofilaments was demonstrated by potassium contractures (EC50 at baseline versus 20 minutes of pyruvate infusion (peak force development) was 701±94 vs. 445±65 nM, p<0.01, n = 6). This study is the first to demonstrate that a leftward shift in myofilament calcium sensitivity is an important mediator of the inotropic effect of pyruvate. This finding can have important implications for future development of therapeutic strategies in the management of heart failure. PMID:23691074

Performance effects of acute β-alanine induced paresthesia in competitive cyclists.

PubMed

Bellinger, Phillip M; Minahan, Clare L

2016-01-01

β-alanine is a common ingredient in supplements consumed by athletes. Indeed, athletes may believe that the β-alanine induced paresthesia, experienced shortly after ingestion, is associated with its ergogenic effect despite no scientific mechanism supporting this notion. The present study examined changes in cycling performance under conditions of β-alanine induced paresthesia. Eight competitive cyclists (VO2max = 61.8 ± 4.2 mL·kg·min(-1)) performed three practices, one baseline and four experimental trials. The experimental trials comprised a 1-km cycling time trial under four conditions with varying information (i.e., athlete informed β-alanine or placebo) and supplement content (athlete received β-alanine or placebo) delivered to the cyclist: informed β-alanine/received β-alanine, informed placebo/received β-alanine, informed β-alanine/received placebo and informed placebo/received placebo. Questionnaires were undertaken exploring the cyclists' experience of the effects of the experimental conditions. A possibly likely increase in mean power was associated with conditions in which β-alanine was administered (±95% CL: 2.2% ± 4.0%), but these results were inconclusive for performance enhancement (p = 0.32, effect size = 0.18, smallest worthwhile change = 56% beneficial). A possibly harmful effect was observed when cyclists were correctly informed that they had ingested a placebo (-1.0% ± 1.9%). Questionnaire data suggested that β-alanine ingestion resulted in evident sensory side effects and six cyclists reported placebo effects. Acute ingestion of β-alanine is not associated with improved 1-km TT performance in competitive cyclists. These findings are in contrast to the athlete's "belief" as cyclists reported improved energy and the ability to sustain a higher power output under conditions of β-alanine induced paresthesia.

β-Cell-specific pyruvate dehydrogenase deficiency impairs glucose-stimulated insulin secretion

PubMed Central

Srinivasan, Malathi; Choi, Cheol S.; Ghoshal, Pushpankur; Pliss, Lioudmila; Pandya, Jignesh D.; Hill, David; Cline, Gary

2010-01-01

Glucose-stimulated insulin secretion (GSIS) by β-cells requires the generation of ATP from oxidation of pyruvate as well as generation of coupling factors involving three different pyruvate cycling shuttles. The roles of several key enzymes involved in pyruvate cycling in β-cells have been documented using isolated islets and β-cell clonal lines. To investigate the role of the pyruvate dehydrogenase (PDH) complex (PDC) in GSIS, a murine model of β-cell-specific PDH deficiency (β-PDHKO) was created. Pancreatic insulin content was decreased in 1-day-old β-PDHKO male pups and adult male mice. The plasma insulin levels were decreased and blood glucose levels increased in β-PDHKO male mice from neonatal life onward. GSIS was reduced in isolated islets from β-PDHKO male mice with about 50% reduction in PDC activity. Impairment in a glucose tolerance test and in vivo insulin secretion during hyperglycemic clamp was evident in β-PDHKO adults. No change in the number or size of islets was found in pancreata from 4-wk-old β-PDHKO male mice. However, an increase in the mean size of individual β-cells in islets of these mice was observed. These findings show a key role of PDC in GSIS by pyruvate oxidation. This β-PDHKO mouse model represents the first mouse model in which a mitochondrial oxidative enzyme deletion by gene knockout has been employed to demonstrate an altered GSIS by β-cells. PMID:20841503

Exogenous lactate supply affects lactate kinetics of rainbow trout, not swimming performance

PubMed Central

Omlin, Teye; Langevin, Karolanne

2014-01-01

Intense swimming causes circulatory lactate accumulation in rainbow trout because lactate disposal (Rd) is not stimulated as strongly as lactate appearance (Ra). This mismatch suggests that maximal Rd is limited by tissue capacity to metabolize lactate. This study uses exogenous lactate to investigate what constrains maximal Rd and minimal Ra. Our goals were to determine how exogenous lactate affects: 1) Ra and Rd of lactate under baseline conditions or during graded swimming, and 2) exercise performance (critical swimming speed, Ucrit) and energetics (cost of transport, COT). Results show that exogenous lactate allows swimming trout to boost maximal Rd lactate by 40% and reach impressive rates of 56 μmol·kg−1·min−1. This shows that the metabolic capacity of tissues for lactate disposal is not responsible for setting the highest Rd normally observed after intense swimming. Baseline endogenous Ra (resting in normoxic water) is not significantly reduced by exogenous lactate supply. Therefore, trout have an obligatory need to produce lactate, either as a fuel for oxidative tissues and/or from organs relying on glycolysis. Exogenous lactate does not affect Ucrit or COT, probably because it acts as a substitute for glucose and lipids rather than extra fuel. We conclude that the observed 40% increase in Rd lactate is made possible by accelerating lactate entry into oxidative tissues via monocarboxylate transporters (MCTs). This observation together with the weak expression of MCTs and the phenomenon of white muscle lactate retention show that lactate metabolism of rainbow trout is significantly constrained by transmembrane transport. PMID:25121611

The impact of ions on allosteric functions in human liver pyruvate kinase

PubMed Central

Alontaga, Aileen Y.

2010-01-01

Experimental designs used to monitor the magnitude of an allosteric response can greatly influence observed values. We report here the impact of buffer, monovalent cation, divalent cation and anion on the magnitude of the allosteric regulation of the affinity of human liver pyruvate kinase (hL-PYK) for substrate, phosphoenolpyruvate (PEP). The magnitudes of the allosteric activation by fructose-1,6-bisphosphate (Fru-1,6-BP) and the allosteric inhibition by alanine are independent of most, but not all buffers tested. However, these magnitudes are dependent on whether Mg2+ or Mn2+ is included as the divalent cation. In the presence of Mn2+, any change in Kapp-PEP caused by Fru-1,6-BP is minimal. hL-PYK activity does not appear to require monovalent cation. Monovalent cation binding in the active site impacts PEP affinity with minimum influence on the magnitude of allosteric coupling. However, Na+ and Li+ reduce the magnitude of the allosteric response to Fru-1,6-BP, likely due to mechanisms outside of the active site. Which anion is used to maintain a constant monovalent cation concentration also influences the magnitude of the allosteric response. The value of determining the impact of ions on allosteric function can be appreciated by considering that representative structures used in comparative studies have often been determined using protein crystals grown in diverse buffer and salt conditions. PMID:21609859

Lactation and reproduction*

PubMed Central

Thomson, A. M.; Hytten, F. E.; Black, A. E.

1975-01-01

The authors review the literature on the effect of lactation on fertility in the absence of contraception and on the effects of contraceptive measures on lactation. They examine data from several countries on the intervals between births and on the return of menstruation and ovulation after childbirth, comparing lactating with nonlactating women. They conclude that lactation is an inefficient contraceptive for the individual, but that in populations sustained lactation is associated with reduced fertility. Possible physiological mechanisms causing lactation amenorrhoea are discussed. Though much of the literature on the effect of contraceptives on lactation is inadequate, there is general agreement that the estrogen component of hormonal preparations has an adverse effect on lactation, but that progestins alone do not. Many questions remain. Is this effect seen in established lactation, or only in the puerperal period? Is it a direct pharmacological effect, or are pill-users the mothers least motivated to maintain breast-feeding? Does a close relationship exist between hormones given and lactation performance? The authors comment on some of the technical deficiencies of previous studies in this field and discuss practical possibilities of, and limitations to, obtaining adequate scientific information in the future. PMID:1084804

Identification of diagnostic biomarkers and metabolic pathway shifts of heat-stressed lactating dairy cows.

PubMed

Tian, He; Wang, Weiyu; Zheng, Nan; Cheng, Jianbo; Li, Songli; Zhang, Yangdong; Wang, Jiaqi

2015-07-01

Controlling heat stress (HS) is a global challenge for the dairy industry. However, simple and reliable biomarkers that aid the diagnoses of HS-induced metabolic disorders have not yet been identified. In this work, an integrated metabolomic and lipidomic approach using (1)H nuclear magnetic resonance and ultra-fast LC-MS was employed to investigate the discrimination of plasma metabolic profiles between HS-free and HS lactating dairy cows. Targeted detection using LC-MS in multiple reaction monitoring mode was used to verify the reliability of the metabolites as biomarker candidates. Overall, 41 metabolites were identified as candidates for lactating dairy cows exposed to HS, among which 13 metabolites, including trimethylamine, glucose, lactate, betaine, creatine, pyruvate, acetoacetate, acetone, β-hydroxybutyrate, C16 sphinganine, lysophosphatidylcholine (18:0), phosphatidylcholine (16:0/14:0), and arachidonic acid, had high sensitivity and specificity in diagnosing HS status, and are likely to be the potential biomarkers of HS dairy cows. All of these potentially diagnostic biomarkers were involved in carbohydrate, amino acid, lipid, or gut microbiome-derived metabolism, indicating that HS affected the metabolic pathways in lactating dairy cows. Further research is warranted to evaluate these biomarkers in practical applications and to elucidate the physiological mechanisms of HS-induced metabolic disorders. Heat stress (HS) annually causes huge losses to global dairy industry, including animal performance decrease, metabolic disorder and health problem. So far, physiological mechanisms underlying HS of dairy cows still remain elusive. To our best knowledge, this is the first attempt to elucidate the HS-induced metabolic disorders of dairy cows using integrated (1)H NMR and LC-MS-based metabolic study. The results not only provided potential diagnostic biomarkers for HS lactating dairy cows, but also significantly explored the related physiological mechanisms

Superior Cardiac Function Via Anaplerotic Pyruvate in the Immature Swine Heart After Cardiopulmonary Bypass and Reperfusion

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

Olson, Aaron; Hyyti, Outi M.; Cohen, Gordon A.

2008-12-01

Pyruvate produces inotropic responses in the adult reperfused heart. Pyruvate oxidation and anaplerotic entry into the citric acid cycle (CAC) via carboxylation are linked to stimulation of contractile function. The goals of this study were to determine if these metabolic pathways operate and are maintained in the developing myocardium after reperfusion. Immature male swine (age 10-18 days) were subjected to cardiopulmonary bypass (CPB). Intracoronary infusion of [2]-13C-pyruvate (to achieve a final concentration of 8 mM) was given for 35 minutes starting either during weaning (Group I), after discontinuation (Group II) or without (Control) CPB. Hemodynamic data was collected. 13C NMRmore » spectroscopy was used to determine the fraction of pyruvate entering the CAC via pyruvate carboxylation (PC) to total CAC entry (PC plus decarboxlyation via pyruvate dehydrogenase). Liquid chromatography-mass spectrometry was used to determine total glutamate enrichment.« less

Prognostic significance of blood lactate and lactate clearance in trauma patients.

