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Sample records for adenosine monophosphate amp-activated

  1. Liver MicroRNA-291b-3p Promotes Hepatic Lipogenesis through Negative Regulation of Adenosine 5'-Monophosphate (AMP)-activated Protein Kinase α1.

    PubMed

    Meng, Xiangyu; Guo, Jun; Fang, Weiwei; Dou, Lin; Li, Meng; Huang, Xiuqing; Zhou, Shutong; Man, Yong; Tang, Weiqing; Yu, Liqing; Li, Jian

    2016-05-13

    In a microarray study, we found that hepatic miR-291b-3p was significantly increased in leptin-receptor-deficient type 2 mice (db/db), a mouse model of diabetes. The function of miR-291b-3p is unknown. The potential role of miR-291b-3p in regulating hepatic lipid metabolism was explored in this study. High-fat diet (HFD)- and chow-fed mice were injected with an adenovirus expressing a miR-291b-3p inhibitor and a miR-291b-3p mimic through the tail vein. Hepatic lipids and lipogenic gene expression were analyzed. Additionally, gain- and loss-of-function studies were performed in vitro to identify direct targets of miR-291b-3p. MiR-291b-3p expression and the protein levels of sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FAS) were increased in the steatotic liver of db/db mice and HFD-fed mice versus their respective controls. Inhibition of hepatic miR-291b-3p expression prevented increases in hepatic lipogenesis and steatosis in HFD-fed mice. The opposite was observed when miR-291b-3p was overexpressed in the livers of chow-fed C57BL/6J wild-type mice. In vitro studies revealed that silencing of miR-291b-3p in NCTC1469 hepatic cells ameliorated oleic acid/palmitic acid mixture-induced elevation of cellular triglycerides. Importantly, we identified AMP-activated protein kinase (AMPK)-α1 as a direct target of miR-291b-3p. Using metformin, an activator of AMPK, we showed that AMPK activation-induced inhibition of hepatic lipid accumulation was accompanied by reduced expression of miR-291b-3p in the liver. Liver miR-291b-3p promoted hepatic lipogenesis and lipid accumulation in mice. AMPKα1 is a direct target of miR-291b-3p. In conclusion, our findings indicate that miR-291b-3p promotes hepatic lipogenesis by suppressing AMPKα1 expression and activity, indicating the therapeutic potential of miR-291b-3p inhibitors in fatty liver disease. PMID:27013659

  2. Genetics Home Reference: adenosine monophosphate deaminase deficiency

    MedlinePlus

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions adenosine monophosphate deaminase deficiency adenosine ...

  3. Cyclic adenosine monophosphate phosphodiesterase in brain: effect on anxiety.

    PubMed

    Beer, B; Chasin, M; Clody, D E; Vogel, J R

    1972-04-28

    Drugs that reduce anxiety may be mediated by cyclic adenosine monophosphate in the brain because (i) potent anxiety-reducing drugs are also potent inhibitors of brain phosphodiesterase activity; (ii) dibutyryl cyclic adenosine monophosphate has the ability to reduce anxiety; (iii) the methylxanthines show significant anxiety-reducing effects; (iv) theophylline and chlordiazepoxide produce additive anxiety-reducing activity; and (v) there is a significant correlation between the anxiety-reducing property of drugs and their ability to inhibit phosphodiesterase activity in the brain. PMID:4402069

  4. Adenosine Monophosphate-Based Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

    Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya

    2009-01-01

    A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

  5. Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury.

    PubMed

    Hwang, Jung Hwan; Kim, Yong-Hoon; Noh, Jung-Ran; Choi, Dong-Hee; Kim, Kyoung-Shim; Lee, Chul-Ho

    2015-10-01

    The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose. PMID:26434492

  6. Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury

    PubMed Central

    Hwang, Jung Hwan; Kim, Yong-Hoon; Noh, Jung-Ran; Choi, Dong-Hee; Kim, Kyoung-Shim; Lee, Chul-Ho

    2015-01-01

    The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose. PMID:26434492

  7. Postirradiation administration of adenosine monophosphate combined with dipyridamole reduces early cellular damage in mice

    SciTech Connect

    Bohacek, J.; Hosek, B.; Pospisil, M. )

    1993-01-01

    The administration of dipyridamole and adenosine 5'-monophosphate (AMP) to mice 5 to 25 min after 1 Gy of total-body gamma irradiation was found to decrease cellular damage, as indicated by the thymidine level in plasma and the amount of saline soluble polynucleotides in the thymus. The drug combination used did not influence similar cytotoxic effects of hydrocortisone. Furthermore, it was shown that the addition of dipyridamole and AMP to in vitro irradiated suspensions of thymocytes enhanced the rejoining processes of DNA strand breaks. Receptor-mediated action of extracellular adenosine may be responsible for the therapeutic effects observed.

  8. Histone deacetylases 6 increases the cyclic adenosine monophosphate level and promotes renal cyst growth.

    PubMed

    Wu, Ming; Mei, Changlin

    2016-07-01

    Autosomal dominant polycystic kidney disease (ADPKD) is characterized by abnormal enhanced cell proliferation and fluid secretion, which are triggered by increased levels of cyclic adenosine monophosphate (cAMP). Cebotaru et al. showed that a HDAC6 inhibitor reduced the cAMP level and inhibited cyst formation in Pkd1 knockout mice, which may become a new potential therapeutic agent for ADPKD. This study also raised several intriguing questions that might advance our understanding of the molecular pathogenesis of ADPKD. PMID:27312442

  9. Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

    PubMed Central

    Yamaguchi, Hiroshi; Maruyama, Toshihiko; Urade, Yoshihiro; Nagata, Shigekazu

    2014-01-01

    Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a ‘calm down’ signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001 PMID:24668173

  10. Adenosine 3',5'-monophosphate waves in dictyostelium discoideum: a demonstration by isotope dilution-fluorography

    SciTech Connect

    Tomchik, K.J.; Devreotes, P.N.

    1981-04-24

    The distribution of adenosine 3',5'-monophosphate (cyclic AMP) in fields of aggregating amoebae of Dictyostelium discoidenum was examined by a novel isotope dilution-fluorographic technique. Cellular cyclic AMP was visualized by its competition with exogenous /sup 3/H-labeled cyclic AMP for high-affinity binding sites on protein kinase immobilized on a Millipore filter used to blot the monolayer. The cyclic AMP was distributed in spiral or concentric circular wave patterns which centered on the foci of the aggregations. These patterns were correlated with those of cell shape change that propagate through the monolayers. These observations support the hypothesis that the aggregation process in Dictyostelium is mediated by the periodic relay of cyclic AMP signals and suggest a simple scheme for the dynamics of the aggregation process.

  11. Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

    PubMed

    Hortle, Elinor; Nijagal, Brunda; Bauer, Denis C; Jensen, Lora M; Ahn, Seong Beom; Cockburn, Ian A; Lampkin, Shelley; Tull, Dedreia; McConville, Malcolm J; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

    2016-09-01

    The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. PMID:27465915

  12. Stimulation of cyclic adenosine monophosphate accumulation causes breakdown of the blood-retinal barrier.

    PubMed

    Sen, H A; Campochiaro, P A

    1991-06-01

    Pigmented rabbits were given an intravitreous injection of 0.1 ml of various concentrations of test drug, and vitreous fluorophotometry was done 6 and 24 hr after injection. Dibutyryl cyclic adenosine monophosphate (AMP) and 8-bromo-cyclic AMP caused reversible intravitreous fluorescein leakage only at relatively high concentrations. Adrenergic agents that are effective stimulators of adenylate cyclase (epinephrine, isoproterenol, and norepinephrine) caused transient intravitreous fluorescein leakage (2.3-3.1-fold above baseline) that was significantly greater than that caused by phenylephrine (1.1-fold above baseline), an adrenergic agent that is a poor stimulator of adenylate cyclase. Prostaglandins E1 and E2, which are good stimulators of adenylate cyclase, caused striking disruption of the blood-ocular barriers, and prostaglandins that are not good stimulators of adenylate cyclase had little or no effect on these barriers. The magnitude of the prostaglandin E1 effect (9.3-fold above baseline) was similar to that of N-ethylcarboxamidoadenosine (NECA), the most potent adenosine agonist, and was greater than one would predict based on its effect on adenylate cyclase in vitro. Prostaglandin E1, like NECA, also caused retinal vasodilation and hemorrhages. These data suggest that stimulation of intracellular cyclic AMP accumulation may be a common feature of mediators that cause breakdown of the blood-retinal barrier, but there may be another as yet unexplained feature shared by PGE1 and NECA that makes them particularly effective and capable of causing retinal vasodilation and hemorrhages. PMID:1647374

  13. Attempts to detect cyclic adenosine 3':5'-monophosphate in higher plants by three assay methods.

    PubMed

    Bressan, R A; Ross, C W

    1976-01-01

    Endogenous levels of cyclic adenosine-3':5'-monophosphate in coleoptile first leaf segments of oat (Avena sativa L.), potato (Solanum tuberosum L.) tubers, tobacco (Nicotiana tabacum L.) callus, and germinating seeds of lettuce (Lactuca sativa L.) were measured with a modified Gilman binding assay and a protein kinase activation assay. The incorporation of adenosine-8-(14)C into compounds with properties similar to those of cyclic AMP was also measured in studies with germinating lettuce seeds. The binding assay proved reliable for mouse and rat liver analyses, but was nonspecific for plant tissues. It responded to various components from lettuce and potato tissues chromatographically similar to but not identical with cyclic AMP. The protein kinase activation assay was much more specific, but it also exhibited positive responses in the presence of compounds not chromatographically identical to cyclic AMP. The concentrations of cyclic AMP in the plant tissues tested were at the lower limits of detection and characterization obtainable with these assays. The estimates of maximal levels were much lower than reported in many previous studies. PMID:16659419

  14. LDL-cholesterol reduction in patients with hypercholesterolemia by modulation of adenosine triphosphate-citrate lyase and adenosine monophosphate-activated protein kinase

    PubMed Central

    Filippov, Sergey; Pinkosky, Stephen L.; Newton, Roger S.

    2014-01-01

    Purpose of review To review the profile of ETC-1002, as shown in preclinical and clinical studies, including LDL-cholesterol (LDL-C)-lowering activity and beneficial effects on other cardiometabolic risk markers as they relate to the inhibition of adenosine triphosphate-citrate lyase and the activation of adenosine monophosphate-activated protein kinase. Recent findings ETC-1002 is an adenosine triphosphate-citrate lyase inhibitor/adenosine monophosphate-activated protein kinase activator currently in Phase 2b clinical development. In seven Phase 1 and Phase 2a clinical studies, ETC-1002 dosed once daily for 2–12 weeks has lowered LDL-C and reduced high-sensitivity C-reactive protein by up to 40%, with neutral to positive effects on glucose levels, blood pressure, and body weight. Importantly, use of ETC-1002 in statin-intolerant patients has shown statin-like lowering of LDL-C without the muscle pain and weakness responsible for discontinuation of statin use by many patients. ETC-1002 has also been shown to produce an incremental benefit, lowering LDL-C as an add-on therapy to a low-dose statin. In over 300 individuals in studies of up to 12 weeks, ETC-1002 has been well tolerated with no serious adverse effects. Summary Because adenosine triphosphate-citrate lyase and adenosine monophosphate-activated protein kinase play central roles in regulating lipid and glucose metabolism, pharmacological modulation of these two enzymes could provide an important therapeutic alternative for statin-intolerant patients with hypercholesterolemia. PMID:24978142

  15. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5‧ monophosphate (AMP)

    NASA Astrophysics Data System (ADS)

    Hammami, K.; Feki, H. El; Marsan, O.; Drouet, C.

    2015-10-01

    This work investigates the interaction between the nucleotide adenosine 5‧ monophosphate molecule (AMP) and a biomimetic nanocrystalline carbonated apatite as a model for bone mineral. The analogy of the apatite phase used in this work with biological apatite was first pointed out by complementary techniques. AMP adsorption isotherms were then investigated. Obtained data were fitted to a Sips isotherm with an exponent greater than one suggesting positive cooperativity among adsorbed molecules. The data were compared to a previous study relative to the adsorption of another nucleotide, cytidine monophosphate (CMP) onto a similar substrate, evidencing some effect of the chemical nature of the nucleic base. An enhanced adsorption was observed under acidic (pH 6) conditions as opposed to pH 7.4, which parallels the case of DNA adsorption on biomimetic apatite. An estimated standard Gibbs free energy associated to the adsorption process (ΔG°ads ≅ -22 kJ/mol) intermediate between "physisorption" and "chemisorption" was found. The analysis of the solids after adsorption pointed to the preservation of the main characteristics of the apatite substrate but shifts or enhancements of Raman bands attributed to AMP showed the existence of chemical interactions involving both the phosphate and adenine parts of AMP. This contribution adds to the works conducted in view of better understanding the interaction of DNA/RNA and their constitutive nucleotides and the surface of biomimetic apatites. It could prove helpful in disciplines such as bone diagenesis (DNA/apatite interface in aged bones) or nanomedicine (setup of DNA- or RNA-loaded apatite systems). Also, the adsorption of nucleic acids on minerals like apatites could have played a role in the preservation of such biomolecules in the varying conditions known to exist at the origin of life on Earth, underlining the importance of dedicated adsorption studies.

  16. Adenosine 5′-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice

    PubMed Central

    Zhan, Y; Wang, Z; Yang, P; Wang, T; Xia, L; Zhou, M; Wang, Y; Wang, S; Hua, Z; Zhang, J

    2014-01-01

    D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5′-monophosphate (5′-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5′-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5′-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5′-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5′-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury. PMID:24407238

  17. The effect of polystyrene beads on cyclic 3',5'-adenosine monophosphate concentration in leukocytes.

    PubMed

    Manganiello, V; Evans, W H; Stossel, T P; Mason, R J; Vaughan, M

    1971-12-01

    After incubation with polystyrene latex beads for 5 min. the cyclic 3',5'-adenosine monophosphate (cyclic AMP) content of human peripheral blood leukocyte suspensions was increased severalfold. Preparations enriched in mononuclear cells and containing only 0-20% polymorphonuclear leukocytes (PMN) and no visible platelets exhibited a quantitatively similar response. Purified fractions of cells containing 85-90% PMN responded to polystyrene beads with a much smaller increase in cyclic AMP content. Phagocytosis of paraffin oil emulsion in the unfractionated mixed human leukocyte preparation was associated with little or no change in cyclic AMP levels. There was no change in cyclic AMP content of rabbit alveolar macrophages or guinea pig PMN during phagocytosis of polystyrene beads. All of these observations are consistent with the view that particle uptake per se does not increase cyclic AMP levels in phagocytic cells. It seems probable that the increase in cyclic AMP concentration that results when unfractionated human blood leukocytes are incubated with polystyrene beads occurs in cells other than PMN. PMID:4331596

  18. Control of cyclic adenosine 3',5'-monophosphate levels by depolarizing agents in fungi.

    PubMed Central

    Trevillyan, J M; Pall, M L

    1979-01-01

    It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity. PMID:220213

  19. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats.

    PubMed

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin

    2016-01-01

    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5'-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

  20. Regulation of Maltodextrin Phosphorylase Synthesis in Escherichia coli by Cyclic Adenosine 3′, 5′-Monophosphate and Glucose1

    PubMed Central

    Chao, Julie; Weathersbee, Carolyn J.

    1974-01-01

    Cyclic adenosine 3′, 5′-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3′-AMP, 5′-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, α-methyl glucoside, 2-deoxyglucose, and pyridoxal 5′-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase. PMID:4358043

  1. Cyclic adenosine 3', 5'-monophosphate in cerebrospinal fluid during thermoregulation and fever.

    PubMed Central

    Dascombe, M J; Milton, A S

    1976-01-01

    1. Samples of cerebrospinal fluid (c.s.f.) have been taken from the cisterna magna of unanaesthetized cats, whilst rectal temperature was recorded, during exposure of the animals to various ambient temperatures and during fever induced by pyrogen. The concentration of adenosine 3', 5'-monophosphate (cyclic AMP) in samples of c.s.f. has been assayed. 2. Cats exposed to low ambient temperatures (-2 to +2 degrees C) for 3 h maintained body temperature by both behavioural and autonomic heat gain activity. Exposure of cats to high ambient temperatures (44 - 45 degrees C) for 3.5 h caused a rise in body temperatures of about 2.5 degrees C, despite behavioural and autonomic heat loss activity. Neither cold nor heat stress had a significant effect on c.s.f. cyclic AMP. 3. Fever induced by intravenous Shigella dysenteriae (2 and 20 mug/kg) was associated with a dose-related increase in the concentration of cyclic AMP in c.s.f. Paracetamol (75 mg/kg) injected I.P. before the onset of fever, suppressed the increase in both temperature and c.s.f. cyclic AMP in response to pyrogen. Paracetamol (50 and 100 mg/kg), injected after the onset of fever, caused a fall in temperature, which was not associated with a decrease in the concentration of cyclic AMP in c.s.f. 4. Fever induced in cats by intravenous Shigella dysenteriae (20 mug/kg) was associated with an increase in the concentration of cyclic AMP in plasma as well as in c.s.f. 5. The sodium salt of cyclic AMP (0.1-10 mg/kg) injected I.V. into unanaesthetized cats caused a dose-related hypothermia, which was associated with autonomic heat loss activity and a dose-related increase in the concentration of cyclic AMP in cisternal c.s.f., which was not mimicked by adenosine. 6. It is concluded that the raised concentrations of cyclic AMP in c.s.f., in response to pyrogen I.V., do not mediate fever in the cat and that the concentration of cyclic AMP in cisternal c.s.f. may be affected by changes in the plasma concentration of the

  2. The effects of cyclic adenosine 3',5'-monophosphate and other adenine nucleotides on body temperature.

    PubMed Central

    Dascombe, M J; Milton, A S

    1975-01-01

    1. Adenosine 3',5'-monophosphate (cAMP), its dibutyryl derivative (Db-cAMP) and other adenine nucleotides have been micro-injected into the hypothalamic region of the unanaesthetized cat and the effects on body temperature, and on behavioural and autonomic thermoregulatory activities observed. 2. Db-cAMP and cAMP both produced hypothermia when applied to the pre-optic anterior hypothalamus. With Db-cAMP the hypothermia was shown to be dose dependent between 50 and 500 mug (0-096-0-96 mumole). 3. AMP, ADP and ATP also produced hypothermia when injected into the pre-optic anterior hypothalamus. 4. The order of relative potencies of the adenine nucleotides with respect both to the hypothermia produced and to the autonomic thermoregulatory effects observed were similar. Db-cAMP was most potent and cAMP least. 5. Micro-injection into the pre-optic anterior hypothalamus of many substances including saline produced in most cats a non-specific rise in body temperature apparently the result of tissue damage. Intraperitoneal injection of 4-acetamidophenol (paracetamol 50 mg/kg) reduced or abolished this febrile response. 6. The hypothermic effect of the adenine nucleotides has been compared with the effects produced in these same cats by micro-injections of noradrenaline, 5-hydroxytryptamine, a mixture of acetylcholine and physostigmine (1:1), EDTA and excess Ca2+ ions. 7. It is concluded that as Db-cAMP and cAMP both produce hypothermia, it is unlikely that endogenous cAMP in the pre-optic anterior hypothalamus mediates the hyperthermic responses to pyrogens and prostaglandins. PMID:170396

  3. The effects of cyclic adenosine 3',5'-monophosphate and other adenine nucleotides on body temperature.

    PubMed

    Dascombe, M J; Milton, A S

    1975-08-01

    1. Adenosine 3',5'-monophosphate (cAMP), its dibutyryl derivative (Db-cAMP) and other adenine nucleotides have been micro-injected into the hypothalamic region of the unanaesthetized cat and the effects on body temperature, and on behavioural and autonomic thermoregulatory activities observed. 2. Db-cAMP and cAMP both produced hypothermia when applied to the pre-optic anterior hypothalamus. With Db-cAMP the hypothermia was shown to be dose dependent between 50 and 500 mug (0-096-0-96 mumole). 3. AMP, ADP and ATP also produced hypothermia when injected into the pre-optic anterior hypothalamus. 4. The order of relative potencies of the adenine nucleotides with respect both to the hypothermia produced and to the autonomic thermoregulatory effects observed were similar. Db-cAMP was most potent and cAMP least. 5. Micro-injection into the pre-optic anterior hypothalamus of many substances including saline produced in most cats a non-specific rise in body temperature apparently the result of tissue damage. Intraperitoneal injection of 4-acetamidophenol (paracetamol 50 mg/kg) reduced or abolished this febrile response. 6. The hypothermic effect of the adenine nucleotides has been compared with the effects produced in these same cats by micro-injections of noradrenaline, 5-hydroxytryptamine, a mixture of acetylcholine and physostigmine (1:1), EDTA and excess Ca2+ ions. 7. It is concluded that as Db-cAMP and cAMP both produce hypothermia, it is unlikely that endogenous cAMP in the pre-optic anterior hypothalamus mediates the hyperthermic responses to pyrogens and prostaglandins. PMID:170396

  4. Mitochondrial Oxidative Stress Corrupts Coronary Collateral Growth by Activating Adenosine Monophosphate Activated Kinase-α Signaling

    PubMed Central

    Pung, Yuh Fen; Sam, Wai Johnn; Stevanov, Kelly; Enrick, Molly; Chen, Chwen-Lih; Kolz, Christopher; Thakker, Prashanth; Hardwick, James P.; Chen, Yeong-Renn; Dyck, Jason R.B.; Yin, Liya; Chilian, William M.

    2015-01-01

    Objective Our goal was to determine the mechanism by which mitochondrial oxidative stress impairs collateral growth in the heart. Approach and Results Rats were treated with rotenone (mitochondrial complex I inhibitor that increases reactive oxygen species production) or sham-treated with vehicle and subjected to repetitive ischemia protocol for 10 days to induce coronary collateral growth. In control rats, repetitive ischemia increased flow to the collateral-dependent zone; however, rotenone treatment prevented this increase suggesting that mitochondrial oxidative stress compromises coronary collateral growth. In addition, rotenone also attenuated mitochondrial complex I activity and led to excessive mitochondrial aggregation. To further understand the mechanistic pathway(s) involved, human coronary artery endothelial cells were treated with 50 ng/ mL vascular endothelial growth factor, 1 µmol/L rotenone, and rotenone/vascular endothelial growth factor for 48 hours. Vascular endothelial growth factor induced robust tube formation; however, rotenone completely inhibited this effect (P<0.05 rotenone versus vascular endothelial growth factor treatment). Inhibition of tube formation by rotenone was also associated with significant increase in mitochondrial superoxide generation. Immunoblot analyses of human coronary artery endothelial cells with rotenone treatment showed significant activation of adenosine monophosphate activated kinase (AMPK)-α and inhibition of mammalian target of rapamycin and p70 ribosomal S6 kinase. Activation of AMPK-α suggested impairments in energy production, which was reflected by decrease in O2 consumption and bioenergetic reserve capacity of cultured cells. Knockdown of AMPK-α (siRNA) also preserved tube formation during rotenone, suggesting the negative effects were mediated by the activation of AMPK-α. Conversely, expression of a constitutively active AMPK-α blocked tube formation. Conclusions We conclude that activation of AMPK

  5. Cyclic Adenosine Monophosphate Accumulation and beta-Adrenergic Binding in Unweighted and Denervated Rat Soleus Muscle

    NASA Technical Reports Server (NTRS)

    Kirby, Christopher R.; Woodman, Christopher R.; Woolridge, Dale; Tischler, Marc E.

    1992-01-01

    Unweighting, but not denervation, of muscle reportedly "spares" insulin receptors, increasing insulin sensitivity. Unweighting also increases beta-adrenergic responses of carbohydrate metabolism. These differential characteristics were studied further by comparing cyclic adenosine monophosphate (cAMP) accumulation and beta-adrenergic binding in normal and 3-day unweighted or denervated soleus muscle. Submaximal amounts of isoproterenol, a p-agonist, increased cAMP accumulation in vitro and in vivo (by intramuscular (IM) injection) to a greater degree (P less than .05) in unweighted muscles. Forskolin or maximal isoproterenol had similar in vitro effects in all muscles, suggesting increased beta-adrenergic sensitivity following unweighting. Increased sensitivity was confirmed by a greater receptor density (B(sub max)) for iodo-125(-)-pindolol in particulate preparations of unweighted (420 x 10(exp -18) mol/mg muscle) than of control or denervated muscles (285 x 10(exp-18) mol/mg muscle). The three dissociation constant (Kd) values were similar (20.3 to 25.8 pmol/L). Total binding capacity (11.4 fmol/muscle) did not change during 3 days of unweighting, but diminished by 30% with denervation. This result illustrates the "sparing" and loss of receptors, respectively, in these two atrophy models. In diabetic animals, IM injection of insulin diminished CAMP accumulation in the presence of theophylline in unweighted muscle (-66% +/- 2%) more than in controls (-42% +'- 6%, P less than .001). These results show that insulin affects CAMP formation in muscle, and support a greater in vivo insulin response following unweighting atrophy. These various data support a role for lysosomal proteolysis in denervation, but not in unweighting, atrophy.

  6. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

    PubMed Central

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin

    2016-01-01

    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5’-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

  7. Adenosine 3', 5'-cyclic monophosphate levels in Thermomonospora curvata during cellulase biosynthesis

    SciTech Connect

    Fennington, G.; Neubauer, D.; Stutzenberger, F.

    1983-01-01

    The enzymatic degradation of cellulose requires the synergistic activity of at least three enzymes: exo-beta-1,4-glucanase (EC3.2.1.91), endo-beta-1,4-glucanase (EC3.2.1.4), and beta-glucosidase (EC3.2.1.21). Despite extensive studies on a variety of cellulolytic bacteria and fungi, the mechanism(s) regulating the biosynthesis of this inducible catabolic enzyme complex remains unknown. The intracellular concentrations of cyclic nucleotides such as adenosine 3',5'-cyclic monophosphate (cAMP) have been shown to play a major role in mediating catabolite repression of enzyme biosynthesis. The cAMP acts through a cAMP receptor protein (termed CRP or CAP) which is a dimer having two identical subunits each capable of binding one molecule of cAMP. The N-terminal domain of the CRP binds the cAMP while the C-terminal domain binds to DNA at the promotor region of a cAMP-dependent operon and stimulates transcription by promoting the formation of a preinitiation complex between RNA polymerase and the DNA. Intracellular cAMP levels in E. coli (the prototype organism for such studies) are influenced by the type and availability of carbon source used for growth. High intracellular cAMP levels should lead to higher concentrations of cAMP-CRP complexes which should increase the transcription rates for cAMP-dependent operons (such as the lac operon of beta-galactosidase) and indeed the differential rate of beta-galactosidase biosynthesis correlates to intracellular cAMP levels. In the case of cellulase, catabolite repression by glucose or other readily metabolizable compounds closely controls production in an apparently similar manner and therefore a correlation may exist between enzyme biosynthesis and intracellular cAMP levels. This communication describes the fluctuation in cAMP levels during cellulase induction and repression in the thermophilic actinomycete, Thermomonospora curvata.

  8. Repetitive mechanical strain suppresses macrophage uptake of immunoglobulin G complexes and enhances cyclic adenosine monophosphate synthesis.

    PubMed Central

    Mattana, J.; Sankaran, R. T.; Singhal, P. C.

    1995-01-01

    Uptake of immunoglobulin G (IgG) complexes by macrophages (M phi) may play an important role in disease states characterized by increased levels of circulating immune complexes. In sites such as the glomerular mesangium M phi may be subjected to repetitive mechanical strain, although in vitro studies of M phi endocytosis are typically carried out with cells grown on rigid surfaces. We undertook the present study to determine whether repetitive mechanical strain could modulate M phi endocytosis of IgG complexes. IgG complex uptake was significantly diminished in M phi that were subjected to repetitive mechanical strain using parameters corresponding to peak and minimal intraglomerular pressures compared with control, and uptake varied according to the amount of mechanical strain applied. There was no significant difference in surface binding of IgG between M phi subjected to strain and those not. Mechanical strain did not significantly influence the rate of IgG complex degradation. Inhibition of nitric oxide synthase and guanylate cyclase activity did not alter the effect of mechanical strain, although this effect was potentiated by 3-isobutyl-1-methylxanthine (IBMX). Angiotensin II, which has been shown to reduce adenosine 3',5'-cyclic monophosphate (cAMP) production in M phi, significantly attenuated the suppressive effect of mechanical strain on IgG complex uptake as well as another inhibitor of cAMP generation, indomethacin. Enzyme immunoassay demonstrated significantly enhanced levels of cAMP in M phi that were subjected to mechanical strain compared with control, an effect that was potentiated by IBMX and attenuated by angiotensin II and indomethacin. These results demonstrate that repetitive mechanical strain significantly reduces IgG complex uptake by M phi, most likely by enhancing cAMP synthesis. Such an effect might play a significant role in macromolecule handling by M phi in sites in which they are subjected to repetitive mechanical deformation such as

  9. Novel adenosine 3 prime ,5 prime -cyclic monophosphate dependent protein kinases in a marine diatom

    SciTech Connect

    Lin, P.P.C.; Volcani, B.E. )

    1989-08-08

    Two novel adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg{sup 2+} and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser({sup 32}P)-Ser-Asn-Ala-Arg and have an apparent M{sub r} of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M{sub r} of about 78,000 is photolabeled with 8-azido({sup 32}P)cAMP and is also phosphorylated with ({gamma}-{sup 32}P)ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids.

  10. Adenosine 3′:5′-cyclic monophosphate- and guanosine 3′:5′-cyclic monophosphate-dependent protein kinases: Possible homologous proteins

    PubMed Central

    Lincoln, Thomas M.; Corbin, Jackie D.

    1977-01-01

    The properties of purified mammalian adenosine 3′:5′-cyclic monophosphate (cAMP)- and guanosine 3′:5′-cyclic monophosphate (cGMP)-dependent protein kinases were compared. Several physical characteristics of the two enzymes were similar, including size, shape, affinity for cyclic nucleotide binding, and Km for ATP. In addition, the amino acid composition of the two proteins indicated a close composition homology (70-90%). Both cyclic nucleotide-dependent protein kinases catalyzed phosphorylation of rat liver pyruvate kinase (EC 2.7.1.40) and fructose 1,6-diphosphatase (EC 3.1.3.11), rabbit skeletal muscle glycogen synthase (EC 2.4.1.11) and phosphorylase b kinase (EC 2.7.1.38), and calf thymus histone H2b. The phosphorylation of several synthetic peptides and of trypsin-sensitive and trypsin-insensitive sites in glycogen synthase suggested similar recognition sites on the protein substrates for the two kinases. The cAMP-dependent protein kinase was the better catalyst with each protein or peptides substrate. The results suggest that the two enzymes evolved from a common ancestral protein. Images PMID:198777

  11. Human monocyte killing of Staphylococcus aureus: modulation by agonists of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate.

    PubMed Central

    O'Dorisio, M S; Vandenbark, G R; LoBuglio, A F

    1979-01-01

    This study was designed to test whether cyclic nucleotides play a role in the regulation of bacterial killing by human monocytes. Agents were tested for their ability to activate monocyte adenylate or guanylate cyclase in cell-free preparations, to increase cyclic adenosine 3',5'-monophosphate (cAMP) or cyclic guanosine 3',5'-monophosphate (cGMP) in intact human monocytes, and to modulate monocyte-induced killing of Staphylococcus aureus in vitro. Prostaglandin E1 and cholera toxin activated monocyte adenylate cyclase and inhibited monocyte killing of S. aureus. An adenylate cyclase inhibitor, RMI 12330A, reversed the prostaglandin E1-mediated inhibition of bacterial killing, thus implicating cAMP as the intracellular mediator of this inhibition. In contrast, monocyte cGMP levels were increased 5- and 17-fold by 5-hydroxytryptamine and N-methyl-N' -nitro-N-nitrosoguanidine, respectively, but neither agent was effective in modulating monocyte bactericidal activity. Thus, modulation of bactericidal activity in human monocytes did not conform to the yin/yang theory of opposing actions by cAMP and cGMP, for although monocyte-mediated killing of S. aureus was inhibited by cAMP agonists, it was not enhanced by cGMP agonists. PMID:44704

  12. Dielectric spectra broadening as a signature for dipole-matrix interaction. III. Water in adenosine monophosphate/adenosine-5'-triphosphate solutions.

    PubMed

    Puzenko, Alexander; Levy, Evgeniya; Shendrik, Andrey; Talary, Mark S; Caduff, Andreas; Feldman, Yuri

    2012-11-21

    In this, the third part of our series on the dielectric spectrum symmetrical broadening of water, we consider the nucleotide aqueous solutions. Where in Parts I [E. Levy et al., J. Chem. Phys. 136, 114502 (2012)] and II [E. Levy et al., J. Chem. Phys. 136, 114503 (2012)], the dipole-dipole or ion-dipole interaction had a dominant feature, now the interplay between these two types of dipole-matrix interactions will be considered. We present the results of high frequency dielectric measurements of different concentrations of adenosine monophosphate/adenosine-5'-triphosphate aqueous solutions. We observed the Cole-Cole broadening of the main relaxation peak of the solvent in the solutions. Moreover, depending on the nucleotide concentration, we observed both types of dipole-matrix interaction. The 3D trajectory approach (described in detail in Part I) is applied in order to highlight the differences between the two types of interaction. PMID:23181321

  13. Gas-Phase Conformations and Energetics of Protonated 2^'-DEOXYADENOSINE-5^'-MONOPHOSPHATE and ADENOSINE-5^'-MONOPHOSPHATE: Irmpd Action Spectroscopy and Theoretical Studies

    NASA Astrophysics Data System (ADS)

    Wu, Ranran; Nei, Y.-W.; He, Chenchen; Hamlow, Lucas; Berden, Giel; Oomens, J.; Rodgers, M. T.

    2015-06-01

    Nature uses protonation to alter the structures and reactivities of molecules to facilitate various biological functions and chemical transformations. For example, in nucleobase repair and salvage processes, protonation facilitates nucleobase removal by lowering the activation barrier for glycosidic bond cleavage. Systematic studies of the structures of protonated 2'-deoxyribonucleotides and ribonucleotides may provide insight into the roles protonation plays in altering the nucleobase orientation relative to the glycosidic bond and sugar puckering. In this study, infrared multiple photon dissociation (IRMPD) action spectroscopy experiments in conjunction with electronic structure calculations are performed to probe the effects of protonation on the structures and stabilities of 2^'-deoxyadenosine-5^'-monophosphate (pdAdo) and adenosine-5^'-monophosphate (pAdo). Photodissociation as a function of IR wavelength is measured to generate the IRMPD action spectra. Geometry optimizations and frequency analyses performed at the B3LYP/6-311+G(d,p) level of theory are used to characterize the stable low-energy structures and to generate their linear IR spectra. Single point energy calculations performed at the B3LYP/6-311+G(2d,2p) and MP2(full)/6-311+G(2d,2p) levels of theory provide relative stabilities of the optimized conformations. The structures accessed in the experiments are determined by comparing the calculated linear IR spectra for the stable low-energy conformers computed to the measured IRMPD action spectra. The effects of the 2^'-hydroxyl moiety are elucidated by comparing the structures and IRMPD spectra of [pAdo+H]+ to those of its DNA analogue. Comparisons are also made to the deprotonated forms of these nucleotides and the protonated forms of the analogous nucleosides to elucidate the effects of protonation and the phosphate group on the structures.

  14. Lanthanum inhibition of Vibrio cholerae and Escherichia coli enterotoxin-induced enterosorption and its effects on intestinal mucosa cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels.

    PubMed Central

    Leitch, G J; Amer, M S

    1975-01-01

    Several trivalent cations, including lanthanum (La3+), inhibited the secretion (enterosorption) induced by the enterotoxins of Vibrio cholerae and Escherichia coli in the rabbit ileum in vivo. High concentrations (greater than 10 mM) of La3+ were required to inhibit cholera enterotoxin (CE)-induced enterosorption, probably because of the adsorption of the La3+ often potentiated the CE-induced enterosorption. If luminal La3+ exposure followed CE exposure, some recovery of the enterosorptive response was observed. The longer the lag between the CE exposure and the La3+ exposure, the greater was the recovery of the enterosorptive response. Lanthanum inhibited HCO3- secretion more than Cl- secretion. By altering the luminal fluid pH at the time of La3+ exposure, it was found that La3+ was adsorbed to negatively charged luminal sites, having an apparent pK between 2.5 and 3.0. Although La3+ antagonized the enterosorptive response to CE, it mimicked rather than antagonized the cyclic adenosine 3',5'-monophosphate elevation and cyclic guanosine 3',5'-monophosphate depression induced by the toxin. It is therefore concluded that the La3+ inhibition of the CE-induced enterosorption must have occurred at a site following the generation of the cyclic nucleotides. Cholera enterotoxin caused complex time-dependent changes in the mucosal cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels, as revealed by studying tissue cyclic adenosine 3',5'-monophosphate/cyclic guanosine 3',5'-monophosphate ratios. The possible roles these two cyclic nucleotides may play in the pathogenesis of the cholera diarrhea are discussed. PMID:164410

  15. In Silico Design for Adenosine Monophosphate-Activated Protein Kinase Agonist from Traditional Chinese Medicine for Treatment of Metabolic Syndromes

    PubMed Central

    Tang, Hsin-Chieh

    2014-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) acts as a master mediator of metabolic homeostasis. It is considered as a significant millstone to treat metabolic syndromes including obesity, diabetes, and fatty liver. It can sense cellular energy or nutrient status by switching on the catabolic pathways. Investigation of AMPK has new findings recently. AMPK can inhibit cell growth by the way of autophagy. Thus AMPK has become a hot target for small molecular drug design of tumor inhibition. Activation of AMPK must undergo certain extent change of the structure. Through the methods of structure-based virtual screening and molecular dynamics simulation, we attempted to find out appropriate small compounds from the world's largest TCM Database@Taiwan that had the ability to activate the function of AMPK. Finally, we found that two TCM compounds, eugenyl_beta-D-glucopyranoside and 6-O-cinnamoyl-D-glucopyranose, had the qualification to be AMPK agonist. PMID:24899913

  16. Investigation on the occurrence and significance of cyclic adenosine 3':5'-monophosphate in phytoplankton and natural aquatic communities

    SciTech Connect

    Francko, D.A.

    1980-01-01

    This study demonstrates, on the basis of several analyanalytical criteria, that the production and extracellular release of cyclic adenosine 3':5'-monophosphate (cAMP) is widespread among phytoplankton species. The production and release of CAMP varied markedly among different species grown under similar environmental conditions, and intraspecifically during the life cycle of a given algal species. This investigation marks the first time cAMP has been investigated in natural aquatic systems. An examination of epilimnetic lakewater samples from Lawrence Lake, a hardwater oligotrophic lake, and Wintergreen Lake, a hardwater hypereutrophic lake, both in southwestern Michigan, demonstrated that cAMP existed in both particulate-associated and dissolved forms in these systems.

  17. Adenosine 3',5'-monophosphate in relation to inhibition of cervical smooth muscle activity in early pregnant women.

    PubMed

    Norström, A; Bryman, I

    1991-08-01

    Contractile activity was registered in strips of cervical tissue obtained by needle biopsy from women in the first trimester of pregnancy. Dibutyryl cyclic adenosine-3',5'-monophosphate (5 x 10(-6) mol/l), isobutyryl methylxanthine (10(-4) mol/l), and forskolin (10(-5)-10(-4) mol/l), the latter two drugs known to increase the levels of endogenous cAMP, inhibited spontaneous muscle activity. The levels of tissue cAMP were determined in strips during relaxation induced by prostaglandin E2 or purified porcine relaxin and compared with cAMP levels in strips from the same women during contractile activity. Exposure to prostaglandin E2 but not to relaxin was followed by increased levels of cAMP. It is suggested that cAMP has a role as a second messenger in the prostaglandin E2-mediated relaxation of cervical smooth muscle. PMID:1654721

  18. Measuring the dynamics of cyclic adenosine monophosphate level in living cells induced by low-level laser irradiation using bioluminescence resonance energy transfer.

    PubMed

    Huang, Yimei; Zheng, Liqin; Yang, Hongqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen; Zeng, Haishan

    2015-05-01

    Several studies demonstrated that the cyclic adenosine monophosphate (cAMP), an important second messenger, is involved in the mechanism of low-level laser irradiation (LLLI) treatment. However, most of these studies obtained the cAMP level in cell culture extracts or supernatant. In this study, the cAMP level in living cells was measured with bioluminescence resonance energy transfer (BRET). The effect of LLLI on cAMP level in living cells with adenosine receptors blocked was explored to identify the role of adenosine receptors in LLLI. The results showed that LLLI increased the cAMP level. Moreover, the rise of cAMP level was light dose dependent but wavelength independent for 658-, 785-, and 830-nm laser light. The results also exhibited that the adenosine receptors, a class of G protein-coupled receptor (GPCR), modulated the increase of cAMP level induced by LLLI. The cAMP level increased more significantly when the A3 adenosine receptors (A3R) were blocked by A3R antagonist compared with A1 adenosine receptor or A2a adenosine receptor blocked in HEK293T cells after LLLI, which was in good agreement with the adenosine receptors’ expressions. All these results suggested that measuring the cAMP level with BRET could be a useful technique to study the role of GPCRs in living cells under LLLI. PMID:25611980

  19. Measuring the dynamics of cyclic adenosine monophosphate level in living cells induced by low-level laser irradiation using bioluminescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Zheng, Liqin; Yang, Hongqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen; Zeng, Haishan

    2015-05-01

    Several studies demonstrated that the cyclic adenosine monophosphate (cAMP), an important second messenger, is involved in the mechanism of low-level laser irradiation (LLLI) treatment. However, most of these studies obtained the cAMP level in cell culture extracts or supernatant. In this study, the cAMP level in living cells was measured with bioluminescence resonance energy transfer (BRET). The effect of LLLI on cAMP level in living cells with adenosine receptors blocked was explored to identify the role of adenosine receptors in LLLI. The results showed that LLLI increased the cAMP level. Moreover, the rise of cAMP level was light dose dependent but wavelength independent for 658-, 785-, and 830-nm laser light. The results also exhibited that the adenosine receptors, a class of G protein-coupled receptor (GPCR), modulated the increase of cAMP level induced by LLLI. The cAMP level increased more significantly when the A3 adenosine receptors (A3R) were blocked by A3R antagonist compared with A1 adenosine receptor or A2a adenosine receptor blocked in HEK293T cells after LLLI, which was in good agreement with the adenosine receptors' expressions. All these results suggested that measuring the cAMP level with BRET could be a useful technique to study the role of GPCRs in living cells under LLLI.

  20. Calf-ovary protein kinases dependent on adenosine 3':5' -monophosphate. Analysis by electrophoresis and electro-focusing on polyacrylamide get.

    PubMed

    Salokangas, A; Talmadge, K; Bechtel, E; Eppenberger, U; Chrambach, A

    1977-03-01

    High resolving power and quantitative application polyacrylamide-gel electrophopresis at various pore sizes and electrofocusing provide resolution of a calf-ovarian protein-kinase system at an increased level of magnification, as well as optimal preparative routes. Three protein kinases dependent on adenosine 3':5' -monophosphate are distinguished by polyacrylamide gel electrophoresis in calf ovarian cytosol. These enzymes which are observed in the pH range 7.5--10.2, appear to be aggregates of a commonsubmit or monomer. The three kinases are, by the criteria of polyacylamide gel electrophoresis, distinct from three adenosine-3':5' -monophosphate-binding proteins found in the calf ovarian system. Analysis by electrofocusing on polyacrylamide gel shows that conventionally purified preparations of the major kinase of cytosol contain an overwhelming majority of contaminant proteins. PMID:191253

  1. Online cleanup of accelerated solvent extractions for determination of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly using high-performance liquid chromatography.

    PubMed

    Xue, Xiaofeng; Wang, Feng; Zhou, Jinhui; Chen, Fang; Li, Yi; Zhao, Jing

    2009-06-10

    Determination of the levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly is important for the study of its pharmacological activities, health benefits, and adenosine phosphate degradation. In this study was developed a novel method to determine ATP, ADP, and AMP levels in royal jelly using accelerated solvent extraction (ASE) followed by online cleanup and high-performance liquid chromatography (HPLC) with diode array detection (DAD). The optimum extraction conditions were obtained using an 11 mL ASE cell, ethanol/water (5:5 v/v) as the extraction solvent, 1500 psi, 80 degrees C, a 5 min static time, and a 60% flush volume. Optimum separation of the three compounds was achieved in <25 min using a Waters XBridge Shield RP18 column with 0.05 mol L(-1) NH(4)H(2)PO(4) (pH 5.70) and acetonitrile as the mobile phase. Detection was performed at 257 nm. The method was sensitive (LOD adenosine phosphate extraction procedures (perchloric acid). The results indicate that the two techniques are similar in terms of recovery and reproducibility, but when other factors such as extraction time, environmental protection, and worker's health are considered, ASE is preferable to the classical extraction method. With this ASE-HPLC method, a minisurvey of ATP, ADP, and AMP levels in 15 samples of royal jelly of different origins was performed. Sample results indicated that the AMP concentration was 24.2-2214.4 mg kg(-1), whereas ATP and ADP were not detectable or present only at low levels. PMID:19435312

  2. Selective Phosphonylation of 5'-Adenosine Monophosphate (5'-AMP) via Pyrophosphite [PPi(III)

    NASA Astrophysics Data System (ADS)

    Kaye, Karl; Bryant, David E.; Marriott, Katie E. R.; Ohara, Shohei; Fishwick, Colin W. G.; Kee, Terence P.

    2016-05-01

    We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H2P2O5 2-; PPi(III)], to the phosphate group of 5'-adenosine mono phosphate (5'-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M2+) and by organic co-factors such as acetate (AcO-). In this specific case of P-transfer from PPi(III) to 5'-AMP, we show a synergistic enhancement of transfer in the combined presence of M2+ & AcO-. Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5'-AMP, [P(III)P(V)-5'-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.

  3. Metabolite gene regulation: imidazole and imidazole derivatives which circumvent cyclic adenosine 3',5'-monophosphate in induction of the Escherichia coli L-arabinose operon.

    PubMed Central

    Kline, E L; Bankaitis, V A; Brown, C S; Montefiori, D C

    1980-01-01

    Imidazole, histidine, histamine, histidinol phosphate, urocanic acid, or imidazolepropionic acid were shown to induce the L-arabinose operon in the absence of cyclic adenosine 3',5'-monophosphate. Induction was quantitated by measuring the increased differential rate of synthesis of L-arabinose isomerase in Escherichia coli strains which carried a deletion of the adenyl cyclase gene. The crp gene product (cyclic adenosine 3',5'-monophosphate receptor protein) and the araC gene product (P2) were essential for induction of the L-arabinose operon by imidazole and its derivatives. These compounds were unable to circumvent the cyclic adenosine 3',5'-monophosphate in the induction of the lactose or the maltose operons. The L-arabinose regulon was catabolite repressed upon the addition of glucose to a strain carrying an adenyl cyclase deletion growing in the presence of L-arabinose with imidazole. These results demonstrated that several imidazole derivatives may be involved in metabolite gene regulation (23). Images PMID:6245056

  4. Regulation of ciliary reversal in triton-extracted Paramecium by calcium and cyclic adenosine monophosphate.

    PubMed

    Nakaoka, Y; Ooi, H

    1985-08-01

    A Triton-extracted model of Paramecium swims forwards when the Ca2+ concentration in the reactivation medium containing ATP is below 10(-6) M and swims backwards when Ca2+ concentration is above 10(-6) M. We found that cAMP (adenosine 3':5'-cyclic monophosphoric acid) inhibited Ca-induced backward swimming of the model and caused forward swimming even when the [Ca2+] was above 10(-6) M. This effect of cAMP was abolished by an inhibitor of cAMP-dependent protein kinase. In order to study the possible role of phosphorylation in the regulation of ciliary orientation, ATP in the reactivation medium was replaced by an ATP analogue, ARP gamma S (adenosine 5'-O-3-thiotriphosphate), which irreversibly thiophosphorylates proteins. In ATP gamma S medium, the model ceased both swimming and ciliary beating, but the orientation of cilia was dependent on [Ca2+]. At low [Ca2+], cilia were perpendicular to the cell surface and, with increase in [Ca2+], their orientation gradually changed towards the cell anterior. Such a change in ciliary orientation corresponds roughly to the change in the swimming direction observed in ATP medium. The ciliary orientation towards the anterior of the cell in ATP gamma S medium at high [Ca2+] was maintained when [Ca2+] was decreased. In contrast, in ATP medium, the swimming direction was reversibly changed with changes in [Ca2+]. These results suggest that the ciliary orientation is regulated not only by Ca2+ but also by cAMP, probably via protein phosphorylation. PMID:3003129

  5. Cilostazol Improves Developmental Competence of Pig Oocytes by Increasing Intraoocyte Cyclic Adenosine Monophosphate Level and Delaying Meiotic Resumption.

    PubMed

    Elahi, F; Lee, H; Lee, Y; Park, B; Lee, J; Hyun, S-H; Lee, E

    2016-04-01

    Cilostazol (CLZ) is a cyclic adenosine monophosphate (cAMP) modulator that influences the steady state of the meiotic stage. This study was conducted to determine the effects of CLZ treatment during in vitro maturation (IVM) on developmental competence of pig oocytes. Immature oocytes were exposed to 0 (control), 0.5, 2 and 4 μm CLZ during the first 22 h of IVM. Nuclear maturation, intraoocyte glutathione content and embryo cleavage after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were not influenced by CLZ at any concentrations. However, 4 μm CLZ significantly (p < 0.05) improved blastocyst formation after PA (52.1% vs 38.7-46.0%) and SCNT relative to other concentrations (40.8% vs 25.0-30.7%). The mean cell numbers of SCNT blastocysts were significantly increased by 4 μm CLZ compared to the control (42.6 cells vs 35.3 cells/blastocyst). CLZ treatment significantly increased the intraoocyte cAMP level and effectively arrested oocytes at the germinal vesicle (GV) and GV break down stages compared to the control (74.5% vs 45.4%). Our results demonstrated that improved developmental competence of PA and SCNT pig embryos occurred via better synchronization of nuclear and cytoplasmic maturation induced by increased cAMP and delayed meiotic resumption after CLZ treatment. PMID:26834044

  6. Coordination properties of adenosine-5'-monophosphate and related ligands towards Me2Sn(IV)2+ in aqueous solution.

    PubMed

    Jankovics, H; Nagy, L; Buzás, N; Pellerito, L; Barbieri, R

    2002-09-30

    The coordination of Me2Sn(IV)2+ to adenosine-5'-monophosphate (AMP) and the related compounds D-ribose-5-phosphate (R5P), D-glucose-1-phosphate (G1P) and D-glucose-6-phosphate (G6P) in aqueous solution was investigated by means of potentiometric titration, and 1H-, 31P-NMR and Mössbauer spectroscopic methods in the pH range 2-11 (I=0.1 M NaClO4, 298 K). The complex of AMP and Me2Sn(IV)2+ precipitated at low pH was characterised by elemental analysis, FT-IR and Mössbauer spectroscopic methods. From a comparison of the pK values obtained in the presence and absence of metal ion and the stability constants for the different systems, the coordination of [N] is excluded, while bidentate coordination of the phosphate group is presumed. Mössbauer spectroscopic measurements recorded in the glassy state confirmed bidentate coordination of the phosphate and the formation of mixed hydroxo complexes in the weakly acidic, neutral and strongly basic pH range. With increasing pH, the phosphate groups were replaced by the deprotonated alcoholic [O] atoms of the sugar moiety. The solid complex proved to be tbp structure with bidentate phosphate coordination. PMID:12230988

  7. Directed breeding of an Arthrobacter mutant for high-yield production of cyclic adenosine monophosphate by N + ion implantation

    NASA Astrophysics Data System (ADS)

    Song, He; Chen, Xiaochun; Cao, Jiaming; Fang, Ting; Bai, Jianxin; Xiong, Jian; Ying, Hanjie

    2010-08-01

    To obtain a cyclic adenosine monophosphate (cAMP) high-yield production strain, Arthrobacter NG-1 was mutated by N + ion implantation with an energy level of 10 keV and dose of 7×10 15 ions/cm 2. Combined with directed screening methods, a xanthine-defective and 8-azaguanine (8-AG)-resistant mutant Arthrobacter A302 was selected. The concentration of cAMP produced by this mutant was 41.7% higher than that of the original strain and reached 9.78 g/L. Through ten-generation investigation, the capability of cAMP production of A302 was found to be stable. Compared with the original strain, the special activities of key enzymes in A302, which influenced the cAMP biosynthesis, was analyzed. IMP dehydrogenase activity was defective, whereas PRPP amidotransferase, sAMP synthetase and adenylate cyclase activities were increased by 61.5%, 147% and 21.7%, respecitively, which might explain the mutagenesis mechanism by N + ions implantation under the enzymatic level.

  8. An evaluation of short-term corticosteroid response in perennial allergic rhinitis using histamine and adenosine monophosphate nasal challenge

    PubMed Central

    Wilson, Andrew M; Sims, Erika J; Orr, Linda C; Robb, Fiona; Lipworth, Brian J

    2003-01-01

    Aims To evaluate the role of AMP nasal challenge as a measure of short-term treatment response in patients receiving intranasal corticosteroids. Adenosine monophosphate (AMP) challenge has been shown to be a good inflammatory surrogate in the lower airways, but it has not been properly evaluated as a nasal challenge test. Methods Fourteen patients with perennial allergic rhinitis (PAR) were randomized to receive 2 weeks treatment with placebo (PL) or 200 µg intranasal mometasone furoate (MF) once daily in a randomized single-blind crossover study. AMP (25–800 mg ml−1) and histamine (0.25–8 mg ml−1) nasal challenge testing were performed after each treatment period with 30% decrease in minimal cross-sectional area (MCA). Domiciliary symptom data were collected. Results There was a significant (P < 0.05) improvement in PC30 MCA and nasal volume with AMP but not with histamine comparing MF vs PL. This amounted to a 2.8 (95% CI 1.5, 4.0) and 0.7 (95% CI −0.5, 1.9) doubling-dose change for AMP and histamine challenges, respectively. There were significant (P < 0.05) improvements in nasal symptoms and quality of life. Conclusions AMP nasal challenge using acoustic rhinometry may be a useful test to assess short-term treatment response in patient with PAR. PMID:12680883

  9. Activation of Cyclic Adenosine Monophosphate Pathway Increases the Sensitivity of Cancer Cells to the Oncolytic Virus M1.

    PubMed

    Li, Kai; Zhang, Haipeng; Qiu, Jianguang; Lin, Yuan; Liang, Jiankai; Xiao, Xiao; Fu, Liwu; Wang, Fang; Cai, Jing; Tan, Yaqian; Zhu, Wenbo; Yin, Wei; Lu, Bingzheng; Xing, Fan; Tang, Lipeng; Yan, Min; Mai, Jialuo; Li, Yuan; Chen, Wenli; Qiu, Pengxin; Su, Xingwen; Gao, Guangping; Tai, Phillip W L; Hu, Jun; Yan, Guangmei

    2016-02-01

    Oncolytic virotherapy is a novel and emerging treatment modality that uses replication-competent viruses to destroy cancer cells. Although diverse cancer cell types are sensitive to oncolytic viruses, one of the major challenges of oncolytic virotherapy is that the sensitivity to oncolysis ranges among different cancer cell types. Furthermore, the underlying mechanism of action is not fully understood. Here, we report that activation of cyclic adenosine monophosphate (cAMP) signaling significantly sensitizes refractory cancer cells to alphavirus M1 in vitro, in vivo, and ex vivo. We find that activation of the cAMP signaling pathway inhibits M1-induced expression of antiviral factors in refractory cancer cells, leading to prolonged and severe endoplasmic reticulum (ER) stress, and cell apoptosis. We also demonstrate that M1-mediated oncolysis, which is enhanced by cAMP signaling, involves the factor, exchange protein directly activated by cAMP 1 (Epac1), but not the classical cAMP-dependent protein kinase A (PKA). Taken together, cAMP/Epac1 signaling pathway activation inhibits antiviral factors and improves responsiveness of refractory cancer cells to M1-mediated virotherapy. PMID:26373347

  10. Triiodothyronine causes rapid reversal of alpha 1/cyclic adenosine monophosphate synergism on brown adipocyte respiration and type II deiodinase activity.

    PubMed

    Noronha, M; Raasmaja, A; Moolten, N; Larsen, P R

    1991-12-01

    Previous studies have shown that thyroid status affects the response of brown adipose tissue (BAT) to the sympathetic nervous system. For example, hypothyroidism is associated with the development of a marked synergism between alpha 1- and beta-adrenergic pathways to stimulate type II iodothyronine 5'-deiodinase activity. Hypothyroidism also attenuates the respiratory response (thermogenesis) of isolated brown adipocytes to norepinephrine. To explore the interactions of the sympathetic nervous system and thyroid status in these cells, we compared the thermogenic and 5'-deiodinase responses to adrenergic agonists in isolated brown adipocytes from hypothyroid rats during treatment with 3,5,3'-triiodothyronine (T3). The fivefold synergism of alpha 1- and beta-adrenergic catecholamines to increase the deiodinase activity was progressively reduced, reaching a control euthyroid value of unity after 5 days of T3 treatment. Hypothyroidism reduced both the O2max (twofold to threefold) and increased the concentration of agonist required for 50% stimulation (10-fold) for both norepinephrine and forskolin. In hypothyroid cells, there was a twofold synergism between the alpha 1-agonist cirazoline and forskolin to increase respiration, which was blocked by prazosin and reproduced by the calcium ionophore, A23187. This synergistic effect of the alpha 1-agonist was lost within 2 days of T3 administration. These studies identify a second Ca(2+)-dependent intra-adrenergic synergism, which functions to ameliorate the reduced cyclic adenosine monophosphate (cAMP) responsiveness of the hypothyroid brown adipocyte. PMID:1683679

  11. Investigation on the occurrence and significance of cyclic adenosine 3':5'-monophosphate in phytoplankton and natural aquatic communities

    SciTech Connect

    Francko, D.A.

    1980-01-01

    This study is an investigation into the occurrence and potential functions of cyclic adenosine 3':5'-monophosphate (cAMP), a potent and ubiquitous metabolic regulatory molecule in heterotrophic organisms, in phytoplankton and in natural aquatic communities. Laboratory-cultured phytoplankton were grown under both optimal and suboptimal nutrient regimes under constant temperature and illumination regimes. Cellular and extracellular cAMP production, characterized by a number of biochemical techniques, was correlated with growth rate dynamics, chlorophyll a synthesis, /sup 14/C-bicarbonate uptake, alkaline phosphatase activity, and heterocyst formation. The blue-green alga Anabaena flos-aquae was used as a model system in the examination of these metabolic variables. Additionally, this alga was used to test the effects of perturbation of cAMP levels on the aforementioned metabolic variables. Investigations on the occurrence and seasonal dynamics of cAMP in aquatic systems were conducted on Lawrence Lake, a hardwater oligotrophic lake, and on Wintergreen Lake, a hardwater hypereutrophic lake, both in southwestern Michigan. Putative cAMP from both systems was characterized by several biochemical techniques. Weekly sampling of particulate and dissolved cAMP in the epilimnia of both lakes was correlated with data on the rates of primary productivity, alkaline phosphatase activity, chlorophyll a synthesis and changes in phytoplankton community structure.

  12. Cyclic adenosine monophosphate phosphodiesterase activity in peripheral blood mononuclear leucocytes from patients with atopic dermatitis: correlation with respiratory atopy.

    PubMed

    Sawai, T; Ikai, K; Uehara, M

    1998-05-01

    We determined the cyclic adenosine monophosphate phosphodiesterase (cAMP-PDE) activity in peripheral blood mononuclear leucocytes from 100 patients with atopic dermatitis (AD) aged 13-57 years (mean +/- SD, 29.8 +/- 17.7 years). The correlation between cAMP-PDE activity and clinical parameters such as the severity of eczema and a personal or family predisposition to atopic respiratory diseases (ARD) (asthma or allergic rhinitis) was examined. Although the enzymic activity varied from normal to very high in the AD patients, cAMP-PDE activity was significantly (P < 0.005) elevated in AD patients (42.1 +/- 22.0 units) as compared with the normal controls (12.4 +/- 5.6) and clinical control subjects (13.4 +/- 9.5). In contrast, we found no correlation between cAMP-PDE activity and the severity of eczema when AD patients were classified into four categories (remission, mild, moderate and severe) according to the extent of their skin involvement. Furthermore, we found that systemic corticosteroid therapy in severe AD patients did not alter the cAMP-PDE activity. cAMP-PDE activity was significantly (P < 0.01) higher in those AD patients who had a personal history of ARD (47.2 +/- 11.2) than in AD patients with a family history of ARD (37.2 +/- 17.4) and those without a personal or family history ('pure' AD) (34.4 +/- 19.8). Nevertheless, the cAMP-PDE activity was significantly higher even in 'pure' AD patients than in the controls. These results suggest that an elevation of cAMP-PDE activity is closely related to a predisposition to respiratory atopy, and does not follow inflammation in AD patients. PMID:9666832

  13. Cyclic adenosine monophosphate response element-binding protein transcriptionally regulates CHCHD2 associated with the molecular pathogenesis of hepatocellular carcinoma.

    PubMed

    Song, Rui; Yang, Biao; Gao, Xuesong; Zhang, Jinqian; Sun, Lei; Wang, Peng; Meng, Yixing; Wang, Qi; Liu, Shunai; Cheng, Jun

    2015-06-01

    The function of the novel cell migration‑promoting factor, coiled‑coil‑helix‑coiled‑coil‑helix domain containing 2 (CHCHD2) in liver cancer remains to be elucidated. The aim of the present study was to elucidate the role of CHCHD2 in liver carcinogenesis. Immunohistochemistry was performed on patients with hepatocellular carcinoma (HCC) and suppression subtractive hybridization (SSH) was used for screening differentially expressed genes in the HepG2 cell cDNA library. Chronic hepatitis C virus (HCV) infection frequently leads to liver cancer. The HCV NS2 protein is a hydrophobic transmembrane protein that is associated with certain cellular proteins. Detailed characterization of the nonstructural protein 2 (NS2) of the HCV was performed with respect to its role in transregulatory activity in the HepG2 cell lines. A gel electrophoresis mobility shift assay and a chromatin immunoprecipitation assay were used to confirm the presence of cyclic adenosine monophosphate response element‑binding protein (CREB), a transcriptional factor, which specifically interacts with the CHCHD2 promoter. CHCHD2 was highly expressed in the HCC specimens and was consistent with tumor markers of HCC. CHCHD2 was identified by SSH in the HepG2 cells. NS2 upregulated the expression of CHCHD2 by monitoring its promoter activities. The promoter of CHCHD2 contained 350 bp between nucleotides ‑257 and +93 and was positively regulated by CREB. In conclusion, the results of the present study indicated that CHCHD2 may be a novel biomarker for HCC and that CREB is important in the transcriptional activation of CHCHD2 by HCV NS2. PMID:25625293

  14. Adenosine monophosphate-activated protein kinase (AMPK) activators for the prevention, treatment and potential reversal of pathological pain

    PubMed Central

    Price, Theodore J.; Das, Vaskar; Dussor, Gregory

    2015-01-01

    Pathological pain is an enormous medical problem that places a significant burden on patients and can result from an injury that has long since healed or be due to an unidentifiable cause. Although treatments exist, they often either lack efficacy or have intolerable side effects. More importantly, they do not reverse the changes in the nervous system mediating pathological pain, and thus symptoms often return when therapies are discontinued. Consequently, novel therapies are urgently needed that have both improved efficacy and disease-modifying properties. Here we highlight an emerging target for novel pain therapies, adenosine monophosphate-activated protein kinase (AMPK). AMPK is capable of regulating a variety of cellular processes including protein translation, activity of other kinases, and mitochondrial metabolism, many of which are thought to contribute to pathological pain. Consistent with these properties, preclinical studies show positive, and in some cases disease-modifying effects of either pharmacological activation or genetic regulation of AMPK in models of nerve injury, chemotherapy-induced peripheral neuropathy (CIPN), postsurgical pain, inflammatory pain, and diabetic neuropathy. Given the AMPK-activating ability of metformin, a widely prescribed and well-tolerated drug, these preclinical studies provide a strong rationale for both retrospective and prospective human pain trials with this drug. They also argue for the development of novel AMPK activators, whether orthosteric, allosteric, or modulators of events upstream of the kinase. Together, this review will present the case for AMPK as a novel therapeutic target for pain and will discuss future challenges in the path toward development of AMPK-based pain therapeutics. PMID:26521775

  15. Curcumin inhibits aerobic glycolysis in hepatic stellate cells associated with activation of adenosine monophosphate-activated protein kinase.

    PubMed

    Lian, Naqi; Jin, Huanhuan; Zhang, Feng; Wu, Li; Shao, Jiangjuan; Lu, Yin; Zheng, Shizhong

    2016-07-01

    Activation of hepatic stellate cells (HSCs) is characterized by expression of extracellular matrix and loss of adipogenic phenotype during liver fibrogenesis. Emerging evidence suggests that HSCs adopt aerobic glycolysis during activation. The present work aimed at investigating whether the anti-fibrogenic effects of curcumin was associated with interfering with glycolysis in HSCs. Primary rat HSCs were cultured in vitro. We demonstrated that inhibition of glycolysis by 2-deoxyglucose or galloflavin reduced the expression of α-smooth muscle actin (α-SMA) and α1(I)procollagen at both mRNA and protein levels, and increased the intracellular lipid contents and upregulated the gene and protein expression of adipogenic transcription factors C/EBPα and PPAR-γ in HSCs. Curcumin at 20 μM produced similar effects. Moreover, curcumin decreased the expression of hexokinase (HK), phosphofructokinase-2 (PFK2), and glucose transporter 4 (glut4), three key glycolytic parameters, at both mRNA and protein levels. Curcumin also reduced lactate production concentration-dependently in HSCs. Furthermore, curcumin increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), but AMPK inhibitor BML-275 significantly abolished the curcumin downregulation of HK, PFK2, and glut4. In addition, curcumin inhibition of α-SMA and α1(I)procollagen was rescued by BML-275, and curcumin upregulation of C/EBPα and PPAR-γ was abrogated by BML-275. These results collectively indicated that curcumin inhibited glycolysis in an AMPK activation-dependent manner in HSCs. We revealed a novel mechanism for curcumin suppression of HSC activation implicated in antifibrotic therapy. © 2016 IUBMB Life, 68(7):589-596, 2016. PMID:27278959

  16. Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

    SciTech Connect

    Browne, E.S.; Bhalla, V.K. )

    1991-02-01

    Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10{sup {minus} 10} M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable {sup 125}I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the {sup 125}I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

  17. Isolation of novel ribozymes that ligate AMP-activated RNA substrates

    NASA Technical Reports Server (NTRS)

    Hager, A. J.; Szostak, J. W.

    1997-01-01

    BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

  18. Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress.

    PubMed

    Toyama, Erin Quan; Herzig, Sébastien; Courchet, Julien; Lewis, Tommy L; Losón, Oliver C; Hellberg, Kristina; Young, Nathan P; Chen, Hsiuchen; Polleux, Franck; Chan, David C; Shaw, Reuben J

    2016-01-15

    Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA-linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission. PMID:26816379

  19. Generation of cAMP-Activated Chloride Currents by Expression of CFTR

    NASA Astrophysics Data System (ADS)

    Anderson, Matthew P.; Rich, Devra P.; Gregory, Richard J.; Smith, Alan E.; Welsh, Michael J.

    1991-02-01

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. In order to evaluate its function, CFTR was expressed in HeLa, Chinese hamster ovary (CHO), and NIH 3T3 fibroblast cells, and anion permeability was assessed with a fluorescence microscopic assay and the whole-cell patch-clamp technique. Adenosine 3',5'-monophosphate (cAMP) increased anion permeability and chloride currents in cells expressing CFTR, but not in cells expressing a mutant CFTR (ΔF508) or in nontransfected cells. The simplest interpretation of these observations is that CFTR is itself a cAMP-activated chloride channel. The alternative interpretation, that CFTR directly or indirectly regulates chloride channels, requires that these cells have endogenous cryptic, chloride channels that are stimulated by cAMP only in the presence of CFTR.

  20. Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

    PubMed Central

    Newby, A C

    1980-01-01

    1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells. PMID:6249264

  1. Mode of action of parathyroid hormone and cyclic adenosine 3′,5′-monophosphate on renal tubular phosphate reabsorption in the dog

    PubMed Central

    Agus, Zalman S.; Puschett, Jules B.; Senesky, Dorothy; Goldberg, Martin

    1971-01-01

    To evaluate the effects of parathyroid hormone and cyclic adenosine monophosphate on proximal tubular sodium and phosphate reabsorption, micropuncture studies were performed on dogs that received a highly purified preparation of parathyroid hormone (PTH), dibutyryl cyclic 3′,5′-adenosine monophosphate (cyclic AMP), 5′-AMP, and saline. PTH resulted in a 30-40% inhibition of sodium and phosphate reabsorption in the proximal tubule unassociated with a rise in either total kidney or single nephron glomerular filtration rate (GFR). The bulk of the phosphate rejected proximally was excreted in the final urine while sodium excretion rose minimally despite the marked proximal inhibition, consistent with the presence of reabsorptive sites in the distal nephron for sodium but not phosphate. The infusion of dibutyryl cyclic AMP either systemically or directly into the renal artery inhibited proximal sodium and phosphate reabsorption in the absence of changes in either total kidney or single nephron GFR, resembling the effects of PTH quantitatively and qualitatively. In contrast, another adenine nucleotide, 5′-AMP, did not inhibit the reabsorption of either sodium or phosphate. These observations support the thesis that renal effects of PTH are mediated via stimulation of renal cortical adenyl cyclase. The infusion of a moderate saline load, 25 ml/kg, also produced a similar inhibition of proximal tubular fractional sodium and phosphate reabsorption with a marked phosphaturia but only minimal natriuresis. Thus, changes in sodium and phosphate reabsorption occur in parallel in the proximal tubule when sodium reabsorption is inhibited either with volume expansion or with administration of “specific” phosphaturic agents such as PTH or cyclic AMP. These data are consistent with the thesis that phosphate reabsorption is dependent upon proximal tubular sodium reabsorption wherein the phosphaturic effect of PTH might be the result of a primary inhibition of proximal

  2. A short review on structure and role of cyclic-3’,5’-adenosine monophosphate-specific phosphodiesterase 4 as a treatment tool

    PubMed Central

    Eskandari, Nahid; Mirmosayyeb, Omid; Bordbari, Gazaleh; Bastan, Reza; Yousefi, Zahra; Andalib, Alireza

    2015-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) are known as a super-family of enzymes which catalyze the metabolism of the intracellular cyclic nucleotides, cyclic-3’,5’-adenosine monophosphate (cAMP), and cyclic-3’,5’-guanosine monophosphate that are expressed in a variety of cell types that can exert various functions based on their cells distribution. The PDE4 family has been the focus of vast research efforts over recent years because this family is considered as a prime target for therapeutic intervention in a number of inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and rheumatoid arthritis, and it should be used and researched by pharmacists. This is because the major isoform of PDE that regulates inflammatory cell activity is the cAMP-specific PDE, PDE4. This review discusses the relationship between PDE4 and its inhibitor drugs based on structures, cells distribution, and pharmacological properties of PDE4 which can be informative for all pharmacy specialists. PMID:26645022

  3. Evaluation of the metal-ion-coordinating differences between the 2'-, 3'- and 5'-monophosphates of adenosine.

    PubMed

    Massoud, S S; Sigel, H

    1989-02-01

    The stability constants of the 1:1 complexes formed between Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+ and 2'AMP2-, 3'AMP2- or 5'AMP2- were determined by potentiometric pH titration in aqueous solution (I = 0.1 M, NaNO3; 25 degrees C). The experimental conditions were carefully selected such that self-association of the nucleotides and their complexes is negligibly small; i.e. it was made certain that the properties of the monomeric divalent-metal-ion--AMP [M(AMP)] complexes were studied. Based on recent measurements with simple phosphate monoesters, R-MP2- where R is a non-coordinating residue [Massoud, S. S. & Sigel, H. (1988) Inorg. Chem. 27, 1447-1453], it is shown that all the M(AMP) complexes of the alkaline earth ions, with the possible exception of Mg(5'AMP), have exactly the stability expected for a sole-phosphate coordination of the metal ion. The same property is revealed for the complexes with Mn2+, Co2+, Zn2+ or Cd2+ and 3'AMP2-; in case of Ni(3'AMP) and Cu(3'AMP) a slight stability increase just at the edge of the experimental-error limits is indicated. This slight stability increase is attributed to the formation of a macrochelate (possibly with N-3); in fact, additional information confirms macrochelation for Cu(3'AMP). About 45% of Cu(2'AMP) exists in aqueous solution as a macrochelate (probably involving N-3); the other M(2'AMP) complexes (M2+ = Mn2+, Co2+, Ni2+, Zn2+, Cd2+) form (if at all) only traces of a base-backbound species. Most pronounced is macrochelate formation with 5'AMP2-: all mentioned 3d ions and Zn2+ or Cd2+ form to some extent macrochelates via N-7 (the structures of these closed species are indicated). In case of M(5'AMP) the base-binding site is certain: replacement of N-7 by a CH unit (tubercidin 5'-monophosphate) eliminates any increased complex stability, whereas formation of the 1,N6-etheno bridge to form 1,N6-ethenoadenosine 5'-monophosphate results in the phenanthroline-like N-6,N-7 site which

  4. Adenosine monophosphate-activated protein kinase activation, substrate transporter translocation, and metabolism in the contracting hyperthyroid rat heart.

    PubMed

    Heather, Lisa C; Cole, Mark A; Atherton, Helen J; Coumans, Will A; Evans, Rhys D; Tyler, Damian J; Glatz, Jan F C; Luiken, Joost J F P; Clarke, Kieran

    2010-01-01

    Thyroid hormones can modify cardiac metabolism via multiple molecular mechanisms, yet their integrated effect on overall substrate metabolism is poorly understood. Here we determined the effect of hyperthyroidism on substrate metabolism in the isolated, perfused, contracting rat heart. Male Wistar rats were injected for 7 d with T(3) (0.2 mg/kg x d ip). Plasma free fatty acids increased by 97%, heart weights increased by 33%, and cardiac rate pressure product, an indicator of contractile function, increased by 33% in hyperthyroid rats. Insulin-stimulated glycolytic rates and lactate efflux rates were increased by 33% in hyperthyroid rat hearts, mediated by an increased insulin-stimulated translocation of the glucose transporter GLUT4 to the sarcolemma. This was accompanied by a 70% increase in phosphorylated AMP-activated protein kinase (AMPK) and a 100% increase in phosphorylated acetyl CoA carboxylase, confirming downstream signaling from AMPK. Fatty acid oxidation rates increased in direct proportion to the increased heart weight and rate pressure product in the hyperthyroid heart, mediated by synchronized changes in mitochondrial enzymes and respiration. Protein levels of the fatty acid transporter, fatty acid translocase (FAT/CD36), were reduced by 24% but were accompanied by a 19% increase in the sarcolemmal content of fatty acid transport protein 1 (FATP1). Thus, the relationship between fatty acid metabolism, cardiac mass, and contractile function was maintained in the hyperthyroid heart, associated with a sarcolemmal reorganization of fatty acid transporters. The combined effects of T(3)-induced AMPK activation and insulin stimulation were associated with increased sarcolemmal GLUT4 localization and glycolytic flux in the hyperthyroid heart. PMID:19940039

  5. Role of hypothalamic adenosine 5'-monophosphate-activated protein kinase in the impaired counterregulatory response induced by repetitive neuroglucopenia.

    PubMed

    Alquier, Thierry; Kawashima, Junji; Tsuji, Youki; Kahn, Barbara B

    2007-03-01

    Antecedent hypoglycemia blunts counterregulatory responses that normally restore glycemia, a phenomenon known as hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to impaired counterregulatory responses are largely unknown. Hypothalamic AMP-activated protein kinase (AMPK) acts as a glucose sensor. To determine whether failure to activate AMPK could be involved in the etiology of HAAF, we developed a model of HAAF using repetitive intracerebroventricular (icv) injection of 2-deoxy-D-glucose (2DG) resulting in transient neuroglucopenia in normal rats. Ten minutes after a single icv injection of 2DG, both alpha1- and alpha2-AMPK activities were increased 30-50% in arcuate and ventromedial/dorsomedial hypothalamus but not in other hypothalamic regions, hindbrain, or cortex. Increased AMPK activity persisted in arcuate hypothalamus at 60 min after 2DG injection when serum glucagon and corticosterone levels were increased 2.5- to 3.4-fold. When 2DG was injected icv daily for 4 d, hypothalamic alpha1- and alpha2-AMPK responses were markedly blunted in arcuate hypothalamus, and alpha1-AMPK was also blunted in mediobasal hypothalamus 10 min after 2DG on d 4. Both AMPK isoforms were activated normally in arcuate hypothalamus at 60 min. Counterregulatory hormone responses were impaired by recurrent neuroglucopenia and were partially restored by icv injection of 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside, an AMPK activator, before 2DG. Glycogen content increased 2-fold in hypothalamus after recurrent neuroglucopenia, suggesting that glycogen supercompensation could be involved in down-regulating the AMPK glucose-sensing pathway in HAAF. Thus, activation of hypothalamic AMPK may be important for the full counterregulatory hormone response to neuroglucopenia. Furthermore, impaired or delayed AMPK activation in specific hypothalamic regions may play a critical role in the etiology of HAAF. PMID:17185376

  6. Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation

    PubMed Central

    Ezoe, Kenji; Yabuuchi, Akiko; Tani, Tetsuya; Mori, Chiemi; Miki, Tetsuya; Takayama, Yuko; Beyhan, Zeki; Kato, Yoko; Okuno, Takashi; Kobayashi, Tamotsu; Kato, Keiichi

    2015-01-01

    Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming. PMID:25965267

  7. Effect of prostaglandins and cyclic adenosine 3',5'-monophosphate modulators on herpes simplex virus growth and interferon response in human cells.

    PubMed Central

    Trofatter, K F; Daniels, C A

    1980-01-01

    Mechanisms whereby prostaglandins and other cyclic adenosine 3',5'-monophosphate (cAMP) modulators might enhance the growth of herpes simplex virus (HSV) in human skin fibroblasts were explored. Prostaglandins A1, B1, E1, E2, and F2 alpha, as well as isoproterenol, imidazole, carbamylcholine, and dibutyryl cAMP had no effect on HSV growth. On the other hand, the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine and theophylline delayed the growth, suppressed the cell-to-cell spread, but inhibited neither the adsorption nor the penetration of the virus. Although none of the cAMP-elevating reagents directly enhanced HSV growth, they were found to inhibit dose dependently the antiviral action of both type I and HSV antigen-induced human interferon preparations. Furthermore, these reagents suppressed the production of HSV antigen-induced interferon by immune human mononuclear leukocytes. These data support the hypothesis that prostaglandin elaboration in vivo could contribute to exacerbations of HSV infections by compromising the host's interferon defense system. PMID:6244226

  8. Application and optimization of the tenderization of pig Longissimus dorsi muscle by adenosine 5'-monophosphate (AMP) using the response surface methodology.