PubMed

Régnier, Marie-Alix; Raux, Mathieu; Le Manach, Yannick; Asencio, Yves; Gaillard, Johann; Devilliers, Catherine; Langeron, Olivier; Riou, Bruno

2012-12-01

Lactate has been shown to be a prognostic biomarker in trauma. Although lactate clearance has already been proposed as an intermediate endpoint in randomized trials, its precise role in trauma patients remains to be determined. Blood lactate levels and lactate clearance (LC) were calculated at admission and 2 and 4 h later in trauma patients. The association of initial blood lactate level and lactate clearance with mortality was tested using receiver-operating characteristics curve, logistic regression using triage scores, Trauma Related Injury Severity Score as a reference standard, and reclassification method. The authors evaluated 586 trauma patients (mean age 38±16 yr, 84% blunt and 16% penetrating, mortality 13%). Blood lactate levels at admission were elevated in 327 (56%) patients. The lactate clearance should be calculated within the first 2 h after admission as LC0-2 h was correlated with LC0-4 h (R=0.55, P<0.001) but not with LC2-4 h (R=0.04, not significant). The lactate clearance provides additional predictive information to initial blood lactate levels and triage scores and the reference score. This additional information may be summarized using a categorical approach (i.e., less than or equal to -20 %/h) in contrast to initial blood lactate. The results were comparable in patients with high (5 mM/l or more) initial blood lactate. Early (0-2 h) lactate clearance is an important and independent prognostic variable that should probably be incorporated in future decision schemes for the resuscitation of trauma patients.

The Role of Pyruvate in Protecting 661W Photoreceptor-Like Cells Against Light-Induced Cell Death.

PubMed

Natoli, Riccardo; Rutar, Matt; Lu, Yen-Zhen; Chu-Tan, Joshua A; Chen, Yuwei; Saxena, Kartik; Madigan, Michele; Valter, Krisztina; Provis, Jan M

2016-11-01

Light is a requirement for the function of photoreceptors in visual processing. However, prolonged light exposure can be toxic to photoreceptors, leading to increased reactive oxygen species (ROS), lipid peroxidation, and photoreceptor cell death. We used the 661W mouse cone photoreceptor-like cell line to study the effects of pyruvate in protecting these cells from light-induced toxicity. 661W cells were exposed to 15,000 lux continuous bright light for 5 hours and incubated in Dulbecco's modified eagle medium (DMEM) with various concentrations of pyruvate. Following light damage, cells were assessed for changes in morphology, cell toxicity, viability, and ROS production. Mitochondrial respiration and anaerobic glycolysis were also assessed using a Seahorse Xfe96 extracellular flux analyzer. We found that cell death caused by light damage in 661W cells was dramatically reduced in the presence of pyruvate. Cells with pyruvate-supplemented media also showed attenuation of oxidative stress and maintained normal levels of ATP. We also found that alterations in the concentrations of pyruvate had no effect on mitochondrial respiration or glycolysis in light-damaged cells. Taken together, the results show that pyruvate is protective against light damage but does not alter the metabolic output of the cells, indicating an alternative role for pyruvate in reducing oxidative stress. Thus, sodium pyruvate is a possible candidate for the treatment against the oxidative stress component of retinal degenerations.

Occurrence of the malate-aspartate shuttle in various tumor types.

PubMed

Greenhouse, W V; Lehninger, A L

1976-04-01

The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm.

Nonconvulsive electrographic seizures after traumatic brain injury result in a delayed, prolonged increase in intracranial pressure and metabolic crisis.

PubMed

Vespa, Paul M; Miller, Chad; McArthur, David; Eliseo, Mathew; Etchepare, Maria; Hirt, Daniel; Glenn, Thomas C; Martin, Neil; Hovda, David

2007-12-01

To determine whether nonconvulsive electrographic post-traumatic seizures result in increases in intracranial pressure and microdialysis lactate/pyruvate ratio. Prospective monitoring with retrospective data analysis. Single center academic neurologic intensive care unit. Twenty moderate to severe traumatic brain injury patients (Glasgow Coma Score 3-13). Continuous electroencephalography and cerebral microdialysis were performed for 7 days after injury. Ten patients had seizures and were compared with a matched cohort of traumatic brain injury patients without seizures. The seizures were repetitive and constituted status epilepticus in seven of ten patients. Using a within-subject design, post-traumatic seizures resulted in episodic increases in intracranial pressure (22.4 +/- 7 vs. 12.8 +/- 4.3 mm Hg; p < .001) and an episodic increase in lactate/pyruvate ratio (49.4 +/- 16 vs. 23.8 +/- 7.6; p < .001) in the seizure group. Using a between-subjects comparison, the seizure group demonstrated a higher mean intracranial pressure (17.6 +/- 6.5 vs. 12.2 +/- 4.2 mm Hg; p < .001), a higher mean lactate/pyruvate ratio (38.6 +/- 18 vs. 27 +/- 9; p < .001) compared with nonseizure patients. The intracranial pressure and lactate/pyruvate ratio remained elevated beyond postinjury hour 100 in the seizure group but not the nonseizure group (p < .02). Post-traumatic seizures result in episodic as well as long-lasting increases in intracranial pressure and microdialysis lactate/pyruvate ratio. These data suggest that post-traumatic seizures represent a therapeutic target for patients with traumatic brain injury.

Inhibition of lactation.

PubMed

Llewellyn-Jones, D

1975-01-01

The mechanism and hormonal regulation of lactation is explained and illustrated with a schematic representation. Circulating estrogen above a critical amount seems to be the inhibitory factor controlling lactation during pregnancy. Once delivery occurs, the level of estrogen falls, that of prolactin rises, and lactation begins. Nonsuckling can be used to inhibit lactation. Estrogens can also be used to inhibit lactation more quickly and with less pain. The reported association between estrogens and puerperal thromboembolism cannot be considered conclusive due to defects in the reporting studies. There is no reason not to use estrogens in lactation inhibition except for women over 35 who experienced a surgical delivery. Alternative therapy is available for these women. The recently-developed drug, brom-ergocryptine, may replace other methods of lactation inhibition.

Does the 1H-NMR plasma metabolome reflect the host-tumor interactions in human breast cancer?

PubMed

Richard, Vincent; Conotte, Raphaël; Mayne, David; Colet, Jean-Marie

2017-07-25

Breast cancer (BC) is the most common diagnosed cancer and the leading cause of cancer death in women worldwide. There is an obvious need for a better understanding of BC biology. Alterations in the serum metabolome of BC patients have been identified but their clinical significance remains elusive. We evaluated by 1H-Nuclear Magnetic Resonance (1H-NMR) spectroscopy, filtered plasma metabolome of 50 early (EBC) and 15 metastatic BC (MBC) patients. Using Principal Component Analysis, Partial Least-Squares Discriminant Analysis and Hierarchical Clustering we show that plasma levels of glucose, lactate, pyruvate, alanine, leucine, isoleucine, glutamate, glutamine, valine, lysine, glycine, threonine, tyrosine, phenylalanine, acetate, acetoacetate, β-hydroxy-butyrate, urea, creatine and creatinine are modulated across patients clusters. In particular lactate levels are inversely correlated with the tumor size in the EBC cohort (Pearson correlation r = -0.309; p = 0.044). We suggest that, in BC patients, tumor cells could induce modulation of the whole patient's metabolism even at early stages. If confirmed in a lager study these observations could be of clinical importance.

Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons

PubMed Central

Bak, Lasse K.; Obel, Linea F.; Walls, Anne B.; Schousboe, Arne; Faek, Sevan A.A.; Jajo, Farah S.; Waagepetersen, Helle S.

2012-01-01

We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca2+ during activation cause influx of Ca2+ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate–aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca2+ (i.e. independent of synaptic activity) on neuronal energy metabolism employing 13C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca2+ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca2+ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca2+-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca2+ signalling. PMID:22385215

Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

NASA Technical Reports Server (NTRS)

2003-01-01

Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

EPR/alanine dosimetry for two therapeutic proton beams

NASA Astrophysics Data System (ADS)

Marrale, Maurizio; Carlino, Antonio; Gallo, Salvatore; Longo, Anna; Panzeca, Salvatore; Bolsi, Alessandra; Hrbacek, Jan; Lomax, Tony

2016-02-01

In this work the analysis of the electron paramagnetic resonance (EPR) response of alanine pellets exposed to two different clinical proton beams employed for radiotherapy is performed. One beam is characterized by a passive delivery technique and is dedicated to the eyes treatment (OPTIS2 beam line). Alanine pellets were irradiated with a 70 MeV proton beam corresponding to 35 mm range in eye tissue. We investigated how collimators with different sizes and shape used to conform the dose to the planned target volume influence the delivered dose. For this purpose we performed measurements with varying the collimator size (Output Factor) and the results were compared with those obtained with other dosimetric techniques (such as Markus chamber and diode detector). This analysis showed that the dosimeter response is independent of collimator diameter if this is larger than or equal to 10 mm. The other beam is characterized by an active spot-scanning technique, the Gantry1 beam line (maximum energy 230 MeV), and is used to treat deep-seated tumors. The dose linearity of alanine response in the clinical dose range was tested and the alanine dose response at selected locations in depth was measured and compared with the TPS planned dose in a quasi-clinical scenario. The alanine response was found to be linear in the dose in the clinical explored range (from 10 to 70 Gy). Furthermore, a depth dose profile in a quasi-clinical scenario was measured and compared to the dose computed by the Treatment Planning System PSIPLAN. The comparison of calibrated proton alanine measurements and TPS dose shows a difference under 1% in the SOBP and a "quenching" effect up to 4% in the distal part of SOBP. The positive dosimetric characteristics of the alanine pellets confirm the feasibility to use these detectors for "in vivo" dosimetry in clinical proton beams.

SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells.