    PubMed

    Deng, Shaoying; Wang, Daoying; Zhang, Muhan; Geng, Zhiming; Sun, Chong; Bian, Huan; Xu, Weimin; Zhu, Yongzhi; Liu, Fang; Wu, Haihong

    2016-03-01

    Based on single factor experiments, NaCl concentration, adenosine 5'-monophosphate (AMP) concentration and temperature were selected as independent variables for a three-level Box-Behnken experimental design, and the shear force and cooking loss were response values for regression analysis. According to the statistical models, it showed that all independent variables had significant effects on shear force and cooking loss, and optimal values were at the NaCl concentration of 4.15%, AMP concentration of 22.27 mmol/L and temperature of 16.70°C, which was determined with three-dimensional response surface diagrams and contour plots. Under this condition, the observed shear force and cooking loss were 0.625 kg and 8.07%, respectively, exhibiting a good agreement with their predicted values, showing the good applicability and feasibility of response surface methodology (RSM) for improving pork tenderness. Compared with control pig muscles, AMP combined with NaCl treatment demonstrated significant effects on improvement of meat tenderness and reduction of cooking loss. Therefore, AMP could be regarded as an effective tenderization agent for pork. PMID:26212625

  9. Sensitivity of fructose-1,6-biphosphatase from yeast, liver and skeletal muscle to fructose-2,6-biphosphate and 5'-adenosine monophosphate.

    PubMed

    von Herrath, M; Holzer, H

    1988-05-01

    As a prerequisite for future studies on the possible effect of sulphite, an anti-microbial agent, on gluconeogenesis in yeast, a comparative study of fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis, from yeast, liver and skeletal muscle is reported. In contrast to FBPase from yeast or liver, FBPase from skeletal muscle is approximately 1000-fold more sensitive to inhibition by 5' adenosine monophosphate and 30 to 250-fold less sensitive to inhibition by fructose-2,6-bisphosphate. The kinetic properties of the FBPases, determined by the ratios R(Mg2+/Mn2+) and R (pH 7/9) of the enzyme activities, measured at 10 mM Mg2+ and 2 mM Mn2+ and at pH 7.0 and 9.0, respectively, show a drastic difference between the skeletal muscle and the yeast or liver enzymes. The data support the idea that the enzymes from yeast and liver function in gluconeogenesis, whereas the enzyme from skeletal muscle is involved in other biological functions. PMID:3291467

  10. Adenosine Monophosphate-activated Protein Kinase Regulates Interleukin-1β Expression and Glial Glutamate Transporter Function in Rodents with Neuropathic Pain

    PubMed Central

    Maixner, Dylan W.; Yan, Xisheng; Gao, Mei; Yadav, Ruchi; Weng, Han-Rong

    2015-01-01

    Background Neuroinflammation and dysfunctional glial glutamate transporters (GTs) in the spinal dorsal horn (SDH) are implicated in the genesis of neuropathic pain. We determined if adenosine monophosphate-activated protein kinase (AMPK) in the SDH regulates these processes in rodents with neuropathic pain. Methods Hind paw withdrawal responses to radiant heat and mechanical stimuli were used to assess nociceptive behaviors. Spinal markers related to neuroinflammation and glial GTs were determined by Western blotting. AMPK activities were manipulated pharmacologically and genetically. Regulation of glial GTs was determined by measuring protein expression and activities of glial GTs. Results AMPK activities were reduced in the SDH of rats (n = 5) with thermal hyperalgesia induced by nerve injury, which were accompanied with the activation of astrocytes, increased production of interleukin-1beta and activities of glycogen synthase kinase 3β, and suppressed protein expression of glial glutamate transporter-1. Thermal hyperalgesia was reversed by spinal activation of AMPK in neuropathic rats (n = 10), and induced by inhibiting spinal AMPK in naïve rats (n = 7 to 8). Spinal AMPKα knockdown (n = 6) and AMPKα1 conditional knockout (n = 6) induced thermal hyperalgesia and mechanical allodynia. These genetic alterations mimicked the changes of molecular markers induced by nerve injury. Pharmacological activation of AMPK enhanced glial GT activity in mice with neuropathic pain (n = 8) and attenuated glial glutamate transporter-1 internalization induced by interleukin-1β (n = 4). Conclusion These findings suggest enhancing spinal AMPK activities could be an effective approach for the treatment of neuropathic pain. PMID:25710409

  11. Compartmentalized Cyclic Adenosine 3′,5′-Monophosphate at the Plasma Membrane Clusters PDE3A and Cystic Fibrosis Transmembrane Conductance Regulator into Microdomains

    PubMed Central

    Penmatsa, Himabindu; Zhang, Weiqiang; Yarlagadda, Sunitha; Li, Chunying; Conoley, Veronica G.; Yue, Junming; Bahouth, Suleiman W.; Buddington, Randal K.; Zhang, Guangping; Nelson, Deborah J.; Sonecha, Monal D.; Manganiello, Vincent; Wine, Jeffrey J.

    2010-01-01

    Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3′,5′-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A–CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A–containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A–CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion. PMID:20089840

  12. The role of nitric oxide in the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein in the spinal cord after intradermal injection of capsaicin.

    PubMed

    Wu, Jing; Fang, Li; Lin, Qing; Willis, William D

    2002-06-01

    We investigated the involvement of nitric oxide (NO) in the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein (CREB) in the spinal cord of rats during central sensitization after intradermal capsaicin injection. CREB and phosphorylated CREB (p-CREB) were measured by immunoblotting. The level of p-CREB increased by 20 minutes, peaked between 20 and 60 minutes after capsaicin injection, and started to decrease after 150 minutes. CREB itself did not show an obvious change after capsaicin injection. The p-CREB expression on the ipsilateral side of the spinal dorsal horn, but not on the contralateral side, increased significantly after capsaicin injection. The increase in p-CREB induced by capsaicin injection was partially blocked by pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, administered through a microdialysis fiber placed across the spinal cord. D-NAME, an inactive form of L-NAME, had no effect. CREB phosphorylation, not the level of CREB, was induced within 20 minutes by microdialysis administration of SIN-1, an NO donor. These results indicate that CREB phosphorylation in the spinal cord results from both endogenous and exogenous NO release and that p-CREB may play a role in central sensitization or in longer-term changes in gene expression induced by strong peripheral noxious stimulation. PMID:14622772

  13. Inhibition by morphine of prostaglandin E1-stimulated secretion and cyclic adenosine 3',5'-monophosphate formation in the rat jejunum in vivo.

    PubMed Central

    Beubler, E.; Lembeck, F.

    1980-01-01

    1 The effects were studied of prostaglandin E1 (PGE1), theophylline and morphine on net water flux and mucosal cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels in the jejunum of anaesthetized rats in vivo. 2 Infusion of PGE1 (3.2 micrograms/min, i.a.) caused a reversal from net water absorption to net secretion and enhanced the mucosal cyclic AMP content by 54%. 3 Theophylline (5 mg/ml, intraluminal) similarly produced a reversal from net water absorption to net secretion and increased mucosal cyclic AMP content by 54%. Additional intra-arterial infusion of PGE1 resulted in a massive increase in net water secretion and an increase in mucosal cyclic AMP content by about 200%. 4 Pretreatment with morphine (10 mg/kg, s.c.) reduced the effect of PGE1 on net water flux and completely inhibited its effect on the mucosal cyclic AMP content. Naloxone (10 mg/kg, s.c.) abolished both effects of morphine. 5 A good correlation (r = 0.99) was demonstrated between mucosal cyclic AMP levels and net water flux. 6 The present results demonstrate that PGE1 stimulates intestinal fluid secretion by increasing mucosal cyclic AMP levels. The antidiarrhoeal effect of morphine can be explained by its inhibition of the PGE-mediated increase in cyclic AMP levels, which, in turn, leads to a reduction in intestinal secretion. PMID:6301596

  14. 5′-Adenosine Monophosphate-Induced Hypothermia Attenuates Brain Ischemia/Reperfusion Injury in a Rat Model by Inhibiting the Inflammatory Response

    PubMed Central

    Miao, Yi-Feng; Wu, Hui; Yang, Shao-Feng; Dai, Jiong; Qiu, Yong-Ming; Tao, Zhen-Yi; Zhang, Xiao-Hua

    2015-01-01

    Hypothermia treatment is a promising therapeutic strategy for brain injury. We previously demonstrated that 5′-adenosine monophosphate (5′-AMP), a ribonucleic acid nucleotide, produces reversible deep hypothermia in rats when the ambient temperature is appropriately controlled. Thus, we hypothesized that 5′-AMP-induced hypothermia (AIH) may attenuate brain ischemia/reperfusion injury. Transient cerebral ischemia was induced by using the middle cerebral artery occlusion (MCAO) model in rats. Rats that underwent AIH treatment exhibited a significant reduction in neutrophil elastase infiltration into neuronal cells and matrix metalloproteinase 9 (MMP-9), interleukin-1 receptor (IL-1R), tumor necrosis factor receptor (TNFR), and Toll-like receptor (TLR) protein expression in the infarcted area compared to euthermic controls. AIH treatment also decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL-) positive neuronal cells. The overall infarct volume was significantly smaller in AIH-treated rats, and neurological function was improved. By contrast, rats with ischemic brain injury that were administered 5′-AMP without inducing hypothermia had ischemia/reperfusion injuries similar to those in euthermic controls. Thus, the neuroprotective effects of AIH were primarily related to hypothermia. PMID:25873763

  15. Adenosine 5'-monophosphate-activated protein kinase regulates IL-10-mediated anti-inflammatory signaling pathways in macrophages.

    PubMed

    Zhu, Yanfang Peipei; Brown, Jonathan R; Sag, Duygu; Zhang, Lihua; Suttles, Jill

    2015-01-15

    AMP-activated protein kinase (AMPK) is a conserved serine/threonine kinase with a critical function in the regulation of metabolic pathways in eukaryotic cells. Recently, AMPK has been shown to play an additional role as a regulator of inflammatory activity in leukocytes. Treatment of macrophages with chemical AMPK activators, or forced expression of a constitutively active form of AMPK, results in polarization to an anti-inflammatory phenotype. In addition, we reported previously that stimulation of macrophages with anti-inflammatory cytokines such as IL-10, IL-4, and TGF-β results in rapid activation of AMPK, suggesting that AMPK contributes to the suppressive function of these cytokines. In this study, we investigated the role of AMPK in IL-10-induced gene expression and anti-inflammatory function. IL-10-stimulated wild-type macrophages displayed rapid activation of PI3K and its downstream targets Akt and mammalian target of rapamycin complex (mTORC1), an effect that was not seen in macrophages generated from AMPKα1-deficient mice. AMPK activation was not impacted by treatment with either the PI3K inhibitor LY294002 or the JAK inhibitor CP-690550, suggesting that IL-10-mediated activation of AMPK is independent of PI3K and JAK activity. IL-10 induced phosphorylation of both Tyr(705) and Ser(727) residues of STAT3 in an AMPKα1-dependent manner, and these phosphorylation events were blocked by inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase β, an upstream activator of AMPK, and by the mTORC1 inhibitor rapamycin, respectively. The impaired STAT3 phosphorylation in response to IL-10 observed in AMPKα1-deficient macrophages was accompanied by reduced suppressor of cytokine signaling 3 expression and an inadequacy of IL-10 to suppress LPS-induced proinflammatory cytokine production. Overall, our data demonstrate that AMPKα1 is required for IL-10 activation of the PI3K/Akt/mTORC1 and STAT3-mediated anti-inflammatory pathways regulating macrophage

  16. Metformin inhibits growth of human non-small cell lung cancer cells via liver kinase B-1-independent activation of adenosine monophosphate-activated protein kinase

    PubMed Central

    GUO, QIANQIAN; LIU, ZHIYAN; JIANG, LILI; LIU, MENGJIE; MA, JIEQUN; YANG, CHENGCHENG; HAN, LILI; NAN, KEJUN; LIANG, XUAN

    2016-01-01

    Metformin, the most widely administered oral anti-diabetic therapeutic agent, exerts its glucose-lowering effect predominantly via liver kinase B1 (LKB1)-dependent activation of adenosine monophosphate-activated protein kinase (AMPK). Accumulating evidence has demonstrated that metformin possesses potential antitumor effects. However, whether the antitumor effect of metformin is via the LKB1/AMPK signaling pathway remains to be determined. In the current study, the effects of metformin on proliferation, cell cycle progression, and apoptosis of human non-small cell lung cancer (NSCLC) H460 (LKB1-null) and H1299 (LKB1-positive) cells were assessed, and the role of LKB1/AMPK signaling in the anti-growth effects of metformin were investigated. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle distribution and apoptosis were assessed by flow cytometry, and protein expression levels were measured by western blotting. Metformin inhibited proliferation, induced significant cell cycle arrest at the G0–G1 phase and increased apoptosis in NSCLC cells in a time- and concentration-dependent manner, regardless of the level of LKB1 protein expression. Furthermore, knockdown of LKB1 with short hairpin RNA (shRNA) did not affect the antiproliferative effect of metformin in the H1299 cells. Metformin stimulated AMPK phosphorylation and subsequently suppressed the phosphorylation of mammalian target of rapamycin and its downstream effector, 70-kDa ribosomal protein S6 kinase in the two cell lines. These effects were abrogated by silencing AMPK with small interfering RNA (siRNA). In addition, knockdown of AMPK with siRNA inhibited the effect of metformin on cell proliferation in the two cell lines. These results provide evidence that the growth inhibition of metformin in NSCLC cells is mediated by LKB1-independent activation of AMPK, indicating that metformin may be a potential therapeutic agent for the treatment of

  17. Human gonadotropin-releasing hormone receptor gene transcription: up-regulation by 3',5'-cyclic adenosine monophosphate/protein kinase A pathway.

    PubMed

    Cheng, K W; Leung, P C

    2001-07-01

    Transient transfection of mouse gonadotrope-derived (alphaT3-1) cells with a 2297 bp human GnRHR promoter-luciferase construct (p2300-LucF) showed a dose- and time-dependent increase in the human gonodotropin-releasing hormone receptor (GnRHR) promoter activity after forskolin treatment. An average of 4.8-fold increase in promoter activity was observed after 12 h of 10 microM forskolin treatment. This effect was mimicked by administration of cholera toxin, cAMP analog or pituitary adenylate cyclase activating polypeptide 38 (PACAP). A specific adenylate cyclase (AC) inhibitor (ACI) or protein kinase A (PKA) inhibitor (PKAI) pretreatment reversed the forskolin- and PACAP-induced increase in the human GnRHR promoter activity. These results not only confirm the stimulatory effect of Cyclic adenosine monophosphate (cAMP) in human GnRHR promoter activation, but also suggest that hormones or neurotransmitters that activate adenylate cyclase in pituitary gonadotropes may increase the expression of human GnRHR gene in transcriptional level. Progressive 5' deletion assays identified a 412 bp fragment (-577 to 167) in the human GnRHR 5'-flanking region that is essential in maintaining the basal responsiveness to cAMP. Mutagenesis coupled with functional studies have identified two putative AP-1/CREB binding sites, namely hGR-AP/CRE-1 and hGR-AP/CRE-2 that participated in mediating the cAMP-stimulatory effect. Mutation of the putative hGR-AP/CRE-1 and hGR-CRE-2 resulted in a 38 and 32% decrease in the forskolin-induced stimulation. However, mutation of both binding sites did not completely abolish the cAMP-stimulatory effect, suggesting that multiple transcription factor binding sites were involved in full response in cAMP stimulation. The binding of CREB to these motifs was confirmed by gel mobility shift assay and antibody supershift assay. PMID:11476937

  18. Vasoactive intestinal peptide attenuates liver ischemia/reperfusion injury in mice via the cyclic adenosine monophosphate-protein kinase a pathway.

    PubMed

    Ji, Haofeng; Zhang, Yu; Liu, Yuanxing; Shen, Xiu-Da; Gao, Feng; Nguyen, Terry T; Busuttil, Ronald W; Waschek, James A; Kupiec-Weglinski, Jerzy W

    2013-09-01

    Hepatic ischemia/reperfusion injury (IRI), an exogenous, antigen-independent, local inflammation response, occurs in multiple clinical settings, including liver transplantation, hepatic resection, trauma, and shock. The nervous system maintains extensive crosstalk with the immune system through neuropeptide and peptide hormone networks. This study examined the function and therapeutic potential of the vasoactive intestinal peptide (VIP) neuropeptide in a murine model of liver warm ischemia (90 minutes) followed by reperfusion. Liver ischemia/reperfusion (IR) triggered an induction of gene expression of intrinsic VIP; this peaked at 24 hours of reperfusion and coincided with a hepatic self-healing phase. Treatment with the VIP neuropeptide protected livers from IRI; this was evidenced by diminished serum alanine aminotransferase levels and well-preserved tissue architecture and was associated with elevated intracellular cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling. The hepatocellular protection rendered by VIP was accompanied by diminished neutrophil/macrophage infiltration and activation, reduced hepatocyte necrosis/apoptosis, and increased hepatic interleukin-10 (IL-10) expression. Strikingly, PKA inhibition restored liver damage in otherwise IR-resistant VIP-treated mice. In vitro, VIP not only diminished macrophage tumor necrosis factor α/IL-6/IL-12 expression in a PKA-dependent manner but also prevented necrosis/apoptosis in primary mouse hepatocyte cultures. In conclusion, our findings document the importance of VIP neuropeptide-mediated cAMP-PKA signaling in hepatic homeostasis and cytoprotection in vivo. Because the enhancement of neural modulation differentially regulates local inflammation and prevents hepatocyte death, these results provide the rationale for novel approaches to managing liver IRI in transplant patients. PMID:23744729

  19. Vasoactive intestinal peptide: A potent stimulator of adenosine 3′:5′-cyclic monophosphate accumulation in gut carcinoma cell lines in culture*

    PubMed Central

    Laburthe, M.; Rousset, M.; Boissard, C.; Chevalier, G.; Zweibaum, A.; Rosselin, G.

    1978-01-01

    Vasoactive intestinal peptide (VIP) is a potent and efficient stimulator of adenosine 3′:5′-cyclic monophosphate (cAMP) accumulation in a human colon carcinoma cell line, HT 29. cAMP accumulation is sensitive to a concentration of VIP as low as 3×10-12 M. Maximum VIP-induced cAMP levels were observed with 10-9 M VIP and are about 200 times above the basal levels. Half-maximum cAMP production was obtained at 3×10-10 M VIP. 125I-Labeled VIP was found to bind to HT 29 cells; this binding was competitively inhibited by concentrations of unlabeled VIP between 10-10 and 10-7 M. Half-maximum inhibition of binding was observed with 2×10-9 M VIP. Secretin also stimulated cAMP accumulation in HT 29 cells, but its effectiveness was 1/1000 that of VIP. The other peptides tested at 10-7 M, such as insulin, glucagon, bovine pancreatic polypeptide, somatostatin, octapeptide of cholecystokinin, neurotensin, and substance P, did not stimulate cAMP accumulation. Prostaglandin E1 and catecholamines stimulated cAMP production but were 1/2.3 and 1/5.5 as efficient as VIP, respectively. Another malignant cell line from the gut, the human rectal tumor cell line HRT 18, is also sensitive to VIP. In HRT 18 cells, VIP stimulated cAMP accumulation with a maximal effect at 10-8 M; half-maximum stimulation was observed at about 10-9 M. These results demonstrate the presence of VIP receptors in two malignant human intestinal cell lines (HT 29 and HRT 18) in culture and provide a model for studying the action of VIP on cell proliferation. PMID:208077

  20. On the interaction of caffeine with nucleic acids. III. 1H NMR studies of caffeine--5'-adenosine monophosphate and caffeine-poly(riboadenylate) interactions.

    PubMed

    Fritzsche, H; Petri, I; Schütz, H; Weller, K; Sedmera, P; Lang, H

    1980-02-01

    1) The self-association of both caffeine (Cf) and 5'-adenosine monophosphate (AMP) in aqueous solution has been reinvestigated by 1H NMR. The self-association process is characterized by an isodesmic model. The apparent self-association constants of the vertical stacking process are KCf = (10.6 +/- 1.0) M-1 and KAMP = (1.67 +/- 0.17) M-1. The arrangement of the monomeric units in the stacked aggregates is discussed in terms of isoshielding curves theoretically calculated by Giessner-Prettre and Pullman. Models are proposed which are consistent with these and further previous NMR data. 2) The interaction of Cf and AMP has been studied by 1H NMR. The apparent association constant of the complex Cf-AMP is KC-A = (7.3 +/- 1.2) M-1. Two models of the mutual arrangement of AMP and Cf in the complex are proposed on the basis of the calculated isoshielding curves considering both ring current and local atomic diamagnetic anisotropy effects. 3) The interaction of Cf and poly(riboadenylate), (rA)n, is indicated by a downfield shift of the H-8 line but an upfield shifts of the H-2 line in the 1H NMR spectra of (rA)n. The concentration dependence of the 1H NMR shifts of both Cf and (rA)n can be explained by the existence of two binding mechanisms. We suggest (i) partial insertion of Cf between adjacent base residues of ordered single-stranded regions of (rA)n and (ii) outside binding of Cf in form of monomeric Cf as well as of self-associated aggregates. The complex geometry of insertion proposed on the basis of the calculated isoshielding curves is characterized by a stronger overlapping of the Cf ring and the H-2 proton of (rA)n as compared to the H-8 proton. PMID:7357061

  1. Adenosine Monophosphate-Activated Protein Kinase (AMPK) as a New Target for Antidiabetic Drugs: A Review on Metabolic, Pharmacological and Chemical Considerations

    PubMed Central

    Gruzman, Arie; Babai, Gali; Sasson, Shlomo

    2009-01-01

    In view of the epidemic nature of type 2 diabetes and the substantial rate of failure of current oral antidiabetic drugs the quest for new therapeutics is intensive. The adenosine monophosphate-activated protein kinase (AMPK) is an important regulatory protein for cellular energy balance and is considered a master switch of glucose and lipid metabolism in various organs, especially in skeletal muscle and liver. In skeletal muscles, AMPK stimulates glucose transport and fatty acid oxidation. In the liver, it augments fatty acid oxidation and decreases glucose output, cholesterol and triglyceride synthesis. These metabolic effects induced by AMPK are associated with lowering blood glucose levels in hyperglycemic individuals. Two classes of oral antihyperglycemic drugs (biguanidines and thiazolidinediones) have been shown to exert some of their therapeutic effects by directly or indirectly activating AMPK. However, side effects and an acquired resistance to these drugs emphasize the need for the development of novel and efficacious AMPK activators. We have recently discovered a new class of hydrophobic D-xylose derivatives that activates AMPK in skeletal muscles in a non insulin-dependent manner. One of these derivatives (2,4;3,5-dibenzylidene-D-xylose-diethyl-dithioacetal) stimulates the rate of hexose transport in skeletal muscle cells by increasing the abundance of glucose transporter-4 (GLUT-4) in the plasma membrane through activation of AMPK. This compound reduces blood glucose levels in diabetic mice and therefore offers a novel strategy of therapeutic intervention strategy in type 2 diabetes. The present review describes various classes of chemically-related compounds that activate AMPK by direct or indirect interactions and discusses their potential for candidate antihyperglycemic drug development. PMID:19557293

  2. Effect of the growth stage and cultivar on policosanol profiles of barley sprouts and their adenosine 5'-monophosphate-activated protein kinase activation.

    PubMed

    Seo, Woo Duck; Yuk, Heung Joo; Curtis-Long, Marcus J; Jang, Ki Chang; Lee, Jin Hwan; Han, Sang-Ik; Kang, Hang Won; Nam, Min Hee; Lee, Sung-Joon; Lee, Ji Hae; Park, Ki Hun

    2013-02-01

    Adenosine 5'-monophosphate-activated protein kinase (AMPK) is an intracellular sensor that can regulate glucose levels within the cell. For this reason, it is well-known to be a target for drugs against diabetes and obesity. AMPK was activated significantly by the hexane extract of barley sprouts. This AMPK activation emerges across the growth stages of the sprout, becoming most significant (3 times above the initial stages) 10 days after sprouting. After this time, the activation decreased between 13 and 20 days post-sprouting. Analysis of the hexane extracts by gas chromatography-mass spectrometry showed that the amounts of policosanols (PCs, which are linear, primary aliphatic alcohols with 20-30 carbons) in the plant dramatically increased between 5 days (109.7 mg/100 g) and 10 days (343.7 mg/100 g) post-sprouting and then levels fell back down, reaching 76.4 mg/100 g at 20 days post-sprouting. This trend is consistent with PCs being the active ingredient in the barley plants. We validate this by showing that hexacosanol is an activator of AMPK. The richest cultivar for PCs was found to be the Daejin cultivar. Cultivars had a significant effect on the total PC content (113.2-183.5 mg/100 g) within the plant up to 5 days post-sprouting. However this dependence upon the cultivar was not so apparent at peak stages of PC production (10 days post-sprouting). The most abundant PC in barley sprout, hexacosanol, contributed 62-80% of the total PC content at every stage. These results are valuable to determine the optimal times of harvest to obtain the highest yield of PCs. PMID:23301834

  3. Targeting energy metabolic and oncogenic signaling pathways in triple-negative breast cancer by a novel adenosine monophosphate-activated protein kinase (AMPK) activator.

    PubMed

    Lee, Kuen-Haur; Hsu, En-Chi; Guh, Jih-Hwa; Yang, Hsiao-Ching; Wang, Dasheng; Kulp, Samuel K; Shapiro, Charles L; Chen, Ching-Shih

    2011-11-11

    The antitumor activities of the novel adenosine monophosphate-activated protein kinase (AMPK) activator, OSU-53, were assessed in in vitro and in vivo models of triple-negative breast cancer. OSU-53 directly stimulated recombinant AMPK kinase activity (EC(50), 0.3 μM) and inhibited the viability and clonogenic growth of MDA-MB-231 and MDA-MB-468 cells with equal potency (IC(50), 5 and 2 μM, respectively) despite lack of LKB1 expression in MDA-MB-231 cells. Nonmalignant MCF-10A cells, however, were unaffected. Beyond AMPK-mediated effects on mammalian target of rapamycin signaling and lipogenesis, OSU-53 also targeted multiple AMPK downstream pathways. Among these, the protein phosphatase 2A-dependent dephosphorylation of Akt is noteworthy because it circumvents the feedback activation of Akt that results from mammalian target of rapamycin inhibition. OSU-53 also modulated energy homeostasis by suppressing fatty acid biosynthesis and shifting the metabolism to oxidation by up-regulating the expression of key regulators of mitochondrial biogenesis, such as a peroxisome proliferator-activated receptor γ coactivator 1α and the transcription factor nuclear respiratory factor 1. Moreover, OSU-53 suppressed LPS-induced IL-6 production, thereby blocking subsequent Stat3 activation, and inhibited hypoxia-induced epithelial-mesenchymal transition in association with the silencing of hypoxia-inducible factor 1a and the E-cadherin repressor Snail. In MDA-MB-231 tumor-bearing mice, daily oral administration of OSU-53 (50 and 100 mg/kg) suppressed tumor growth by 47-49% and modulated relevant intratumoral biomarkers of drug activity. However, OSU-53 also induced protective autophagy that attenuated its antiproliferative potency. Accordingly, cotreatment with the autophagy inhibitor chloroquine increased the in vivo tumor-suppressive activity of OSU-53. OSU-53 is a potent, orally bioavailable AMPK activator that acts through a broad spectrum of antitumor activities. PMID

  4. The adipokine adiponectin has potent anti-fibrotic effects mediated via adenosine monophosphate-activated protein kinase: novel target for fibrosis therapy

    PubMed Central

    2012-01-01

    Introduction Fibrosis in scleroderma is associated with collagen deposition and myofibroblast accumulation. Peroxisome proliferator activated receptor gamma (PPAR-γ), a master regulator of adipogenesis, inhibits profibrotic responses induced by transforming growth factor-ß (TGF-β), and its expression is impaired in scleroderma. The roles of adiponectin, a PPAR-γ regulated pleiotropic adipokine, in regulating the response of fibroblasts and in mediating the effects of PPAR-γ are unknown. Methods Regulation of fibrotic gene expression and TGF-ß signaling by adiponectin and adenosine monophosphate protein-activated (AMP) kinase agonists were examined in normal fibroblasts in monolayer cultures and in three-dimensional skin equivalents. AdipoR1/2 expression on skin fibroblasts was determined by real-time quantitative PCR. Results Adiponectin, an adipokine directly regulated by PPAR-γ, acts as a potent anti-fibrotic signal in normal and scleroderma fibroblasts that abrogates the stimulatory effects of diverse fibrotic stimuli and reduces elevated collagen gene expression in scleroderma fibroblasts. Adiponectin responses are mediated via AMP kinase, a fuel-sensing cellular enzyme that is necessary and sufficient for down-regulation of fibrotic genes by blocking canonical Smad signaling. Moreover, we demonstrate that endogenous adiponectin accounts, at least in part, for the anti-fibrotic effects exerted by ligands of PPAR-γ. Conclusions These findings reveal a novel link between cellular energy metabolism and extracellular matrix homeostasis converging on AMP kinase. Since the levels of adiponectin as well as its receptor are impaired in scleroderma patients with progressive fibrosis, the present results suggest a potential role for defective adiponectin expression or function in progressive fibrogenesis in scleroderma and other chronic fibrosing conditions. Restoring the adiponectin signaling axis in fibroblasts might, therefore, represent a novel

  5. Dietary effects of adenosine monophosphate to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream, Pagrus major.

    PubMed

    Hossain, Md Sakhawat; Koshio, Shunsuke; Ishikawa, Manabu; Yokoyama, Saichiro; Sony, Nadia Mahjabin

    2016-09-01

    Our study explored the dietary effects of adenosine monophosphate (AMP) to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream. A semi-purified basal diet supplemented with 0% (Control), 0.1% (AMP-0.1), 0.2% (AMP-0.2), 0.4% (AMP-0.4) and 0.8% (AMP-0.8) purified AMP to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish (mean initial weight 3.4 g) for 56 days. The results indicated that dietary AMP supplements tended to improve growth performances. One of the best ones was found in diet group AMP-0.2, followed by diet groups AMP-0.1, AMP-0.4 and AMP-0.8. The Apparent digestibility coefficients (dry matter, protein and lipid) also improved by AMP supplementation and the significantly highest dry matter digestibility was observed in diet group AMP-0.2. Fish fed diet groups AMP-0.2 and AMP-0.4 had significantly higher peroxidase and bactericidal activities than fish fed the control diet. Nitro-blue-tetrazolium (NBT) activity was found to be significantly (P < 0.05) greater in fish fed diet groups AMP-0.4 and AMP-0.8. Total serum protein, lysozyme activity and agglutination antibody titer were also increased (P > 0.05) by dietary supplementation. In contrast, catalase activity decreased with AMP supplementation. Moreover, the fish fed AMP supplemented diets had better improvement (P < 0.05) in body lipid contents, condition factor, hematocrit content and glutamyl oxaloacetic transaminase (GOT) level than the control group. Supplementation also improved both freshwater and oxidative stress resistances. Interestingly, the fish fed diet groups AMP-0.2 and AMP-0.4 showed the least oxidative stress condition. Finally it is concluded that, dietary AMP supplementation enhanced the growth, digestibility, immune response and stress resistance of red sea bream. The regression analysis revealed that a dietary AMP supplementation between 0.2 and 0.4% supported weight gain and

  6. Effects of single or combined histamine H1-receptor and leukotriene CysLT1-receptor antagonism on nasal adenosine monophosphate challenge in persistent allergic rhinitis

    PubMed Central

    Lee, Daniel K C; Jackson, Catherine M; Soutar, Patricia C; Fardon, Thomas C; Lipworth, Brian J

    2004-01-01

    Background The effects of single or combined histamine H1-receptor and leukotriene CysLT1-receptor antagonism on nasal adenosine monophosphate (AMP) challenge in allergic rhinitis are unknown. Objective We elected to study the effects of usual clinically recommended doses of fexofenadine (FEX), montelukast (ML) and FEX + ML combination, compared with placebo (PL), on nasal AMP challenge in patients with persistent allergic rhinitis. Methods Twelve patients with persistent allergic rhinitis (all skin prick positive to house dust mite) were randomized in a double-blind cross-over fashion to receive for 1 week either FEX 180 mg, ML 10 mg, FEX 180 mg +ML 10 mg combination, or PL, with nasal AMP challenge performed 12 h after dosing. There was a 1-week washout period between each randomized treatment. The primary outcome measure was the maximum percentage peak nasal inspiratory flow (PNIF) fall from baseline over a 60-min period after nasal challenge with a single 400 mg ml−1 dose of AMP. The area under the 60-min time–response curve (AUC) and nasal symptoms were measured as secondary outcomes. Results There was significant attenuation (P < 0.05) of the mean maximum percentage PNIF fall from baseline after nasal AMP challenge vs. PL, 48; with FEX, 37; 95% confidence interval for difference 2, 20; ML, 35 (4, 22); and FEX + ML, 32 (7, 24). The AUC (%.min) was also significantly attenuated (P < 0.05) vs. PL, 1893; with FEX, 1306 (30, 1143); ML, 1246 (214, 1078); and FEX + ML, 1153 (251, 1227). There were no significant differences for FEX vs. ML vs. FEX + ML comparing either the maximum or AUC response. The total nasal symptom score (out of 12) was also significantly improved (P < 0.05) vs. PL, 3.3; with FEX, 2.1 (0.3, 2.0); ML, 2.0 (0.5, 1.9); and FEX + ML, 2.5 (0.1, 1.4). Conclusion FEX and ML as monotherapy significantly attenuated the response to nasal AMP challenge and improved nasal symptoms compared with PL, while combination therapy conferred no additional

  7. Feedback inhibition of cyclic adenosine monophosphate-stimulated Na+ transport in the rabbit cortical collecting duct via Na(+)-dependent basolateral Ca++ entry.

    PubMed Central

    Breyer, M D

    1991-01-01

    Arginine vasopressin (AVP) transiently stimulates Na+ transport in the rabbit cortical collecting duct (CCD). However, the sustained effect of both AVP and its putative second messenger, cyclic adenosine monophosphate (cAMP), on Na+ transport in the rabbit CCD is inhibitory. Because maneuvers that increase [Ca++]i inhibit Na+ transport, the effects of AVP and cell-permeable cAMP analogues, on [Ca++]i were investigated in fura-2-loaded in vitro microperfused rabbit CCDs. Low-dose AVP (23-230 pM) selectively stimulated Ca++ influx, whereas 23 nM AVP additionally released calcium from intracellular stores. 8-chlorophenylthio-cAMP (8CPTcAMP) and 8-bromo-cAMP (8-Br-cAMP) also increased CCD [Ca++]i. The 8CPTcAMP-stimulated [Ca++]i increase was totally dependent on basolateral [Ca++]. In the absence of cAMP, peritubular Na+ removal produced a marked increase in [Ca++]i, which was also dependent on bath [Ca++], suggesting the existence of basolateral Na+/Ca++ exchange. Luminal Na+ removal in the absence of cAMP did not alter CCD [Ca++]i, but it completely blocked the cAMP-stimulated [Ca++]i increase. Thus the cAMP-dependent Ca++ increase is totally dependent on both luminal Na+ and basolateral Ca++, suggesting the [Ca++]i increase is secondary to cAMP effects on luminal Na+ entry and its coupling to basolateral Na+/Ca++ exchange. 8CPTcAMP inhibits lumen-to-bath 22Na flux [JNa(l-b)] in CCDs bathed in a normal Ca++ bath (2.4 mM). However, when bath Ca++ was lowered to 100 nM, a maneuver that also blocks the 8CPTcAMP [Ca++]i increase, 8CPTcAMP stimulated, rather than inhibited JNa(l-b). These results suggest that cAMP formation initially stimulates CCD Na+ transport, and that increased apical Na+ entry secondarily activates basolateral Ca++ entry. The cAMP-dependent [Ca++]i increase leads to inhibition Na+ transport in the rabbit CCD. PMID:1658041

  8. Comparison of three inhaled non-steroidal anti-inflammatory drugs on the airway response to sodium metabisulphite and adenosine 5'-monophosphate challenge in asthma.

    PubMed Central

    Wang, M.; Wisniewski, A.; Pavord, I.; Knox, A.; Tattersfield, A.

    1996-01-01

    BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) are used to assess the role of prostaglandins in asthma but their effects on bronchoconstrictor challenges have been inconsistent. The effects of three nebulised nonsteroidal anti-inflammatory drugs on the airway response to inhaled sodium metabisulphite (MBS) and adenosine 5'-monophosphate (AMP) were compared in the same asthmatic subjects to see whether contractile prostaglandins were involved in MBS or AMP induced bronchoconstriction. A possible protective effect of the osmolarity or pH of the inhaled solutions was also assessed. METHODS: Two double blind placebo controlled studies were carried out. In study 1, 15 non-aspirin sensitive patients with mild asthma attended on four occasions and inhaled 5 ml of lysine aspirin (L-aspirin) 900 mg, indomethacin 50 mg, sodium salicylate 800 mg, or saline 20 minutes before an inhaled MBS challenge. On four further occasions 14 of the patients inhaled the same solutions followed by an inhaled AMP challenge. In study 2, 10 of the patients attended on four additional occasions and inhaled 5 ml of 0.9%, 3%, 10%, or 9.5% saline with indomethacin 50 mg 20 minutes before an inhaled MBS challenge. RESULTS: In study 1 inhaled lysine aspirin had a similar effect on MBS and AMP induced bronchoconstriction, increasing the provocative dose causing a 20% fall in FEV1 (PD20) by 1.29 (95% CI 0.54 to 2.03) and 1.23 (95% CI 0.53 to 1.93) doubling doses, respectively. Indomethacin increased the MBS PD20 and AMP PD20 by 0.64 (95% CI -0.1 to 1.38) and 0.99 (95% CI 0.29 to 1.69) doubling doses, respectively. Sodium salicylate had no significant effect on either challenge. The two solutions causing most inhibition were the most acidic and the most alkaline. In study 2 inhaled 9.5% saline with indomethacin (osmolarity 3005 mOsm/kg) increased the MBS PD20 by 1.1 doubling doses (95% CI 0.2 to 2.0) compared with only 0.09 (95% CI -0.83 to 1.0) and 0.04 (95% CI -0.88 to 0.95) doubling doses

  9. Inhibitory effects of total saponin from Korean Red Ginseng on [Ca2+]i mobilization through phosphorylation of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and inositol 1,4,5-trisphosphate receptor type I in human platelets

    PubMed Central

    Shin, Jung-Hae; Kwon, Hyuk-Woo; Cho, Hyun-Jeong; Rhee, Man Hee; Park, Hwa-Jin

    2015-01-01

    Background Intracellular Ca2+([Ca2+]i) is a platelet aggregation-inducing molecule. Therefore, understanding the inhibitory mechanism of [Ca2+]i mobilization is very important to evaluate the antiplatelet effect of a substance. This study was carried out to understand the Ca2+-antagonistic effect of total saponin from Korean Red Ginseng (KRG-TS). Methods We investigated the Ca2+-antagonistic effect of KRG-TS on cyclic nucleotides-associated phosphorylation of inositol 1,4,5-trisphosphate receptor type I (IP3RI) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation. Results The inhibition of [Ca2+]i mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-Br-cAMPS), which was more stronger than the inhibition by a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) (Thr197) by KRG-TS. The phosphorylation of IP3RI (Ser1756) by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS compared with that by Rp-8-Br-cGMPS. These results suggest that the inhibitory effect of [Ca2+]i mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. KRG-TS also inhibited the release of adenosine triphosphate and serotonin. In addition, only G-Rg3 of protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation. Conclusion These results strongly indicate that KRG-TS is a potent beneficial compound that inhibits [Ca2+]i mobilization in thrombin–platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease. PMID:26869828

  10. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors

    SciTech Connect

    Lund, Kaleb C. Wallace, Kendall B.