PubMed

Ozden, Ozkan; Park, Seong-Hoon; Wagner, Brett A; Song, Ha Yong; Zhu, Yueming; Vassilopoulos, Athanassios; Jung, Barbara; Buettner, Garry R; Gius, David

2014-11-01

Pyruvate dehydrogenase E1α (PDHA1) is the first component enzyme of the pyruvate dehydrogenase (PDH) complex that transforms pyruvate, via pyruvate decarboxylation, into acetyl-CoA that is subsequently used by both the citric acid cycle and oxidative phosphorylation to generate ATP. As such, PDH links glycolysis and oxidative phosphorylation in normal as well as cancer cells. Herein we report that SIRT3 interacts with PDHA1 and directs its enzymatic activity via changes in protein acetylation. SIRT3 deacetylates PDHA1 lysine 321 (K321), and a PDHA1 mutant mimicking a deacetylated lysine (PDHA1(K321R)) increases PDH activity, compared to the K321 acetylation mimic (PDHA1(K321Q)) or wild-type PDHA1. Finally, PDHA1(K321Q) exhibited a more transformed in vitro cellular phenotype compared to PDHA1(K321R). These results suggest that the acetylation of PDHA1 provides another layer of enzymatic regulation, in addition to phosphorylation, involving a reversible acetyllysine, suggesting that the acetylome, as well as the kinome, links glycolysis to respiration. Copyright © 2014 Elsevier Inc. All rights reserved.

Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export.

PubMed

Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

2017-01-01

The Escherichia coli alaE gene encodes the L-alanine exporter, AlaE, that catalyzes active export of L-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient L-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to L-alanyl-L-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of L-alanine. To elucidate the mechanism by which AlaE exports L-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the L-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in L-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on L-alanine export.

Brain lactate kinetics: Modeling evidence for neuronal lactate uptake upon activation.

PubMed

Aubert, Agnès; Costalat, Robert; Magistretti, Pierre J; Pellerin, Luc

2005-11-08

A critical issue in brain energy metabolism is whether lactate produced within the brain by astrocytes is taken up and metabolized by neurons upon activation. Although there is ample evidence that neurons can efficiently use lactate as an energy substrate, at least in vitro, few experimental data exist to indicate that it is indeed the case in vivo. To address this question, we used a modeling approach to determine which mechanisms are necessary to explain typical brain lactate kinetics observed upon activation. On the basis of a previously validated model that takes into account the compartmentalization of energy metabolism, we developed a mathematical model of brain lactate kinetics, which was applied to published data describing the changes in extracellular lactate levels upon activation. Results show that the initial dip in the extracellular lactate concentration observed at the onset of stimulation can only be satisfactorily explained by a rapid uptake within an intraparenchymal cellular compartment. In contrast, neither blood flow increase, nor extracellular pH variation can be major causes of the lactate initial dip, whereas tissue lactate diffusion only tends to reduce its amplitude. The kinetic properties of monocarboxylate transporter isoforms strongly suggest that neurons represent the most likely compartment for activation-induced lactate uptake and that neuronal lactate utilization occurring early after activation onset is responsible for the initial dip in brain lactate levels observed in both animals and humans.

STUDIES ON MAMMALIAN AND HUMAN PYRUVATE AND ALPHA-KETOGLUTARATE DEHYDROGENATION COMPLEXES.

DTIC Science & Technology

Enzyme systems that catalyze a coenzyme A- and nicotinamide adenine dinucleotide-linked oxidative decarboxylation of pyruvate and alpha - ketoglutarate ...The pig heart pyruvate dehydrogenase complex was strongly inhibited by EDTA at low concentration, but the pig heart alpha - ketoglutarate ...On the oxidative decarboxylation of alpha -keto acids in pig heart complexes, Ca(2+) was strongly stimulatory to the same or more extent than Mg(2

Triiodothyronine Activates Lactate Oxidation Without Impairing Fatty Acid Oxidation and Improves Weaning From Extracorporeal Membrane Oxygenation

PubMed Central

Kajimoto, Masaki; Ledee, Dolena R.; Xu, Chun; Kajimoto, Hidemi; Isern, Nancy G.; Portman, Michael A.

2017-01-01

Background Extracorporeal membrane oxygenation (ECMO) provides a rescue for children with severe cardiac failure. It has previously been shown that triiodothyronine (T3) improves cardiac function by modulating pyruvate oxidation during weaning. This study focused on fatty acid (FA) metabolism modulated by T3 for weaning from ECMO after cardiac injury. Methods and Results Nineteen immature piglets (9.1–15.3 kg) were separated into 3 groups with ECMO (6.5 h) and wean: normal circulation (Group-C); transient coronary occlusion (10 min) for ischemia-reperfusion (IR) followed by ECMO (Group-IR); and IR with T3 supplementation (Group-IR-T3). 13-Carbon (13C)-labeled lactate, medium-chain and long-chain FAs, was infused as oxidative substrates. Substrate fractional contribution (FC) to the citric acid cycle was analyzed by 13C-nuclear magnetic resonance. ECMO depressed circulating T3 levels to 40% of the baseline at 4 h and were restored in Group-IR-T3. Group-IR decreased cardiac power, which was not fully restorable and 2 pigs were lost because of weaning failure. Group-IR also depressed FC-lactate, while the excellent contractile function and energy efficiency in Group-IR-T3 occurred along with a marked FC-lactate increase and [adenosine triphosphate]/[adenosine diphosphate] without either decreasing FC-FAs or elevating myocardial oxygen consumption over Group-C or -IR. Conclusions T3 releases inhibition of lactate oxidation following IR injury without impairing FA oxidation. These findings indicate that T3 depression during ECMO is maladaptive, and that restoring levels improves metabolic flux and enhances contractile function during weaning. PMID:25421230

Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter.

PubMed

Nagampalli, Raghavendra Sashi Krishna; Quesñay, José Edwin Neciosup; Adamoski, Douglas; Islam, Zeyaul; Birch, James; Sebinelli, Heitor Gobbi; Girard, Richard Marcel Bruno Moreira; Ascenção, Carolline Fernanda Rodrigues; Fala, Angela Maria; Pauletti, Bianca Alves; Consonni, Sílvio Roberto; de Oliveira, Juliana Ferreira; Silva, Amanda Cristina Teixeira; Franchini, Kleber Gomes; Leme, Adriana Franco Paes; Silber, Ariel Mariano; Ciancaglini, Pietro; Moraes, Isabel; Dias, Sandra Martha Gomes; Ambrosio, Andre Luis Berteli

2018-02-22

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

Molecular and Physiological Logics of the Pyruvate-Induced Response of a Novel Transporter in Bacillus subtilis.

PubMed

Charbonnier, Teddy; Le Coq, Dominique; McGovern, Stephen; Calabre, Magali; Delumeau, Olivier; Aymerich, Stéphane; Jules, Matthieu

2017-10-03

At the heart of central carbon metabolism, pyruvate is a pivotal metabolite in all living cells. Bacillus subtilis is able to excrete pyruvate as well as to use it as the sole carbon source. We herein reveal that ysbAB (renamed pftAB ), the only operon specifically induced in pyruvate-grown B. subtilis cells, encodes a hetero-oligomeric membrane complex which operates as a facilitated transport system specific for pyruvate, thereby defining a novel class of transporter. We demonstrate that the LytST two-component system is responsible for the induction of pftAB in the presence of pyruvate by binding of the LytT response regulator to a palindromic region upstream of pftAB We show that both glucose and malate, the preferred carbon sources for B. subtilis , trigger the binding of CcpA upstream of pftAB , which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate. Screening a genome-wide transposon mutant library, we find that an active malic enzyme replenishing the pyruvate pool is required for this repression. We next reveal that the higher the influx of pyruvate, the stronger the CcpA-independent repression of pftAB , which suggests that intracellular pyruvate retroinhibits pftAB induction via LytST. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry. Overall, we provide evidence for a complete system of sensors, feed-forward and feedback controllers that play a major role in environmental growth of B. subtilis IMPORTANCE Pyruvate is a small-molecule metabolite ubiquitous in living cells. Several species also use it as a carbon source as well as excrete it into the environment. The bacterial systems for pyruvate import/export have yet to be discovered. Here, we identified in the model bacterium Bacillus subtilis the first import

Analysis of the Mycoplasma bovis lactate dehydrogenase reveals typical enzymatic activity despite the presence of an atypical catalytic site motif.

PubMed

Masukagami, Yumiko; Tivendale, Kelly Anne; Browning, Glenn Francis; Sansom, Fiona Margaret

2018-02-01

The lactate dehydrogenase (LDH) of Mycoplasma genitalium has been predicted to also act as a malate dehydrogenase (MDH), but there has been no experimental validation of this hypothesized dual function for any mollicute. Our analysis of the metabolite profile of Mycoplasma bovis using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) detected malate, suggesting that there may be MDH activity in M. bovis. To investigate whether the putative l-LDH enzyme of M. bovis has a dual function (MDH and LDH), we performed bioinformatic and functional biochemical analyses. Although the amino acid sequence and predicted structural analysis of M. bovisl-LDH revealed unusual residues within the catalytic site, suggesting that it may have the flexibility to possess a dual function, our biochemical studies using recombinant M. bovis -LDH did not detect any MDH activity. However, we did show that the enzyme has typical LDH activity that could be inhibited by both MDH substrates oxaloacetate (OAA) and malate, suggesting that these substrates may be able to bind to M. bovis LDH. Inhibition of the conversion of pyruvate to lactate by OAA may be one method the mycoplasma cell uses to reduce the potential for accumulation of intracellular lactate.

Half-sandwich rhodium(III) transfer hydrogenation catalysts: Reduction of NAD(+) and pyruvate, and antiproliferative activity.

PubMed

Soldevila-Barreda, Joan J; Habtemariam, Abraha; Romero-Canelón, Isolda; Sadler, Peter J

2015-12-01

Organometallic complexes have the potential to behave as catalytic drugs. We investigate here Rh(III) complexes of general formula [(Cp(x))Rh(N,N')(Cl)], where N,N' is ethylenediamine (en), 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen) or N-(2-aminoethyl)-4-(trifluoromethyl)benzenesulfonamide (TfEn), and Cp(x) is pentamethylcyclopentadienyl (Cp*), 1-phenyl-2,3,4,5-tetramethylcyclopentadienyl (Cp(xPh)) or 1-biphenyl-2,3,4,5-tetramethyl cyclopentadienyl (Cp(xPhPh)). These complexes can reduce NAD(+) to NADH using formate as a hydride source under biologically-relevant conditions. The catalytic activity decreased in the order of N,N-chelated ligand bpy > phen > en with Cp* as the η(5)-donor. The en complexes (1-3) became more active with extension to the Cp(X) ring, whereas the activity of the phen (7-9) and bpy (4-6) compounds decreased. [Cp*Rh(bpy)Cl](+) (4) showed the highest catalytic activity, with a TOF of 37.4±2h(-1). Fast hydrolysis of the chlorido complexes 1-10 was observed by (1)H NMR (<10min at 310K). The pKa* values for the aqua adducts were determined to be ca. 8-10. Complexes 1-9 also catalysed the reduction of pyruvate to lactate using formate as the hydride donor. The efficiency of the transfer hydrogenation reactions was highly dependent on the nature of the chelating ligand and the Cp(x) ring. Competition reactions between NAD(+) and pyruvate for reduction by formate catalysed by 4 showed a preference for reduction of NAD(+). The antiproliferative activity of complex 3 towards A2780 human ovarian cancer cells increased by up to 50% when administered in combination with non-toxic doses of formate, suggesting that transfer hydrogenation can induce reductive stress in cancer cells. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Underestimation of pyruvic acid concentrations by fructose and cysteine in 2,4-dinitrophenylhydrazine-mediated onion pungency test.

PubMed

Yoo, Kil Sun; Lee, Eun Jin; Patil, Bhimanagouda S

2011-10-01

Onion pungency has been routinely measured by determining pyruvic acid concentration in onion juice by reacting with 2,4-dinitrophenylhydrazine (DNPH) since 1961. However, the absorbency of the color adduct of the reaction rapidly decreased in onion samples as compared to that of the pyruvic acid standards, resulting in underestimations of the pyruvic acid concentrations. By measuring the absorbency at 1 min, we have demonstrated that accuracy could be substantially improved. As a continuation, the causes of degradation of the color adduct after the reaction and pyruvic acid itself before the reaction were examined in this study. Alliinase action in juice (fresh or cooked) and bulb colors did not influence the degradation. Some organic acids indigenously found in onion, such as ascorbic acid, proline, and glutamic acid, did not reduce the absorbency. However, fructose within the onion juice or supplemented caused the degradation of the color adduct, whereas sucrose and glucose had a lesser effect. Degradation rates increased proportionally as fructose concentrations increased up to 70 mg/mL. Cysteine was found to degrade the pyruvic acid itself before the pyruvic acid could react with DNPH. Approximately 90% of the pyruvic acid was degraded after 60 min in samples of 7 mM pyruvic acid supplemented with 10 mg/mL cysteine. Spectral comparisons of onion juice containing fructose naturally and pyruvic acid solution with supplemented fructose indicated identical patterns and confirmed that the color-adduct degradation was caused by fructose. Our study elucidated that fructose, a major sugar in onion juice, caused the degradation of color adduct in the onion pungency test and resulted in underestimation of the pyruvic acid concentration. © 2011 Institute of Food Technologists®

Cultivation of parasitic leptospires: effect of pyruvate.

PubMed

Johnson, R C; Walby, J; Henry, R A; Auran, N E

1973-07-01

Sodium pyruvate (100 mug/ml) is a useful addition to the Tween 80-albumin medium for the cultivation of parasitic serotypes. It is most effective in promoting growth from small inocula and growth of the nutritionally fastidious serotypes.

NMR-based metabolite profiling of human milk: A pilot study of methods for investigating compositional changes during lactation

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

Wu, Junfang; Domellöf, Magnus; Zivkovic, Angela M.

Low-molecular-weight metabolites in human milk are gaining increasing interest in studies of infant nutrition. In the present study, the milk metabolome from a single mother was explored at different stages of lactation. Metabolites were extracted from sample aliquots using either methanol/water (MeOH/H{sub 2}O) extraction or ultrafiltration. Nuclear magnetic resonance (NMR) spectroscopy was used for metabolite identification and quantification, and multi- and univariate statistical data analyses were used to detect changes over time of lactation. Compared to MeOH/H{sub 2}O extraction, ultrafiltration more efficiently reduced the interference from lipid and protein resonances, thereby enabling the identification and quantification of 36 metabolites. Themore » human milk metabolomes at the early (9–24 days after delivery) and late (31–87 days after delivery) stages of lactation were distinctly different according to multi- and univariate statistics. The late lactation stage was characterized by significantly elevated concentrations of lactose, choline, alanine, glutamate, and glutamine, as well as by reduced levels of citrate, phosphocholine, glycerophosphocholine, and N-acetylglucosamine. Our results indicate that there are significant compositional changes of the human milk metabolome also in different phases of the matured lactation stage. These findings complement temporal studies on the colostrum and transitional metabolome in providing a better understanding of the nutritional variations received by an infant. - Highlights: • 36 metabolites were simultaneously quantified in human milk by NMR. • Ultrafiltration more efficiently reduces interferences than MeOH/H{sub 2}O extraction. • Compositional changes of the human milk exist during the matured lactation stage.« less

Enantioselective oxidation of racemic lactic acid to D-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM.

PubMed

Gao, Chao; Qiu, Jianhua; Li, Jingchen; Ma, Cuiqing; Tang, Hongzhi; Xu, Ping

2009-03-01

D-lactic acid and pyruvic acid are two important building block intermediates. Production of D-lactic acid and pyruvic acid from racemic lactic acid by biotransformation is economically interesting. Biocatalyst prepared from 9 g dry cell wt l(-1) of Pseudomonas stutzeri SDM could catalyze 45.00 g l(-1)DL-lactic acid into 25.23 g l(-1)D-lactic acid and 19.70 g l(-1) pyruvic acid in 10h. Using a simple ion exchange process, D-lactic acid and pyruvic acid were effectively separated from the biotransformation system. Co-production of d-lactic acid and pyruvic acid by enantioselective oxidation of racemic lactic acid is technically feasible.

Brain lactate kinetics: Modeling evidence for neuronal lactate uptake upon activation

PubMed Central

Aubert, Agnès; Costalat, Robert; Magistretti, Pierre J.; Pellerin, Luc

2005-01-01

A critical issue in brain energy metabolism is whether lactate produced within the brain by astrocytes is taken up and metabolized by neurons upon activation. Although there is ample evidence that neurons can efficiently use lactate as an energy substrate, at least in vitro, few experimental data exist to indicate that it is indeed the case in vivo. To address this question, we used a modeling approach to determine which mechanisms are necessary to explain typical brain lactate kinetics observed upon activation. On the basis of a previously validated model that takes into account the compartmentalization of energy metabolism, we developed a mathematical model of brain lactate kinetics, which was applied to published data describing the changes in extracellular lactate levels upon activation. Results show that the initial dip in the extracellular lactate concentration observed at the onset of stimulation can only be satisfactorily explained by a rapid uptake within an intraparenchymal cellular compartment. In contrast, neither blood flow increase, nor extracellular pH variation can be major causes of the lactate initial dip, whereas tissue lactate diffusion only tends to reduce its amplitude. The kinetic properties of monocarboxylate transporter isoforms strongly suggest that neurons represent the most likely compartment for activation-induced lactate uptake and that neuronal lactate utilization occurring early after activation onset is responsible for the initial dip in brain lactate levels observed in both animals and humans. PMID:16260743

Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato.

PubMed Central

Millar, A H; Knorpp, C; Leaver, C J; Hill, S A

1998-01-01

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 micromol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins. PMID:9729464

Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine

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

Bruning, John B.; Murillo, Ana C.; Chacon, Ofelia

D-Alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 {angstrom}. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoinedmore » by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC{sub 50}) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.« less

Interaction Between the Biotin Carboxyl Carrier Domain and the Biotin Carboxylase Domain in Pyruvate Carboxylase from Rhizobium etli†

PubMed Central

Lietzan, Adam D.; Menefee, Ann L.; Zeczycki, Tonya N.; Kumar, Sudhanshu; Attwood, Paul V.; Wallace, John C.; Cleland, W. Wallace; Maurice, Martin St.

2011-01-01

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family. PMID:21958016

Influence of pyruvate on economy of contraction in isolated rabbit myocardium.

PubMed

Keweloh, Boris; Janssen, Paul M L; Siegel, Ulf; Datz, Nicolin; Zeitz, Oliver; Hermann, Hans-Peter

2007-08-01

Treatment of acute heart failure frequently requires positive-inotropic stimulation. However, there is still no inotropic agent available, which combines a favourable haemodynamic profile with low expenditure for energy metabolism. Pyruvate exhibits positive inotropic effects in vitro and in patients with heart failure. The effect on myocardial energy metabolism however remains unclear, but is meaningful in light of a clinical application. We investigated the influence of pyruvate on contractility and oxygen consumption in isolated isometric contracting rabbit myocardium compared to beta-adrenergic stimulation with isoproterenol. Pyruvate (30 mM) increased developed force from 18.7+/-4.1 to 50.8+/-12.1 mN/mm2 (n=10, p<0.01). Force-time integral (FTI) increased by 329%, oxygen consumption assessed by diffusion-microelectrode technique increased from 2.86+/-0.30 mlO2/min*100 g to 6.28+/-1.28 mlO2/min*100 g (n=7, p<0.05). Economy of myocardial contraction calculated as the ratio of total FTI to oxygen consumption remained unchanged. In contrast, while isoproterenol (10 microM) produced a comparable increase in developed force from 21.4+/-8.3 to 67.3+/-15 mN/mm2 (n=7, p<0.01), FTI increased only by 260% and MVO2 increased from 2.96+/-0.43 to 6.12+/-1.01 mlO2/min*100 g (n=7, p<0.01); thus, economy decreased by 23% (n=7, p<0.05). Pyruvate does not impair economy of myocardial contraction while isoproterenol decreases economy. Regarding energy expenditure, pyruvate appears superior to isoproterenol for the purpose of positive inotropic stimulation.

Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis*

PubMed Central

Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E.; Rhee, Kyu Y.; Jacobs, William R.; Berney, Michael; Blanchard, John S.

2016-01-01

Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests that Mtb relies mainly on fatty acid catabolism in the host. However, Mtb also maintains a functional glycolytic pathway and its role in the cellular metabolism of Mtb has yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and the Mtb genome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show that pykA encodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion of pykA prevents Mtb growth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism in Mtb. PMID:26858255

Pyruvate dose response studies targeting the vital signs following hemorrhagic shock

PubMed Central

Sharma, Pushpa; Vyacheslav, Makler; Carissa, Chalut; Vanessa, Rodriguez; Bodo, Mike

2015-01-01

Objectives: To determine the optimal effective dose of sodium pyruvate in maintaining the vital signs following hemorrhagic shock (HS) in rats. Materials and Methods: Anesthetized, male Sprague-Dawley rats underwent computer-controlled HS for 30 minute followed by fluid resuscitation with either hypertonic saline, or sodium pyruvate solutions of 0.5 M, 1.0 M, 2.0 M, and 4.0 M at a rate of 5ml/kg/h (60 minute) and subsequent blood infusion (60 minute). The results were compared with sham and non- resuscitated groups. The animals were continuously monitored for mean arterial pressure, systolic and diastolic pressure, heart rate, pulse pressure, temperature, shock index and Kerdo index (KI). Results: The Sham group remained stable throughout the experiment. Non-resuscitated HS animals did not survive for the entire experiment due to non-viable vital signs and poor shock and KI. All fluids were effective in normalizing the vital signs when shed blood was used adjunctively. Sodium pyruvate 2.0 M was most effective, and 4.0 M solution was least effective in improving the vital signs after HS. Conclusions: Future studies should be directed to use 2.0 M sodium pyruvate adjuvant for resuscitation on multiorgan failure and survival rate in HS. PMID:26229300

Improved tolerance to off-resonance in spectral-spatial EPI of hyperpolarized [1-13 C]pyruvate and metabolites.