    2008-01-01

    Nucleoside analog reverse transcriptase inhibitors (NRTIs) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study, we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3'-azido-3'-deoxythymidine (AZT; 10 and 50 {mu}M), AZT monophosphate (150 {mu}M), and 2',3'-dideoxycytidine (ddC; 1 {mu}M) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2',3'-dideoxyinosine (ddI; 10 {mu}M) and ddC (1 {mu}M). In the presence of succinate + cAMP, AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-{gamma} activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug.

  11. Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

    SciTech Connect

    Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

    2010-09-01

    Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results: IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

  12. Post-Translational Regulation of the Glucose-6-Phosphatase Complex by Cyclic Adenosine Monophosphate Is a Crucial Determinant of Endogenous Glucose Production and Is Controlled by the Glucose-6-Phosphate Transporter.

    PubMed

    Soty, Maud; Chilloux, Julien; Delalande, François; Zitoun, Carine; Bertile, Fabrice; Mithieux, Gilles; Gautier-Stein, Amandine

    2016-04-01

    The excessive endogenous glucose production (EGP) induced by glucagon participates in the development of type 2 diabetes. To further understand this hormonal control, we studied the short-term regulation by cyclic adenosine monophosphate (cAMP) of the glucose-6-phosphatase (G6Pase) enzyme, which catalyzes the last reaction of EGP. In gluconeogenic cell models, a 1-h treatment by the adenylate cyclase activator forskolin increased G6Pase activity and glucose production independently of any change in enzyme protein amount or G6P content. Using specific inhibitors or protein overexpression, we showed that the stimulation of G6Pase activity involved the protein kinase A (PKA). Results of site-directed mutagenesis, mass spectrometry analyses, and in vitro phosphorylation experiments suggested that the PKA stimulation of G6Pase activity did not depend on a direct phosphorylation of the enzyme. However, the temperature-dependent induction of both G6Pase activity and glucose release suggested a membrane-based mechanism. G6Pase is composed of a G6P transporter (G6PT) and a catalytic unit (G6PC). Surprisingly, we demonstrated that the increase in G6PT activity was required for the stimulation of G6Pase activity by forskolin. Our data demonstrate the existence of a post-translational mechanism that regulates G6Pase activity and reveal the key role of G6PT in the hormonal regulation of G6Pase activity and of EGP. PMID:26958868

  13. Metformin attenuates graft-versus-host disease via restricting mammalian target of rapamycin/signal transducer and activator of transcription 3 and promoting adenosine monophosphate-activated protein kinase-autophagy for the balance between T helper 17 and Tregs.

    PubMed

    Park, Min-Jung; Lee, Seon-Yeong; Moon, Su-Jin; Son, Hye-Jin; Lee, Sung-Hee; Kim, Eun-Kyung; Byun, Jae-Kyeong; Shin, Dong Yun; Park, Sung-Hwan; Yang, Chul-Woo; Cho, Mi-La

    2016-07-01

    Acute graft-versus-host disease (aGVHD), caused by donor T cell-mediated injury to host tissues, is a problem in allogeneic bone marrow transplantation. The transition from naïve to effector T cells is accompanied by shift in metabolism main pathway; from glucose oxidative phosphorylation to aerobic glycolysis. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine kinase that is a metabolic sensor that helps maintain cellular energy homeostasis. Although AMPK activation can exert anti-inflammatory properties by negatively regulating pro-inflammatory mediators, its role as a therapeutic potential of graft-versus-host disease development remains unclear. In this study, we found that the intraperitoneal administration of metformin, which activates AMPK signaling significantly, ameliorated the clinical severity of aGHVD and lethality. This was associated with reductions in type I T helper (Th1) and Th17 and rises in Th2 and regulatory T (Treg) cell. The enhanced signal transducer and activator of transcription 3 activation noted during the development of aGVHD was reduced by metformin treatment. Furthermore, metformin-treated Th17 cells became converted into Treg cells via enhanced autophagy. The reduction in mortality associated with metformin treatment was associated with inhibition of the mammalian target of rapamycin/signal transducer and activator of transcription 3 pathway. These results suggest that metformin might be of significant use in the treatment of patients with aGVHD. PMID:27126953

  14. Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

    PubMed

    Ido, Yasuo

    2016-07-01

    Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3. PMID:27181414

  15. Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5'-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells.

    PubMed

    Chang, Wenguang; Zhang, Ming; Meng, Zhaojie; Yu, Yang; Yao, Fan; Hatch, Grant M; Chen, Li

    2015-12-15

    Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy. PMID:26522928

  16. A long-acting β2-adrenergic agonist increases the expression of muscarine cholinergic subtype-3 receptors by activating the β2-adrenoceptor cyclic adenosine monophosphate signaling pathway in airway smooth muscle cells

    PubMed Central

    LIU, YUAN-HUA; WU, SONG-ZE; WANG, GANG; HUANG, NI-WEN; LIU, CHUN-TAO

    2015-01-01

    The persistent administration of β2-adrenergic (β2AR) agonists has been demonstrated to increase the risk of severe asthma, partly due to the induction of tolerance to bronchoprotection via undefined mechanisms. The present study investigated the potential effect of the long-acting β2-adrenergic agonist, formoterol, on the expression of muscarinic M3 receptor (M3R) in rat airway smooth muscle cells (ASMCs). Primary rat ASMCs were isolated and characterized following immunostaining with anti-α-smooth muscle actin antibodies. The protein expression levels of M3R and phospholipase C-β1 (PLCβ1) were characterized by western blot analysis and the production of inositol 1,4,5-trisphosphate (IP3) was determined using an enzyme-linked immunosorbent assay. Formoterol increased the protein expression of M3R in rat ASMCs in a time- and dose-dependent manner, which was significantly inhibited by the β2AR antagonist, ICI118,551 and the cyclic adenosine monophosphate (cAMP) inhibitor, SQ22,536. The increased protein expression of M3R was positively correlated with increased production of PLCβ1 and IP3. Furthermore, treatment with the glucocorticoid, budesonide, and the PLC inhibitor, U73,122, significantly suppressed the formoterol-induced upregulated protein expression levels of M3R and PLCβ1 and production of IP3. The present study demonstrated that formoterol mediated the upregulation of M3R in the rat ASMCs by activating the β2AR-cAMP signaling pathway, resulting in increased expression levels of PLCβ1 and IP3, which are key to inducing bronchoprotection tolerance. Administration of glucocorticoids or a PLC antagonist prevented formoterol-induced bronchoprotection tolerance by suppressing the protein expression of M3R. PMID:25672589

  17. Early glycogen synthase kinase-3β and protein phosphatase 2A independent tau dephosphorylation during global brain ischaemia and reperfusion following cardiac arrest and the role of the adenosine monophosphate kinase pathway.

    PubMed

    Majd, Shohreh; Power, John H T; Koblar, Simon A; Grantham, Hugh J M

    2016-08-01

    Abnormal tau phosphorylation (p-tau) has been shown after hypoxic damage to the brain associated with traumatic brain injury and stroke. As the level of p-tau is controlled by Glycogen Synthase Kinase (GSK)-3β, Protein Phosphatase 2A (PP2A) and Adenosine Monophosphate Kinase (AMPK), different activity levels of these enzymes could be involved in tau phosphorylation following ischaemia. This study assessed the effects of global brain ischaemia/reperfusion on the immediate status of p-tau in a rat model of cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We reported an early dephosphorylation of tau at its AMPK sensitive residues, Ser(396) and Ser(262) after 2 min of ischaemia, which did not recover during the first two hours of reperfusion, while the tau phosphorylation at GSK-3β sensitive but AMPK insensitive residues, Ser(202) /Thr(205) (AT8), as well as the total amount of tau remained unchanged. Our data showed no alteration in the activities of GSK-3β and PP2A during similar episodes of ischaemia of up to 8 min and reperfusion of up to 2 h, and 4 weeks recovery. Dephosphorylation of AMPK followed the same pattern as tau dephosphorylation during ischaemia/reperfusion. Catalase, another AMPK downstream substrate also showed a similar pattern of decline to p-AMPK, in ischaemic/reperfusion groups. This suggests the involvement of AMPK in changing the p-tau levels, indicating that tau dephosphorylation following ischaemia is not dependent on GSK-3β or PP2A activity, but is associated with AMPK dephosphorylation. We propose that a reduction in AMPK activity is a possible early mechanism responsible for tau dephosphorylation. PMID:27177932

  18. Role of 3', 5' cyclic adenosine monophosphate and protein kinase C in the regulation of insulin-like growth factor-binding protein secretion by thyroid-stimulating hormone in isolated ovine thyroid cells.

    PubMed

    Wang, J F; Hill, D J; Becks, G P

    1994-05-01

    Isolated sheep thyroid follicles release insulin-like growth factors (IGF)-I and -II together with IGF-binding proteins (IGFBPs). We previously showed that TSH suppresses the biosynthesis and release of IGFBPs in vitro which may increase the tissue availability of IGFs, allowing a synergy with TSH which potentiates both thyroid growth and function. Many of the actions of TSH on thyroid cell function are dependent upon activation of adenylate cyclase, although increased synthesis of inositol trisphosphate and activation of protein kinase C (PKC) have also been implicated. We have now examined whether probable changes in intracellular cyclic adenosine monophosphate (cAMP) or PKC are involved in TSH-mediated suppression of IGFBP release. Confluent primary cultures of ovine thyroid cells were maintained in serum-free Ham's modified F-12M medium containing transferrin, somatostatin and glycyl-histidyl-lysine (designated 3H), and further supplemented with sodium iodide (10(-8)-10(-3) mol/l), dibutyryl cAMP (0.25-1 mmol/l), forskolin (5-20 mumol/l) or 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-11)-10(-6) mol/l), with or without exposure to TSH (200 microU/ml). The uptake and organification of Na [125I] by cells was examined after test incubations of up to 48 h, and IGFBPs in conditioned media were analysed by ligand blot using 125I-labelled IGF-II. The PKC activity in the cytosol and plasma membrane fractions of cells was measured by phosphorylation of histone using [gamma-32P]ATP, and PKC immunoreactivity was visualized by Western immunoblot analysis. While dibutyryl cAMP or forskolin largely reproduced the stimulatory effect of TSH on iodine organification, they did not mimic the inhibitory effect of TSH on the secretion of IGFBPs of 43, 34, 28 and 19 kDa. Incubation with physiological or pharmacological concentrations of iodide (10(-6)-10(-3) mol/l) for up to 48 h significantly decreased TSH action on iodide uptake and organification but did not alter the

  19. [Adenosine and its role in physiology].

    PubMed

    Novotný, J

    2015-01-01

    Adenosine is not just a major component of adenine nucleotides and ribonucleic acids, but also has its own signaling functions. ExtraceIlular level of adenosine in an organism is strictly maintained through the balance between its formation, degradation and transport. Adenosine is formed by enzymatic degradation of adenosine triphosphate and eliminated by phosphorylation to adenosine monophosphate or by deamination to inosine. Transport of adenosine across the cell membrane is ensured by equilibrative and concentrative nucleoside transporters. All these processes participate in maintenance of adenosine level under normal conditions, but a balanced equilibrium can be disrupted in some pathophysiological situations. Extracellular adenosine as a signaling molecule binds to adenosine receptors, which may trigger via their cognate trimeric G proteins different signaling pathways. In this way, adenosine regulates energy homeostasis and affects the function of various organs. Targeted pharmacological manipulations of specific adenosine receptor subtypes or enzymes involved in its metabolism can potentially be used for therapeutic purposes. PMID:26738245

  20. Purine metabolism in adenosine deaminase deficiency.

    PubMed Central

    Mills, G C; Schmalstieg, F C; Trimmer, K B; Goldman, A S; Goldblum, R M

    1976-01-01

    Purine and pyrimidine metabolites were measured in erythrocytes, plasma, and urine of a 5-month-old infant with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. Adenosine and adenine were measured using newly devised ion exchange separation techniques and a sensitive fluorescence assay. Plasma adenosine levels were increased, whereas adenosine was normal in erythrocytes and not detectable in urine. Increased amounts of adenine were found in erythrocytes and urine as well as in the plasma. Erythrocyte adenosine 5'-monophosphate and adenosine diphosphate concentrations were normal, but adenosine triphosphate content was greatly elevated. Because of the possibility of pyrimidine starvation, pyrimidine nucleotides (pyrimidine coenzymes) in erythrocytes and orotic acid in urine were measured. Pyrimidine nucleotide concentrations were normal, while orotic acid was not detected. These studies suggest that the immune deficiency associated with adenosine deaminase deficiency may be related to increased amounts of adenine, adenosine, or adenine nucleotides. PMID:1066699

  1. Alpha-Lipoic acid increases energy expenditure by enhancing adenosine monophosphate-activated protein kinase-peroxisome proliferator-activated receptor-gamma coactivator-1alpha signaling in the skeletal muscle of aged mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Skeletal muscle mitochondrial dysfunction is associated with aging and diabetes, which decreases respiratory capacity and increases reactive oxygen species. Lipoic acid (LA) possesses antioxidative and antidiabetic properties. Metabolic action of LA is mediated by activation of adenosine monophospha...

  2. A comparison of the effects of divalent and trivalent cations on parathyroid hormone release, 3',5'-cyclic-adenosine monophosphate accumulation, and the levels of inositol phosphates in bovine parathyroid cells.

    PubMed

    Brown, E M; Fuleihan G el-H; Chen, C J; Kifor, O

    1990-09-01

    We compared the effects of a series of di- and trivalent cations on various aspects of parathyroid function to investigate whether these polyvalent cations act on the parathyroid cell through a similar mechanism. Like high extracellular concentrations of Ca2+, high levels of barium (Ba2+), strontium (Sr2+), gadolinium (Gd3+), europium (Eu3+), terbium (Tb3+), and ytterbium (Yb3+) [corrected] each inhibited low calcium-stimulated PTH release and showed IC50 values (the concentration producing half of the maximal inhibitory effect) of 1.12 mM, 1.18 mM, 2.2 microM, 2.5 microM, 0.89 microM, and 15 microM, respectively. The inhibitory effects of both divalent (Ca2+ and Ba2+) and trivalent (Gd3+) cations were reversible by 76-100% after removal of the cation, suggesting that the polyvalent cation-mediated reduction in PTH release was not due to nonspecific toxicity. The same di- and trivalent cations produced an 80-90% decrease in agonist-stimulated cAMP accumulation with a similar order of potency as for their effects on PTH release. Preincubation overnight with pertussis toxin totally prevented the inhibitory effects of the trivalent cations on cAMP accumulation. The same di- and trivalent cations also increased the accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. Their effects on this parameter differed from those on PTH release and cAMP accumulation in several respects. First, Ba2+ and Sr2+, rather than being equipotent with Ca2+, were about 2-fold less potent in increasing the levels of inositol phosphates. Second, the trivalent cations were 5-50-fold less potent in raising inositol phosphates than in modulating PTH release and cAMP accumulation, and all were nearly equipotent. These results show that trivalent cations of the lanthanide series mimic the actions of divalent cations on several aspects of parathyroid function, and likely do so by interacting with the cell surface "Ca2(+)-receptor-like mechanism" through which

  3. Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

    PubMed Central

    Azhar, Salman; Menon, K. M. Jairam

    1979-01-01

    uptake and metabolism of cyclic [3H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [3H]AMP, the radioactivity associated with the cells increased almost linearly up to 250μm-cyclic [3H]AMP concentration in the incubation medium. The 3H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation. PMID:226066

  4. (−)-Epicatechin-3-O-β-d-allopyranoside from Davallia formosana, Prevents Diabetes and Hyperlipidemia by Regulation of Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Shih, Chun-Ching; Wu, Jin-Bin; Jian, Jia-Ying; Lin, Cheng-Hsiu; Ho, Hui-Ya

    2015-01-01

    The purpose of this experiment was to determine the antidiabetic and lipid-lowering effects of (−)-epicatechin-3-O-β-d-allopyranoside (BB) from the roots and stems of Davallia formosana in mice. Animal treatment was induced by high-fat diet (HFD) or low-fat diet (control diet, CD). After eight weeks of HFD or CD exposure, the HFD mice were treating with BB or rosiglitazone (Rosi) or fenofibrate (Feno) or water through gavage for another four weeks. However, at 12 weeks, the HFD-fed group had enhanced blood levels of glucose, triglyceride (TG), and insulin. BB treatment significantly decreased blood glucose, TG, and insulin levels. Moreover, visceral fat weights were enhanced in HFD-fed mice, accompanied by increased blood leptin concentrations and decreased adiponectin levels, which were reversed by treatment with BB. Muscular membrane protein levels of glucose transporter 4 (GLUT4) were reduced in HFD-fed mice and significantly enhanced upon administration of BB, Rosi, and Feno. Moreover, BB treatment markedly increased hepatic and skeletal muscular expression levels of phosphorylation of AMP-activated (adenosine monophosphate) protein kinase (phospho-AMPK). BB also decreased hepatic mRNA levels of phosphenolpyruvate carboxykinase (PEPCK), which are associated with a decrease in hepatic glucose production. BB-exerted hypotriglyceridemic activity may be partly associated with increased mRNA levels of peroxisome proliferator activated receptor α (PPARα), and with reduced hepatic glycerol-3-phosphate acyltransferase (GPAT) mRNA levels in the liver, which decreased triacylglycerol synthesis. Nevertheless, we demonstrated BB was a useful approach for the management of type 2 diabetes and dyslipidemia in this animal model. PMID:26492243

  5. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress

    PubMed Central

    Courchet, Julien; Lewis, Tommy L.; Losón, Oliver C.; Hellberg, Kristina; Young, Nathan P.; Chen, Hsiuchen; Polleux, Franck; Chan, David C.; Shaw, Reuben J.

    2016-01-01

    Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA–linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)–activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission. PMID:26816379

  6. Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

    PubMed Central

    Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

    2016-01-01

    Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

  7. Anticancer effect of adenosine on gastric cancer via diverse signaling pathways

    PubMed Central

    Tsuchiya, Ayako; Nishizaki, Tomoyuki

    2015-01-01

    Extracellular adenosine induces apoptosis in a variety of cancer cells via intrinsic and extrinsic pathways. In the former pathway, adenosine uptake into cells triggers apoptosis, and in the latter pathway, adenosine receptors mediate apoptosis. Extracellular adenosine also induces apoptosis of gastric cancer cells. Extracellular adenosine is transported into cells through an adenosine transporter and converted to AMP by adenosine kinase. In turn, AMP activates AMP-activated protein kinase (AMPK). AMPK is the factor responsible for caspase-independent apoptosis of GT3-TKB gastric cancer cells. Extracellular adenosine, on the other hand, induces caspase-dependent apoptosis of MKN28 and MKN45 gastric cancer cells by two mechanisms. Firstly, AMP, converted from intracellularly transported adenosine, initiates apoptosis, regardless of AMPK. Secondly, the A3 adenosine receptor, linked to Gi/Gq proteins, mediates apoptosis by activating the Gq protein effector, phospholipase Cγ, to produce inositol 1,4,5-trisphosphate and diacylglycerol, which activate protein kinase C. Consequently, the mechanisms underlying adenosine-induced apoptosis vary, depending upon gastric cancer cell types. Understand the contribution of each downstream target molecule of adenosine to apoptosis induction may aid the establishment of tailor-made chemotherapy for gastric cancer. PMID:26494951

  8. Anticancer effect of adenosine on gastric cancer via diverse signaling pathways.

    PubMed

    Tsuchiya, Ayako; Nishizaki, Tomoyuki

    2015-10-21

    Extracellular adenosine induces apoptosis in a variety of cancer cells via intrinsic and extrinsic pathways. In the former pathway, adenosine uptake into cells triggers apoptosis, and in the latter pathway, adenosine receptors mediate apoptosis. Extracellular adenosine also induces apoptosis of gastric cancer cells. Extracellular adenosine is transported into cells through an adenosine transporter and converted to AMP by adenosine kinase. In turn, AMP activates AMP-activated protein kinase (AMPK). AMPK is the factor responsible for caspase-independent apoptosis of GT3-TKB gastric cancer cells. Extracellular adenosine, on the other hand, induces caspase-dependent apoptosis of MKN28 and MKN45 gastric cancer cells by two mechanisms. Firstly, AMP, converted from intracellularly transported adenosine, initiates apoptosis, regardless of AMPK. Secondly, the A3 adenosine receptor, linked to Gi/Gq proteins, mediates apoptosis by activating the Gq protein effector, phospholipase Cγ, to produce inositol 1,4,5-trisphosphate and diacylglycerol, which activate protein kinase C. Consequently, the mechanisms underlying adenosine-induced apoptosis vary, depending upon gastric cancer cell types. Understand the contribution of each downstream target molecule of adenosine to apoptosis induction may aid the establishment of tailor-made chemotherapy for gastric cancer. PMID:26494951

  9. AMP-activated protein kinase is activated by non-steroidal anti-inflammatory drugs.

    PubMed

    King, Tanya S; Russe, Otto Quintus; Möser, Christine V; Ferreirós, Nerea; Kynast, Katharina L; Knothe, Claudia; Olbrich, Katrin; Geisslinger, Gerd; Niederberger, Ellen

    2015-09-01

    AMP-activated kinase (AMPK) is a cellular energy sensor, which is activated in stages of increased adenosine triphosphate (ATP) consumption. Its activation has been associated with a number of beneficial effects such as decrease of inflammatory processes and inhibition of disease progression of diabetes and obesity. A recent study suggested that salicylate, the active metabolite of the non-steroidal anti-inflammatory drug (NSAID) acetyl-salicylic acid (aspirin), is able to activate AMPK pharmacologically. This observation raised the question whether or not other NSAIDs might also act as AMPK activators and whether this action might contribute to their cyclooxygenase (COX)-independent anti-inflammatory properties. In this study, we investigated mouse and human neuronal cells and liver tissue of mice after treatment with various NSAIDs. Our results showed that the non-selective acidic NSAIDs ibuprofen and diclofenac induced AMPK activation similar to aspirin while the COX-2 selective drug etoricoxib and the non-opioid analgesic paracetamol, both drugs have no acidic structure, failed to activate AMPK. In conclusion, our results revealed that AMPK can be activated by specific non-steroidal anti-inflammatory drugs such as salicylic acid, ibuprofen or diclofenac possibly depending on the acidic structure of the drugs. AMPK might therefore contribute to their antinociceptive and anti-inflammatory properties. PMID:26049010

  10. N3 Protonation Induces Base Rotation of 2'-Deoxyadenosine-5'-monophosphate and Adenosine-5'-monophosphate.

    PubMed

    Wu, R R; He, C C; Hamlow, L A; Nei, Y-W; Berden, G; Oomens, J; Rodgers, M T

    2016-05-26

    Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments combined with theoretical calculations are performed to investigate the stable gas-phase conformations of the protonated adenine mononucleotides, [pdAdo+H](+) and [pAdo+H](+). Conformations that are present in the experiments are elucidated via comparative analyses of the experimental IRMPD spectra and the B3LYP/6-311+G(d,p) IR spectra predicted for the conformers optimized at this level of theory. N3 protonation is preferred as it induces base rotation, which allows a strong hydrogen bond to be formed between the excess proton of adenine and the phosphate moiety. In contrast, both N1 and N7 protonation are predicted to be >35 kJ/mol less favorable than N3 protonation. Only N3 protonated conformers are present in the experiments in measurable abundance. Both the low-energy conformers computed and the experimental IRMPD spectra of [pdAdo+H](+) and [pAdo+H](+) indicate that the 2'-hydroxyl moiety does not significantly impact the structure of the most stable conformer or the IRMPD spectral profile of [pAdo+H](+) vs that of [pdAdo+H](+). However, the 2'-hydroxyl leads to a 3-fold enhancement in the IRMPD yield of [pAdo+H](+) in the fingerprint region. Comparison of present results to those reported in a previous IRMPD study of the analogous protonated adenine nucleosides allows the effects of the phosphate moiety on the gas-phase conformations to be elucidated. PMID:27138137

  11. Regulation of the creatine transporter by AMP-activated protein kinase in kidney epithelial cells

    PubMed Central

    Li, Hui; Thali, Ramon F.; Smolak, Christy; Gong, Fan; Alzamora, Rodrigo; Wallimann, Theo; Scholz, Roland; Pastor-Soler, Núria M.; Neumann, Dietbert

    2010-01-01

    The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na+-dependent creatine uptake into CRT-expressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten Vmax parameter. [14C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local

  12. The adenosine system modulates Toll-like receptor function: basic mechanisms, clinical correlates and translational opportunities

    PubMed Central

    Coombs, Melanie R. Power; Belderbos, Mirjam E.; Gallington, Leighanne C.; Bont, Louis; Levy, Ofer

    2014-01-01

    Adenosine is an endogenous purine metabolite whose concentration in human blood plasma rises from nanomolar to micromolar during stress or hypoxia. Leukocytes express seven-transmembrane adenosine receptors whose engagement modulates Toll-like receptor-mediated cytokine responses, in part via modulation of intracellular cyclic adenosine monophosphate (cAMP). Adenosine congeners are used clinically to treat arrhythmias and apnea of prematurity. Herein we consider the potential of adenosine congeners as innate immune response modifiers to prevent and/or treat infection. PMID:21342073

  13. Adenosine transporters.

    PubMed

    Thorn, J A; Jarvis, S M

    1996-06-01

    1. In mammals, nucleoside transport is an important determinant of the pharmacokinetics, plasma and tissue concentration, disposition and in vivo biological activity of adenosine as well as nucleoside analogues used in antiviral and anticancer therapies. 2. Two broad types of adenosine transporter exist, facilitated-diffusion carriers and active processes driven by the transmembrane sodium gradient. 3. Facilitated-diffusion adenosine carriers may be sensitive (es) or insensitive (ei) to nanomolar concentrations of the transport inhibitor nitrobenzylthioinosine (NBMPR). Dipyridamole, dilazep and lidoflazine analogues are also more potent inhibitors of the es carrier than the ei transporter in cells other than those derived from rat tissues. 4. The es transporter has a broad substrate specificity (apparent Km for adenosine approximately 25 microM in many cells at 25 degrees C), is a glycoprotein with an average apparent Mr of 57,000 in human erythrocytes that has been purified to near homogeneity and may exist in situ as a dimer. However, there is increasing evidence to suggest the presence of isoforms of the es transporter in different cells and species, based on kinetic and molecular properties. 5. The ei transporter also has a broad substrate specificity with a lower affinity for some nucleoside permeants than the es carrier, is genetically distinct from es but little information exists as to the molecular properties of the protein. 6. Sodium-dependent adenosine transport is present in many cell types and catalysed by four distinct systems, N1-N4, distinguished by substrate specificity, sodium coupling and tissue distribution. 7. Two genes have been identified which encode sodium-dependent adenosine transport proteins, SNST1 from the sodium/glucose cotransporter (SGLT1) gene family and the rat intestinal N2 transporter (cNT1) from a novel gene family including a bacterial nucleoside carrier (NupC). Transcripts of cNT1, which encodes a 648-residue protein, are

  14. Adenosine Kinase: Exploitation for Therapeutic Gain

    PubMed Central

    2013-01-01

    Adenosine kinase (ADK; EC 2.7.1.20) is an evolutionarily conserved phosphotransferase that converts the purine ribonucleoside adenosine into 5′-adenosine-monophosphate. This enzymatic reaction plays a fundamental role in determining the tone of adenosine, which fulfills essential functions as a homeostatic and metabolic regulator in all living systems. Adenosine not only activates specific signaling pathways by activation of four types of adenosine receptors but it is also a primordial metabolite and regulator of biochemical enzyme reactions that couple to bioenergetic and epigenetic functions. By regulating adenosine, ADK can thus be identified as an upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. Consequently, ADK emerges as a rational therapeutic target, and adenosine-regulating drugs have been tested extensively. In recent attempts to improve specificity of treatment, localized therapies have been developed to augment adenosine signaling at sites of injury or pathology; those approaches include transplantation of stem cells with deletions of ADK or the use of gene therapy vectors to downregulate ADK expression. More recently, the first human mutations in ADK have been described, and novel findings suggest an unexpected role of ADK in a wider range of pathologies. ADK-regulating strategies thus represent innovative therapeutic opportunities to reconstruct network homeostasis in a multitude of conditions. This review will provide a comprehensive overview of the genetics, biochemistry, and pharmacology of ADK and will then focus on pathologies and therapeutic interventions. Challenges to translate ADK-based therapies into clinical use will be discussed critically. PMID:23592612

  15. Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

    2013-02-01

    Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

  16. 5-aminoimidazole-4-carboxamide ribonucleoside and AMP-activated protein kinase inhibit signalling through NF-κB.

    PubMed

    Katerelos, Marina; Mudge, Stuart J; Stapleton, David; Auwardt, Russell B; Fraser, Scott A; Chen, C-G; Kemp, Bruce E; Power, David A

    2010-10-01

    Activation of nuclear factor-kappa B (NF-κB) is one of the most important pro-inflammatory mechanisms in disease. In this study, we show that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an intermediate in nucleoside metabolism, inhibits signalling by NF-κB in three cell types, including bovine aortic endothelial cells (BAEC). The block in the NF-κB signalling pathway occurred beyond degradation of IκB-α and movement of p65 into the nucleus of BAEC. There was, however, reduced binding of NF-κB from AICAR-treated cells to a κB-consensus oligonucleotide, suggesting that part of the mechanism was a reduction in NF-κB DNA-binding activity. Although AICAR is metabolized to ZMP and then adenosine, adenosine had no effect on activation of an NF-κB reporter. ZMP, however, activates the metabolic stress-sensing AMP-activated protein kinase (AMPK). Transfection of active AMPK into BAEC reduced NF-κB reporter activity compared with a kinase-dead mutant, suggesting that part of the ability of AICAR to inhibit NF-κB signalling is due to activation of AMPK. Inhibition of NF-κB signalling may be important in the anti-inflammatory action of drugs such as sulfasalazine and methotrexate, which led to the accumulation of AICAR within target cells. PMID:20404837

  17. Functions of AMP-activated protein kinase in adipose tissue

    PubMed Central

    Daval, Marie; Foufelle, Fabienne; Ferré, Pascal

    2006-01-01

    AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Its functions have been extensively studied in muscles and liver. AMPK stimulates pathways which increase energy production (glucose transport, fatty acid oxidation) and switches off pathways which consume energy (lipogenesis, protein synthesis, gluconeogenesis). This has led to the concept that AMPK has an interesting pharmaceutical potential in situations of insulin resistance and it is indeed the target of existing drugs and hormones which improve insulin sensitivity. Adipose tissue is a key player in energy metabolism through the release of substrates and hormones involved in metabolism and insulin sensitivity. Activation of AMPK in adipose tissue can be achieved through situations such as fasting and exercise. Leptin and adiponectin as well as hypoglycaemic drugs are activators of adipose tissue AMPK. This activation probably involves changes in the AMP/ATP ratio and the upstream kinase LKB1. When activated, AMPK limits fatty acid efflux from adipocytes and favours local fatty acid oxidation. Since fatty acids have a key role in insulin resistance, especially in muscles, activating AMPK in adipose tissue might be found to be beneficial in insulin-resistant states, particularly as AMPK activation also reduces cytokine secretion in adipocytes. PMID:16709632

  18. Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis.

    PubMed

    Takiar, Vinita; Nishio, Saori; Seo-Mayer, Patricia; King, J Darwin; Li, Hui; Zhang, Li; Karihaloo, Anil; Hallows, Kenneth R; Somlo, Stefan; Caplan, Michael J

    2011-02-01

    Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD. PMID:21262823

  19. Perivascular fat, AMP-activated protein kinase and vascular diseases

    PubMed Central

    Almabrouk, T A M; Ewart, M A; Salt, I P; Kennedy, S

    2014-01-01

    Perivascular adipose tissue (PVAT) is an active endocrine and paracrine organ that modulates vascular function, with implications for the pathophysiology of cardiovascular disease (CVD). Adipocytes and stromal cells contained within PVAT produce mediators (adipokines, cytokines, reactive oxygen species and gaseous compounds) with a range of paracrine effects modulating vascular smooth muscle cell contraction, proliferation and migration. However, the modulatory effect of PVAT on the vascular system in diseases, such as obesity, hypertension and atherosclerosis, remains poorly characterized. AMP-activated protein kinase (AMPK) regulates adipocyte metabolism, adipose biology and vascular function, and hence may be a potential therapeutic target for metabolic disorders such as type 2 diabetes mellitus (T2DM) and the vascular complications associated with obesity and T2DM. The role of AMPK in PVAT or the actions of PVAT have yet to be established, however. Activation of AMPK by pharmacological agents, such as metformin and thiazolidinediones, may modulate the activity of PVAT surrounding blood vessels and thereby contribute to their beneficial effect in cardiometabolic diseases. This review will provide a current perspective on how PVAT may influence vascular function via AMPK. We will also attempt to demonstrate how modulating AMPK activity using pharmacological agents could be exploited therapeutically to treat cardiometabolic diseases. PMID:24490856

  20. p-HPEA-EDA, a phenolic compound of virgin olive oil, activates AMP-activated protein kinase to inhibit carcinogenesis.

    PubMed

    Khanal, Prem; Oh, Won-Keun; Yun, Hyo Jeong; Namgoong, Gwang Mo; Ahn, Sang-Gun; Kwon, Seong-Min; Choi, Hoo-Kyun; Choi, Hong Seok

    2011-04-01

    Phenolic constituents of virgin olive oil are reported to have antitumor activity. However, the underlying molecular mechanisms and specific target proteins of virgin olive oil remain to be elucidated. Here, we report that dialdehydic form of decarboxymethyl ligstroside aglycone (p-HPEA-EDA), a phenolic compound of virgin olive oil, inhibits tumor promoter-induced cell transformation in JB6 Cl41 cells and suppress cyclooxygenase-2 (COX-2) and tumorigenicity by adenosine monophosphate-activated protein kinase (AMPK) activation in HT-29 cells. p-HPEA-EDA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of extracellular signal-regulated kinases 1/2 and p90RSK in JB6 Cl41 cells, resulting in the inhibition of cell proliferation, activator protein-1 transactivation and cell transformation promoted by TPA. Moreover, p-HPEA-EDA strongly inhibited the cell viability and COX-2 expression by activation of AMPK activity in HT-29 cells, resulted from depletion of intracellular adenosine triphosphate. p-HPEA-EDA-induced activation of caspase-3 and poly-adenosine diphosphate-ribose polymerase, phosphorylation of p53 (Ser15) and DNA fragmentation in HT-29 cells, leading to apoptosis. Importantly, p-HPEA-EDA suppressed the colony formation of HT-29 cells in soft agar. In contrast, Compound C, an AMPK inhibitor, and Z-DEVD-FMK, a caspase-3 inhibitor, blocked the p-HPEA-EDA-inhibited colony formation in HT-29 cells. In vivo chorioallantoic membrane assay also showed that p-HPEA-EDA-inhibited tumorigenicity of HT-29 cells. These findings revealed that targeted activation of AMPK and inhibition of COX-2 expression by p-HPEA-EDA contribute to the chemopreventive and chemotherapeutic potential of virgin olive oil against colon cancer cells. PMID:21216846

  1. Dietary adenine controls adult lifespan via adenosine nucleotide biosynthesis and AMPK, and regulates the longevity benefit of caloric restriction

    PubMed Central

    Stenesen, Drew; Suh, Jae Myoung; Seo, Jin; Yu, Kweon; Lee, Kyu-Sun; Kim, Jong-Seok; Min, Kyung-Jin; Graff, Jonathan M.

    2012-01-01

    SUMMARY A common thread among conserved lifespan regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of adenosine monophosphate (AMP) biosynthetic enzymes extend Drosophila lifespan. The lifespan benefit of these mutations depends upon increased AMP to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended lifespan, while AMPK RNAi reduced lifespan. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed lifespan extension. Remarkably, this simple change in diet also blocked the pro-longevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult lifespan, provide a mechanistic link between cellular anabolism and energy sensing pathways, and indicate that dietary adenine manipulations might alter metabolism to influence animal lifespan. PMID:23312286

  2. Analysis of Nucleotide 5'-Monophosphates in Infant Formulas by HPLC-UV: Collaborative Study, Final Action 2011.20.

    PubMed

    Gill, Brendon D; Indyk, Harvey E

    2015-01-01

    A collaborative study was conducted on AOAC First Action Method 2011.20: 5'-Mononucleotides in Infant Formula and Adult/Pediatric Nutritional Formula. After the successful analysis of National Institute of Standards and Technology (NIST) 1849a Standard Reference Material (SRM) as a practice sample, 12 laboratories participated in the analysis of duplicate samples of six different infant formula products. The samples were dissolved in high-salt solution to inhibit protein and fat interactions, with the nucleotides [uridine 5'-monophosphate (UMP), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), and cytidine 5'-monophosphate (CMP)] separated from the sample matrix by strong-anion exchange SPE, followed by chromatographic analysis using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique using thymidine 5'-monophosphate. For nucleotide-supplemented products, precision is within the Standard Method Performance RequirementsSM (SMPR) 2011.008 target reproducibility limit of ≤11%, with the reproducibility RSD (RSDR) estimated at 7.1-8.7% for CMP, 7.9-9.0% for UMP, 2.8-7.7% for GMP, 5.5-10.3% for IMP, and 2.7-6.2% for AMP, and Horwitz ratio (HorRat) values of 0.9-1.0 for CMP, 0.9-1.0 for UMP, 0.3-0.7 for GMP, 0.6-1.0 for IMP, and 0.3-0.7 for AMP. PMID:26268980

  3. Adenosine and Ischemic Preconditioning

    PubMed Central

    Liang, Bruce T.; Swierkosz, Tomasz A.; Herrmann, Howard C.; Kimmel, Stephen; Jacobson, Kenneth A.

    2012-01-01

    Adenosine is released in large amounts during myocardial ischemia and is capable of exerting potent cardioprotective effects in the heart. Although these observations on adenosine have been known for a long time, how adenosine acts to achieve its anti-ischemic effect remains incompletely understood. However, recent advances on the chemistry and pharmacology of adenosine receptor ligands have provided important and novel information on the function of adenosine receptor subtypes in the cardiovascular system. The development of model systems for the cardiac actions of adenosine has yielded important insights into its mechanism of action and have begun to elucidate the sequence of signalling events from receptor activation to the actual exertion of its cardioprotective effect. The present review will focus on the adenosine receptors that mediate the potent anti-ischemic effect of adenosine, new ligands at the receptors, potential molecular signalling mechanisms downstream of the receptor, mediators for cardioprotection, and possible clinical applications in cardiovascular disorders. PMID:10607860

  4. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass.

    PubMed

    Shah, M; Kola, B; Bataveljic, A; Arnett, T R; Viollet, B; Saxon, L; Korbonits, M; Chenu, C

    2010-08-01

    Adenosine 5'-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cultured in the presence of AMPK activators (AICAR and metformin), AMPK inhibitor (compound C), the gastric peptide hormone ghrelin and the beta-adrenergic blocker propranolol. AMPK activity was measured in cell lysates by a functional kinase assay and AMPK protein phosphorylation was studied by Western Blotting using an antibody recognizing AMPK Thr-172 residue. We demonstrated that treatment of ROS 17/2.8 cells with AICAR and metformin stimulates Thr-172 phosphorylation of AMPK and dose-dependently increases its activity. In contrast, treatment of ROS 17/2.8 cells with compound C inhibited AMPK phosphorylation. Ghrelin and propranolol dose-dependently increased AMPK phosphorylation and activity. Cell proliferation and alkaline phosphatase activity were not affected by metformin treatment while AICAR significantly inhibited ROS 17/2.8 cell proliferation and alkaline phosphatase activity at high concentrations. To study the effect of AMPK activation on bone formation in vitro, primary osteoblasts obtained from rat calvaria were cultured for 14-17days in the presence of AICAR, metformin and compound C. Formation of 'trabecular-shaped' bone nodules was evaluated following alizarin red staining. We demonstrated that both AICAR and metformin dose-dependently increase trabecular bone nodule formation, while compound C inhibits bone formation. When primary osteoblasts were co-treated with AICAR and compound C, compound C suppressed the stimulatory effect of AICAR on bone nodule formation

  5. Metformin revisited: Does this regulator of AMP-activated protein kinase secondarily affect bone metabolism and prevent diabetic osteopathy

    PubMed Central

    McCarthy, Antonio Desmond; Cortizo, Ana María; Sedlinsky, Claudia

    2016-01-01

    Patients with long-term type 1 and type 2 diabetes mellitus (DM) can develop skeletal complications or “diabetic osteopathy”. These include osteopenia, osteoporosis and an increased incidence of low-stress fractures. In this context, it is important to evaluate whether current anti-diabetic treatments can secondarily affect bone metabolism. Adenosine monophosphate-activated protein kinase (AMPK) modulates multiple metabolic pathways and acts as a sensor of the cellular energy status; recent evidence suggests a critical role for AMPK in bone homeostasis. In addition, AMPK activation is believed to mediate most clinical effects of the insulin-sensitizer metformin. Over the past decade, several research groups have investigated the effects of metformin on bone, providing a considerable body of pre-clinical (in vitro, ex vivo and in vivo) as well as clinical evidence for an anabolic action of metformin on bone. However, two caveats should be kept in mind when considering metformin treatment for a patient with type 2 DM at risk for diabetic osteopathy. In the first place, metformin should probably not be considered an anti-osteoporotic drug; it is an insulin sensitizer with proven macrovascular benefits that can secondarily improve bone metabolism in the context of DM. Secondly, we are still awaiting the results of randomized placebo-controlled studies in humans that evaluate the effects of metformin on bone metabolism as a primary endpoint. PMID:27022443

  6. Metformin revisited: Does this regulator of AMP-activated protein kinase secondarily affect bone metabolism and prevent diabetic osteopathy.

    PubMed

    McCarthy, Antonio Desmond; Cortizo, Ana María; Sedlinsky, Claudia

    2016-03-25

    Patients with long-term type 1 and type 2 diabetes mellitus (DM) can develop skeletal complications or "diabetic osteopathy". These include osteopenia, osteoporosis and an increased incidence of low-stress fractures. In this context, it is important to evaluate whether current anti-diabetic treatments can secondarily affect bone metabolism. Adenosine monophosphate-activated protein kinase (AMPK) modulates multiple metabolic pathways and acts as a sensor of the cellular energy status; recent evidence suggests a critical role for AMPK in bone homeostasis. In addition, AMPK activation is believed to mediate most clinical effects of the insulin-sensitizer metformin. Over the past decade, several research groups have investigated the effects of metformin on bone, providing a considerable body of pre-clinical (in vitro, ex vivo and in vivo) as well as clinical evidence for an anabolic action of metformin on bone. However, two caveats should be kept in mind when considering metformin treatment for a patient with type 2 DM at risk for diabetic osteopathy. In the first place, metformin should probably not be considered an anti-osteoporotic drug; it is an insulin sensitizer with proven macrovascular benefits that can secondarily improve bone metabolism in the context of DM. Secondly, we are still awaiting the results of randomized placebo-controlled studies in humans that evaluate the effects of metformin on bone metabolism as a primary endpoint. PMID:27022443

  7. Characterization of adenosine receptors involved in adenosine-induced bronchoconstriction in allergic rabbits.

    PubMed Central

    el-Hashim, A.; D'Agostino, B.; Matera, M. G.; Page, C.

    1996-01-01

    1. Recent work has suggested that adenosine may be involved in asthma via the activation of A1 receptors. However, the role of the recently cloned A3 receptor in airways is largely unknown. In the present study, we have investigated the role of the A3 receptor in adenosine-induced bronchoconstriction in allergic rabbits. 2. Aerosol challenge of antigen (Ag) immunized rabbits with the adenosine precursor, adenosine 5'-monophosphate (AMP), resulted in a dose-dependent fall in dynamic compliance (Cdyn). The maximum fall in Cdyn in these rabbits was significantly greater than that in litter matched, sham immunized animals (P < 0.05). However, there was no significant difference in the maximum increase in airways resistance (Rt) between Ag and sham immunized rabbits (P > 0.05). 3. Aerosol challenge of Ag immunized rabbits with cyclopentyl-adenosine (CPA) (A1-receptor agonist) elicited a dose-dependent fall in Cdyn in Ag immunized rabbits and the maximum fall in Cdyn in these rabbits was significantly greater than that observed in sham immunized rabbits (P < 0.05). Similarly, CPA induced dose-dependent increases in R1 in Ag immunized rabbits whereas sham immunized rabbits failed to respond to CPA within the same dose range. The maximum increase in RL in Ag immunized rabbits was significantly greater than that of sham immunized rabbits (P < 0.05). 4. Aerosol challenge of either Ag or sham immunized rabbits with the A3 agonist aminophenylethyladenosine (APNEA) did not elicit dose-dependent changes in either RL or Cdyn. Moreover, there was no significant difference in the maximum response, measured by either parameter, between the two animal groups (P > 0.05). 5. These data provide further evidence for a role of the A1 receptor in the airways, but do not support a role for the A3 receptor in adenosine-induced bronchoconstriction in the allergic rabbit. PMID:8937732

  8. AMP-activated Protein Kinase Is Activated as a Consequence of Lipolysis in the Adipocyte

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AMP-activated protein kinase (AMPK) is activated in adipocytes during exercise and other states in which lipolysis is stimulated. However, the mechanism(s) responsible for this effect and its physiological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with agents...

  9. Expression of AMP-activated protein kinase subunits during chicken embryonic and post-hatch development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine protein kinase that senses cellular energy status (AMP/ATP ratio) and acts to maintain energy homeostasis by regulating the activities of energy-consuming and energy-generating metabolic pathways. AMPK is a heterotrimeric en...

  10. Effect of transportation and pre-slaughter water shower spray with resting on AMP-activated protein kinase, glycolysis and meat quality of broilers during summer.

    PubMed

    Xing, Tong; Xu, Xinglian; Jiang, Nannan; Deng, ShaoLin

    2016-02-01

    The aim of this study was to determine the effects of pre-slaughter transport during summer and subsequent water shower spray on stress, postmortem glycolysis, energy metabolism and adenosine monophosphate-activated protein kinase (AMPK) in Pectoralis major (PM) muscle of broilers. Results indicated that transport during high ambient temperature induced the release of plasma corticosterone, which significantly affected stress conditions. Moreover, we found a lower energy status in the early postmortem period compared to the control group. AMPK was activated in this situation, following by the rapid glycolysis and accumulation of lactic acid, leading to a high incidence of pale, soft, exudative (PSE)-like meat. Water shower spray with resting after transport relieved the stress situation, recovered energy homeostasis and lessened the deterioration of meat quality. As a key molecular target for the control of energy status, AMPK has a similar potential in regulating postmortem muscle glycolysis of broilers as in mammals. PMID:26315350

  11. Downregulation of the renal outer medullary K(+) channel ROMK by the AMP-activated protein kinase.

    PubMed

    Siraskar, Balasaheb; Huang, Dan Yang; Pakladok, Tatsiana; Siraskar, Gulab; Sopjani, Mentor; Alesutan, Ioana; Kucherenko, Yulia; Almilaji, Ahmad; Devanathan, Vasudharani; Shumilina, Ekaterina; Föller, Michael; Munoz, Carlos; Lang, Florian

    2013-02-01

    The 5'-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca(2+) activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na(+) channel ENaC and the voltage-gated K(+) channel KCNE1/KCNQ1. It is partially effective by decreasing PIP(2) formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K(+) channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK(γR70Q) (AMPK(α1)-HA+AMPK(β1)-Flag+AMPKγ1(R70Q)), or of inactive AMPK(αK45R) (AMPK(α1K45R)+AMPK(β1)-Flag+AMPK(γ1)-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence in Xenopus oocytes and western blot in whole kidney tissue. Moreover, renal Na(+) and K(+) excretion were determined in AMPK(α1)-deficient mice (ampk ( -/- )) and wild-type mice (ampk ( +/+ )) prior to and following an acute K(+) load (111 mM KCl, 30 mM NaHCO(3), 4.7 mM NaCl, and 2.25 g/dl BSA) at a rate of 500 μl/h. As a result, coexpression of AMPK(γR70Q) but not of AMPK(αK45R) significantly decreased the current in ROMK1-expressing Xenopus oocytes. Injection of phosphatidylinositol PI((4,5))P(2) significantly increased the current in ROMK1-expressing Xenopus oocytes, an effect reversed in the presence of AMPK(γR70Q). Under control conditions, no significant differences between ampk ( -/- ) and ampk ( +/+ ) mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentrations as well as absolute and fractional Na(+) and K(+) excretion. Following an acute K(+) load, GFR, urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentration were again similar in both genotypes, but renal absolute

  12. Response of AMP-activated protein kinase and energy metabolism to acute nitrite exposure in the Nile tilapia Oreochromis niloticus.

    PubMed

    Xu, Zhixin; Li, Erchao; Xu, Chang; Gan, Lei; Qin, Jian G; Chen, Liqiao

    2016-08-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a prevalent mammalian energy metabolism sensor, but little is known about its role as an energy sensor in fish experiencing stress. We aimed to study AMPK in Oreochromis niloticus on both the molecular and the physical level. We found that the cDNAs encoding the AMPKα1 and AMPKα2 variants of the O. niloticus catalytic α subunit were 1753bp and 2563 bp long and encoded 571 and 557 amino acids, respectively. Both the AMPKα1 and the AMPKα2 isoform possess structural features similar to mammalian AMPKα, including a phosphorylation site at Thr172 in the N-terminus, and exhibit high homology with other fish and vertebrate AMPKα sequences (81.3%-98.1%). mRNA encoding the AMPKα isoforms was widely expressed in various tissues with distinctive patterns. AMPKα1 and AMPKα2 were primarily expressed in the intestines and brain, respectively. Under acute nitrite challenge, the mRNA encoding the AMPKα isoforms, as well as AMPK activity, changed over time. Its recovery period in freshwater, combined with the fact that it is highly conserved, suggests that fish AMPK, like its mammalian orthologues, acts as an energy metabolism sensor. Furthermore, subsequent decreases in AMPK mRNA levels and activity suggested that its action was transient but efficient. Physically, glucose, lactic acid and TGs in plasma, as well as energy materials in the hepatopancreas and muscle, were significantly altered over time, indicating changes in energy metabolism during the experimental period. These data have enabled us to characterize energy utilization in O. niloticus and further illustrate the role of fish AMPK as an energy sensor. This study provides new insight into energy metabolism and sensing by AMPK in teleost and necessitates further study of the multiple physiologic roles of AMPK in fish. PMID:27262938

  13. Adenosine inhibition of gamma-aminobutyric acid release from slices of rat cerebral cortex.

    PubMed Central

    Hollins, C.; Stone, T. W.

    1980-01-01

    1 The effect of purine compounds on the potassium-evoked release of 14C-labelled gamma-aminobutyric acid (GABA) has been studied in 400 micrometers slices of rat cerebral cortex in vitro. 2 Adenosine and adenosine 5' monophosphate (AMP) inhibited the release of GABA at 10(-5) to 10(-3) M. Adenosine triphosphate (ATP) produced a significant inhibition of release only at 10(-3) M. 3 Theophylline 10(-4) or 10(-3) M reduced the inhibitory effect of adenosine, but did not change basal release of GABA. 4 Dipyridamole 10(-5) M itself reduced evoked GABA release, but did not prevent the inhibitory effect of adenosine, implying that adenosine was acting at an extracellularly directed receptor. 5 Calcium removal or antagonism by verapamil reduced the evoked release of GABA, but adenosine did not produce any further reduction of the calcium-independent release. This may indicate that the inhibitory effect of adenosine on GABA release results from interference with calcium influx or availability within the terminals. PMID:7378648

  14. Hypertonic NaCl enhances adenosine release and hormonal cAMP production in mouse thick ascending limb.

    PubMed

    Baudouin-Legros, M; Badou, A; Paulais, M; Hammet, M; Teulon, J

    1995-07-01

    Adenosine 3',5'-cyclic monophosphate (cAMP), accumulated in the presence of adenosine, was measured in medullary portions of mouse thick ascending limbs of Henle's loop, suspended either in classic extracellular buffer or in the presence of added NaCl. Under control conditions (140 mmol/l NaCl), adenosine (< 10(-5) mol/l) and N6-cyclohexyladenosine, an A1 adenosine receptor agonist, inhibit the cAMP accumulation induced by arginine vasopressin (AVP). On the other hand, high concentrations of adenosine and CGS-21680, an A2 adenosine receptor agonist, stimulate cAMP formation. Addition of NaCl (+300 mmol/l) to extracellular buffer stimulates the release of endogenous adenosine. It also enhances A2 receptor-induced cAMP accumulation but suppresses A1 receptor-mediated inhibition of adenylyl cyclase. This hypertonic NaCl medium also potentiates the stimulatory action of AVP on adenylyl cyclase. The modifications of tubular responses to both AVP and A1 and A2 agonists, brought about by hypertonic NaCl, were all inhibited by adenosine deaminase, thereby demonstrating the involvement of endogenous adenosine. Adenosine, the release and the effects of which are modulated by hypertonic NaCl, thus appears to act as an endogenous physiological modulator of kidney medulla function. PMID:7631823

  15. Pranlukast inhibits renal epithelial cyst progression via activation of AMP-activated protein kinase.

    PubMed

    Pathomthongtaweechai, Nutthapoom; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-02-01

    Cysteinyl leukotriene receptor 1 (CysLT1 receptor) antagonists were found to inhibit chloride secretion in human airway epithelial cells. Since chloride secretion in renal epithelial cells, which shares common mechanisms with airway epithelial cells, plays important roles in renal cyst progression in polycystic kidney disease (PKD), this study was aimed to investigate effects of drugs acting as CysLT1 receptor antagonists on renal cyst progression and its underlying mechanisms. Effects of CysLT1 receptor antagonists on renal cyst growth and formation were determined using Madine Darby canine kidney (MDCK) cyst models. Mechanisms of actions of CysLT1 receptor antagonists were determined using short-circuit current measurement, assays of cell viability and cell proliferation, and immunoblot analysis of signaling proteins. Of the three drugs acting as CysLT1 receptor antagonists (montelukast, pranlukast and zafirlukast) tested, pranlukast was the most promising drug that inhibited MDCK cyst growth and formation without affecting cell viability. Its effect was independent of the inhibition of CysLT1 receptors. Instead, it reduced cAMP-activated chloride secretion and proliferation of MDCK cells in an AMP-activated protein kinase (AMPK)-dependent manner and had no effect on CFTR protein expression. Interestingly, pranlukast enhanced AMPK activation via calcium/calmodulin-dependent protein kinase kinase beta (CaMKKβ) with consequent activation of acetyl-CoA carboxylase (ACC) and suppression of mammalian target of rapamycin (mTOR) pathway. These results indicate that pranlukast retards renal epithelial cyst progression by inhibiting cAMP-activated chloride secretion and cell proliferation via CaMKKβ-AMPK-mTOR pathway. Therefore, pranlukast represents a class of known drugs that may have potential utility in PKD treatment. PMID:24360935

  16. Activation of AMP-activated kinase as a strategy for managing autosomal dominant polycystic kidney disease.

    PubMed

    McCarty, Mark F; Barroso-Aranda, Jorge; Contreras, Francisco

    2009-12-01

    There is evidence that overactivity of both mammalian target of rapamycin (mTOR) and cystic fibrosis transmembrane conductance regulator (CFTR) contributes importantly to the progressive expansion of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). Recent research has established that AMP-activated kinase (AMPK) can suppress the activity of each of these proteins. Clinical AMPK activators such as metformin and berberine may thus have potential in the clinical management of ADPKD. The traditional use of berberine in diarrhea associated with bacterial infections may reflect, in part, the inhibitory impact of AMPK on chloride extrusion by small intestinal enterocytes. PMID:19570618

  17. Relaxation of isolated taenia coli of guinea-pig by enantiomers of 2-azido analogues of adenosine and adenine nucleotides.

    PubMed Central

    Cusack, N. J.; Planker, M.

    1979-01-01

    1 2-Azido photoaffinity analogues of adenosine 5'triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), and adenosine have been synthesized and tested on guinea-pig taenia coli. 2 2-Azido-ATP and 2-azido-ADP were approximately 20 times more potent than ATP as relaxants of taenia coli, and required prolonged washout times before recovery of the muscle. 3 2-Azido-AMP and 2-azidoadenosine were 2 to 12 times more potent than ATP, but took much longer (up to 100 s) to reach maximal relaxation. This behaviour is different from that of AMP and adenosine which were much less potent than ATP. 4 L-Enantiomers of adenosine and adenine nucleotides were also tested. L-ATP and L-ADP were 3 to 6 times less potent than ATP and ADP, and L-AMP and L-adenosine were inactive. 2-Azido-L-ATP and 2-azido-L-ADP were approximately 120 times less potent than 2-Azido-ATP and 6 times less potent than ATP as relaxants of taenia coli. 2-Azido-L-AMP and 2-azidio-L-adenosine were almost inactive. 5 2-Azido derivatives are photolysed by u.v. irradiation to reactive intermediates. 2-Azido-ATP and 2-azidoadenosine might be suitable photoaffinity ligands for labelling putative P2 and P1 purine receptors respectively. 2-Azido-L-ATP and 2-azido-L-adenosine could be useful controls for nonspecific labelling. PMID:497519

  18. Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

    PubMed Central

    Davis, William M.; White, David C.

    1980-01-01

    Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N6-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge. PMID:16345633

  19. Fluorometric determination of adenosine nucleotide derivatives as measures of the microfouling, detrital, and sedimentary microbial biomass and physiological status.

    PubMed

    Davis, W M; White, D C

    1980-09-01

    Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge. PMID:16345633

  20. Adenosine and Bone Metabolism

    PubMed Central

    Mediero, Aránzazu; Cronstein, Bruce N.

    2013-01-01

    Bone is a dynamic organ that undergoes continuous remodeling whilst maintaining a balance between bone formation and resorption. Osteoblasts, which synthesize and mineralize new bone, and osteoclasts, the cells that resorb bone, act in concert to maintain bone homeostasis. In recent years, there has been increasing appreciation of purinergic regulation of bone metabolism. Adenosine, released locally, mediates its physiologic and pharmacologic actions via interactions with G-protein coupled receptors and recent work has indicated that these receptors are involved in the regulation of osteoclast differentiation and function, as well as osteoblast differentiation and bone formation. Moreover, adenosine receptors also regulate chondrocyte and cartilage homeostasis. These recent findings underscore the potential therapeutic importance of adenosine receptors in regulating bone physiology and pathology. PMID:23499155

  1. Adenosine receptor interactions and anxiolytics.

    PubMed

    Bruns, R F; Katims, J J; Annau, Z; Snyder, S H; Daly, J W

    1983-12-01

    [3H]-N6-cyclohexyladenosine and [3H]-1,3-diethyl-8-phenylxanthine label the A1 subtype of adenosine receptor in brain membranes. The affinities of methylxanthines in competing for A1 adenosine receptors parallel their potencies as locomotor stimulants. The adenosine agonist N6-(phenylisopropyl) adenosine is a potent locomotor depressant. Both diazepam and N6-(L-phenylisopropyl)adenosine cause locomotor stimulation in a narrow range of subdepressant doses. Combined stimulant doses of the two agents depress motor activity, as do larger doses of either one, given separately. Evidence supporting and against the hypothesis that some of the actions of benzodiazepines are mediated via the adenosine system is reviewed. A number of compounds interact with both systems, probably because of physico-chemical similarities between adenosine and diazepam. It is concluded that of the four classic actions of benzodiazepines, the sedative and muscle relaxant (but not anxiolytic or anticonvulsant) actions could possibly be mediated by adenosine. PMID:6199685

  2. Adenosine in fibrosis

    PubMed Central

    Chan, Edwin S. L.

    2011-01-01

    Adenosine is an endogenous autocoid that regulates a multitude of bodily functions. Its anti-inflammatory actions are well known to rheumatologists since it mediates many of the anti-inflammatory effects of a number of antirheumatic drugs such as methotrexate. However, inflammatory and tissue regenerative responses are intricately linked, with wound healing being a prime example. It has only recently been appreciated that adenosine has a key role in tissue regenerative and fibrotic processes. An understanding of these processes may shed new light on potential therapeutic options in diseases such as scleroderma where tissue fibrosis features prominently. PMID:19949965

  3. Bitter melon juice activates cellular energy sensor AMP-activated protein kinase causing apoptotic death of human pancreatic carcinoma cells

    PubMed Central

    Agarwal, Rajesh

    2013-01-01

    Prognosis of pancreatic cancer is extremely poor, suggesting critical needs for additional drugs to improve disease outcome. In this study, we examined efficacy and associated mechanism of a novel agent bitter melon juice (BMJ) against pancreatic carcinoma cells both in culture and nude mice. BMJ anticancer efficacy was analyzed in human pancreatic carcinoma BxPC-3, MiaPaCa-2, AsPC-1 and Capan-2 cells by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide, cell death enzyme-linked immunosorbent assay and annexin/propidium iodide assays. BMJ effect on apoptosis regulators was assessed by immunoblotting. In vivo BMJ efficacy was evaluated against MiaPaCa-2 tumors in nude mice, and xenograft was analyzed for biomarkers by immunohistochemistry (IHC). Results showed that BMJ (2–5% v/v) decreases cell viability in all four pancreatic carcinoma cell lines by inducing strong apoptotic death. At molecular level, BMJ caused caspases activation, altered expression of Bcl-2 family members and cytochrome-c release into the cytosol. Additionally, BMJ decreased survivin and X-linked inhibitor of apoptosis protein but increased p21, CHOP and phosphorylated mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2 and p38) levels. Importantly, BMJ activated adenosine monophosphate-activated protein kinase (AMPK), a biomarker for cellular energy status, and an AMPK inhibitor (Compound C) reversed BMJ-induced caspase-3 activation suggesting activated AMPK involvement in BMJ-induced apoptosis. In vivo, oral administration of lyophilized BMJ (5mg in 100 µl water/day/mouse) for 6 weeks inhibited MiaPaCa-2 tumor xenograft growth by 60% (P < 0.01) without noticeable toxicity in nude mice. IHC analyses of MiaPaCa-2 xenografts showed that BMJ also inhibits proliferation, induces apoptosis and activates AMPK in vivo. Overall, BMJ exerts strong anticancer efficacy against human pancreatic carcinoma cells, both in vitro and in vivo, suggesting its clinical

  4. Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis.

    PubMed Central

    Rutter, Guy A; Da Silva Xavier, Gabriela; Leclerc, Isabelle

    2003-01-01

    AMPK (5'-AMP-activated protein kinase) is emerging as a metabolic master switch, by which cells in both mammals and lower organisms sense and decode changes in energy status. Changes in AMPK activity have been shown to regulate glucose transport in muscle and glucose production by the liver. Moreover, AMPK appears to be a key regulator of at least one transcription factor linked to a monogenic form of diabetes mellitus. As a result, considerable efforts are now under way to explore the usefulness of AMPK as a therapeutic target for other forms of this disease. Here we review this topic, and discuss new findings which suggest that AMPK may play roles in regulating insulin release and the survival of pancreatic islet beta-cells, and nutrient sensing by the brain. PMID:12839490

  5. Pharmacological Targeting of AMP-Activated Protein Kinase and Opportunities for Computer-Aided Drug Design.

    PubMed

    Miglianico, Marie; Nicolaes, Gerry A F; Neumann, Dietbert

    2016-04-14

    As a central regulator of metabolism, the AMP-activated protein kinase (AMPK) is an established therapeutic target for metabolic diseases. Beyond the metabolic area, the number of medical fields that involve AMPK grows continuously, expanding the potential applications for AMPK modulators. Even though indirect AMPK activators are used in the clinics for their beneficial metabolic outcome, the few described direct agonists all failed to reach the market to date, which leaves options open for novel targeting methods. As AMPK is not actually a single molecule and has different roles depending on its isoform composition, the opportunity for isoform-specific targeting has notably come forward, but the currently available modulators fall short of expectations. In this review, we argue that with the amount of available structural and ligand data, computer-based drug design offers a number of opportunities to undertake novel and isoform-specific targeting of AMPK. PMID:26510622

  6. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

  7. Past Strategies and Future Directions for Identifying AMP-Activated Protein Kinase (AMPK) Modulators

    PubMed Central

    Sinnett, Sarah E.; Brenman, Jay E.

    2014-01-01

    AMP-activated protein kinase (AMPK) is a promising therapeutic target for cancer, type II diabetes, and other illnesses characterized by abnormal energy utilization. During the last decade, numerous labs have published a range of methods for identifying novel AMPK modulators. The current understanding of AMPK structure and regulation, however, has propelled a paradigm shift in which many researchers now consider ADP to be an additional regulatory nucleotide of AMPK. How can the AMPK community apply this new understanding of AMPK signaling to translational research? Recent insights into AMPK structure, regulation, and holoenzyme-sensitive signaling may provide the hindsight needed to clearly evaluate the strengths and weaknesses of past AMPK drug discovery efforts. Improving future strategies for AMPK drug discovery will require pairing the current understanding of AMPK signaling with improved experimental designs. PMID:24583089

  8. Adenosine and sleep

    SciTech Connect

    Yanik, G.M. Jr.

    1987-01-01

    Behavioral and biochemical approaches have been used to determine the relative contribution of endogenous adenosine and adenosine receptors to the sleep-wake cycle in the rat. Adenosine concentrations in specific areas of the rat brain were not affected by 24 hours of total sleep deprivation, or by 24 or 48 hours of REM sleep deprivation. In order to assess the effect of REM sleep deprivation on adenosine A/sub 1/ receptors, /sup 3/H-L-PIA binding was measured. The Bmax values for /sup 3/H-L-PIA binding to membrane preparations of the cortices and corpus striata from 48 hour REM sleep-deprived animals were increased 14.8% and 23%, respectively. These increases were not maintained following the cessation of sleep deprivation and recovered within 2 hours. The results of a 96 hour REM deprivation experiment were similar to those of the 48 hour REM sleep deprivation experiment. However, these increases were not evident in similar structures taken from stress control animals, and conclusively demonstrated that the changes in /sup 3/H-L-PIA binding resulted from REM sleep deprivation and not from stress.

  9. Potassium and chloride conductances in rat Leydig cells: effects of gonadotrophins and cyclic adenosine monophosphate.

    PubMed Central

    Duchatelle, P; Joffre, M

    1990-01-01

    1. The effects of gonadotrophins (luteinizing hormone and human chorionic gonadotrophin) and cyclic AMP on ionic conductances were investigated using the tight-seal whole-cell recording technique in Leydig cells freshly isolated from nature rat testis by enzymatic treatment. 2. In resting cells, the predominant ionic conductance is a voltage-dependent K+ conductance resembling the delayed rectifier K+ conductance of T-lymphocytes. This conductance is characterized by: (1) a time-dependent inactivation for potentials more positive than +20 mV, (2) a reversal potential near -65 mV, (3) a sensitivity to intracellular Cs+, and (4) a sensitivity to extracellular TEA and 4-aminopyridine. 3. A Cl- conductance is also present resembling the Cl- background conductance in squid axons and heart cells. In resting cells, this conductance contributes only a small component of the total outward current obtained with depolarizing pulses. 4. Gonadotrophins (human chorionic gonadotrophin, porcine luteinizing hormone and ovine luteinizing hormone) have little effect on the K+ conductance. They transiently increase a Cl- conductance after a delay of up to 30 s. This response does not occur if the hormones are applied late in the whole-cell recording. Gonadoliberine (GnRH) does not affect the Cl- or K+ conductance. 5. Internal cyclic AMP (100 microM) mimics all these effects while internal application of a GTP-ATP mixture induces a similar response, which is, however, sustained rather than transient. 6. The Cl- conductance was studied quantitatively with a GTP-ATP internal solution. This conductance is activated by depolarizing voltage steps to test potentials of -40 mV or more. Under these conditions, the instantaneous current observed as soon as the depolarizing pulse is applied displays outward rectification and reverses near ECl. During the pulses, a strong inactivation is observed for potentials greater than +40 mV. This conductance is independent of external and internal calcium. 7. It is concluded that the gonadotrophins act through a cyclic AMP-dependent process to activate a Cl- conductance. This conductance is different to the hyperpolarization-activated Cl- conductance and the calcium-activated Cl-conductance also present in the membrane of resting cells. PMID:1700109

  10. 3',5'-cyclic adenosine monophosphate and adenylate cyclase in phototransduction by limulus ventral photoreceptors.

    PubMed Central

    Brown, J E; Kaupp, U B; Malbon, C C

    1984-01-01

    Biochemical and electrophysiological measurements were made on photoreceptor cells from Limulus ventral eyes to investigate the possible role of cyclic AMP and adenylate cyclase in the visual transduction mechanism. Cyclic AMP content in a photoreceptor-enriched fraction (the end organs) of Limulus ventral eyes was approximately 15 pmol/mg protein. The cyclic AMP content was increased by bathing eyes in 1-methyl-3-isobutyl xanthine or forskolin and was increased almost 100-fold when bathed in both. Illumination did not change cyclic AMP content significantly in any of these conditions. Discrete events that can be recorded electrophysiologically occur spontaneously in darkness. An increase in the frequency of discrete events is evoked by dim illumination. The discrete events are a sign of excitation of Limulus photoreceptor cells. Drug-induced changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness were not correlated with changes in cyclic AMP content. Adenylate cyclase activity measured from a small number of pooled photoreceptor clusters was stimulated by fluoride and vanadate ions, hydrolysis-resistant analogues of GTP, cholera toxin and forskolin. The Limulus enzyme is similar pharmacologically to mammalian and avian adenylate cyclases. Activation of adenylate cyclase by drugs was not correlated with changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness. A heat-treated Lubrol extract of membranes from Limulus ventral eyes reconstituted the adenylate cyclase activity of membranes from S49 mouse lymphoma cyc- mutant cells which lack a functional regulatory protein. These findings suggest that Limulus ventral eye photoreceptors contain a regulatory protein that mediates the activation of adenylate cyclase by guanine nucleotides, fluoride or cholera toxin. This regulatory protein is homologous with that found in mammalian and avian adenylate cyclases. Our findings suggest that neither cyclic AMP nor adenylate cyclase activation is a necessary or obligatory component of the excitation mechanism in Limulus ventral photoreceptors. PMID:6207288

  11. Coordinatively Unsaturated Lanthanide(III) Helicates: Luminescence Sensors for Adenosine Monophosphate in Aqueous Media.

    PubMed

    Sahoo, Jashobanta; Arunachalam, Rajendran; Subramanian, Palani S; Suresh, Eringathodi; Valkonen, Arto; Rissanen, Kari; Albrecht, Markus

    2016-08-01

    Coordinatively unsaturated double-stranded helicates [(H2 L)2 Eu2 (NO3 )2 (H2 O)4 ](NO3 )4 , [(H2 L)2 Tb2 (H2 O)6 ](NO3 )6 , and [(H2 L)2 Tb2 (H2 O)6 ]Cl6 (H2 L=butanedioicacid-1,4-bis[2-(2-pyridinylmethylene)hydrazide]) are easily obtained by self-assembly from the ligand and the corresponding lanthanide(III) salts. The complexes are characterized by X-ray crystallography showing the helical arrangement of the ligands. Co-ligands at the metal ions can be easily substituted by appropriate anions. A specific luminescence response of AMP in presence of ADP, ATP, and other anions is observed. Specificity is assigned to the perfect size match of AMP to bridge the two metal centers and to replace quenching co-ligands in the coordination sphere. PMID:27346062

  12. Microheterogeneity of adenosine cyclic monophosphate-dependent protein kinases from mouse brain and heart.

    PubMed Central

    Malkinson, A M; Gharrett, A J; Hogy, L

    1978-01-01

    1. DEAE-cellulose chromatography of mouse brain cytosol indicated the presence of only the type II isoenzyme of cyclic AMP-dependent protein kinase. Mouse heart cytosol contained approximately equal amounts of the type I and type II isoenzymes. 2. Both brain and heart type II isoenzymes reassociated after a transient exposure to cyclic AMP, but the heart type I isoenzyme remained dissociated. 3. Elution of brain cytosol continuously exposed to cyclic AMP resolved multiple peaks of protein kinase and cyclic AMP-binding activities. A single peak of kinase and multiple peaks of cyclic AMP-binding activities were found under the same conditions with heart cytosol. Various control experiments suggested that the heterogeneity within the brain type II isoenzymic class had not been caused by proteolysis. 4. Kinetic experiments with unfractionated brain cytosol showed that the binding of cyclic AMP, the dissociation of cyclic AMP from protein and the rate of heat denaturation of the cyclic AMP-binding activity gave results consistent with the presence of multiple binding species. 5. It concluded that the type II isoenzymic peak obtained by DEAE-cellulose chromatography of mouse brain cytosol represents a class of enzymes containing multiple regulatory and catalytic subunits. The two heart cytosol isoenzymes contain a common catalytic subunit. The degree of protein kinase 'microheterogeneity", defined as the presence of multiple regulatory and/or catalytic subunits within a single isoenzymic class, appears to be tissue-specific. PMID:217338

  13. Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

    PubMed Central

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-01-01

    Cyclic AMP-activated intestinal Cl− secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl− secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl− secretion in human intestinal epithelial (T84) cells with IC50 of ∼20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl− current showed that diclofenac reversibly inhibited CFTR Cl− channel activity (IC50∼10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na+-K+ ATPases and Na+-K+-Cl− cotransporters, but inhibited cAMP-activated basolateral K+ channels with IC50 of ∼3 µM. In addition, diclofenac suppressed Ca2+-activated Cl− channels, inwardly rectifying Cl− channels, and Ca2+-activated basolateral K+ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl− secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca2+-activated Cl− secretion by inhibiting both apical Cl− channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  14. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

    PubMed

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  15. Structure of the DNA Ligase-Adenylate Intermediate: Lysine (ε-amino)-Linked Adenosine Monophosphoramidate*

    PubMed Central

    Gumport, Richard I.; Lehman, I. R.

    1971-01-01

    Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5′-monophosphate linked to the ε-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (ε-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the ε-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (εamino)-linked adenosine monophosphoramidate. PMID:4944632

  16. AMP-activated protein kinase: an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases.

    PubMed

    Srivastava, Rai Ajit K; Pinkosky, Stephen L; Filippov, Sergey; Hanselman, Jeffrey C; Cramer, Clay T; Newton, Roger S

    2012-12-01

    The adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor of energy metabolism at the cellular as well as whole-body level. It is activated by low energy status that triggers a switch from ATP-consuming anabolic pathways to ATP-producing catabolic pathways. AMPK is involved in a wide range of biological activities that normalizes lipid, glucose, and energy imbalances. These pathways are dysregulated in patients with metabolic syndrome (MetS), which represents a clustering of major cardiovascular risk factors including diabetes, lipid abnormalities, and energy imbalances. Clearly, there is an unmet medical need to find a molecule to treat alarming number of patients with MetS. AMPK, with multifaceted activities in various tissues, has emerged as an attractive drug target to manage lipid and glucose abnormalities and maintain energy homeostasis. A number of AMPK activators have been tested in preclinical models, but many of them have yet to reach to the clinic. This review focuses on the structure-function and role of AMPK in lipid, carbohydrate, and energy metabolism. The mode of action of AMPK activators, mechanism of anti-inflammatory activities, and preclinical and clinical findings as well as future prospects of AMPK as a drug target in treating cardio-metabolic disease are discussed. PMID:22798688

  17. An SNP in Exon 11 of Chicken 5'-AMP-Activated Protein Kinase Gamma 3 Subunit Gene was Associated with Meat Water Holding Capacity.

    PubMed

    Yang, Yunzhou; Xiong, Dan; Yao, Ling; Zhao, Chunjiang

    2016-01-01

    The 5'-Adenosine-monophosphate -activated protein kinase plays a key role in regulating cellular energy homeostasis, and it was reported that nucleotide variants in the genes coding the protein were associated with meat quality. In the present study, genetic variations in the exons of gamma non-catalytic subunit genes of the protein kinase were screened among 284 White Plymouth Rock chickens from 7 families with denaturing gradient gel electrophoresis, and their meat quality traits including drip loss and cooked meat rate which reflect water holding capacity and serum biochemical indices were also measured, and the association between the genotypes and the traits was analyzed with a SAS GLM procedure. Our results showed that there were three G/A nucleotide variants including one in exon 6 of the gamma subunit 2 gene and two in exon 11 of the gamma subunit 3 gene, which resulted in amino acid substitutions V150I, V315 M, and A337 T, respectively. And locus V315 M was associated with water holding capacity significantly (P < 0.05). The studied polymorphic locus has the potential to be used as a genetic marker for poultry breeding work. PMID:26597656

  18. Effects of water-misting sprays with forced ventilation on post mortem glycolysis, AMP-activated protein kinase and meat quality of broilers after transport during summer.

    PubMed

    Jiang, Nannan; Xing, Tong; Han, Minyi; Deng, Shaolin; Xu, Xinglian

    2016-05-01

    Effects of water-misting sprays with forced ventilation on post mortem glycolysis, adenosine monophosphate-activated protein kinase (AMPK) and meat quality of broilers after transport during summer were investigated in the present paper. A total of 105 mixed-sex Arbor Acres broilers were divided into three treatment groups: (i) 45 min transport without rest (T); (ii) 45 min transport with 1 h rest (TR); and (iii) 45 min transport with 15 min water-misting sprays with forced ventilation and 45 min rest (TWFR). Each treatment consisted of five replicates with seven birds each. The results indicated that the water-misting sprays with forced ventilation could mitigate the stress caused by transport under high temperature conditions during summer, which reduced the energy depletion in post mortem Pectoralis major (PM) muscle. This resulted in a higher energy status compared to the T group, which would decrease the expression of phosphorylation of AMPK (p-AMPK). Furthermore, decreased the expression of p-AMPK then slowed down the rate of glycolysis in post mortem PM muscle during the early post mortem period, which in turn lessened the negative effects caused by transport on meat quality. In conclusion, water-misting sprays with forced ventilation may be a better method to control the incidence of the pale, soft and exudative meat in broilers. PMID:26712455

  19. Coffee polyphenol caffeic acid but not chlorogenic acid increases 5'AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle.

    PubMed

    Tsuda, Satoshi; Egawa, Tatsuro; Ma, Xiao; Oshima, Rieko; Kurogi, Eriko; Hayashi, Tatsuya

    2012-11-01

    Chlorogenic acid is an ester of caffeic and quinic acids, and is one of the most widely consumed polyphenols because it is abundant in foods, especially coffee. We explored whether chlorogenic acid and its metabolite, caffeic acid, act directly on skeletal muscle to stimulate 5'-adenosine monophosphate-activated protein kinase (AMPK). Incubation of rat epitrochlearis muscles with Krebs buffer containing caffeic acid (≥0.1 mM, ≥30 min) but not chlorogenic acid increased the phosphorylation of AMPKα Thr(172), an essential step for kinase activation, and acetyl CoA carboxylase Ser(79), a downstream target of AMPK, in a dose- and time-dependent manner. Analysis of isoform-specific AMPK activity revealed that AMPKα2 activity increased significantly, whereas AMPKα1 activity did not change. This enzyme activation was associated with a reduction in phosphocreatine content and an increased rate of 3-O-methyl-d-glucose transport activity in the absence of insulin. These results suggest that caffeic acid but not chlorogenic acid acutely stimulates skeletal muscle AMPK activity and insulin-independent glucose transport with a reduction of the intracellular energy status. PMID:22227267

  20. AMP-activated protein kinase: an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases

    PubMed Central

    Srivastava, Rai Ajit K.; Pinkosky, Stephen L.; Filippov, Sergey; Hanselman, Jeffrey C.; Cramer, Clay T.; Newton, Roger S.