PubMed

Lau, Justin Y C; Geraghty, Benjamin J; Chen, Albert P; Cunningham, Charles H

2018-09-01

For 13 C echo-planar imaging (EPI) with spectral-spatial excitation, main field inhomogeneity can result in reduced flip angle and spatial artifacts. A hybrid time-resolved pulse sequence, multi-echo spectral-spatial EPI, is proposed combining broader spectral-spatial passbands for greater off-resonance tolerance with a multi-echo acquisition to separate signals from potentially co-excited resonances. The performance of the imaging sequence and the reconstruction pipeline were evaluated for 1 H imaging using a series of increasingly dilute 1,4-dioxane solutions and for 13 C imaging using an ethylene glycol phantom. Hyperpolarized [1- 13 C]pyruvate was administered to two healthy rats. Multi-echo data of the rat kidneys were acquired to test realistic cases of off-resonance. Analysis of separated images of water and 1,4-dioxane following multi-echo signal decomposition showed water-to-dioxane 1 H signal ratios that were in agreement with the independent measurements by 1 H spectroscopy for all four concentrations of 1,4-dioxane. The 13 C signal ratio of two co-excited resonances of ethylene glycol was accurately recovered after correction for the spectral profile of the redesigned spectral-spatial pulse. In vivo, successful separation of lactate and pyruvate-hydrate signals was achieved for all except the early time points during which signal variations exceeded the temporal resolution of the multi-echo acquisition. Improved tolerance to off-resonance in the new 13 C data acquisition pipeline was demonstrated in vitro and in vivo. Magn Reson Med 80:925-934, 2018. © 2018 International Society for Magnetic Resonance in Medicine. © 2018 International Society for Magnetic Resonance in Medicine.

Measurements of lactate in exhaled breath condensate at rest and after maximal exercise in young and healthy subjects.

PubMed

Marek, E M; Volke, J; Hawener, I; Platen, P; Mückenhoff, K; Marek, W

2010-03-01

Arterial lactate concentrations, taken as indicators of physical fitness, in athletes as well as in patients with cardio-respiratory or metabolic diseases, are measured invasively from arterialized ear lobe blood. Currently developed micro enzyme detectors permit a non-invasive measurement of hypoxia-related metabolites such as lactate in exhaled breath condensate (EBC). The aim of our study is to prove whether this technology will replace the traditional measurement of lactate in arterialized blood. Therefore, we determined the functional relation between lactate release in EBC and lactate concentration in blood in young and healthy subjects at rest and after exhausting bicycle exercise. During resting conditions as well as after exhausting bicycle exercise, 100 L of exhaled air along with blood samples from the ear lobe was collected after stationary load conditions in 16 healthy subjects. EBC was obtained by cooling the expired air volume with an ECoScreen I (FILT GmbH, Berlin) condenser. The analysis was performed within 90 min using an ECoCheck ampere meter (FILT GmbH, Berlin). Lactate measurements were performed using a bi-enzyme sensor after lactate oxidase-induced oxidation of lactate to pyruvate and H2O2. The rates of lactate release via the exhaled air were calculated from the lactate concentration, the volume and the collection time of the EBC. The functional relation of lactate release in exhaled air and lactate concentration of arterial blood was computed. At rest, the mean lactate concentration in arterialized blood was 0.93 ± 0.30 mmol L(-1). At a resting ventilation of 11.5 ± 3.4 L min(-1), the collection time for 100 L of exhaled air, Ts, was 8.4 ± 2.9 min, and 1.68 ± 0.40 mL EBC was obtained. In EBC, the lactate concentration was 21.4 ± 7.7 µmol L(-1), and the rate of lactate release rate in collected EBC was 4.5 ± 1.7 nmol min(-1). After maximal exercise load (220 ± 20 W), the blood lactate concentration increased to 10.9 ± 1.8 mmol L(-1

Cultivation of Parasitic Leptospires: Effect of Pyruvate

PubMed Central

Johnson, R. C.; Walby, J.; Henry, R. A.; Auran, N. E.

1973-01-01

Sodium pyruvate (100 μg/ml) is a useful addition to the Tween 80-albumin medium for the cultivation of parasitic serotypes. It is most effective in promoting growth from small inocula and growth of the nutritionally fastidious serotypes. Images PMID:4580191

Immobilization of R-ω-transaminase on MnO2 nanorods for catalyzing the conversion of (R)-1-phenylethylamine.

PubMed

Sun, Jian; Cui, Wen-Hui; Du, Kun; Gao, Qian; Du, Mengmeng; Ji, Peijun; Feng, Wei

2017-03-10

R-ɷ-transaminases transfer an amino group from an amino donor (e.g. (R)-1-phenylethylamine) onto an amino acceptor (e.g. pyruvate), resulting a co-product (e.g. d-alanine). This work intends to immobilize R-ɷ-Transaminase on MnO 2 nanorods to achieve multienzyme catalysis. R-ɷ-Transaminase (RTA) and d-amino acid oxidase (DAAO) have been fused to an elastin-like polypeptide (ELP) separately through genetic engineering of the enzymes. ELP-RTA and ELP-DAAO have been separately immobilized on polydopamine-coated MnO 2 nanorods. When the two immobilized enzymes were used together in one pot, the transformation of (R)-1-phenylethylamine was catalyzed by the immobilized ELP-RTA, and the co-product d-alanine was converted back to pyruvate under the catalysis of the immobilized ELP-DAAO, achieving the recycling of pyruvate in situ. Thus pyruvate was maintained at a low concentration in order to reduce its negative effect. On the other hand, the generated H 2 O 2 of ELP-DAAO was decomposed by the MnO 2 nanorods, and the evolved oxygen oxidized the reduced cofactors of ELP-DAAO. Forming the circles of hydrogen peroxide→oxygen→hydrogen peroxide accelerated the deamination reaction. The highly efficient conversion of the co-product d-alanine back to pyruvate accelerated the forming of the pyruvate→d-alanine→pyruvate cycle between the two immobilized enzymes. The coordination of the pyruvate→d-alanine→pyruvate and hydrogen peroxide→oxygen→hydrogen peroxide cycles accelerated the transformation of (R)-1-phenylethylamine. As a result, As a result, the immobilized enzymes achieved a conversion of 98±1.8% in comparison to 69.6±1.2% by free enzymes. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of high and low blood lactate concentrations on sweat lactate response.

PubMed

Green, J M; Bishop, P A; Muir, I H; McLester, J R; Heath, H E

2000-11-01

Sweat lactate results from eccrine gland metabolism, however, the possible clearance of blood lactate through sweat has not been resolved. On separate days in an environmental chamber (32 +/- 1 C) 12 subjects completed a constant load (CON) (30 min at 40% VO2 max) and an interval cycling trial (INT) (15 one-min intervals at 80% VO2 max, each separated by one min rest) each designed to elicit different blood lactate responses. Each 30 min cycling trial was preceded by 15 min warm-up (30 watts) and followed by 15 min passive rest. Sweat and blood were analyzed for lactate concentration at 15, 25, 35, 45, and 60 min during CON and INT. Total body water loss was used to calculate sweat rate (ml/hr). Blood lactate was significantly greater (p < or = 0.05) at 25, 35, 45, and 60 min during INT compared to CON (approximately 5 mmol/L vs 1.5 mmol/L). Sweat lactate was not significantly different (p>0.05) between trials at any time (approximately 10 mmol/L). Sweat rates (approximately 600ml/hr) and estimated total lactate secretion were not significantly different (CON vs. INT) (p > 0.05). Elevated blood lactate was not associated with changes in sweat lactate concentration. Sweat lactate seems to originate in eccrine glands independent of blood lactate.

Tricarboxylic acid cycle inhibition by Li+ in the human neuroblastoma SH-SY5Y cell line: a 13C NMR isotopomer analysis.

PubMed

Fonseca, Carla P; Jones, John G; Carvalho, Rui A; Jeffrey, F Mark H; Montezinho, Liliana P; Geraldes, Carlos F G C; Castro, M M C A

2005-11-01

Li+ effects on glucose metabolism and on the competitive metabolism of glucose and lactate were investigated in the human neuroblastoma SH-SY5Y cell line using 13C NMR spectroscopy. The metabolic model proposed for glucose and lactate metabolism in these cells, based on tcaCALC best fitting solutions, for both control and Li+ conditions, was consistent with: (i) a single pyruvate pool; (ii) anaplerotic flux from endogenous unlabelled substrates; (iii) no cycling between pyruvate and oxaloacetate. Li+ was shown to induce a 38 and 53% decrease, for 1 and 15 mM Li+, respectively, in the rate of glucose conversion into pyruvate, when [U-13C]glucose was present, while no effects on lactate production were observed. Pyruvate oxidation by the tricarboxylic acid cycle and citrate synthase flux were shown to be significantly reduced by 64 and 84% in the presence of 1 and 15 mM Li+, respectively, suggesting a direct inhibitory effect of Li+ on tricarboxylic acid cycle flux. This work also showed that when both glucose and lactate are present as energetic substrates, SH-SY5Y cells preferentially consumed exogenous lactate over glucose, as 62% of the acetyl-CoA was derived from [3-13C]lactate while only 26% was derived from [U-13C]glucose. Li+ did not significantly affect the relative utilisation of these two substrates by the cells or the residual contribution of unlabelled endogenous sources for the acetyl-CoA pool.