    2012-01-01

    The adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor of energy metabolism at the cellular as well as whole-body level. It is activated by low energy status that triggers a switch from ATP-consuming anabolic pathways to ATP-producing catabolic pathways. AMPK is involved in a wide range of biological activities that normalizes lipid, glucose, and energy imbalances. These pathways are dysregulated in patients with metabolic syndrome (MetS), which represents a clustering of major cardiovascular risk factors including diabetes, lipid abnormalities, and energy imbalances. Clearly, there is an unmet medical need to find a molecule to treat alarming number of patients with MetS. AMPK, with multifaceted activities in various tissues, has emerged as an attractive drug target to manage lipid and glucose abnormalities and maintain energy homeostasis. A number of AMPK activators have been tested in preclinical models, but many of them have yet to reach to the clinic. This review focuses on the structure-function and role of AMPK in lipid, carbohydrate, and energy metabolism. The mode of action of AMPK activators, mechanism of anti-inflammatory activities, and preclinical and clinical findings as well as future prospects of AMPK as a drug target in treating cardio-metabolic disease are discussed. PMID:22798688

  1. GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

    1992-11-01

    Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[β-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[γ-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[β-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

  2. Expression and activity of the 5'-AMP-activated protein kinase pathway in selected tissues during chicken embryonic development.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 5’-AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine protein kinase and a key part of a kinase signaling cascade that senses cellular energy status (AMP/ATP ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating m...

  3. Adenosine kinase inhibitors attenuate opiate withdrawal via adenosine receptor activation.

    PubMed

    Kaplan, G B; Coyle, T S

    1998-11-27

    Previous studies have demonstrated a role for adenosine in mediating opiate effects. This study examines the effects of indirect activation of adenosine receptors, via treatment with adenosine kinase inhibitors, on the expression of opiate withdrawal in mice. Mice receive chronic morphine treatment via implantation of subcutaneous morphine pellets (75 mg) for 72 h. Mice then receive parenteral treatment with adenosine kinase inhibitors, either 5'-amino-5'-deoxyadenosine (2, 5, 20, 40 mg/kg, intraperitoneal or i.p.) or iodotubericidin (1, 2, 5 mg/kg, i.p.), followed by naloxone injection and opiate withdrawal signs are measured over 20 min. Both adenosine kinase inhibitors significantly reduce the following opiate withdrawal signs in a dose-dependent manner compared to vehicle: withdrawal jumps, teeth chattering, forepaw tremors, and forepaw treads. Additionally, 5'-amino-5'-deoxyadenosine significantly reduces withdrawal-induced diarrhea and weight loss. Effects of 5'-amino-5'-deoxyadenosine (40 mg/kg) on opiate withdrawal signs appear to be mediated via adenosine receptor activation as they are reversed by pretreatment by adenosine receptor antagonist caffeine (20 mg, i.p.) but not by selective phosphodiesterase inhibitor Ro 20-1724 (10 mg/kg, i.p.). Adenosine receptor activation via adenosine kinase inhibitor treatment attenuates opiate withdrawal and these agents may be generally useful in the treatment of drug withdrawal syndromes. PMID:9865523

  4. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    SciTech Connect

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  5. Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

    SciTech Connect

    Nakatsu, Yusuke; Kotake, Yaichiro Hino, Atsuko; Ohta, Shigeru

    2008-08-01

    AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release.

  6. AMP Activated Protein Kinase Is Indispensable for Myocardial Adaptation to Caloric Restriction in Mice

    PubMed Central

    Chen, Kai; Kobayashi, Satoru; Xu, Xianmin; Viollet, Benoit; Liang, Qiangrong

    2013-01-01

    Caloric restriction (CR) is a robust dietary intervention known to enhance cardiovascular health. AMP activated protein kinase (AMPK) has been suggested to mediate the cardioprotective effects of CR. However, this hypothesis remains to be tested by using definitive loss-of-function animal models. In the present study, we subjected AMPKα2 knockout (KO) mice and their wild type (WT) littermates to a CR regimen that reduces caloric intake by 20%–40% for 4 weeks. CR decreased body weight, heart weight and serum levels of insulin in both WT and KO mice to the same degree, indicating the effectiveness of the CR protocol. CR activated cardiac AMPK signaling in WT mice, but not in AMPKα2 KO mice. Correspondingly, AMPKα2 KO mice had markedly reduced cardiac function during CR as determined by echocardiography and hemodynamic measurements. The compromised cardiac function was associated with increased markers of oxidative stress, endoplasmic reticulum stress and myocyte apoptosis. Mechanistically, CR down-regulated the expression of ATP5g2, a subunit of mitochondrial ATP synthase, and reduced ATP content in AMPKα2 KO hearts, but not in WT hearts. In addition, CR accelerated cardiac autophagic flux in WT mice, but failed to do so in AMPKα2 KO mice. These results demonstrated that without AMPK, CR triggers adverse effects that can lead to cardiac dysfunction, suggesting that AMPK signaling pathway is indispensible for energy homeostasis and myocardial adaptation to CR, a dietary intervention that normally produces beneficial cardiac effects. PMID:23527250

  7. Isodihydrocapsiate stimulates plasma glucose uptake by activation of AMP-activated protein kinase.

    PubMed

    Hwang, Seung-Lark; Yang, Byung-Keun; Lee, Jai-Youl; Kim, Jeong-Han; Kim, Byung-Dong; Kim, Byung-Hong; Suh, Ki-Hyoung; Kim, Dae Young; Kim, Dae-Yong; Kim, Moon Sung; Song, Hebok; Park, Byeoung-Soo; Huh, Tae-Lin

    2008-06-27

    AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is implicated as a key factor in controlling whole body homeostasis, including fatty acid oxidation and glucose uptake. We report that a synthetic structural isomer of dihydrocapsiate, isodihydrocapsiate (8-methylnonanoic acid 3-hydroxy-4-methoxy benzyl ester) improves type 2 diabetes by activating AMPK through the LKB1 pathway. In L6 myotube cells, phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) and glucose uptake were significantly increased, whereas these effects were attenuated by an AMPK inhibitor, compound C. In addition, increased phosphorylation of AMPK and ACC by isodihydrocapsiate was significantly reduced by radicicol, an LKB1 destabilizer, suggesting that increased glucose uptake in L6 cells with isodihydrocapsiate treatment is predominantly accomplished by a LKB1-mediated AMPK activation pathway. Oral administration of isodihydrocapsiate to diabetic (db/db) mice reduced blood glucose levels by 40% after a 4-week treatment period. Our results support the development of isodihydrocapsiate as a potential therapeutic agent to target AMPK in type 2 diabetes. PMID:18435912

  8. Stimulation of IGF-binding protein-1 secretion by AMP-activated protein kinase.

    PubMed

    Lewitt, M S

    2001-04-20

    Insulin-like growth factor-binding protein-1 (IGFBP-1) is stimulated during intensive exercise and in catabolic conditions to very high concentrations, which are not completely explained by known regulators such as insulin and glucocorticoids. The role of AMP-activated protein kinase (AMPK), an important signaling system in lipid and carbohydrate metabolism, in regulating IGFBP-1 was studied in H4-II-E rat hepatoma cells. Arsenic(III) oxide and 5-aminoimidazole-4-carboxamide-riboside (AICAR) were used as activators. AICAR (150 microM) stimulated IGFBP-1 secretion twofold during a 5-h incubation (P = 0.002). Insulin (100 ng/ml) inhibited IGFBP-1 by 80% (P < 0.001), but this was completely abolished in the presence of 150 microM AICAR. The effect of dexamethasone in stimulating IGFBP-1 threefold was additive to the effect of AICAR (P < 0.001) and, in the presence of AICAR, was incompletely inhibited by insulin. In conclusion AMPK is identified as a novel regulatory pathway for IGFBP-1, stimulating secretion and blocking the inhibitory effect of insulin. PMID:11302732

  9. Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase

    PubMed Central

    Lipovka, Yulia; Chen, Hao; Vagner, Josef; Price, Theodore J.; Tsao, Tsu-Shuen; Konhilas, John P.

    2015-01-01

    Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signalling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity and cancer. AMPK is targeted by 17β-oestradiol (E2), the main circulating oestrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an oestrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit-binding domain. Finally, both ERs interacted with the upstream liver kinase B 1 (LKB1) kinase complex, which is required for E2-dependent activation of AMPK. We conclude that E2 activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα. PMID:26374855

  10. Suppression of Chemically Induced and Spontaneous Mouse Oocyte Activation by AMP-Activated Protein Kinase1

    PubMed Central

    Ya, Ru; Downs, Stephen M.

    2013-01-01

    ABSTRACT Oocyte activation is an important process triggered by fertilization that initiates embryonic development. However, parthenogenetic activation can occur either spontaneously or with chemical treatments. The LT/Sv mouse strain is genetically predisposed to spontaneous activation. LT oocytes have a cell cycle defect and are ovulated at the metaphase I stage instead of metaphase II. A thorough understanding of the female meiosis defects in this strain remains elusive. We have reported that AMP-activated protein kinase (PRKA) has an important role in stimulating meiotic resumption and promoting completion of meiosis I while suppressing premature parthenogenetic activation. Here we show that early activation of PRKA during the oocyte maturation period blocked chemically induced activation in B6SJL oocytes and spontaneous activation in LT/SvEiJ oocytes. This inhibitory effect was associated with high levels of MAPK1/3 activity. Furthermore, stimulation of PRKA partially rescued the meiotic defects of LT/Sv mouse oocytes in concert with correction of abnormal spindle pole localization of PRKA and loss of prolonged spindle assembly checkpoint activity. Altogether, these results confirm a role for PRKA in helping sustain the MII arrest in mature oocytes and suggest that dysfunctional PRKA contributes to meiotic defects in LT/SvEiJ oocytes. PMID:23390161

  11. Regulation of AMP-activated protein kinase by natural and synthetic activators

    PubMed Central

    Grahame Hardie, David

    2015-01-01

    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function. PMID:26904394

  12. AMP-activated kinase α2 deficiency protects mice from denervation-induced skeletal muscle atrophy.

    PubMed

    Guo, Yuting; Meng, Jin; Tang, Yinglong; Wang, Ting; Wei, Bin; Feng, Run; Gong, Bing; Wang, Huiwen; Ji, Guangju; Lu, Zhongbing

    2016-06-15

    AMP-activated protein kinase (AMPK) is a master regulator of skeletal muscle metabolic pathways. Recently, AMPK activation by AICAR has been shown to increase myofibrillar protein degradation in C2C12 myotubes via stimulating autophagy and ubiquitin proteasome system. However, the impact of AMPKα on denervation induced muscle atrophy has not been tested. In this study, we performed sciatic denervation on hind limb muscles in both wild type (WT) and AMPKα2(-/-) mice. We found that AMPKα was phosphorylated in atrophic muscles following denervation. In addition, deletion of AMPKα2 significantly attenuated denervation induced skeletal muscle wasting and protein degradation, as evidenced by preserved muscle mass and myofiber area, as well as lower levels of ubiquitinated protein, Atrogin-1 and MuRF-1 expression, and LC3-II/I ratio in tibial anterior (TA) muscles. Interestingly, the phosphorylated FoxO3a at Ser253 was significantly decreased in atrophic TA muscles, which was preserved in AMPKα2(-/-) mice. Collectively, our data support the notion that the activation of AMPKα2 contributes to the atrophic effects of denervation. PMID:27136709

  13. AMP-activated protein kinase regulates the assembly of epithelial tight junctions.

    PubMed

    Zhang, Li; Li, Ji; Young, Lawrence H; Caplan, Michael J

    2006-11-14

    AMP activated protein kinase (AMPK), a sensor of cellular energy status in all eukaryotic cells, is activated by LKB1-dependent phosphorylation. Recent studies indicate that activated LKB1 induces polarity in epithelial cells and that this polarization is accompanied by the formation of tight junction structures. We wished to determine whether AMPK also contributes to the assembly of tight junctions in the epithelial cell polarization process. We found that AMPK is activated during calcium-induced tight junction assembly. Activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside facilitates tight junction assembly under conditions of normal extracellular Ca2+ concentrations and initiates tight junction assembly in the absence of Ca2+ as revealed by the relocation of zonula occludens 1, the establishment of transepithelial electrical resistance, and the paracellular flux assay. Expression of a dominant negative AMPK construct inhibits tight junction assembly in MDCK cells, and this defect in tight junction assembly can be partially ameliorated by rapamycin. These results suggest that AMPK plays a role in the regulation of tight junction assembly. PMID:17088526

  14. Ghrelin protects infarcted myocardium by induction of autophagy and AMP-activated protein kinase pathway.

    PubMed

    Yuan, Ming-Jie; Kong, Bin; Wang, Tao; Wang, Xin; Huang, He; Maghsoudi, Taneen

    2016-08-01

    The majority of studies have reported that enhancing autophagy in the myocardium is cardioprotective. Here, we tested the hypothesis that ghrelin, a growth hormone-releasing peptide, will protect infarcted myocardium by inducing of autophagy. Myocardial infarction was induced in mice by left coronary artery ligation the surviving mice 24 h after surgical were started on 2 week treatments with one of the following: vehicle, acylated ghrelin(50 mg/kg per day) or acylated ghrelin plus 3-MA(an autophagy inhibitor, 15 mg/kg, per day). We found that ghrelin significantly improved the cardiac function, and autophagy was enhanced by elevated LC3-II/LC-I ratio and mRNA expression of autophagy related protein. In vitro, cultured neonatal rat ventricular cardiomyocytes were subjected to simulate ischemia/reperfusion, 3-MA significantly attenuated ghrelin-induced autophagy, which was associated with activated AMP-activated protein kinase (AMPK) signal pathway. Moreover, ghrelin reduced cell death, and RNAi-mediated knockdown of autophagy protein 5 (Atg5) partly abolished ghrelin's cardioprotective effect. It is the first time to demonstrate that the cardioprotective effect of ghrelin on ischemia myocardium in part through regulating of autophagy signal pathway. PMID:27235554

  15. Unraveling the actions of AMP-activated protein kinase in metabolic diseases: Systemic to molecular insights.

    PubMed

    Weikel, Karen A; Ruderman, Neil B; Cacicedo, José M

    2016-05-01

    AMP-activated protein kinase (AMPK) plays a critical role both in sensing and regulating cellular energy state. In experimental animals, its activation has been shown to reduce the risk of obesity and diabetes-related co-morbidities such as insulin resistance, the metabolic syndrome and atherosclerotic cardiovascular disease. However, in humans, AMPK activation alone often does not completely resolve these conditions. Thus, an improved understanding of AMPK action and regulation in metabolic and other diseases is needed. Herein, we provide a brief description of the enzymatic regulation of AMPK and review its role in maintaining energy homeostasis. We then discuss tissue-specific actions of AMPK that become distorted during such conditions as obesity, type 2 diabetes and certain cancers. Finally, we explore recent findings regarding the interactions of AMPK with mammalian target of rapamycin complex 1 and the lysosome and discuss how changes in these relationships during overnutrition may lead to AMPK dysfunction. A more thorough understanding of AMPK's molecular interactions during diseases of overnutrition may provide key insights for the development of AMPK-based combinatorial treatments for metabolic disease. PMID:27085772

  16. Regulation of AMP-activated protein kinase by natural and synthetic activators.

    PubMed

    Grahame Hardie, David

    2016-01-01

    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function. PMID:26904394

  17. Sasa borealis extract exerts an antidiabetic effect via activation of the AMP-activated protein kinase.

    PubMed

    Nam, Jung Soo; Chung, Hee Jin; Jang, Min Kyung; Jung, In Ah; Park, Seong Ha; Cho, Su In; Jung, Myeong Ho

    2013-02-01

    Leaf of Sasa borealis, a species of bamboo, has been reported to exhibit anti-hyperglycemic effect. However, its antidiabetic mechanism is not fully understood. In this study, we examined whether an extract of S. borealis activates AMP-activated protein kinase (AMPK) and exerts anti-hyperglycemic effects. Treatment with the S. borealis extract increased insulin signaling and phosphorylation of AMPK and stimulated the expression of its downstream targets, including PPARα, ACO, and CPT-1 in C2C12 cells and PPARα in HepG2 cells. However, inhibition of AMPK activation attenuated insulin signaling and prevented the stimulation of AMPK target genes. The S. borealis extract increased glucose uptake in C2C12 cells and suppressed expression of the gluconeogenic gene, PEPCK in HepG2 cells. The extract significantly reduced blood glucose and triglyceride levels in STZ-induced diabetic mice. The extract enhanced AMPK phosphorylation and increased Glut-4 expression in the skeletal muscle of the mice. These findings demonstrated that the S. borealis extract exerts its anti-hyperglycemic effect through activation of AMPK and enhancement of insulin signaling. PMID:23423690

  18. Sasa borealis extract exerts an antidiabetic effect via activation of the AMP-activated protein kinase

    PubMed Central

    Nam, Jung Soo; Chung, Hee Jin; Jang, Min Kyung; Jung, In Ah; Park, Seong Ha; Cho, Su In

    2013-01-01

    Leaf of Sasa borealis, a species of bamboo, has been reported to exhibit anti-hyperglycemic effect. However, its antidiabetic mechanism is not fully understood. In this study, we examined whether an extract of S. borealis activates AMP-activated protein kinase (AMPK) and exerts anti-hyperglycemic effects. Treatment with the S. borealis extract increased insulin signaling and phosphorylation of AMPK and stimulated the expression of its downstream targets, including PPARα, ACO, and CPT-1 in C2C12 cells and PPARα in HepG2 cells. However, inhibition of AMPK activation attenuated insulin signaling and prevented the stimulation of AMPK target genes. The S. borealis extract increased glucose uptake in C2C12 cells and suppressed expression of the gluconeogenic gene, PEPCK in HepG2 cells. The extract significantly reduced blood glucose and triglyceride levels in STZ-induced diabetic mice. The extract enhanced AMPK phosphorylation and increased Glut-4 expression in the skeletal muscle of the mice. These findings demonstrated that the S. borealis extract exerts its anti-hyperglycemic effect through activation of AMPK and enhancement of insulin signaling. PMID:23423690

  19. AMP-activated Protein Kinase Signaling Activation by Resveratrol Modulates Amyloid-β Peptide Metabolism*

    PubMed Central

    Vingtdeux, Valérie; Giliberto, Luca; Zhao, Haitian; Chandakkar, Pallavi; Wu, Qingli; Simon, James E.; Janle, Elsa M.; Lobo, Jessica; Ferruzzi, Mario G.; Davies, Peter; Marambaud, Philippe

    2010-01-01

    Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) peptide deposition into cerebral amyloid plaques. The natural polyphenol resveratrol promotes anti-aging pathways via the activation of several metabolic sensors, including the AMP-activated protein kinase (AMPK). Resveratrol also lowers Aβ levels in cell lines; however, the underlying mechanism responsible for this effect is largely unknown. Moreover, the bioavailability of resveratrol in the brain remains uncertain. Here we show that AMPK signaling controls Aβ metabolism and mediates the anti-amyloidogenic effect of resveratrol in non-neuronal and neuronal cells, including in mouse primary neurons. Resveratrol increased cytosolic calcium levels and promoted AMPK activation by the calcium/calmodulin-dependent protein kinase kinase-β. Direct pharmacological and genetic activation of AMPK lowered extracellular Aβ accumulation, whereas AMPK inhibition reduced the effect of resveratrol on Aβ levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Aβ. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral Aβ levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease. PMID:20080969

  20. AMP-activated protein kinase activation protects gastric epithelial cells from Helicobacter pylori-induced apoptosis.

    PubMed

    Lv, Guoqiang; Zhu, Huanhuan; Zhou, Feng; Lin, Zhou; Lin, Gang; Li, Chenwan

    2014-10-10

    Helicobacter pylori (H pylori), infecting half of the world's population, causes gastritis, duodenal and gastric ulcer, and gastric cancers. AMP-activated protein kinase (AMPK) is a highly conserved regulator of cellular energy and metabolism. Recent studies indicated an important role for AMPK in promoting cell survival. In this study, we discovered that H Pylori induced AMPK activation in transformed (GEC-1 line) and primary human gastric epithelial cells (GECs). Inhibition of H Pylori-stimulated AMPK kinase activity by AMPK inhibitor compound C exacerbated apoptosis in transformed and primary GECs. Meanwhile, downregulation of AMPK expression by targeted shRNAs promoted apoptosis in H pylori-infected GECs. In contrast, A-769662 and resveratrol, two known AMPK activators, or AMPKα1 over-expression, enhanced H Pylori-induced AMPK activation, and inhibited GEC apoptosis. Our data suggested that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) could be the upstream kinase for AMPK activation by H pylori. Partial depletion of TAK1 by shRNAs not only inhibited AMPK activation, but also suppressed survival of H pylori-infected GECs. Taken together, these results suggest that TAK1-dependent AMPK activation protects GECs from H pylori-Induced apoptosis. PMID:25229685

  1. AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity

    SciTech Connect

    Kim, Jae-Hwan; Choi, Soo-Youn; Kang, Byung-Hee; Lee, Soon-Min; Cho, Eun-Jung; Youn, Hong-Duk

    2013-02-01

    Highlights: ► AMPK phosphorylates CtBP1 on serine 158. ► AMPK-mediated phosphorylation of CtBP1 causes the ubiquitination and nuclear export of CtBP1. ► AMPK downregulates the CtBP1-mediated repression of Bax transcription. -- Abstract: CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

  2. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo

    PubMed Central

    Domise, Manon; Didier, Sébastien; Marinangeli, Claudia; Zhao, Haitian; Chandakkar, Pallavi; Buée, Luc; Viollet, Benoit; Davies, Peter; Marambaud, Philippe; Vingtdeux, Valérie

    2016-01-01

    Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer’s disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology. PMID:27230293

  3. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo.

    PubMed

    Domise, Manon; Didier, Sébastien; Marinangeli, Claudia; Zhao, Haitian; Chandakkar, Pallavi; Buée, Luc; Viollet, Benoit; Davies, Peter; Marambaud, Philippe; Vingtdeux, Valérie

    2016-01-01

    Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer's disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology. PMID:27230293

  4. AMP-activated Protein Kinase As a Target For Pathogens: Friends Or Foes?

    PubMed

    Moreira, Diana; Silvestre, Ricardo; Cordeiro-da-Silva, Anabela; Estaquier, Jérôme; Foretz, Marc; Viollet, Benoit

    2016-01-01

    Intracellular pathogens are known to manipulate host cell regulatory pathways to establish an optimal environment for their growth and survival. Pathogens employ active mechanisms to hijack host cell metabolism and acquire existing nutrient and energy store. The role of the cellular energy sensor AMP-activated protein kinase (AMPK) in the regulation of cellular energy homeostasis is well documented. Here, we highlight recent advances showing the importance of AMPK signaling in pathogen-host interactions. Pathogens interact with AMPK by a variety of mechanisms aimed at reprogramming host cell metabolism to their own benefit. Stimulation of AMPK activity provides an efficient process to rapidly adapt pathogen metabolism to the major nutritional changes often encountered during the different phases of infection. However, inhibition of AMPK is also used by pathogens to manipulate innate host response, indicating that AMPK appears relevant to restriction of pathogen infection. We also document the effects of pharmacological AMPK modulators on pathogen proliferation and survival. This review illustrates intricate pathogen-AMPK interactions that may be exploited to the development of novel anti-pathogen therapies. PMID:25882224

  5. GSK621 Targets Glioma Cells via Activating AMP-Activated Protein Kinase Signalings

    PubMed Central

    Jiang, Hong; Liu, Wei; Zhan, Shi-Kun; Pan, Yi-Xin; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang; Pan, Si-Jian

    2016-01-01

    Here, we studied the anti-glioma cell activity by a novel AMP-activated protein kinase (AMPK) activator GSK621. We showed that GSK621 was cytotoxic to human glioma cells (U87MG and U251MG lines), possibly via provoking caspase-dependent apoptotic cell death. Its cytotoxicity was alleviated by caspase inhibitors. GSK621 activated AMPK to inhibit mammalian target of rapamycin (mTOR) and downregulate Tetraspanin 8 (Tspan8) in glioma cells. AMPK inhibition, through shRNA knockdown of AMPKα or introduction of a dominant negative (T172A) AMPKα, almost reversed GSK621-induced AMPK activation, mTOR inhibition and Tspan8 degradation. Consequently, GSK621’s cytotoxicity in glioma cells was also significantly attenuated by AMPKα knockdown or mutation. Further studies showed that GSK621, at a relatively low concentration, significantly potentiated temozolomide (TMZ)’s sensitivity and lethality against glioma cells. We summarized that GSK621 inhibits human glioma cells possibly via activating AMPK signaling. This novel AMPK activator could be a novel and promising anti-glioma cell agent. PMID:27532105

  6. The dynamical mechanism of auto-inhibition of AMP-activated protein kinase.

    PubMed

    Peng, Cheng; Head-Gordon, Teresa

    2011-07-01

    We use a novel normal mode analysis of an elastic network model drawn from configurations generated during microsecond all-atom molecular dynamics simulations to analyze the mechanism of auto-inhibition of AMP-activated protein kinase (AMPK). A recent X-ray and mutagenesis experiment (Chen, et al Nature 2009, 459, 1146) of the AMPK homolog S. Pombe sucrose non-fermenting 1 (SNF1) has proposed a new conformational switch model involving the movement of the kinase domain (KD) between an inactive unphosphorylated open state and an active or semi-active phosphorylated closed state, mediated by the autoinhibitory domain (AID), and a similar mutagenesis study showed that rat AMPK has the same auto-inhibition mechanism. However, there is no direct dynamical evidence to support this model and it is not clear whether other functionally important local structural components are equally inhibited. By using the same SNF1 KD-AID fragment as that used in experiment, we show that AID inhibits the catalytic function by restraining the KD into an unproductive open conformation, thereby limiting local structural rearrangements, while mutations that disrupt the interactions between the KD and AID allow for both the local structural rearrangement and global interlobe conformational transition. Our calculations further show that the AID also greatly impacts the structuring and mobility of the activation loop. PMID:21814500

  7. A Cell-Autonomous Molecular Cascade Initiated by AMP-Activated Protein Kinase Represses Steroidogenesis

    PubMed Central

    Abdou, Houssein S.; Bergeron, Francis

    2014-01-01

    Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production. PMID:25225331

  8. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    PubMed Central

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2014-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit. PMID:24286368

  9. Adenosine receptor desensitization and trafficking.

    PubMed

    Mundell, Stuart; Kelly, Eamonn

    2011-05-01

    As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. PMID:20550943

  10. Genetics Home Reference: adenosine deaminase 2 deficiency

    MedlinePlus

    ... Health Conditions adenosine deaminase 2 deficiency adenosine deaminase 2 deficiency Enable Javascript to view the expand/collapse ... PDF Open All Close All Description Adenosine deaminase 2 (ADA2) deficiency is a disorder characterized by abnormal ...

  11. Adenosine and the adenosine A2A receptor agonist, CGS21680, upregulate CD39 and CD73 expression through E2F-1 and CREB in regulatory T cells isolated from septic mice.

    PubMed

    Bao, Rui; Shui, Xianqi; Hou, Jiong; Li, Jinbao; Deng, Xiaoming; Zhu, Xiaoyan; Yang, Tao

    2016-09-01

    The number of regulatory T cells (Treg cells) and the expression of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1; also known as CD39) and 5'-ectonucleotidase (NT5E; also known as CD73) on the Treg cell surface are increased during sepsis. In this study, to determine the factors leading to the high expression of CD39 and CD73, and the regulation of the CD39/CD73/adenosine pathway in Treg cells under septic conditions, we constructed a mouse model of sepsis and separated the Treg cells using a flow cytometer. The Treg cells isolated from the peritoneal lavage and splenocytes of the mice were treated with adenosine or the specific adenosine A2A receptor agonist, CGS21680, and were transfected with specific siRNA targeting E2F transcription factor 1 (E2F-1) or cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), which are predicted transcription regulatory factors of CD39 or CD73. The regulatory relationships among these factors were then determined by western blot analysis and dual-luciferase reporter assay. In addition, changes in adenosine metabolism were measured in the treated cells. The results revealed that adenosine and CGS21680 significantly upregulated CD39 and CD73 expression (P<0.01). E2F-1 and CREB induced CD39 and CD73 expression, and were upregulated by adenosine and CGS21680. Adenosine triphosphate (ATP) hydrolysis and adenosine generation were inhibited by the knockdown of E2F-1 or CREB, and were accelerated in the presence of CGS21680. Based on these results, it can be inferred that adenosine, the adenosine A2A receptor agonist, E2F-1 and CREB are the possible factors contributing to the high expression of CD39 and CD73 on the Treg cell surface during sepsis. Adenosine and its A2A receptor agonist served as the signal transducer factors of the CD39/CD73/adenosine pathway, accelerating adenosine generation. Our study may benefit further research on adenosine metabolism for the treatment of sepsis

  12. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    PubMed Central

    Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

    2014-01-01

    The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

  13. Augmenting energy expenditure by mitochondrial uncoupling: a role of AMP-activated protein kinase.

    PubMed

    Klaus, Susanne; Keipert, Susanne; Rossmeisl, Martin; Kopecky, Jan

    2012-07-01

    Strategies to prevent and treat obesity aim to decrease energy intake and/or increase energy expenditure. Regarding the increase of energy expenditure, two key intracellular targets may be considered (1) mitochondrial oxidative phosphorylation, the major site of ATP production, and (2) AMP-activated protein kinase (AMPK), the master regulator of cellular energy homeostasis. Experiments performed mainly in transgenic mice revealed a possibility to ameliorate obesity and associated disorders by mitochondrial uncoupling in metabolically relevant tissues, especially in white adipose tissue (WAT), skeletal muscle (SM), and liver. Thus, ectopic expression of brown fat-specific mitochondrial uncoupling protein 1 (UCP1) elicited major metabolic effects both at the cellular/tissue level and at the whole-body level. In addition to expected increases in energy expenditure, surprisingly complex phenotypic effects were detected. The consequences of mitochondrial uncoupling in WAT and SM are not identical, showing robust and stable obesity resistance accompanied by improvement of lipid metabolism in the case of ectopic UCP1 in WAT, while preservation of insulin sensitivity in the context of high-fat feeding represents the major outcome of muscle UCP1 expression. These complex responses could be largely explained by tissue-specific activation of AMPK, triggered by a depression of cellular energy charge. Experimental data support the idea that (1) while being always activated in response to mitochondrial uncoupling and compromised intracellular energy status in general, AMPK could augment energy expenditure and mediate local as well as whole-body effects; and (2) activation of AMPK alone does not lead to induction of energy expenditure and weight reduction. PMID:22139637

  14. AMP-activated protein kinase is involved in perfluorohexanesulfonate -induced apoptosis of neuronal cells.

    PubMed

    Lee, Youn Ju; Choi, So-Young; Yang, Jae-Ho

    2016-04-01

    Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds (PFCs), has been used in a variety of industrial and consumer applications and detected in serum in the general population. This raised a concern over its possible detrimental health effects, including neurotoxic effects. We have previously shown that PFHxS induced neuronal apoptosis via the NMDA receptor-mediated extracellular signal-regulated kinase (ERK) pathway. Recently, it has been reported that AMP-activated protein kinase (AMPK) acts as a key signal molecule in neuronal excitotoxicity as well as providing a neuroprotective function. In the present study, we have examined the involvement of AMPK in PFHxS-induced neuronal apoptosis using neuronal differentiated PC12 cells. PFHxS induced significant increases in intracellular [Ca(2+)] via the NMDA receptor and the L-type voltage-gated calcium channel (L-VGCC). The inhibition of Ca(2+) loading by the NMDA receptor antagonist, MK801 and the L-VGCC blockers, nifedipine and diltiazem significantly reduced PFHxS-induced apoptosis. PFHxS induced sustained activation of AMPK and the inhibition of AMPK activation by compound C and AMPK siRNA significantly reduced PFHxS-induced caspase-3 activity. These results indicate the pro-apoptotic role of AMPK. The activation of AMPK was attenuated by MK801, nifedipine and diltiazem. However, the activation of AMPK was not affected by the ERK inhibitor, PD98059. Likewise, ERK activation was not affected by compound C but was substantially reduced by MK801, nifedipine or diltiazem. This suggests that the activation of AMPK and ERK is regulated by intracellular Ca(2+) loading in distinct pathways. Taken together, PFHxS-induced neuronal apoptosis is mediated by AMPK and ERK pathways, which are distinctly regulated by increased intracellular Ca(2+) via the NMDA receptor and L-VGCC. PMID:26826296

  15. AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit.

    PubMed

    Hamilton, Stephen R; O'Donnell, John B; Hammet, Andrew; Stapleton, David; Habinowski, Susan A; Means, Anthony R; Kemp, Bruce E; Witters, Lee A

    2002-05-10

    The AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase important for the responses to metabolic stress. It consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, and is regulated both by the allosteric action of AMP and by phosphorylation of the alpha and beta subunits catalyzed by AMPKK(s) and autophosphorylation. The Thr172 site on the alpha subunit has been previously characterized as an activating phosphorylation site. Using bacterially expressed AMPK alpha1 subunit proteins, we have explored the role of Thr172-directed AMPKKs in alpha subunit regulation. Recombinant alpha1 subunit proteins, representing the N-terminus, have been expressed as maltose binding protein (MBP) 6x His fusion proteins and purified to homogeneity by Ni(2+) chromatography. Both wild-type alpha1(1-312) and alpha1(1-312)T172D are inactive when expressed in bacteria, but the former can be fully phosphorylated (1 mol/mol) on Thr172 and activated by a surrogate AMPKK, CaMKKbeta. The corresponding AMPKalpha1(1-392), an alpha construct containing its autoinhibitory sequence, can be similarly phosphorylated, but it remains inactive. In an insulinoma cell line, either low glucose or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) treatment leads to activation and T172 phosphorylation of endogenous AMPK. Under the same conditions of cell incubation, we have identified an AMPKK activity that both phosphorylates and activates the recombinant alpha1(1-312), but this Thr172-directed AMPKK activity is unaltered by low glucose or AICAR, indicating that it is constitutively active. PMID:12051742

  16. Small Molecule Antagonizes Autoinhibition and Activates AMP-activated Protein Kinase in Cells*

    PubMed Central

    Pang, Tao; Zhang, Zhen-Shan; Gu, Min; Qiu, Bei-Ying; Yu, Li-Fang; Cao, Peng-Rong; Shao, Wei; Su, Ming-Bo; Li, Jing-Ya; Nan, Fa-Jun; Li, Jia

    2008-01-01

    AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK α1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in α subunits, we screened a chemical library with inactive human α1394 (α1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK α1394, α1335, α2398, and even heterotrimer α1β1γ1. Based on PT1-docked AMPK α1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK. PMID:18321858

  17. Activation of AMP-Activated Protein Kinase by Interleukin-6 in Rat Skeletal Muscle

    PubMed Central

    Kelly, Meghan; Gauthier, Marie-Soleil; Saha, Asish K.; Ruderman, Neil B.

    2009-01-01

    OBJECTIVE Interleukin-6 (IL-6) directly activates AMP-activated protein kinase (AMPK) in vivo and in vitro; however, the mechanism by which it does so is unknown. RESEARCH DESIGN AND METHODS We examined this question in skeletal muscle using an incubated rat extensor digitorum longus (EDL) muscle preparation as a tool. RESULTS AMPK activation by IL-6 coincided temporally with a nearly threefold increase in the AMP:ATP ratio in the EDL. The effects of IL-6 on both AMPK activity and energy state were inhibited by coincubation with propranolol, suggesting involvement of β-adrenergic signaling. In keeping with this notion, IL-6 concurrently induced a transient increase in cAMP, and its ability to activate AMPK was blocked by the adenyl cyclase inhibitor 2′5′-dideoxyadenosine. In addition, like other β-adrenergic stimuli, IL-6 increased glycogen breakdown and lipolysis in the EDL. Similar effects of IL-6 on AMPK, energy state, and cAMP content were observed in C2C12 myotubes and gastrocnemius muscle in vivo, indicating that they were not unique to the incubated EDL. CONCLUSIONS These studies demonstrate that IL-6 activates AMPK in skeletal muscle by increasing the concentration of cAMP and, secondarily, the AMP:ATP ratio. They also suggest that substantial increases in IL-6 concentrations, such as those that can result from its synthesis by muscles during exercise, may play a role in the mobilization of fuel stores within skeletal muscle as an added means of restoring energy balance. PMID:19502419

  18. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    PubMed Central

    Brandauer, Josef; Vienberg, Sara G; Andersen, Marianne A; Ringholm, Stine; Risis, Steve; Larsen, Per S; Kristensen, Jonas M; Frøsig, Christian; Leick, Lotte; Fentz, Joachim; Jørgensen, Sebastian; Kiens, Bente; Wojtaszewski, Jørgen F P; Richter, Erik A; Zierath, Juleen R; Goodyear, Laurie J; Pilegaard, Henriette; Treebak, Jonas T

    2013-01-01

    Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild-type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2-containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post-translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related. PMID:23918774

  19. Osthole enhances glucose uptake through activation of AMP-activated protein kinase in skeletal muscle cells.

    PubMed

    Lee, Wei-Hwa; Lin, Ren-Jye; Lin, Shyr-Yi; Chen, Yu-Chien; Lin, Hsiu-Ming; Liang, Yu-Chih

    2011-12-28

    AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Activation of AMPK in skeletal muscles, the liver, and adipose tissues results in a favorable metabolic milieu for preventing and treating type 2 diabetes, i.e., decreased levels of circulating glucose, plasma lipids, and ectopic fat accumulation and enhanced insulin sensitivity. Osthole was extracted from a Chinese herbal medicine, and we found that it had glucose lowering activity in our previous study. However, the detailed glucose lowering mechanisms of osthole are still unclear. In this study, we used skeletal muscle cells to examine the underlying molecular mechanisms of osthole's glucose lowering activity. A Western blot analysis revealed that osthole significantly induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Next, we found that osthole significantly increased the level of translocation of glucose transporter 4 (GLUT4) to plasma membranes and glucose uptake in a dose-dependent manner. Osthole-induced glucose uptake was reversed by treatment with Compound C, an AMPK inhibitor, suggesting that osthole-induced glucose uptake was mediated in an AMPK-dependent manner. The increase in the AMP:ATP ratio was involved in osthole's activation of AMPK. Finally, we found that osthole counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that the increase in the AMP:ATP ratio by osthole triggered activation of the AMPK signaling pathway and led to increases in plasma membrane GLUT4 content and glucose uptake level. Therefore, osthole might have potential as an antidiabetic agent for treating diabetes. PMID:22098542

  20. Lymphocytes accelerate epithelial tight junction assembly: role of AMP-activated protein kinase (AMPK).

    PubMed

    Tang, Xiao Xiao; Chen, Hao; Yu, Sidney; Zhang, Li; Caplan, Michael J; Chan, Hsiao Chang

    2010-01-01

    The tight junctions (TJs), characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK) cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK). AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK. PMID:20808811

  1. Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2016-02-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.

  2. Antithrombin Up-regulates AMP-activated Protein Kinase Signaling during Myocardial Ischemia/Reperfusion Injury

    PubMed Central

    Ma, Yina; Wang, Jinli; Gao, Junjie; Yang, Hui; Wang, Yanqing; Manithody, Chandrashekhara; Li, Ji; Rezaie, Alireza R.

    2014-01-01

    Summary Antithrombin (AT) is a protein of the serpin superfamily involved in regulation of the proteolytic activity of the serine proteases of the coagulation system. AT is known to exhibit anti-inflammatory and cardioprotective properties when it binds to heparan sulfate proteoglycans (HSPGs) on vascular cells. AMP-activated protein kinase (AMPK) plays an important cardioprotective role during myocardial ischemia and reperfusion (I/R). To determine whether the cardioprotective signaling function of AT is mediated through the AMPK pathway, we evaluated the cardioprotective activities of wild-type AT and its two derivatives, one having high affinity and the other no affinity for heparin, in an acute I/R injury model in C57BL/6J mice in which the left anterior descending coronary artery was occluded. The serpin derivatives were given 5 min before reperfusion. The results showed that AT-WT can activate AMPK in both in vivo and ex vivo conditions. Blocking AMPK activity abolished the cardioprotective function of AT against I/R injury. The AT derivative having high affinity for heparin was more effective in activating AMPK and in limiting infraction, but the derivative lacking affinity for heparin was inactive in eliciting AMPK-dependent cardioprotective activity. Activation of AMPK by AT inhibited the inflammatory c-Jun N-terminal protein kinase (JNK) pathway during I/R. Further studies revealed that the AMPK activity induced by AT also modulates cardiac substrate metabolism by increasing glucose oxidation but inhibiting fatty acid oxidation during I/R. These results suggest that AT binds to HSPGs on heart tissues to invoke a cardioprotective function by triggering cardiac AMPK activation, thereby attenuating JNK inflammatory signaling pathways and modulating substrate metabolism during I/R. PMID:25230600

  3. AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides*

    PubMed Central

    Ishibashi, Yohei; Hirabayashi, Yoshio

    2015-01-01

    The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer. PMID:26048992

  4. AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

    PubMed Central

    Thavarajah, Thanusi; Medvedev, Sergei; Bowden, Peter; Marshall, John G.; Antonescu, Costin N.

    2015-01-01

    The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute

  5. AMP-activated protein kinase suppresses matrix metalloproteinase-9 expression in mouse embryonic fibroblasts.

    PubMed

    Morizane, Yuki; Thanos, Aristomenis; Takeuchi, Kimio; Murakami, Yusuke; Kayama, Maki; Trichonas, George; Miller, Joan; Foretz, Marc; Viollet, Benoit; Vavvas, Demetrios G

    2011-05-01

    Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPKα deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPKα1 and AMPKα2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. The AMPK activators 5-amino-4-imidazole carboxamide riboside and A769662 suppressed MMP-9 expression in WT MEFs, and AMPK inhibition by the overexpression of dominant negative (DN) AMPKα elevated MMP-9 expression. However, in AMPKα(-/-) MEFs transduced with DN AMPKα, MMP-9 expression was suppressed. AMPKα(-/-) MEFs showed increased phosphorylation of IκBα, expression of IκBα mRNA, nuclear localization of nuclear factor-κB (NF-κB), and DNA-binding activity of NF-κB compared with WT. Consistently, selective NF-κB inhibitors BMS345541 and SM7368 decreased MMP-9 expression in AMPKα(-/-) MEFs. Overall, our results suggest that both AMPKα isoforms suppress MMP-9 expression and that both the activity and presence of AMPKα contribute to its function as a regulator of MMP-9 expression by inhibiting the NF-κB pathway. PMID:21402702

  6. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  7. Hypoxia promotes drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling

    PubMed Central

    Zhao, Changfu; Zhang, Qiao; Yu, Tao; Sun, Shudong; Wang, Wenjun; Liu, Guangyao

    2016-01-01

    Purpose Drug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling. Experimental design We investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia. Results It was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin. Conclusion The present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy. PMID

  8. Neurochemical measurement of adenosine in discrete brain regions of five strains of inbred mice.

    PubMed

    Pani, Amar K; Jiao, Yun; Sample, Kenneth J; Smeyne, Richard J

    2014-01-01

    Adenosine (ADO), a non-classical neurotransmitter and neuromodulator, and its metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), have been shown to play an important role in a number of biochemical processes. Although their signaling is well described, it has been difficult to directly, accurately and simultaneously quantitate these purines in tissue or fluids. Here, we describe a novel method for measuring adenosine (ADO) and its metabolites using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Using this chromatographic technique, we examined baseline levels of ADO and ATP, ADP and AMP in 6 different brain regions of the C57BL/6J mouse: stratum, cortex, hippocampus, olfactory bulb, substantia nigra and cerebellum and compared ADO levels in 5 different strains of mice (C57BL/6J, Swiss-Webster, FVB/NJ, 129P/J, and BALB/c). These studies demonstrate that baseline levels of purines vary significantly among the brain regions as well as between different mouse strains. These dissimilarities in purine concentrations may explain the variable phenotypes among background strains described in neurological disease models. PMID:24642754

  9. Profiling Hepatic microRNAs in Zebrafish: Fluoxetine Exposure Mimics a Fasting Response That Targets AMP-Activated Protein Kinase (AMPK)

    PubMed Central

    Craig, Paul M.; Trudeau, Vance L.; Moon, Thomas W.

    2014-01-01

    This study examined the similarities in microRNA profiles between fasted and fluoxetine (FLX) exposed zebrafish and downstream target transcripts and biological pathways. Using a custom designed microarray targeting 270 zebrafish miRNAs, we identified 9 differentially expressed miRNAs targeting transcripts in biological pathways associated with anabolic metabolism, such as adipogenesis, cholesterol biosynthesis, triacylglycerol synthesis, and insulin signaling. Exposure of female zebrafish to 540 ng/L FLX, an environmentally relevant concentration and a known metabolic repressor, increased specific miRNAs indicating greater inhibition of these pathways in spite of continued feeding. Further examination revealed two specific miRNAs, dre-let-7d and dre-miR-140-5p, were predicted in silico to bind to a primary regulator of metabolism, adenosine monophosphate-activated protein kinase (AMPK), and more specifically the two isoforms of the catalytic subunit, AMPKα1 and α2, respectively. Real-time analysis of the relative transcript abundance of the α1 and α2 mRNAs indicated a significant inverse relationship between specific miRNA and target transcript. This suggests that AMPK-related pathways may be compromised during FLX exposure as a result of increased miRNA abundance. The mechanism by which FLX regulates miRNA abundance is unknown but may be direct at the liver. The serotonin transporter, slc6a4, is the target of FLX and other selective serotonin reuptake inhibitors (SSRI) and it was found to be expressed in the liver, although treatment did not alter expression of this transporter. Exposure to FLX disrupts key hepatic metabolic pathways, which may be indicative of reduced overall fitness and these effects may be linked to specific miRNA abundance. This has important implications for the heath of fish because concentrations of SSRIs in aquatic ecosystems are continually increasing. PMID:24751937

  10. Houttuynia cordata attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells.

    PubMed

    Kang, Hyun; Koppula, Sushruta

    2014-01-01

    Houttuynia cordata (H. cordata) from the family Saururaceae is a perennial herb native to Southeast Asia. It possesses a range of medicinal properties to treat several disease symptoms including allergic inflammation and anaphylaxis. In the present investigation, we provided the molecular mechanisms underlying the role of H. cordata extract (HCE) in the prevention of high glucose-induced lipid accumulation in human HepG2 hepatocytes. HepG2 cells were pre-treated with various concentrations of HCE (0, 10, 20, 40, and 80 μg/mL) and treated with serum-free medium with normal glucose (5 mM) for 1 h, followed by exposure to high glucose (25 mM D-glucose) for 24 h. HCE significantly and dose-dependently attenuated lipid accumulation in human HepG2 hepatocytes when exposed to high glucose (25 mM D-glucose) (p < 0.05, p < 0.01 and p < 0.001 at 20, 40, and 80 μg/mL concentrations, respectively). Further, HCE attenuated the expression of fatty acid synthase (FAS), sterol regulatory element-binding protein-1 and glycerol 3-phosphate acyltransferases (GPATs). The adenosine monophosphate-activated protein kinase (AMPK) was also activated by HCE treatment when exposed to high glucose (25 mM D-glucose) in human HepG2 hepatocytes. This study suggests the hypolipidemic effects of HCE by the inhibition of lipid biosynthesis mediated through AMPK signaling, which may play an active role and can be developed as an anti-obesity agent. PMID:24871657

  11. Caffeine and contraction synergistically stimulate 5′-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle

    PubMed Central

    Tsuda, Satoshi; Egawa, Tatsuro; Kitani, Kazuto; Oshima, Rieko; Ma, Xiao; Hayashi, Tatsuya

    2015-01-01

    5′-Adenosine monophosphate-activated protein kinase (AMPK) has been identified as a key mediator of contraction-stimulated insulin-independent glucose transport in skeletal muscle. Caffeine acutely stimulates AMPK in resting skeletal muscle, but it is unknown whether caffeine affects AMPK in contracting muscle. Isolated rat epitrochlearis muscle was preincubated and then incubated in the absence or presence of 3 mmol/L caffeine for 30 or 120 min. Electrical stimulation (ES) was used to evoke tetanic contractions during the last 10 min of the incubation period. The combination of caffeine plus contraction had additive effects on AMPKα Thr172 phosphorylation, α-isoform-specific AMPK activity, and 3-O-methylglucose (3MG) transport. In contrast, caffeine inhibited basal and contraction-stimulated Akt Ser473 phosphorylation. Caffeine significantly delayed muscle fatigue during contraction, and the combination of caffeine and contraction additively decreased ATP and phosphocreatine contents. Caffeine did not affect resting tension. Next, rats were given an intraperitoneal injection of caffeine (60 mg/kg body weight) or saline, and the extensor digitorum longus muscle was dissected 15 min later. ES of the sciatic nerve was performed to evoke tetanic contractions for 5 min before dissection. Similar to the findings from isolated muscles incubated in vitro, the combination of caffeine plus contraction in vivo had additive effects on AMPK phosphorylation, AMPK activity, and 3MG transport. Caffeine also inhibited basal and contraction-stimulated Akt phosphorylation in vivo. These findings suggest that caffeine and contraction synergistically stimulate AMPK activity and insulin-independent glucose transport, at least in part by decreasing muscle fatigue and thereby promoting energy consumption during contraction. PMID:26471759

  12. Effects of an High-Fat Diet Enriched in Lard or in Fish Oil on the Hypothalamic Amp-Activated Protein Kinase and Inflammatory Mediators

    PubMed Central

    Viggiano, Emanuela; Mollica, Maria Pina; Lionetti, Lillà; Cavaliere, Gina; Trinchese, Giovanna; De Filippo, Chiara; Chieffi, Sergio; Gaita, Marcello; Barletta, Antonio; De Luca, Bruno; Crispino, Marianna; Monda, Marcellino

    2016-01-01

    The high fat diet (HFD) rich in lard induces obesity, inflammation and oxidative stress, and the deregulation of hypothalamic nuclei plays an important role in this mechanism. One important factor involved in the food intake and inflammation is adenosine monophosphate-dependent kinase (AMPK), a serine/threonine kinase activated by phosphorylation. Omega (ω)3-polyunsaturated fatty acids (PUFA) are dietary compounds known to attenuate the obesity-related diseases, although the molecular mechanisms underlying their actions in the hypothalamus are not completely understood. We hypothesized that the beneficial effects of PUFA may be mediated by AMPK in the hypothalamus. To this aim, rats were fed a control diet (CD), or isocaloric HFD containing either fish oil (FD; rich in ω3-PUFA) or lard for 6 weeks, and the activation of AMPK, inflammatory state (IKKβ, TNF-α) and oxidative stress were analyzed in the hypothalamus. In addition, we also studied serum lipid profile, homeostatic model assessment (HOMA) index, and pro-inflammatory parameters. Our results showed, at the hypothalamic level of LD-fed rats, an increase of AMPK activation, inflammation and oxidative stress, while no modifications were detected in FD-fed animals compared to CD. In addition body weight gain, serum lipid profile, pro-inflammatory parameters and insulin resistance were reduced in FD animals compared to LD. In conclusion, our data indicate that the substitution of saturated by unsaturated fatty acids in the diet has beneficial effects on modulation of hypothalamic inflammation and function in obesity, underlying, at hypothalamic level, the interaction among insulin and/or leptin resistance, AMPK activation and hyperphagia. PMID:27375435

  13. Adenosine-Associated Delivery Systems

    PubMed Central

    Kazemzadeh-Narbat, Mehdi; Annabi, Nasim; Tamayol, Ali; Oklu, Rahmi; Ghanem, Amyl; Khademhosseini, Ali

    2016-01-01

    Adenosine is a naturally occurring purine nucleoside in every cell. Many critical treatments such as modulating irregular heartbeat (arrhythmias), regulation of central nervous system (CNS) activity, and inhibiting seizural episodes can be carried out using adenosine. Despite the significant potential therapeutic impact of adenosine and its derivatives, the severe side effects caused by their systemic administration have significantly limited their clinical use. In addition, due to adenosine’s extremely short half-life in human blood (less than 10 s), there is an unmet need for sustained delivery systems to enhance efficacy and reduce side effects. In this paper, various adenosine delivery techniques, including encapsulation into biodegradable polymers, cell-based delivery, implantable biomaterials, and mechanical-based delivery systems, are critically reviewed and the existing challenges are highlighted. PMID:26453156

  14. AMP-activated protein kinase regulates L-arginine mediated cellular responses

    PubMed Central

    2013-01-01

    Background Our prior study revealed the loss in short-term L-Arginine (ARG) therapeutic efficacy after continuous exposure; resulting in tolerance development, mediated by endothelial nitric oxide synthase (eNOS) down-regulation, secondary to oxidative stress and induced glucose accumulation. However, the potential factor regulating ARG cellular response is presently unknown. Method Human umbilical vein endothelial cells were incubated with 100 μM ARG for 2 h in buffer (short-term or acute), or for 7 days in culture medium and challenged for 2 h in buffer (continuous or chronic), in the presence or absence of other agents. eNOS activity was determined by analyzing cellular nitrite/nitrate (NO2–/NO3–), and AMP-activated protein kinase (AMPK) activity was assayed using SAMS peptide. 13C6 glucose was added to medium to measure glucose uptake during cellular treatments, which were determined by LC-MS/MS. Cellular glucose was identified by o-toluidine method. Superoxide (O2•–) was identified by EPR-spin-trap, and peroxynitrite (ONOO–) was measured by flow-cytometer using aminophenyl fluorescein dye. Results Short-term incubation of cells with 100 μM ARG in the presence or absence of 30 μM L-NG-Nitroarginine methyl ester (L-NAME) or 30 μM AMPK inhibitor (compound C, CMP-C) increased cellular oxidative stress and overall glucose accumulation with no variation in glucose transporter-1 (GLUT-1), or AMPK activity from control. The increase in total NO2–/NO3– after 2 h 100 μM ARG exposure, was suppressed in cells co-incubated with 30 μM CMP-C or L-NAME. Long-term exposure of ARG with or without CMP-C or L-NAME suppressed NO2–/NO3–, glucose uptake, GLUT-1, AMPK expression and activity below control, and increased overall cellular glucose, O2•– and ONOO–. Gluconeogenesis inhibition with 30 μM 5-Chloro-2-N-2,5-dichlorobenzenesulfonamido-benzoxazole (CDB) during ARG exposure for 2 h maintained overall cellular glucose to control, but increased

  15. Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders.

    PubMed

    Viollet, B; Mounier, R; Leclerc, J; Yazigi, A; Foretz, M; Andreelli, F

    2007-12-01

    In the light of recent studies in humans and rodents, AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, has been described as an integrator of regulatory signals monitoring systemic and cellular energy status. AMP-activated protein kinase (AMPK) has been proposed to function as a 'fuel gauge' to monitor cellular energy status in response to nutritional environmental variations. Recently, it has been proposed that AMPK could provide a link in metabolic defects underlying progression to the metabolic syndrome. AMPK is a heterotrimeric enzyme complex consisting of a catalytic subunit alpha and two regulatory subunits beta and gamma. AMPK is activated by rising AMP and falling ATP. AMP activates the system by binding to the gamma subunit that triggers phosphorylation of the catalytic alpha subunit by the upstream kinases LKB1 and CaMKKbeta (calmodulin-dependent protein kinase kinase). AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. As well as acting at the level of the individual cell, the system also regulates food intake and energy expenditure at the whole body level, in particular by mediating the effects of insulin sensitizing adipokines leptin and adiponectin. AMPK is robustly activated during skeletal muscle contraction and myocardial ischaemia playing a role in glucose transport and fatty acid oxidation. In liver, activation of AMPK results in enhanced fatty acid oxidation as well as decreased glucose production. Moreover, the AMPK system is one of the probable targets for the anti-diabetic drugs biguanides and thiazolidinediones. Thus, the relationship between AMPK activation and beneficial metabolic

  16. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5′-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa)

    PubMed Central

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E.; Gallo-Reynoso, Juan P.

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5′-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5′-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP, guanosine 5′-diphosphate (GDP), guanosine 5′-triphosphate (GTP), and xanthosine 5′-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  17. Role of extracellular cysteine residues in the adenosine A2A receptor.

    PubMed

    De Filippo, Elisabetta; Namasivayam, Vigneshwaran; Zappe, Lukas; El-Tayeb, Ali; Schiedel, Anke C; Müller, Christa E

    2016-06-01

    The G protein-coupled A2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71(2.69)-Cys159(45.43); II, Cys74(3.22)-Cys146(45.30), and III, Cys77(3.25)-Cys166(45.50)). However, the A2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists' efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A2A and A2BARs. PMID:26969588

  18. Extracellular guanosine regulates extracellular adenosine levels

    PubMed Central

    Cheng, Dongmei; Jackson, Travis C.; Verrier, Jonathan D.; Gillespie, Delbert G.

    2013-01-01

    The aim of this investigation was to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels. Rat preglomerular vascular smooth muscle cells were incubated with adenosine, guanosine, or both. Guanosine (30 μmol/l) per se had little effect on extracellular adenosine levels. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) were 0.125 ± 0.020 μmol/l, indicating rapid disposition of extracellular adenosine. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) plus guanosine (30 μmol/l) were 1.173 ± 0.061 μmol/l, indicating slow disposition of extracellular adenosine. Cell injury increased extracellular levels of endogenous adenosine and guanosine, and the effects of cell injury on endogenous extracellular adenosine were modulated by altering the levels of endogenous extracellular guanosine with exogenous purine nucleoside phosphorylase (converts guanosine to guanine) or 8-aminoguanosine (inhibits purine nucleoside phosphorylase). Extracellular guanosine also slowed the disposition of extracellular adenosine in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts, and kidney epithelial cells and in human aortic and coronary artery vascular smooth muscle cells and coronary artery endothelial cells. The effects of guanosine on adenosine levels were not mimicked or attenuated by 5-iodotubericidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)-adenine (adenosine deaminase inhibitor), 5-aminoimidazole-4-carboxamide (guanine deaminase inhibitor), aristeromycin (S-adenosylhomocysteine hydrolase inhibitor), low sodium (inhibits concentrative nucleoside transporters), S-(4-nitrobenzyl)−6-thioinosine [inhibits equilibrative nucleoside transporter (ENT) type 1], zidovudine (inhibits ENT type 2), or acadesine (known modulator of adenosine levels). Guanosine also increases extracellular inosine, uridine, thymidine, and cytidine, yet decreases

  19. Activation of AMP-activated protein kinase reduces collagen production via p38 MAPK in cardiac fibroblasts induced by coxsackievirus B3.

    PubMed

    Jiang, Shengyang; Jiang, Donglin; Zhao, Peng; He, Xinlong; Tian, Shunli; Wu, Xueming; Tao, Yijia

    2016-07-01

    Collagen deposition is the major cause of myocardial fibrosis, contributing to impaired cardiac contractile function in coxsackie virus B3 (CVB3)-infected hearts. Adenosine monophosphate-activated protein kinase (AMPK) has been considered as a cellular fuel gauge and super metabolic regulator, however, whether AMPK has an effect on collagen production in CVB3‑infected heart remains to be elucidated. In the present study, the association between AMPK activation and CVB3‑infected neonatal rat cardiac fibroblasts (NRCFs) was investigated. Collagen production was determined by the hydroxyproline content of the supernatant and by the expression of type I/IV collagen in the cell lysate. Rat hydroxyproline ELISA was used to detect hydroxyproline content in the supernatant. The expression of type I/IV collagen, and the phosphorylation of AMPKα‑Thr172 and p38 in the cell lysate were evaluated using western blotting. As expected, it was found that the hydroxyproline content in the supernatant, and the production of collagen I/IV in the cell lysate were significantly promoted at 48 h post‑CVB3‑infection. However, this effect was inhibited in a dose‑dependent manner when pretreated with 5‑aminoimidazole‑4‑carboxamide‑1‑4‑ribofuranoside (AICAR) for 2 h prior to CVB3‑infection. However, if the cells were preincubated with compound C or SB203580 for 30 min prior the treatment with AICAR, the inhibitive effects of AICAR were reversed. The results of the western blotting indicated that the phosphorylation of AMPKα‑Thr172 and p38 were significantly increased by AICAR in the NRCFs. However, only the phosphorylation of p38 mitogen‑activated protein kinase (MAPK) was inhibited by SB203580. In conclusion, AMPK activation reduced collagen production via the p38 MAPK‑dependent pathway in the cardiac fibroblasts induced by CVB3. The results of the present study may contribute to identifying an effective therapy for CVB3‑induced myocarditis and CVB3

  20. AMP-activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma

    PubMed Central

    Li, Yen-Hsing; Luo, Jia; Mosley, Yung-Yi C.; Hedrick, Victoria E.; Paul, Lake N.; Chang, Julia; Zhang, GuangJun; Wang, Yu-Kuo; Banko, Max R.; Brunet, Anne; Kuang, Shihuan; Wu, Jen-Leih; Chang, Chun-Ju; Scott, Matthew P.; Yang, Jer-Yen

    2015-01-01

    Summary The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated and how energy sensor regulates Hh pathway is not clear. AMP-activated Protein Kinase (AMPK) is an important sensor of energy stores that controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This in turn leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency. PMID:26190112

  1. Piperlongumine as a potential activator of AMP-activated protein kinase in HepG2 cells.

    PubMed

    Ryu, Jahee; Kim, Myoung-Jin; Kim, Tae-Oh; Huh, Tae-Lin; Lee, Sung-Eun

    2014-01-01

    AMP-activated protein kinase (AMPK) is a key regulator of fatty acid biosynthesis and fatty acid oxidation throughout the body. Piperlongumine (PL) isolated from Piper longum (L.) was shown to potently upregulate activation of AMPK via phosphorylation and inactivation of acetyl-CoA carboxylases in cultured HepG2 cells, presumably enhancing the transfer of fatty acids into mitochondrial cells by inhibiting malonyl-CoA production. PL showed cytotoxicity on HepG2 cell growth at the concentration of 5 μM of PL, while more than 80% of HepG2 cells were survived at the concentration of 2 μM of PL. Overall, the results of this study indicate that PL activates AMPK phosphorylation and possesses cytotoxicity in HepG2 cells. PMID:24853732

  2. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    PubMed

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα. PMID:27151216

  3. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    PubMed

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-01

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase. PMID:24815694

  4. Activation of AMP-activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib

    PubMed Central

    Ishijima, Naoki; Kanki, Keita; Shimizu, Hiroki; Shiota, Goshi

    2015-01-01

    To improve the outcome of cancer chemotherapy, strategies to enhance the efficacy of anticancer drugs are required. Sorafenib is the only drug to prolong overall survival of the patients with hepatocellular carcinoma (HCC), however, the outcome is still not satisfactory. Retinoids, vitamin A derivatives, have been known to exhibit inhibitory effects on various cancers including HCC. In this study, we investigated the effects of combined treatment using sorafenib and retinoids including all-trans retinoic acid (ATRA), NIK-333, and Am80 on HCC cells. Cell viability assays in six HCC cell lines, HepG2, PLC/PRF/5, HuH6, HLE, HLF, and Hep3B, revealed that 5 and 10 μM ATRA, concentrations that do not exert cytotoxic effects, enhanced the cytotoxicity of sorafenib, being much more effective than NIK-333 and Am80. We found that ATRA induced AMP-activated protein kinase activation, which was followed by reduced intracellular ATP level. Gene expression analysis revealed that ATRA decreased the expression of glycolytic genes such as GLUT-1 and LDHA. In the combination treatment using ATRA and sorafenib, increased apoptosis, followed by the activation of p38 MAPK and JNK, the upregulation and translocation of Bax to mitochondria, and the activation of caspase-3, was observed. Suppression of AMP-activated protein kinase by siRNA restored the viability of the cells treated with ATRA and sorafenib. Our results thus indicate that ATRA is useful for enhancing the cytotoxicity of sorafenib against HCC cells by regulating the energy metabolism of HCC cells. PMID:25683251

  5. Activation of AMP-activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib.

    PubMed

    Ishijima, Naoki; Kanki, Keita; Shimizu, Hiroki; Shiota, Goshi

    2015-05-01

    To improve the outcome of cancer chemotherapy, strategies to enhance the efficacy of anticancer drugs are required. Sorafenib is the only drug to prolong overall survival of the patients with hepatocellular carcinoma (HCC), however, the outcome is still not satisfactory. Retinoids, vitamin A derivatives, have been known to exhibit inhibitory effects on various cancers including HCC. In this study, we investigated the effects of combined treatment using sorafenib and retinoids including all-trans retinoic acid (ATRA), NIK-333, and Am80 on HCC cells. Cell viability assays in six HCC cell lines, HepG2, PLC/PRF/5, HuH6, HLE, HLF, and Hep3B, revealed that 5 and 10 μM ATRA, concentrations that do not exert cytotoxic effects, enhanced the cytotoxicity of sorafenib, being much more effective than NIK-333 and Am80. We found that ATRA induced AMP-activated protein kinase activation, which was followed by reduced intracellular ATP level. Gene expression analysis revealed that ATRA decreased the expression of glycolytic genes such as GLUT-1 and LDHA. In the combination treatment using ATRA and sorafenib, increased apoptosis, followed by the activation of p38 MAPK and JNK, the upregulation and translocation of Bax to mitochondria, and the activation of caspase-3, was observed. Suppression of AMP-activated protein kinase by siRNA restored the viability of the cells treated with ATRA and sorafenib. Our results thus indicate that ATRA is useful for enhancing the cytotoxicity of sorafenib against HCC cells by regulating the energy metabolism of HCC cells. PMID:25683251

  6. A dual role for AMP-activated protein kinase (AMPK) during neonatal hypoxic-ischaemic brain injury in mice.

    PubMed

    Rousset, Catherine I; Leiper, Fiona C; Kichev, Anton; Gressens, Pierre; Carling, David; Hagberg, Henrik; Thornton, Claire

    2015-04-01

    Perinatal hypoxic-ischaemic encephalopathy (HIE) occurs in 1-2 in every 1000 term infants and the devastating consequences range from cerebral palsy, epilepsy and neurological deficit to death. Cellular damage post insult occurs after a delay and is mediated by a secondary neural energy failure. AMP-activated protein kinase (AMPK) is a sensor of cellular stress resulting from ATP depletion and/or calcium dysregulation, hallmarks of the neuronal cell death observed after HIE. AMPK activation has been implicated in the models of adult ischaemic injury but, as yet, there have been no studies defining its role in neonatal asphyxia. Here, we find that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is pathological activation of the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMPKα1 signalling pathway. Pharmacological inhibition of AMPK during the insult promotes neuronal survival but, conversely, inhibiting AMPK activity prior to the insult sensitizes neurons, exacerbating cell death. Our data have pathological relevance for neonatal HIE as prior sensitization such as exposure to bacterial infection (reported to reduce AMPK activity) produces a significant increase in injury. We show that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is a pathological activation of the CaMKKβ-AMPKα1 signalling pathway. Inhibiting AMPK during OGD promotes neuronal survival; conversely, inhibiting AMPK prior to OGD exacerbates cell death. Our data have clinical relevance as prior sensitization (e.g. exposure to bacterial infection reducing AMPK activity) increases injury. AMPK, AMP-activated protein kinase; HI, hypoxia-ischaemia; OGD, oxygen-glucose deprivation. PMID:25598140

  7. Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression

    PubMed Central

    Osawa, Yosuke; Seki, Ekihiro; Kodama, Yuzo; Suetsugu, Atsushi; Miura, Kouichi; Adachi, Masayuki; Ito, Hiroyasu; Shiratori, Yoshimune; Banno, Yoshiko; Olefsky, Jerrold M.; Nagaki, Masahito; Moriwaki, Hisataka; Brenner, David A.; Seishima, Mitsuru

    2011-01-01

    Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine-1-phosphate (S1P). Because sphingolipids regulate AKT activation, we investigated the role of ASM in hepatic glucose and lipid metabolism. Initially, we overexpressed ASM in the livers of wild-type and diabetic db/db mice by adenovirus vector (Ad5ASM). In these mice, glucose tolerance was improved, and glycogen and lipid accumulation in the liver were increased. Using primary cultured hepatocytes, we confirmed that ASM increased glucose uptake, glycogen deposition, and lipid accumulation through activation of AKT and glycogen synthase kinase-3β. In addition, ASM induced up-regulation of glucose transporter 2 accompanied by suppression of AMP-activated protein kinase (AMPK) phosphorylation. Loss of sphingosine kinase-1 (SphK1) diminished ASM-mediated AKT phosphorylation, but exogenous S1P induced AKT activation in hepatocytes. In contrast, SphK1 deficiency did not affect AMPK activation. These results suggest that the SphK/S1P pathway is required for ASM-mediated AKT activation but not for AMPK inactivation. Finally, we found that treatment with high-dose glucose increased glycogen deposition and lipid accumulation in wild-type hepatocytes but not in ASM−/− cells. This result is consistent with glucose intolerance in ASM−/− mice. In conclusion, ASM modulates AKT activation and AMPK inactivation, thus regulating glucose and lipid metabolism in the liver.—Osawa, Y., Seki, E., Kodama, Y., Suetsugu, A., Miura, K., Adachi, M., Ito, H., Shiratori, Y., Banno, Y., Olefsky, J. M., Nagaki, M., Moriwaki, H., Brenner, D. A., Seishima, M. Acid sphingomyelinase regulates glucose and lipid metabolism in hepatocytes through AKT activation and AMP-activated protein kinase suppression. PMID:21163859

  8. Adenosine Receptors and Membrane Microdomains

    PubMed Central

    Lasley, Robert D.

    2010-01-01

    Adenosine receptors are a member of the large family of seven transmembrane spanning G protein coupled receptors (GPCR). The four adenosine receptor subtypes – A1, A2a, A2b, A3 – exert their effects via the activation of one or more heterotrimeric G proteins resulting in the modulation of intracellular signaling. Numerous studies over the past decade have documented the complexity of GPCR signaling at the level of protein-protein interactions as well as through signaling crosstalk. With respect to adenosine receptors the activation of one receptor subtype can have profound direct effects in one cell type, but little or no effect in other cells. There is significant evidence that the compartmentation of subcellular signaling plays a physiological role in the fidelity of GPCR signaling. This compartmentation is evident at the level of the plasma membrane in the form of membrane microdomains such as caveolae and lipid rafts. This review will summarize and critically assess our current understanding of the role of membrane microdomains in regulating adenosine receptor signaling. PMID:20888790

  9. Xanthines as Adenosine Receptor Antagonists

    PubMed Central

    Jacobson, Kenneth A.

    2013-01-01

    The natural plant alkaloids caffeine and theophylline were the first adenosine receptor (AR) antagonists described in the literature. They exhibit micromolar affinities and are non-selective. A large number of derivatives and analogs have subsequently been synthesized and evaluated as AR antagonists. Very potent antagonists have thus been developed with selectivity for each of the four AR subtypes. PMID:20859796

  10. Regulation of adenosine levels during cerebral ischemia

    PubMed Central

    Chu, Stephanie; Xiong, Wei; Zhang, Dali; Soylu, Hanifi; Sun, Chao; Albensi, Benedict C; Parkinson, Fiona E

    2013-01-01

    Adenosine is a neuromodulator with its level increasing up to 100-fold during ischemic events, and attenuates the excitotoxic neuronal injury. Adenosine is produced both intracellularly and extracellularly, and nucleoside transport proteins transfer adenosine across plasma membranes. Adenosine levels and receptor-mediated effects of adenosine are regulated by intracellular ATP consumption, cellular release of ATP, metabolism of extracellular ATP (and other adenine nucleotides), adenosine influx, adenosine efflux and adenosine metabolism. Recent studies have used genetically modified mice to investigate the relative contributions of intra- and extracellular pathways for adenosine formation. The importance of cortical or hippocampal neurons as a source or a sink of adenosine under basal and hypoxic/ischemic conditions was addressed through the use of transgenic mice expressing human equilibrative nucleoside transporter 1 (hENT1) under the control of a promoter for neuron-specific enolase. From these studies, we conclude that ATP consumption within neurons is the primary source of adenosine in neuronal cultures, but not in hippocampal slices or in vivo mice exposed to ischemic conditions. PMID:23064722

  11. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    PubMed Central

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  12. The crystal structure and mechanism of orotidine 5'-monophosphate decarboxylase.

    PubMed

    Appleby, T C; Kinsland, C; Begley, T P; Ealick, S E

    2000-02-29

    The crystal structure of Bacillus subtilis orotidine 5'-monophosphate (OMP) decarboxylase with bound uridine 5'-monophosphate has been determined by multiple wavelength anomalous diffraction phasing techniques and refined to an R-factor of 19.3% at 2.4 A resolution. OMP decarboxylase is a dimer of two identical subunits. Each monomer consists of a triosephosphate isomerase barrel and contains an active site that is located across one end of the barrel and near the dimer interface. For each active site, most of the residues are contributed by one monomer with a few residues contributed from the adjacent monomer. The most highly conserved residues are located in the active site and suggest a novel catalytic mechanism for decarboxylation that is different from any previously proposed OMP decarboxylase mechanism. The uridine 5'-monophosphate molecule is bound to the active site such that the phosphate group is most exposed and the C5-C6 edge of the pyrimidine base is most buried. In the proposed catalytic mechanism, the ground state of the substrate is destabilized by electrostatic repulsion between the carboxylate of the substrate and the carboxylate of Asp60. This repulsion is reduced in the transition state by shifting negative charge from the carboxylate to C6 of the pyrimidine, which is close to the protonated amine of Lys62. We propose that the decarboxylation of OMP proceeds by an electrophilic substitution mechanism in which decarboxylation and carbon-carbon bond protonation by Lys62 occur in a concerted reaction. PMID:10681442

  13. Selective activation of adenosine A2A receptors on immune cells by a CD73-dependent prodrug suppresses joint inflammation in experimental rheumatoid arthritis.

    PubMed

    Flögel, Ulrich; Burghoff, Sandra; van Lent, Peter L E M; Temme, Sebastian; Galbarz, Lisa; Ding, Zhaoping; El-Tayeb, Ali; Huels, Sandra; Bönner, Florian; Borg, Nadine; Jacoby, Christoph; Müller, Christa E; van den Berg, Wim B; Schrader, Jürgen

    2012-08-01

    Adenosine A(2A) receptor (A(2A)R) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A(2A)R agonists (prodrugs) that require the presence of ecto-5'-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5'-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 ((19)F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A(2A)R. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A(2A)R expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A(2A)R agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation. PMID:22875828

  14. Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes.

    PubMed

    Ohta, Mitsuhiro; Fujinami, Aya; Kobayashi, Norihiro; Amano, Akiko; Ishigami, Akihito; Tokuda, Harukuni; Suzuki, Nobutaka; Ito, Fumitake; Mori, Taisuke; Sawada, Morio; Iwasa, Koichi; Kitawaki, Jo; Ohnishi, Katsunori; Tsujikawa, Muneo; Obayashi, Hiroshi

    2015-07-01

    4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are major components of n-hexane/ethyl acetate (5:1) extract of the yellow-colored stem juice of Angelica keiskei. 4-Hydroxyderricin and XAG have been reported to increase glucose transporter 4 (GLUT4)-dependent glucose uptake in 3T3-L1 adipocytes, but the detailed mechanism of this phenomenon remains unknown. This present study was aimed at clarifying the detailed mechanism by which 4HD and XAG increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes. Both 4HD and XAG increased glucose uptake and GLUT4 translocation to the plasma membrane. 4-Hydroxyderricin and XAG also stimulated the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase. In addition, phosphorylation of liver kinase B1 (LKB1), which acts upstream of AMPK, was also increased by 4HD and XAG treatment. Small interfering RNA knockdown of LKB1 attenuated 4HD- and XAG-stimulated AMPK phosphorylation and suppressed glucose uptake. These findings demonstrate that 4HD and XAG can increase GLUT4-dependent glucose uptake through the LKB1/AMPK signaling pathway in 3T3-L1 adipocytes. PMID:26077869

  15. Electron transfer between the QmoABC membrane complex and adenosine 5'-phosphosulfate reductase.

    PubMed

    Duarte, Américo G; Santos, André A; Pereira, Inês A C

    2016-04-01

    The dissimilatory adenosine 5'-phosphosulfate reductase (AprAB) is a key enzyme in the sulfate reduction pathway that catalyzes the reversible two electron reduction of adenosine 5'-phosphosulfate (APS) to sulfite and adenosine monophosphate (AMP). The physiological electron donor for AprAB is proposed to be the QmoABC membrane complex, coupling the quinone-pool to sulfate reduction. However, direct electron transfer between these two proteins has never been observed. In this work we demonstrate for the first time direct electron transfer between the Desulfovibrio desulfuricans ATCC 27774 QmoABC complex and AprAB. Cyclic voltammetry conducted with the modified Qmo electrode and AprAB in the electrolyte solution presented the Qmo electrochemical signature with two additional well-defined one electron redox processes, attributed to the AprAB FAD redox behavior. Moreover, experiments performed under catalytic conditions using the QmoABC modified electrode, with AprAB and APS in solution, show a catalytic current peak develop in the cathodic wave, attributed to substrate reduction, and which is not observed in the absence of QmoABC. Substrate dependence conducted with different electrode preparations (with and without immobilized Qmo) demonstrated that the QmoABC complex is essential for efficient electron delivery to AprAB, in order to sustain catalysis. These results confirm the role of Qmo in electron transfer to AprAB. PMID:26768116

  16. Nucleic acid molecules encoding isopentenyl monophosphate kinase, and methods of use

    DOEpatents

    Croteau, Rodney B.; Lange, Bernd M.

    2001-01-01

    A cDNA encoding isopentenyl monophosphate kinase (IPK) from peppermint (Mentha x piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of isopentenyl monophosphate kinase (SEQ ID NO:2), from peppermint (Mentha x piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for isopentenyl monophosphate kinase, or for a base sequence sufficiently complementary to at least a portion of isopentenyl monophosphate kinase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding isopentenyl monophosphate kinase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant isopentenyl monophosphate kinase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant isopentenyl monophosphate kinase may be used to obtain expression or enhanced expression of isopentenyl monophosphate kinase in plants in order to enhance the production of isopentenyl monophosphate kinase, or isoprenoids derived therefrom, or may be otherwise employed for the regulation or expression of isopentenyl monophosphate kinase, or the production of its products.

  17. Fluorescent Ligands for Adenosine Receptors

    PubMed Central

    Kozma, Eszter; Jayasekara, P Suresh; Squarcialupi, Lucia; Paoletta, Silvia; Moro, Stefano; Federico, Stephanie; Spalluto, Giampiero; Jacobson, Kenneth A.