Pyruvate Administration Reduces Recurrent/Moderate Hypoglycemia-Induced Cortical Neuron Death in Diabetic Rats

PubMed Central

Choi, Bo Young; Kim, Jin Hee; Kim, Hyun Jung; Yoo, Jin Hyuk; Song, Hong Ki; Sohn, Min; Won, Seok Joon; Suh, Sang Won

2013-01-01

Recurrent/moderate (R/M) hypoglycemia is common in type 1 diabetes patients. Moderate hypoglycemia is not life-threatening, but if experienced recurrently it may present several clinical complications. Activated PARP-1 consumes cytosolic NAD, and because NAD is required for glycolysis, hypoglycemia-induced PARP-1 activation may render cells unable to use glucose even when glucose availability is restored. Pyruvate, however, can be metabolized in the absence of cytosolic NAD. We therefore hypothesized that pyruvate may be able to improve the outcome in diabetic rats subjected to insulin-induced R/M hypoglycemia by terminating hypoglycemia with glucose plus pyruvate, as compared with delivering just glucose alone. In an effort to mimic juvenile type 1 diabetes the experiments were conducted in one-month-old young rats that were rendered diabetic by streptozotocin (STZ, 50mg/kg, i.p.) injection. One week after STZ injection, rats were subjected to moderate hypoglycemia by insulin injection (10U/kg, i.p.) without anesthesia for five consecutive days. Pyruvate (500mg/kg) was given by intraperitoneal injection after each R/M hypoglycemia. Three hours after last R/M hypoglycemia, zinc accumulation was evaluated. Three days after R/M hypoglycemia, neuronal death, oxidative stress, microglial activation and GSH concentrations in the cerebral cortex were analyzed. Sparse neuronal death was observed in the cortex. Zinc accumulation, oxidative injury, microglial activation and GSH loss in the cortex after R/M hypoglycemia were all reduced by pyruvate injection. These findings suggest that when delivered alongside glucose, pyruvate may significantly improve the outcome after R/M hypoglycemia by circumventing a sustained impairment in neuronal glucose utilization resulting from PARP-1 activation. PMID:24278448

DdlN from Vancomycin-Producing Amycolatopsis orientalis C329.2 Is a VanA Homologue with d-Alanyl-d-Lactate Ligase Activity

PubMed Central

Marshall, C. Gary; Wright, Gerard D.

1998-01-01

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in d-alanyl-d-lactate. A key enzyme in this process is a d-alanyl-d-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide d-alanyl-d-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for d-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria. PMID:9791137

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).

Molecular and Physiological Logics of the Pyruvate-Induced Response of a Novel Transporter in Bacillus subtilis

PubMed Central

Charbonnier, Teddy; Le Coq, Dominique; McGovern, Stephen; Calabre, Magali; Delumeau, Olivier; Aymerich, Stéphane

2017-01-01

ABSTRACT At the heart of central carbon metabolism, pyruvate is a pivotal metabolite in all living cells. Bacillus subtilis is able to excrete pyruvate as well as to use it as the sole carbon source. We herein reveal that ysbAB (renamed pftAB), the only operon specifically induced in pyruvate-grown B. subtilis cells, encodes a hetero-oligomeric membrane complex which operates as a facilitated transport system specific for pyruvate, thereby defining a novel class of transporter. We demonstrate that the LytST two-component system is responsible for the induction of pftAB in the presence of pyruvate by binding of the LytT response regulator to a palindromic region upstream of pftAB. We show that both glucose and malate, the preferred carbon sources for B. subtilis, trigger the binding of CcpA upstream of pftAB, which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate. Screening a genome-wide transposon mutant library, we find that an active malic enzyme replenishing the pyruvate pool is required for this repression. We next reveal that the higher the influx of pyruvate, the stronger the CcpA-independent repression of pftAB, which suggests that intracellular pyruvate retroinhibits pftAB induction via LytST. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry. Overall, we provide evidence for a complete system of sensors, feed-forward and feedback controllers that play a major role in environmental growth of B. subtilis. PMID:28974613

Dose response of alanine detectors irradiated with carbon ion beams

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

Herrmann, Rochus; Jaekel, Oliver; Palmans, Hugo

Purpose: The dose response of the alanine detector shows a dependence on particle energy and type when irradiated with ion beams. The purpose of this study is to investigate the response behavior of the alanine detector in clinical carbon ion beams and compare the results to model predictions. Methods: Alanine detectors have been irradiated with carbon ions with an energy range of 89-400 MeV/u. The relative effectiveness of alanine has been measured in this regime. Pristine and spread out Bragg peak depth-dose curves have been measured with alanine dosimeters. The track structure based alanine response model developed by Hansen andmore » Olsen has been implemented in the Monte Carlo code FLUKA and calculations were compared to experimental results. Results: Calculations of the relative effectiveness deviate less than 5% from the measured values for monoenergetic beams. Measured depth-dose curves deviate from predictions in the peak region, most pronounced at the distal edge of the peak. Conclusions: The used model and its implementation show a good overall agreement for quasimonoenergetic measurements. Deviations in depth-dose measurements are mainly attributed to uncertainties of the detector geometry implemented in the Monte Carlo simulations.« less

Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death

PubMed Central

Wallace, Martina; Buren, Caodu; Martyniuk, Kelly; Andreyev, Alexander Y.; Li, Edward; Fields, Jerel A.; Cordes, Thekla; Reynolds, Ian J.; Bloodgood, Brenda L.; Metallo, Christian M.

2017-01-01

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity. PMID:28254829

Structural properties of pyruvate carboxylases from chicken liver and other sources

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

Barden, R.E.; Taylor, B.L.; Isohashi, F.

1975-11-01

Varieties of pyruvate carboxylase (pyruvate: CO/sub 2/ ligase (ADP-forming), EC 6.4.1.1) obtained from the livers of several species of vertebrates, including humans, all show the same basic structure. They are composed of large polypeptide chains of molecular weights ranging from 1.2 to 1.3 x 10/sup 5/ for the different varieties of the enzyme. The native form of the enzyme appears to be a tetramer with a molecular weight of about 5 x 10/sup 5/. In the case of pyruvate carboxylase from chicken liver each polypeptide chain contains a biotin moiety, thus supporting the thesis that the tetramer contains four identicalmore » polypeptide chains. Pyruvate carboxylase from yeast appears to be basically similar to those from the vertebrate species and has a tetrameric structure. Each protomer contains a single polypeptide chain with a molecular weight of 1.25 x 10/sup 5/. In contrast, pyruvate carboxylase from two bacterial species, Pseudomonas citronellolis and Azotobacter vinelandii, appears to be a dimer with a molecular weight (2.5 x 10/sup 5/) about half that of the animal and yeast species. As a further difference, each of the protomers of the bacterial enzymes contain two polypeptides of 6.5 and 5.4 x 10/sup 5/ molecular weight in the case of the Pseudomonas enzyme. The larger of the two polypeptides contains the biotin moiety. The functional units of the bacterial enzyme thus appear to contain two polypeptides while that of the liver and yeast enzymes is made up of a single chain. Neither of these arrangements corresponds with those of other biotin enzymes whose structure has been extensively studied (acetyl-CoA carboxylases from liver or Escherichia coli, and transcarboxylase from Propionibacterium). (auth)« less

Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy.

PubMed

Escalera-Fanjul, Ximena; Campero-Basaldua, Carlos; Colón, Maritrini; González, James; Márquez, Dariel; González, Alicia

2017-01-01

Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, Sc Alt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1 , alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display Lk Alt1 and Kl Alt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s). Furthermore, phenotypic analysis of null mutants uncovered the fact that Kl Alt1 and Lk Alt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that Sc Alt2 conserves 64% identity with

Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy

PubMed Central

Escalera-Fanjul, Ximena; Campero-Basaldua, Carlos; Colón, Maritrini; González, James; Márquez, Dariel; González, Alicia

2017-01-01

Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, ScAlt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1, alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display LkAlt1 and KlAlt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s). Furthermore, phenotypic analysis of null mutants uncovered the fact that KlAlt1 and LkAlt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that ScAlt2 conserves 64% identity with LkAlt1

Trimethylamine-N-oxide counteracts urea effects on rabbit muscle lactate dehydrogenase function: a test of the counteraction hypothesis.

PubMed Central

Baskakov, I; Wang, A; Bolen, D W

1998-01-01

Trimethylamine-N-oxide (TMAO) in the cells of sharks and rays is believed to counteract the deleterious effects of the high intracellular concentrations of urea in these animals. It has been hypothesized that TMAO has the generic ability to counteract the effects of urea on protein structure and function, regardless of whether that protein actually evolved in the presence of these two solutes. Rabbit muscle lactate dehydrogenase (LDH) did not evolve in the presence of either solute, and it is used here to test the validity of the counteraction hypothesis. With pyruvate as substrate, results show that its Km and the combined Km of pyruvate and NADH are increased by urea, decreased by TMAO, and in 1:1 and 2:1 mixtures of urea:TMAO the Km values are essentially equivalent to the Km values obtained in the absence of the two solutes. In contrast, values of k(cat) and the Km for NADH as a substrate are unperturbed by urea, TMAO, or urea:TMAO mixtures. All of these effects are consistent with TMAO counteraction of the effects of urea on LDH kinetic parameters, supporting the premise that counteraction is a property of the solvent system and is independent of the evolutionary history of the protein. PMID:9591690

A comparative study of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in the saliva of diabetic and normal patients.

PubMed

Verma, M; Metgud, R; Madhusudan, A S; Verma, N; Saxena, M; Soni, A

2014-10-01

Diabetes has been reported to affect salivary glands adversely in humans and experimental models. Glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) are salivary enzymes that also are widely distributed in animal tissues. We determined GOT and GPT levels in saliva samples of 100 type 1 and 30 type 2 diabetic patients using reflectance spectrophotometry and compared them to 30 age and sex matched healthy controls. Statistically significant differences were observed in the mean values of GOT and GPT in type 1 diabetics compared to type 2 and control groups. Significantly higher GOT levels were found in the 1-20 year age group of type 1 diabetics. Our findings suggest that salivary gland damage is due to the same immunological attack that affects pancreatic β cells and results in type 1 diabetes.

Compound-specific nitrogen isotope analysis of D-alanine, L-alanine, and valine: application of diastereomer separation to delta15N and microbial peptidoglycan studies.

PubMed

Takano, Yoshinori; Chikaraishi, Yoshito; Ogawa, Nanako O; Kitazato, Hiroshi; Ohkouchi, Naohiko

2009-01-01

We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.

Regulation of ATP sensitive potassium channel of isolated guinea pig ventricular myocytes by sarcolemmal monocarboxylate transport.

PubMed

Coetzee, W A

1992-11-01

The aim was to describe the effects of extracellular application of monocarboxylates (pyruvate, lactate, or acetate) on current through KATP channels (iK,ATP) in isolated guinea pig ventricular myocytes. The iK,ATP was elicited during whole cell voltage clamping by application of metabolic poisons, 2,4-dinitrophenol (150 microM) or glucose free cyanide (1 mM) and could be blocked by glibenclamide (3 microM). Extracellular application of monocarboxylates, pyruvate (0.1-10 mM), L-lactate (0.1-10 mM), and acetate (10 mM) led to a rapid inhibition of iK,ATP--an effect which was fully reversible upon washout. Substances without any effect on iK,ATP were (10 mM each) gluconate, citrate, glutamate, creatine, succinate, and glycine. The mechanism underlying the effects of monocarboxylates on iK,ATP was unlikely to be related to an increased ATP production, since D-lactate (10 mM) essentially had the same effect on iK,ATP as the L-isomer of lactate. Furthermore, with intracellular dialysis of alpha-cyano-4-hydroxycinnamate (0.1-0.5 mM), which inhibits pyruvate uptake into mitochondria, extracellular pyruvate exerted the same inhibitory effect on iK,ATP. High concentrations of extracellular alpha-cyano-4-hydroxycinnamate (4 mM), which blocks the sarcolemmal monocarboxylate carrier, prevented the effects on iK,ATP by pyruvate, L-lactate, D-lactate, and acetate. Furthermore, intracellular dialysis with D-lactate (10 mM) led to a more rapid onset of iK,ATP when activated by ATP free dialysis. Activity of isolated KATP channels, measured in isolated membrane patches in the inside out or outside out configuration, typically had a single channel conductance of around 80 pS and was blocked by glibenclamide (3-9 microM). No significant effect of pyruvate was observed in either patch configuration. In cardiac tissue there may be some modulatory role involving monocarboxylate transport on KATP channel activity, the nature of which is unclear at present but which may involve cytosolic

Role of beta-alanine supplementation on muscle carnosine and exercise performance.

PubMed

Artioli, Guilherme Giannini; Gualano, Bruno; Smith, Abbie; Stout, Jeffrey; Lancha, Antonio Herbert

2010-06-01

In this narrative review, we present and discuss the current knowledge available on carnosine and beta-alanine metabolism as well as the effects of beta-alanine supplementation on exercise performance. Intramuscular acidosis has been attributed to be one of the main causes of fatigue during intense exercise. Carnosine has been shown to play a significant role in muscle pH regulation. Carnosine is synthesized in skeletal muscle from the amino acids l-histidine and beta-alanine. The rate-limiting factor of carnosine synthesis is beta-alanine availability. Supplementation with beta-alanine has been shown to increase muscle carnosine content and therefore total muscle buffer capacity, with the potential to elicit improvements in physical performance during high-intensity exercise. Studies on beta-alanine supplementation and exercise performance have demonstrated improvements in performance during multiple bouts of high-intensity exercise and in single bouts of exercise lasting more than 60 s. Similarly, beta-alanine supplementation has been shown to delay the onset of neuromuscular fatigue. Although beta-alanine does not improve maximal strength or VO2max, some aspects of endurance performance, such as anaerobic threshold and time to exhaustion, can be enhanced. Symptoms of paresthesia may be observed if a single dose higher than 800 mg is ingested. The symptoms, however, are transient and related to the increase in plasma concentration. They can be prevented by using controlled release capsules and smaller dosing strategies. No important side effect was related to the use of this amino acid so far. In conclusion, beta-alanine supplementation seems to be a safe nutritional strategy capable of improving high-intensity anaerobic performance.

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides of DL-alanine and N-(tert-butoxycarbonyl)-DL-alanine

NASA Technical Reports Server (NTRS)

Profy, A. T.; Usher, D. A.

1984-01-01

The aminoacylation of diinosine monophosphate was studied experimentally. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-DL-alanine, a 40 percent enantiomeric excess of the isomer was incorporated at the 2' site and the positions of equilibrium for the reversible 2'-3' migration reaction differed for the D and L enantiomers. The reactivity of the nucleoside hydroxyl groups was found to decrease on the order 2'(3') less than internal 2' and less than 5', and the extent of the reaction was affected by the concentration of the imidazole buffer. Reaction of IpI with imidazolide of unprotected DL-alanine, by contrast, led to an excess of the D isomer at the internal 2' site. Finally, reaction with the N-carboxy anhydride of DL-alanine occurred without stereoselection. These results are found to be relevant to the study of the evolution of optical chemical activity and the origin of genetically directed protein synthesis.

Effect of hyperbaric oxygenation on carbohydrate metabolism protein synthesis in the myocardium during sustained hypodynamia

NASA Technical Reports Server (NTRS)

Makarov, G. A.

1980-01-01

Glycolysis and the intensity of protein synthesis were studied in 140 white male rats in subcellular fractions of the myocardium during 45 day hypodynamia and hyperbaric oxygenation. Hypodynamia increased: (1) the amount of lactic acids; (2) the amount of pyruvic acid; (3) the lactate/pyruvate coefficient; and (4) the activities of aldolase and lactate dehydrogenase. Hyperbaric oxygenation was found to have a favorable metabolic effect on the animals with hypodynamia.

In Quest of the Alanine R3 Radical: Multivariate EPR Spectral Analyses of X-Irradiated Alanine in the Solid State.

PubMed

Jåstad, Eirik O; Torheim, Turid; Villeneuve, Kathleen M; Kvaal, Knut; Hole, Eli O; Sagstuen, Einar; Malinen, Eirik; Futsaether, Cecilia M

2017-09-28

The amino acid l-α-alanine is the most commonly used material for solid-state electron paramagnetic resonance (EPR) dosimetry, due to the formation of highly stable radicals upon irradiation, with yields proportional to the radiation dose. Two major alanine radical components designated R1 and R2 have previously been uniquely characterized from EPR and electron-nuclear double resonance (ENDOR) studies as well as from quantum chemical calculations. There is also convincing experimental evidence of a third minor radical component R3, and a tentative radical structure has been suggested, even though no well-defined spectral signature has been observed experimentally. In the present study, temperature dependent EPR spectra of X-ray irradiated polycrystalline alanine were analyzed using five multivariate methods in further attempts to understand the composite nature of the alanine dosimeter EPR spectrum. Principal component analysis (PCA), maximum likelihood common factor analysis (MLCFA), independent component analysis (ICA), self-modeling mixture analysis (SMA), and multivariate curve resolution (MCR) were used to extract pure radical spectra and their fractional contributions from the experimental EPR spectra. All methods yielded spectral estimates resembling the established R1 spectrum. Furthermore, SMA and MCR consistently predicted both the established R2 spectrum and the shape of the R3 spectrum. The predicted shape of the R3 spectrum corresponded well with the proposed tentative spectrum derived from spectrum simulations. Thus, results from two independent multivariate data analysis techniques strongly support the previous evidence that three radicals are indeed present in irradiated alanine samples.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

PubMed Central

Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

2012-01-01

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism. PMID:22922757

Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death.

PubMed

Divakaruni, Ajit S; Wallace, Martina; Buren, Caodu; Martyniuk, Kelly; Andreyev, Alexander Y; Li, Edward; Fields, Jerel A; Cordes, Thekla; Reynolds, Ian J; Bloodgood, Brenda L; Raymond, Lynn A; Metallo, Christian M; Murphy, Anne N

2017-04-03

Glutamate is the dominant excitatory neurotransmitter in the brain, but under conditions of metabolic stress it can accumulate to excitotoxic levels. Although pharmacologic modulation of excitatory amino acid receptors is well studied, minimal consideration has been given to targeting mitochondrial glutamate metabolism to control neurotransmitter levels. Here we demonstrate that chemical inhibition of the mitochondrial pyruvate carrier (MPC) protects primary cortical neurons from excitotoxic death. Reductions in mitochondrial pyruvate uptake do not compromise cellular energy metabolism, suggesting neuronal metabolic flexibility. Rather, MPC inhibition rewires mitochondrial substrate metabolism to preferentially increase reliance on glutamate to fuel energetics and anaplerosis. Mobilizing the neuronal glutamate pool for oxidation decreases the quantity of glutamate released upon depolarization and, in turn, limits the positive-feedback cascade of excitotoxic neuronal injury. The finding links mitochondrial pyruvate metabolism to glutamatergic neurotransmission and establishes the MPC as a therapeutic target to treat neurodegenerative diseases characterized by excitotoxicity. © 2017 Divakaruni et al.

40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for the...

40 CFR 721.520 - Alanine, N-(2-carboxyethyl)-N-alkyl-, salt.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Alanine, N-(2-carboxyethyl)-N-alkyl... Specific Chemical Substances § 721.520 Alanine, N-(2-carboxyethyl)-N-alkyl-, salt. (a) Chemical substance... alanine, N-(2-carboxyethyl)-N- alkyl-, salt (P-89-336) is subject to reporting under this section for the...

New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

PubMed Central

De la Vega-Ruíz, Gustavo; Domínguez-Ramírez, Lenin; Riveros-Rosas, Héctor; Guerrero-Mendiola, Carlos; Torres-Larios, Alfredo; Hernández-Alcántara, Gloria; García-Trejo, José J.; Ramírez-Silva, Leticia

2015-01-01

Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge

Atomic Layer Deposition of L-Alanine Polypeptide

DOE PAGES

Fu, Yaqin; Li, Binsong; Jiang, Ying-Bing; ...

2014-10-30

L-Alanine polypeptide thin films were synthesized via atomic layer deposition (ALD). Rather, instead of using an amino acid monomer as the precursor, an L-alanine amino acid derivatized with a protecting group was used to prevent self-polymerization, increase the vapor pressure, and allow linear cycle-by-cycle growth emblematic of ALD. Moreover, the successful deposition of a conformal polypeptide film has been confirmed by FTIR, TEM, and Mass Spectrometry, and the ALD process has been extended to polyvaline.

Variation in metabolic enzymatic activity in white muscle and liver of blue tilapia, Oreochromis aureus, in response to long-term thermal acclimatization

NASA Astrophysics Data System (ADS)

Younis, Elsayed M.

2015-05-01

The effects of rearing temperature on white muscle and hepatic phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were examined in fingerlings of blue tilapia, Oreochromis aureus. The experiment was conducted for 14 weeks at temperatures of 18, 22, 26, 30, and 34°C. The activity of the glycolytic enzymes PFK, PK, and LDH in white muscle increased significantly with increase in water temperature. A reverse trend was observed for these enzymes in the liver, except for LDH, which behaved in the same manner as in white muscle. Cytosolic AST and ALT activity increased in both white muscle and liver in response to warm thermal acclimatization, while a reduction in mitochondrial AST and ALT activity was noticed at high temperatures in comparison with those at a lower temperature.

Valproate causes metabolic disturbance in normal man.

PubMed Central

Turnbull, D M; Dick, D J; Wilson, L; Sherratt, H S; Alberti, K G

1986-01-01

Valproate is an important anticonvulsant which is rarely associated with fatal hepatotoxicity. Previous experiments have shown that valproate inhibits several metabolic processes in isolated rat hepatocytes and when administered to starved rats causes a fall in the blood concentrations of glucose and ketone bodies. Since these changes may be related to the hepatotoxicity, the effect of valproate administration on intermediary metabolism in man was studied. One gram of valproate given orally to fasted normal humans caused a 78% fall in the concentration of 3-hydroxybutyrate and a 60% fall in total ketones. Also the concentrations of lactate, pyruvate, alanine and glycerol increased after valproate administration. Similar changes were observed after intravenous administration of 400 mg of valproate. Valproate clearly has a significant effect on intermediary metabolism in the liver and this is probably related to the mechanism of the hepatotoxicity. PMID:3084712

Identification of a polysaccharide produced by the pyruvate overproducer Candida glabrata CCTCC M202019.

PubMed

Luo, Zhengshan; Liu, Song; Du, Guocheng; Zhou, Jingwen; Chen, Jian

2017-06-01

Candida glabrata has great potential for the accumulation of pyruvate as a preferred strain in pyruvate production by fermentation. However, its substrate conversion rate is relatively low. In this study, a novel polysaccharide containing α-1,4-glucosidic bonds was observed accidentally in screening a high-titer pyruvate strain by atmospheric and room temperature plasma mutagenesis of C. glabrata. Chemical analysis of the partially purified polysaccharide S 4-C10 showed the main components were 1.2% (w/w) protein and 94.2% (w/w) total sugar. Fourier transform infrared and molecular mass distribution analysis indicated that the main component (PSG-2) of S 4-C10 was a small molecular homogeneous protein-bound polysaccharide. Monosaccharide analysis of PSG-2 showed it consisted of glucose, mannose, and fructose. By optimizing the vitamin mix content, 77.6 g L -1 S 4-C10 polysaccharide could be obtained after 72 h fermentation at 30 °C in 500-mL flasks. RT-qPCR analysis showed that transcriptional level of some key genes related to polysaccharide biosynthesis was upregulated compared to that of wild-type strain. By knocking out two most significantly upregulated genes, CAGL0H02695g and CAGL0K10626g, in the wild-type strain, the pyruvate consumption rate was significantly reduced in late pyruvate fermentation phase, while the titer of polysaccharides was reduced by 18.0%. Besides the potential applications of the novel identified polysaccharide, this study provided clues for increasing the conversion ratio of glucose to pyruvate in C. glabrata by further decreasing the accumulation of polysaccharides.

Pyruvate Dehydrogenase Kinase-mediated Glycolytic Metabolic Shift in the Dorsal Root Ganglion Drives Painful Diabetic Neuropathy*

PubMed Central

Rahman, Md Habibur; Jha, Mithilesh Kumar; Kim, Jong-Heon; Nam, Youngpyo; Lee, Maan Gee; Go, Younghoon; Harris, Robert A.; Park, Dong Ho; Kook, Hyun; Lee, In-Kyu; Suk, Kyoungho

2016-01-01

The dorsal root ganglion (DRG) is a highly vulnerable site in diabetic neuropathy. Under diabetic conditions, the DRG is subjected to tissue ischemia or lower ambient oxygen tension that leads to aberrant metabolic functions. Metabolic dysfunctions have been documented to play a crucial role in the pathogenesis of diverse pain hypersensitivities. However, the contribution of diabetes-induced metabolic dysfunctions in the DRG to the pathogenesis of painful diabetic neuropathy remains ill-explored. In this study, we report that pyruvate dehydrogenase kinases (PDK2 and PDK4), key regulatory enzymes in glucose metabolism, mediate glycolytic metabolic shift in the DRG leading to painful diabetic neuropathy. Streptozotocin-induced diabetes substantially enhanced the expression and activity of the PDKs in the DRG, and the genetic ablation of Pdk2 and Pdk4 attenuated the hyperglycemia-induced pain hypersensitivity. Mechanistically, Pdk2/4 deficiency inhibited the diabetes-induced lactate surge, expression of pain-related ion channels, activation of satellite glial cells, and infiltration of macrophages in the DRG, in addition to reducing central sensitization and neuroinflammation hallmarks in the spinal cord, which probably accounts for the attenuated pain hypersensitivity. Pdk2/4-deficient mice were partly resistant to the diabetes-induced loss of peripheral nerve structure and function. Furthermore, in the experiments using DRG neuron cultures, lactic acid treatment enhanced the expression of the ion channels and compromised cell viability. Finally, the pharmacological inhibition of DRG PDKs or lactic acid production substantially attenuated diabetes-induced pain hypersensitivity. Taken together, PDK2/4 induction and the subsequent lactate surge induce the metabolic shift in the diabetic DRG, thereby contributing to the pathogenesis of painful diabetic neuropathy. PMID:26769971

Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium.

PubMed Central

Stevens, T O; Linkfield, T G; Tiedje, J M

1988-01-01

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium. PMID:3223760

The inhibition of pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei and its metabolic implications.

PubMed

Wiemer, E A; Michels, P A; Opperdoes, F R

1995-12-01

The pyruvate produced by glycolysis in the bloodstream form of the trypanosome is excreted into the host bloodstream by a facilitated diffusion carrier. The sensitivity of pyruvate transport for alpha-cyano-4-hydroxycinnamate and the compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate], which are known to be selective inhibitors of pyruvate (monocarboxylate) transporters present in mitochondria and the plasma membrane of eukaryotic cells, was examined. The trypanosomal pyruvate carrier was found to be rather insensitive to inhibition by alpha-cyano-4-hydroxycinnamate (Ki = 17 mM) but could be completely blocked by UK5099 (Ki = 49 microM). Inhibition of pyruvate transport resulted in the retention, and concomitant accumulation, of pyruvate within the trypanosomes, causing acidification of the cytosol and osmotic destabilization of the cells. Our results indicate that this physiological state has serious metabolic consequences and ultimately leads to cell death; thereby identifying the pyruvate carrier as a possible target for chemotherapeutic intervention.

The metabolic advantage of tumor cells

PubMed Central

2011-01-01

1- Oncogenes express proteins of "Tyrosine kinase receptor pathways", a receptor family including insulin or IGF-Growth Hormone receptors. Other oncogenes alter the PP2A phosphatase brake over these kinases. 2- Experiments on pancreatectomized animals; treated with pure insulin or total pancreatic extracts, showed that choline in the extract, preserved them from hepatomas. Since choline is a methyle donor, and since methylation regulates PP2A, the choline protection may result from PP2A methylation, which then attenuates kinases. 3- Moreover, kinases activated by the boosted signaling pathway inactivate pyruvate kinase and pyruvate dehydrogenase. In addition, demethylated PP2A would no longer dephosphorylate these enzymes. A "bottleneck" between glycolysis and the oxidative-citrate cycle interrupts the glycolytic pyruvate supply now provided via proteolysis and alanine transamination. This pyruvate forms lactate (Warburg effect) and NAD+ for glycolysis. Lipolysis and fatty acids provide acetyl CoA; the citrate condensation increases, unusual oxaloacetate sources are available. ATP citrate lyase follows, supporting aberrant transaminations with glutaminolysis and tumor lipogenesis. Truncated urea cycles, increased polyamine synthesis, consume the methyl donor SAM favoring carcinogenesis. 4- The decrease of butyrate, a histone deacetylase inhibitor, elicits epigenic changes (PETEN, P53, IGFBP decrease; hexokinase, fetal-genes-M2, increase) 5- IGFBP stops binding the IGF - IGFR complex, it is perhaps no longer inherited by a single mitotic daughter cell; leading to two daughter cells with a mitotic capability. 6- An excess of IGF induces a decrease of the major histocompatibility complex MHC1, Natural killer lymphocytes should eliminate such cells that start the tumor, unless the fever prostaglandin PGE2 or inflammation, inhibit them... PMID:21649891

Effect of bicarbonate concentration on aerobic growth of campylobacter in a fumarate-pyruvate medium

USDA-ARS?s Scientific Manuscript database

The purpose of the present study was to examine the effect of sodium bicarbonate (NaHCO3) concentration on aerobic growth of Campylobacter in a fumarate-pyruvate medium. Fumarate-pyruvate broth medium was supplemented with 0.00 to 0.10% NaHCO3 and inoculated with Campylobacter coli 33559, Campyloba...

Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes.

PubMed Central

Allan, E H; Chisholm, A B; Titheradge, M A

1983-01-01

A method is described for measuring rates of mitochondrial pyruvate carboxylation in hepatocytes treated with the polyene antibiotic, filipin, to render the plasma membrane permeable to substrates. With this approach it was possible to demonstrate that treatment of cells with glucagon or catecholamines results in a stimulation of mitochondrial CO2 fixation measured in situ comparable with that observed in the isolated mitochondria, in terms of time of onset of the response, hormone selectivity and sensitivity. In addition, angiotensin II and vasopressin were shown to enhance the activity of pyruvate carboxylase in both the intact mitochondria and filipin-treated cells, thus strengthening the postulate that this site is a major locus of hormone action in the control of gluconeogenesis. Addition of 3-mercaptopicolinic acid, to inhibit gluconeogenesis at the level of phosphoenolpyruvate carboxykinase, had no significant effect on the stimulation of pyruvate carboxylation by adrenaline, suggesting that the effect of the hormone at this site is independent of changes in activity of other enzymes further on in the pathway. The data presented preclude the possibility that acute effects of hormones on mitochondrial metabolism are solely artifacts of the preparation procedure. PMID:6411066

L-alanine-glyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate beta-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes?

PubMed Central

Cooper, Arthur J L; Krasnikov, Boris F; Okuno, Etsuo; Jeitner, Thomas M

2003-01-01

Several halogenated alkenes are metabolized in part to cysteine S-conjugates, which are mitochondrial toxicants of kidney and, to a lesser extent, other organs. Toxicity is due to cysteine S-conjugate beta-lyases, which convert the cysteine S-conjugate into pyruvate, ammonia and a reactive sulphur-containing fragment. A section of the human population is exposed to halogenated alkenes. To understand the health effects of such exposure, it is important to identify cysteine S-conjugate beta-lyases that contribute to mitochondrial damage. Mitochondrial aspartate aminotransferase [Cooper, Bruschi, Iriarte and Martinez-Carrion (2002) Biochem. J. 368, 253-261] and mitochondrial branched-chain aminotransferase [Cooper, Bruschi, Conway and Hutson (2003) Biochem. Pharmacol. 65, 181-192] exhibit beta-lyase activity toward S -(1,2-dichlorovinyl)-L-cysteine (the cysteine S-conjugate of trichloroethylene) and S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene). Turnover leads to eventual inactivation of these enzymes. Here we report that mitochondrial L-alanine-glyoxylate aminotransferase II, which, in the rat, is most active in kidney, catalyses cysteine S-conjugate beta-lyase reactions with S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(benzothiazolyl-L-cysteine); turnover leads to inactivation. Previous workers showed that the reactive-sulphur-containing fragment released from S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and S -(1,2-dichlorovinyl)-L-cysteine is toxic by acting as a thioacylating agent - particularly of lysine residues in nearby proteins. Toxicity, however, may also involve 'self-inactivation' of key enzymes. The present findings suggest that alanine-glyoxylate aminotransferase II may be an important factor in the well-established targeting of rat kidney mitochondria by toxic halogenated cysteine S-conjugates. Previous reports suggest that alanine-glyoxylate aminotransferase II is absent