    2012-01-01

    Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field. PMID:23200243

  18. Adenosine-induced activation of esophageal nociceptors.

    PubMed

    Ru, F; Surdenikova, L; Brozmanova, M; Kollarik, M

    2011-03-01

    Clinical studies implicate adenosine acting on esophageal nociceptive pathways in the pathogenesis of noncardiac chest pain originating from the esophagus. However, the effect of adenosine on esophageal afferent nerve subtypes is incompletely understood. We addressed the hypothesis that adenosine selectively activates esophageal nociceptors. Whole cell perforated patch-clamp recordings and single-cell RT-PCR analysis were performed on the primary afferent neurons retrogradely labeled from the esophagus in the guinea pig. Extracellular recordings were made from the isolated innervated esophagus. In patch-clamp studies, adenosine evoked activation (inward current) in a majority of putative nociceptive (capsaicin-sensitive) vagal nodose, vagal jugular, and spinal dorsal root ganglia (DRG) neurons innervating the esophagus. Single-cell RT-PCR analysis indicated that the majority of the putative nociceptive (transient receptor potential V1-positive) neurons innervating the esophagus express the adenosine receptors. The neural crest-derived (spinal DRG and vagal jugular) esophageal nociceptors expressed predominantly the adenosine A(1) receptor while the placodes-derived vagal nodose nociceptors expressed the adenosine A(1) and/or A(2A) receptors. Consistent with the studies in the cell bodies, adenosine evoked activation (overt action potential discharge) in esophageal nociceptive nerve terminals. Furthermore, the neural crest-derived jugular nociceptors were activated by the selective A(1) receptor agonist CCPA, and the placodes-derived nodose nociceptors were activated by CCPA and/or the selective adenosine A(2A) receptor CGS-21680. In contrast to esophageal nociceptors, adenosine failed to stimulate the vagal esophageal low-threshold (tension) mechanosensors. We conclude that adenosine selectively activates esophageal nociceptors. Our data indicate that the esophageal neural crest-derived nociceptors can be activated via the adenosine A(1) receptor while the placodes

  19. Role of AMP-activated protein kinase and carbohydrate response element binding protein in the regulation of energy balance in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme complex playing a key role in maintaining intracellular energy balance and, on the whole animal level, regulating energy expenditure and food intake. Once activated by phosphorylation, AMPK phosphorylates a variety of protein targets tha...

  20. AMP-activated protein kinase and carbohydrate response element binding protein: A study of two potential regulatory factors in the hepatic lipogenic program of broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study investigated the effects of fasting and refeeding on AMP-activated protein kinase (AMPK) and carbohydrate response element binding protein (ChREBP) mRNA, protein and activity levels; as well as the expression of lipogenic genes involved in regulating lipid synthesis in broiler chicken liv...

  1. Endogenous phosphorylation of microsomal proteins in bovine corpus luteum. Tenfold activation by adenosine 3′:5′-cyclic monophosphate

    PubMed Central

    Hardie, D. Grahame; Stansfield, David A.

    1977-01-01

    Free ribosomes and a smooth-microsomal fraction were prepared from bovine corpus luteum. Both preparations will self-phosphorylate when incubated with Mg2+ and ATP, but at low concentrations of Mg2+ and ATP the self-phosphorylation of the smooth-microsomal fraction was much more dependent on cyclic AMP than was that of free ribosomes, stimulation by the nucleotide being up to 10-fold in the former case. The self-phosphorylation of the smooth-microsomal fraction was studied further. The reaction bears similarities to that brought about by soluble cyclic AMP-dependent protein kinase, being inhibited by Ca2+ and the heat-stable inhibitor protein from skeletal muscle. Cyclic GMP will activate the reaction at concentrations higher than those required for full activation by cyclic AMP. In the presence of cyclic AMP, phosphate bound to protein is found almost exclusively as phosphoserine. Several proteins are phosphorylated, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the phosphorylation of all of them is markedly stimulated by cyclic AMP. If the reaction is carried out at high concentrations of Mg2+ and ATP, a distinct cyclic AMP-independent phosphorylation is observed. This activity is not inhibited by the heat-stable inhibitor protein, and phosphate is found esterified with both threonine and serine residues. PMID:195580

  2. Catecholaminergic activity and 3',5'-cyclic adenosine monophosphate levels in heart right ventricle after naloxone induced withdrawal.

    PubMed

    Milanés, M V; Fuente, T; Laorden, M L

    2000-01-01

    This study evaluated the adaptive changes in noradrenergic neurons and the concomitant production of cAMP during morphine dependence and withdrawal in the right ventricle of the rat. Rats were made dependent on morphine by morphine pellet implantation for 7 days. On the day of sacrifice animals received an acute injection of saline or naloxone (1 mg/kg s.c.) and were decapitated 30 min later. Pretreatment with propranolol 15 min prior to naloxone was conducted to evaluate the possible implication of beta-adrenoceptors. The contents of noradrenaline and dopamine and their metabolites were examined. After naloxone administration to morphine-dependent rats (withdrawal) there was a pronounced increase in the content of normetanephrine and 3,4-dihydroxyphenylacetic acid and increased noradrenaline and dopamine turnover. In addition cAMP levels were increased after naloxone administration to morphine-treated rats. Propranolol did not block the hyperactivity of catecholaminergic neurons or the enhancement of cAMP observed in the heart during withdrawal. The present results indicate that heart catecholaminergic neurons play a significant role in the alterations in heart functions during morphine abstinence syndrome and suggest that those alterations are mediated through cAMP. PMID:10651148

  3. Assay of adenosine 3',5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

    SciTech Connect

    Handa, A.K.; Bressan, R.A.

    1980-03-01

    In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many /sup 32/P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of (..gamma..-/sup 32/P)ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of (..gamma..-/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: (ATP) = (ATP)/sub 0/ e/sup -(cAMP)kt/. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the potein kinase stimulation assay based on transfer of (/sup 32/P) phosphate from (..gamma..-/sup 32/P)ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.

  4. Cyclic adenosine 5'-monophosphate and calcium induce CD152 (CTLA-4) up-regulation in resting CD4+ T lymphocytes.

    PubMed

    Vendetti, Silvia; Riccomi, Antonella; Sacchi, Alessandra; Gatta, Lucia; Pioli, Claudio; De Magistris, Maria Teresa

    2002-12-01

    The CTLA-4 (CD152) molecule is up-regulated upon T cell activation and proliferation, and plays a critical role in the inhibition of immune responses. We show in this study that cAMP induces up-regulation of CD152 in human CD4(+) T lymphocytes. This effect occurs in the absence of the up-regulation of CD69 and CD25 activation markers and T cell proliferation. In addition, we found that the Ca(2+) ionophore ionomycin also up-regulates CD152, and that the combination of a cAMP analog or cAMP inducers with ionomycin further enhances the expression of CD152 in resting CD4(+) T lymphocytes. However, cyclosporin A, which inhibits Ca(2+)/calcineurin signaling pathway, fully prevented the ionomycin- but not the cAMP-induced up-regulation of CD152. The effects of cAMP and ionomycin involve increase of both CD152 mRNA transcripts, coding for the membrane and the soluble forms of CD152. Furthermore, we show that CD152 molecules are translocated to the membrane and are functional, as their engagement by specific mAbs prevented NF-kappaB activation by anti-CD3/CD28 stimulation. These findings demonstrate that at least two novel signal pathways regulate CTLA-4 gene expression and CD152 molecule up-regulation in human CD4(+) T lymphocytes, in the absence of full T cell activation. PMID:12444128

  5. Identification, characterization, and hormonal regulation of 3', 5'-cyclic adenosine monophosphate-dependent protein kinases in rat Sertoli cells.

    PubMed

    Landmark, B F; Fauske, B; Eskild, W; Skålhegg, B; Lohmann, S M; Hansson, V; Jahnsen, T; Beebe, S J

    1991-11-01

    Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels. Unstimulated Sertoli cells contain high levels of R (approximately 9.2 +/- 0.8 pmol/mg protein) and C (approximately 7.3 +/- 0.7 pmol/mg protein). Stimulation with (Bt)2cAMP (0.1 mM) for 24 and 48 h revealed a time-dependent increase in [3H]cAMP-binding activity. During the same time period the catalytic activity remained relatively constant, resulting in an increase in the R/C ratio from approximately 1.3 to 3.0. Using diethylaminoethyl cellulose chromatography, 8-N3-[32P]cAMP photoaffinity labeling, autophosphorylation by gamma-[32P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RI alpha, 25% RII alpha, and very low levels of RII beta. Stimulation of Sertoli cells with (Bt)2cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RI alpha (6.6-14 pmol/mg) and a 10- to 20-fold increase in RII beta (less than 0.1-1.1 pmol/mg), with little or no change in RII alpha (1.9-2.3 pmol/mg). Treatment with cAMP was associated with a slight increase in RI/RII ratio (3.3-4.1). mRNA levels for RII beta increased 30- to 50-fold after (Bt)2cAMP stimulation, whereas only minor changes in mRNA levels for RI alpha, RII alpha, and C alpha were observed (1.5- to 2.0-fold). mRNA levels for RI beta, C beta, and C gamma were not detected in either unstimulated or in cAMP-stimulated Sertoli cells. It is concluded that chronic treatment with cAMP changes the relative proportion of R subunits of PKA in a manner reflecting the changing levels in respective mRNAs. Furthermore, such treatment is associated with the appearance of a new PKA R subunit (RII beta), which is absent in untreated Sertoli cells. PMID:1657573

  6. Micro-electrode studies on the effects of exogenous cyclic adenosine monophosphate on active sodium transport in frog skin.

    PubMed Central

    Els, W J; Mahlangu, A F

    1987-01-01

    1. The electrical parameters of the sodium-transporting cells in frog skin of Rana angolensis were determined under control conditions by using the micro-electrode technique. The data were analysed in terms of an electrical model (Helman, 1979). 2. The control intracellular voltages averaged -84.7 mV while the electromotive force of the inner barrier, E'1, averaged 103.9 mV. The major portion (82%) of the transcellular resistance was situated at the outer, apical, barrier. 3. Exogenous cyclic AMP stimulated active sodium transport and the short-circuit current (Isc) increased by an average 88%. The change in Isc was mediated primarily by decreasing the resistance of the apical barrier (Ro) with little effect on the electromotive force or resistance (Ri) of the inner membranes. 4. Isoprenaline increased the Isc by an average of 165%. The major effect of isoprenaline was to decrease the apical resistance by an average 77%. 5. Forskolin (2.5 microM) stimulated the Isc by an average of 138%. Amiloride would not completely reduce the Isc, but with the low concentration of 0.2 microM-forskolin, the Isc was typically inhibited to values close to zero. The major effect of forskolin was also to reduce the resistance of the apical barrier, although it concurrently also caused the E'1 to decrease by about 13%. 6. Theophylline increased the Isc by reducing the resistance of the apical barrier by an average 61%, with little or no effect on the other parameters. Theophylline augmented the effect of cyclic AMP. 8. Our results are consistent with the theory that cyclic AMP is a second messenger in hormonal control of active sodium transport in frog skin. PMID:2821244

  7. Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct.

    PubMed Central

    Star, R A; Nonoguchi, H; Balaban, R; Knepper, M A

    1988-01-01

    Vasopressin increases both the urea permeability and osmotic water permeability in the terminal part of the renal inner medullary collecting duct (terminal IMCD). To identify the second messengers that mediate these responses, we measured urea permeability, osmotic water permeability, intracellular calcium concentration, and cyclic AMP accumulation in isolated terminal IMCDs. After addition of vasopressin, a transient rise in intracellular calcium occurred that was coincident with increases in cyclic AMP accumulation and urea permeability. Half-maximal increases in urea permeability and osmotic water permeability occurred with 0.01 nM vasopressin. The threshold concentration for a measurable increase in cyclic AMP accumulation was approximately 0.01 nM, while measurable increases in intracellular calcium required much higher vasopressin concentrations (greater than 0.1 nM). Exogenous cyclic AMP (1 mM 8-Br-cAMP) mimicked the effect of vasopressin on urea permeability but did not produce a measurable change in intracellular calcium concentration. Conclusions: (a) Cyclic AMP is the second messenger that mediates the urea permeability response to vasopressin in the rat terminal IMCD. (b) Vasopressin increases the intracellular calcium concentration in the rat terminal IMCD, but the physiological role of this response is not yet known. PMID:2838523

  8. Role of 3'-5'-cyclic adenosine monophosphate on the epidermal growth factor dependent survival in mammary epithelial cells.

    PubMed

    Grinman, Diego Y; Romorini, Leonardo; Presman, Diego M; Rocha-Viegas, Luciana; Coso, Omar A; Davio, Carlos; Pecci, Adali

    2016-01-01

    Epidermal growth factor (EGF) has been suggested to play a key role in the maintenance of epithelial cell survival during lactation. Previously, we demonstrated that EGF dependent activation of PI3K pathway prevents apoptosis in confluent murine HC11 cells cultured under low nutrient conditions. The EGF protective effect is associated with increased levels of the antiapoptotic protein Bcl-XL. Here, we identify the EGF-dependent mechanism involved in cell survival that converges in the regulation of bcl-X expression by activated CREB. EGF induces Bcl-XL expression through activation of a unique bcl-X promoter, the P1; being not only the PI3K/AKT signaling pathway but also the increase in cAMP levels and the concomitant PKA/CREB activation necessary for both bcl-XL upregulation and apoptosis avoidance. Results presented in this work suggest the existence of a novel connection between the EGF receptor and the adenylate cyclase that would have an impact in preventing apoptosis under low nutrient conditions. PMID:26522133

  9. Measurement of adenosine 3':5'-cyclic monophosphate by competitive binding to salt-dissociated protein kinase.

    PubMed Central

    Døskeland, S O; Haga, H J

    1978-01-01

    An assay for cyclic AMP is described which takes advantage of the high affinity of the dissociated receptor moiety of cyclic AMP-dependent protein kinase I for the nucleotide. The kinase is kept dissociated by salt (800 mM-NaCl/30mM-EDTA). In the presence of a simply prepared heat-stable protein fraction the binding reagent is stable for the time needed to reach equilibrium of binding. A simple procedure [precipitation with poly-(ethylene glycol) followed by DEAE-cellulose chromatography] is described for the separation of protein kinase I from other binding proteins for cyclic AMP in rabbit skeletal muscle. The sensitivity, precision, reproducibility and specificity of the assay compared favourably with those of other cyclic AMP assays. The main advantage of the present assay is its resistance towards non-specific interference from a number of salts, tissue-culture media and substances found in crude tissue extracts. The reliability of cyclic AMP measurement directly in crude tissue extracts was ensured by removal of the assayable cyclic AMP with cyclic nucleotide phosphodiesterase digestion or adsorption with antibody against cyclic AMP, by comparison with measurement in tissue extracts purified by chromatography on QAE-Sephadex or sequentially on Dowex 50, and aluminium oxide as well as by dilution and recovery experiments. PMID:213054

  10. Elevated leukocyte phosphodiesterase as a basis for depressed cyclic adenosine monophosphate responses in the Basenji greyhound dog model of asthma

    SciTech Connect

    Chan, S.C.; Hanifin, J.M.; Holden, C.A.; Thompson, W.J.; Hirshman, C.A.

    1985-08-01

    The BG dog manifests various characteristics of human asthma, including airway hyperreactivity to low concentrations of methacholine. Studies have suggested that airway hyperreactivity in asthma is related to inadequate intracellular cAMP responses. The authors studied cAMP characteristics in MNL from 19 BG and 14 mongrel dogs. beta-Adrenergic receptors were assessed by /sup 125/I CYP in the presence and absence of propranolol. The responses of cAMP to ISO were measured by radioimmunoassay. Adenylate cyclase activity was determined in homogenized MNL preparations by cAMP generation. PDE activity was quantitated by radioenzyme assay. Mongrel dog leukocyte ISO-stimulated cAMP levels doubled, whereas there were negligible increases in MNL from BG dogs. Basal PDE levels were higher in BG dogs than in mongrel dogs. The PDE inhibitor Ro 20-1724 restored ISO-stimulated cAMP responses in MNL of BG dogs. Adenylate cyclase activity was not lower in MNL homogenates from BG dogs than in mongrel dogs. Cells from both BG and mongrel dogs demonstrated similar receptor numbers and affinities of saturable, specific beta-adrenergic binding over a 10 pM to 400 pM range. The results suggest that depressed cAMP responses in BG dogs are due to high PDE activity rather than to a defect in the beta-adrenergic receptor adenylate cyclase system.

  11. Altered energy state reversibly controls smooth muscle contractile function in human saphenous vein during acute hypoxia-reoxygenation: Role of glycogen, AMP-activated protein kinase, and insulin-independent glucose uptake.

    PubMed

    Pyla, Rajkumar; Pichavaram, Prahalathan; Fairaq, Arwa; Park, Mary Anne; Kozak, Mark; Kamath, Vinayak; Patel, Vijay S; Segar, Lakshman

    2015-09-01

    Hypoxia is known to promote vasodilation of coronary vessels through several mediators including cardiac-derived adenosine and endothelium-derived prostanoids and nitric oxide. To date, the impact of endogenous glycogen depletion in vascular smooth muscle and the resultant alterations in cellular energy state (e.g., AMP-activated protein kinase, AMPK) on the contractile response to G protein-coupled receptor agonists (e.g., serotonin, 5-HT) has not yet been studied. In the present study, ex vivo exposure of endothelium-denuded human saphenous vein rings to hypoxic and glucose-deprived conditions during KCl-induced contractions for 30 min resulted in a marked depletion of endogenous glycogen by ∼80% (from ∼1.78 μmol/g under normoxia to ∼0.36 μmol/g under hypoxia). Importantly, glycogen-depleted HSV rings, which were maintained under hypoxia/reoxygenation and glucose-deprived conditions, exhibited significant increases in basal AMPK phosphorylation (∼6-fold ↑) and 5-HT-induced AMPK phosphorylation (∼19-fold ↑) with an accompanying suppression of 5-HT-induced maximal contractile response (∼68% ↓), compared with respective controls. Exposure of glycogen-depleted HSV rings to exogenous D-glucose, but not the inactive glucose analogs, prevented the exaggerated increase in 5-HT-induced AMPK phosphorylation and restored 5-HT-induced maximal contractile response. In addition, the ability of exogenous D-glucose to rescue cellular stress and impaired contractile function occurred through GLUT1-mediated but insulin/GLUT4-independent mechanisms. Together, the present findings from clinically-relevant human saphenous vein suggest that the loss of endogenous glycogen in vascular smooth muscle and the resultant accentuation of AMPK phosphorylation by GPCR agonists may constitute a yet another mechanism of metabolic vasodilation of coronary vessels in ischemic heart disease. PMID:26212549

  12. 5′-AMP-activated Protein Kinase (AMPK) Supports the Growth of Aggressive Experimental Human Breast Cancer Tumors*

    PubMed Central

    Laderoute, Keith R.; Calaoagan, Joy M.; Chao, Wan-ru; Dinh, Dominc; Denko, Nicholas; Duellman, Sarah; Kalra, Jessica; Liu, Xiaohe; Papandreou, Ioanna; Sambucetti, Lidia; Boros, Laszlo G.

    2014-01-01

    Rapid tumor growth can establish metabolically stressed microenvironments that activate 5′-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [13C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism. PMID:24993821

  13. [Effects of acute hypobaric hypoxia and exhaustive exercise on AMP-activated protein kinase phosphorylation in rat skeletal muscle].

    PubMed

    Yang, Tao; Huang, Qing-Yuan; Shan, Fa-Bo; Guan, Li-Bin; Cai, Ming-Chun

    2012-04-25

    The present study was aimed to explore the changes of phosphorylated AMP-activated protein kinase (pAMPK) level in skeletal muscle after exposure to acute hypobaric hypoxia and exhaustive exercise. Thirty-two male Sprague-Dawley (SD) rats were randomly divided into sea level and high altitude groups. The rats in high altitude group were submitted to simulated 5 000 m of high altitude in a hypobaric chamber for 24 h, and sea level group was maintained at normal conditions. All the rats were subjected to exhaustive swimming exercise. The exhaustion time was recorded. Before and after the exercise, blood lactate and glycogen content in skeletal muscle were determined; AMPK and pAMPK levels in skeletal muscle were detected by Western blot. The results showed that the exhaustion time was significantly decreased after exposure to high altitude. At the moment of exhaustion, high altitude group had lower blood lactate concentration and higher surplus glycogen content in gastrocnemius compared with sea level group. Exhaustive exercise significantly increased the pAMPK/AMPK ratio in rat skeletal muscles from both sea level and high altitude groups. However, high altitude group showed lower pAMPK/AMPK ratio after exhaustion compared to sea level group. These results suggest that, after exposure to acute hypobaric hypoxia, the decrement in exercise capacity may not be due to running out of glycogen, accumulation of lactate or disturbance in energy status in skeletal muscle. PMID:22513470

  14. Caffeine attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells.

    PubMed

    Quan, Hai Yan; Kim, Do Yeon; Chung, Sung Hyun

    2013-04-01

    The main purpose of this study is to examine the effect of caffeine on lipid accumulation in human hepatoma HepG2 cells. Significant decreases in the accumulation of hepatic lipids, such as triglyceride (TG), and cholesterol were observed when HepG2 cells were treated with caffeine as indicated. Caffeine decreased the mRNA level of lipogenesis-associated genes (SREBP1c, SREBP2, FAS, SCD1, HMGR and LDLR). In contrast, mRNA level of CD36, which is responsible for lipid uptake and catabolism, was increased. Next, the effect of caffeine on AMP-activated protein kinase (AMPK) signaling pathway was examined. Phosphorylation of AMPK and acetyl-CoA carboxylase were evidently increased when the cells were treated with caffeine as indicated for 24 h. These effects were all reversed in the presence of compound C, an AMPK inhibitor. In summary, these data indicate that caffeine effectively depleted TG and cholesterol levels by inhibition of lipogenesis and stimulation of lipolysis through modulating AMPK-SREBP signaling pathways. PMID:23615262

  15. Dibenzoylmethane Exerts Metabolic Activity through Regulation of AMP-Activated Protein Kinase (AMPK)-Mediated Glucose Uptake and Adipogenesis Pathways

    PubMed Central

    Kim, Nami; Kim, Hong Min; Lee, Eun Soo; Lee, Jung Ok; Lee, Hye Jeong; Lee, Soo Kyung; Moon, Ji Wook; Kim, Ji Hae; Kim, Joong Kwan; Kim, Su Jin; Park, Sun Hwa; Chung, Choon Hee; Kim, Hyeon Soo

    2015-01-01

    Dibenzoylmethane (DBM) has been shown to exert a variety of beneficial effects on human health. However, the mechanism of action is poorly understood. In this study, DBM increased phosphorylation of AMP-activated protein kinase (AMPK) and stimulated glucose uptake in a skeletal muscle cell line. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor blocked DBM-induced glucose uptake. DBM increased the concentration of intracellular calcium and glucose uptake due to DBM was abolished by STO-609 (a calcium/calmodulin-dependent protein kinase inhibitor). DBM stimulated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which was blocked by pretreatment with compound C, an AMPK inhibitor. The expression of glucose transporter type 4 (GLUT4) was increased by DBM. The translocation of GLUT4 to the plasma membrane was also increased by DBM in AMPK dependently. In addition, DBM suppressed weight gain and prevented fat accumulation in the liver and abdomen in mice fed a high-fat diet. In pre-adipocyte cells, DBM decreased the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis. Expression of the adipogenic gene, fatty acid synthase (FAS), was suppressed by DBM in an AMPK-dependent manner. These results showed that the beneficial metabolic effects of DBM might be due to regulation of glucose uptake via AMPK in skeletal muscle and inhibition of adipogenesis in pre-adipocytes. PMID:25756788

  16. Adiponectin enhances bone marrow mesenchymal stem cell resistance to flow shear stress through AMP-activated protein kinase signaling

    PubMed Central

    Zhao, Lin; Fan, Chongxi; Zhang, Yu; Yang, Yang; Wang, Dongjin; Deng, Chao; Hu, Wei; Ma, Zhiqiang; Jiang, Shuai; Di, Shouyi; Qin, Zhigang; Lv, Jianjun; Sun, Yang; Yi, Wei

    2016-01-01

    Adiponectin has been demonstrated to protect the cardiovascular system and bone marrow mesenchymal stem cells (BMSCs). However, it is unclear whether adiponectin can protect BMSCs against flow shear stress (FSS). In this study, our aim was to explore the effects of adiponectin on BMSCs and to explore the role of AMP-activated protein kinase (AMPK) signaling in this process. Shear stress significantly inhibits the survival and increases the apoptosis of BMSCs in an intensity-dependent manner. The expression levels of TGF-β, bFGF, VEGF, PDGF, and Bcl2 are simultaneously reduced, and the phosphorylation levels of AMPK and ACC, as well as the expression level of Bax, are increased. Supplementation with adiponectin promotes the survival of BMSCs; reverses the changes in the expression levels of TGF-β, bFGF, VEGF, PDGF, Bcl2, and Bax; and further amplifies the phosphorylation of AMPK and ACC. Furthermore, the protective effects of adiponectin can be partially neutralized by AMPK siRNA. In summary, we have demonstrated for the first time that adiponectin can effectively protect BMSCs from FSS and that this effect depends, at least in part, on the activation of AMPK signaling. PMID:27418435

  17. AMP-Activated Protein Kinase Regulates Oxidative Metabolism in Caenorhabditis elegans through the NHR-49 and MDT-15 Transcriptional Regulators

    PubMed Central

    Moreno-Arriola, Elizabeth; EL Hafidi, Mohammed; Ortega-Cuéllar, Daniel; Carvajal, Karla

    2016-01-01

    Cellular energy regulation relies on complex signaling pathways that respond to fuel availability and metabolic demands. Dysregulation of these networks is implicated in the development of human metabolic diseases such as obesity and metabolic syndrome. In Caenorhabditis elegans the AMP-activated protein kinase, AAK, has been associated with longevity and stress resistance; nevertheless its precise role in energy metabolism remains elusive. In the present study, we find an evolutionary conserved role of AAK in oxidative metabolism. Similar to mammals, AAK is activated by AICAR and metformin and leads to increased glycolytic and oxidative metabolic fluxes evidenced by an increase in lactate levels and mitochondrial oxygen consumption and a decrease in total fatty acids and lipid storage, whereas augmented glucose availability has the opposite effects. We found that these changes were largely dependent on the catalytic subunit AAK-2, since the aak-2 null strain lost the observed metabolic actions. Further results demonstrate that the effects due to AAK activation are associated to SBP-1 and NHR-49 transcriptional factors and MDT-15 transcriptional co-activator, suggesting a regulatory pathway that controls oxidative metabolism. Our findings establish C. elegans as a tractable model system to dissect the relationship between distinct molecules that play a critical role in the regulation of energy metabolism in human metabolic diseases. PMID:26824904

  18. The hypotensive effect of acute and chronic AMP-activated protein kinase activation in normal and hyperlipidemic mice

    PubMed Central

    Greig, Fiona H.; Ewart, Marie-Ann; McNaughton, Eilidh; Cooney, Josephine; Spickett, Corinne M.; Kennedy, Simon

    2015-01-01

    AMP-activated protein kinase (AMPK) is present in the arterial wall and is activated in response to cellular stressors that raise AMP relative to ADP/ATP. Activation of AMPK in vivo lowers blood pressure but the influence of hyperlipidemia on this response has not been studied. ApoE−/− mice on high fat diet for 6 weeks and age-matched controls were treated with the AMPK activator, AICAR daily for two weeks. Under anesthesia, the carotid artery was cannulated for blood pressure measurements. Aortic tissue was removed for in vitro functional experiments and AMPK activity was measured in artery homogenates by Western blotting. ApoE−/− mice had significantly raised mean arterial pressure; chronic AICAR treatment normalized this but had no effect in normolipidemic mice, whereas acute administration of AICAR lowered mean arterial pressure in both groups. Chronic AICAR treatment increased phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase in normolipidemic but not ApoE−/− mice. In aortic rings, AMPK activation induced vasodilation and an anticontractile effect, which was attenuated in ApoE−/− mice. This study demonstrates that hyperlipidemia dysregulates the AMPK pathway in the arterial wall but this effect can be reversed by AMPK activation, possibly through improving vessel compliance. PMID:26196300

  19. Thyroid-stimulating hormone decreases HMG-CoA reductase phosphorylation via AMP-activated protein kinase in the liver

    PubMed Central

    Zhang, Xiujuan; Song, Yongfeng; Feng, Mei; Zhou, Xinli; Lu, Yingli; Gao, Ling; Yu, Chunxiao; Jiang, Xiuyun; Zhao, Jiajun

    2015-01-01

    Cholesterol homeostasis is strictly regulated through the modulation of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of cholesterol synthesis. Phosphorylation of HMGCR inactivates it and dephosphorylation activates it. AMP-activated protein kinase (AMPK) is the major kinase phosphorylating the enzyme. Our previous study found that thyroid-stimulating hormone (TSH) increased the hepatocytic HMGCR expression, but it was still unclear whether TSH affected hepatic HMGCR phosphorylation associated with AMPK. We used bovine TSH (bTSH) to treat the primary mouse hepatocytes and HepG2 cells with or without constitutively active (CA)-AMPK plasmid or protein kinase A inhibitor (H89), and set up the TSH receptor (Tshr)-KO mouse models. The p-HMGCR, p-AMPK, and related molecular expression were tested. The ratios of p-HMGCR/HMGCR and p-AMPK/AMPK decreased in the hepatocytes in a dose-dependent manner following bTSH stimulation. The changes above were inversed when the cells were treated with CA-AMPK plasmid or H89. In Tshr-KO mice, the ratios of liver p-HMGCR/HMGCR and p-AMPK/AMPK were increased relative to the littermate wild-type mice. Consistently, the phosphorylation of acetyl-CoA carboxylase, a downstream target molecule of AMPK, increased. All results suggested that TSH could regulate the phosphorylation of HMGCR via AMPK, which established a potential mechanism for hypercholesterolemia involved in a direct action of the TSH in the liver. PMID:25713102

  20. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells.

    PubMed

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin. PMID:27348124

  1. Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes*

    PubMed Central

    Taïb, Bouchra; Bouyakdan, Khalil; Hryhorczuk, Cécile; Rodaros, Demetra; Fulton, Stephanie; Alquier, Thierry

    2013-01-01

    Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance. PMID:24240094

  2. Adiponectin enhances bone marrow mesenchymal stem cell resistance to flow shear stress through AMP-activated protein kinase signaling.

    PubMed

    Zhao, Lin; Fan, Chongxi; Zhang, Yu; Yang, Yang; Wang, Dongjin; Deng, Chao; Hu, Wei; Ma, Zhiqiang; Jiang, Shuai; Di, Shouyi; Qin, Zhigang; Lv, Jianjun; Sun, Yang; Yi, Wei

    2016-01-01

    Adiponectin has been demonstrated to protect the cardiovascular system and bone marrow mesenchymal stem cells (BMSCs). However, it is unclear whether adiponectin can protect BMSCs against flow shear stress (FSS). In this study, our aim was to explore the effects of adiponectin on BMSCs and to explore the role of AMP-activated protein kinase (AMPK) signaling in this process. Shear stress significantly inhibits the survival and increases the apoptosis of BMSCs in an intensity-dependent manner. The expression levels of TGF-β, bFGF, VEGF, PDGF, and Bcl2 are simultaneously reduced, and the phosphorylation levels of AMPK and ACC, as well as the expression level of Bax, are increased. Supplementation with adiponectin promotes the survival of BMSCs; reverses the changes in the expression levels of TGF-β, bFGF, VEGF, PDGF, Bcl2, and Bax; and further amplifies the phosphorylation of AMPK and ACC. Furthermore, the protective effects of adiponectin can be partially neutralized by AMPK siRNA. In summary, we have demonstrated for the first time that adiponectin can effectively protect BMSCs from FSS and that this effect depends, at least in part, on the activation of AMPK signaling. PMID:27418435

  3. AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae

    PubMed Central

    Xie, Meng; Roy, Richard

    2015-01-01

    Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress. PMID:26098762

  4. The effects of adiponectin and metformin on prostate and colon neoplasia involve activation of AMP-activated protein kinase.

    PubMed

    Zakikhani, Mahvash; Dowling, Ryan J O; Sonenberg, Nahum; Pollak, Michael N

    2008-10-01

    Population studies provide evidence that obesity and insulin resistance are associated not only with elevated serum insulin levels and reduced serum adiponectin levels but also with increased risk of aggressive prostate and colon cancer. We show here that adiponectin activates AMP-activated protein kinase (AMPK) in colon (HT-29) and prostate (PC-3) cancer cells. These results are consistent with prior observations in myocytes, but we show that in epithelial cancer cells AMPK activation is associated with reduction in mammalian target of rapamycin activation as estimated by Ser(2448) phosphorylation, with reduction in p70S6 kinase activation as estimated by Thr(389) phosphorylation, with ribosomal protein S6 activation as estimated by Ser(235/236) phosphorylation, with reduction in protein translation as estimated by [(35)S]methionine incorporation, and with growth inhibition. Adiponectin-induced growth inhibition is significantly attenuated when AMPK level is reduced using small interfering RNA, indicating that AMPK is involved in mediating the antiproliferative action of this adipokine. Thus, adiponectin has the characteristics of a AMPK-dependent growth inhibitor that is deficient in obesity, and this may contribute to the adverse effects of obesity on neoplastic disease. Furthermore, metformin was observed to activate AMPK and to have growth inhibitory actions on prostate and colon cancer cells, suggesting that this compound may be of particular value in attenuating the adverse effects of obesity on neoplasia. PMID:19138981

  5. BRAFV600E inhibition stimulates AMP-activated protein kinase-mediated autophagy in colorectal cancer cells

    PubMed Central

    Sueda, Toshinori; Sakai, Daisuke; Kawamoto, Koichi; Konno, Masamitsu; Nishida, Naohiro; Koseki, Jun; Colvin, Hugh; Takahashi, Hidekazu; Haraguchi, Naotsugu; Nishimura, Junichi; Hata, Taishi; Takemasa, Ichiro; Mizushima, Tsunekazu; Yamamoto, Hirofumi; Satoh, Taroh; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

    2016-01-01

    Although BRAFV600E mutation is associated with adverse clinical outcomes in patients with colorectal cancer (CRC), response and resistance mechanisms for therapeutic BRAFV600E inhibitors remains poorly understood. In the present study, we demonstrate that selective BRAFV600E inhibition activates AMP-activated protein kinase (AMPK), which induces autophagy as a mechanism of therapeutic resistance in human cancers. The present data show AMPK-dependent cytoprotective roles of autophagy under conditions of therapeutic BRAFV600E inhibition, and AMPK was negatively correlated with BRAFV600E-dependent activation of MEK-ERK-RSK signaling and positively correlated with unc-51-like kinase 1 (ULK1), a key initiator of autophagy. Furthermore, selective BRAFV600E inhibition and concomitant suppression of autophagy led to the induction of apoptosis. Taken together, present experiments indicate that AMPK plays a role in the survival of BRAFV600E CRC cells by selective inhibition and suggest that the control of autophagy contributes to overcome the chemoresistance of BRAFV600E CRC cells. PMID:26750638

  6. AMP-Activated Protein Kinase and Glycogen Synthase Kinase 3β Modulate the Severity of Sepsis-Induced Lung Injury

    PubMed Central

    Liu, Zhongyu; Bone, Nathaniel; Jiang, Shaoning; Park, Dae Won; Tadie, Jean-Marc; Deshane, Jessy; Rodriguez, Cilina Ann; Pittet, Jean-Francois; Abraham, Edward; Zmijewski, Jaroslaw W

    2015-01-01

    Alterations in metabolic and bioenergetic homeostasis contribute to sepsis-mediated organ injury. However, how AMP-activated protein kinase (AMPK), a major sensor and regulator of energy expenditure and production, affects development of organ injury and loss of innate capacity during polymicrobial sepsis remains unclear. In the present experiments, we found that cross-talk between the AMPK and GSK3β signaling pathways controls chemotaxis and the ability of neutrophils and macrophages to kill bacteria ex vivo. In mice with polymicrobial abdominal sepsis or more severe sepsis induced by the combination of hemorrhage and intraabdominal infection, administration of the AMPK activator metformin or the GSK3β inhibitor SB216763 reduced the severity of acute lung injury (ALI). Improved survival in metformin-treated septic mice was correlated with preservation of mitochondrial complex V (ATP synthase) function and increased amounts of ETC complex III and IV. Although immunosuppression is a consequence of sepsis, metformin effectively increased innate immune capacity to eradicate P. aeruginosa in the lungs of septic mice. We also found that AMPK activation diminished accumulation of the immunosuppressive transcriptional factor HIF-1α as well as the development of endotoxin tolerance in LPS-treated macrophages. Furthermore, AMPK-dependent preservation of mitochondrial membrane potential also prevented LPS-mediated dysfunction of neutrophil chemotaxis. These results indicate that AMPK activation reduces the severity of polymicrobial sepsis-induced lung injury and prevents the development of sepsis-associated immunosuppression. PMID:26650187

  7. Activation of AMP-activated kinase modulates sensitivity of glioma cells against epidermal growth factor receptor inhibition.

    PubMed

    Hartel, Ines; Ronellenfitsch, Michael; Wanka, Christina; Wolking, Stefan; Steinbach, Joachim P; Rieger, Johannes

    2016-07-01

    The epidermal growth factor (EGFR) pathway is frequently activated in glioblastoma but the clinical efficacy of EGFR inhibitors in malignant glioma has been disappointing. The reasons for the failure of the mechanisms of resistance of these inhibitors are unclear, but may involve factors of the tumor microenvironment such as limited glucose availability and hypoxia. It was therefore examined whether glucose and oxygen influenced the response of glioma cells to EGFR inhibition. Decreased levels of glucose and oxygen led to resistance against the EGFR inhibitor PD153035, whereas high glucose amounts and normoxia sensitised glioma cells towards the inhibitor. Low levels of glucose and oxygen stimulated AMP-activated kinase (AMPK) in glioma cells. 2DG, an inhibitor of glycolysis, and the AMPK activator A769662 reduced glucose consumption, induced phosphorylation of AMPK and mimicked the effects of low glucose availability on the toxicity of PD153035. Similarly, 2DG reduced toxicity of imatinib in K562 leukemia cells. In contrast, inhibition of AMPK by compound C or by short-hairpin (sh)-mediated gene suppression increased cell death induced by the EGFR inhibitor and reverted the protective effects of 2DG and A769662. In conclusion, cytotoxicity of EGFR inhibition can be diminished by AMPK activation in glioma cells. These results may provide one explanation for the low activity of EGFR inhibitors in clinical trials and suggest antagonism of AMPK or of AMPK-regulated metabolic alterations as a promising approach to enhance their therapeutic efficacy. PMID:27121290

  8. Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

    PubMed Central

    Feng, Qin; Gou, Xiao-jun; Meng, Sheng-xi; Huang, Cheng; Zhang, Yu-quan; Tang, Ya-jun; Wang, Wen-jing; Xu, Lin; Peng, Jing-hua; Hu, Yi-yang

    2013-01-01

    Qushi Huayu Decoction (QHD), a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD) in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK) in vivo and in vitro. Nonalcoholic fatty liver (NAFL) model was duplicated with high-fat diet in rats and with free fatty acid (FFA) in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC) and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1) and carbohydrate-responsive element-binding protein (ChREBP) in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro. PMID:23573117

  9. Absence of humoral mediated 5'AMP-activated protein kinase activation in human skeletal muscle and adipose tissue during exercise.

    PubMed

    Kristensen, Jonas Møller; Johnsen, Anders Bo; Birk, Jesper B; Nielsen, Jakob Nis; Jensen, Bente Rona; Hellsten, Ylva; Richter, Erik A; Wojtaszewski, Jørgen F P

    2007-12-15

    5'AMP-activated protein kinase (AMPK) exists as a heterotrimer comprising a catalytic alpha subunit and regulatory beta and gamma subunits. The AMPK system is activated under conditions of cellular stress, indicated by an increase in the AMP/ATP ratio, as observed, e.g. in muscles during contractile activity. AMPK was originally thought to be activated only by local intracellular mechanisms. However, recently it has become apparent that AMPK in mammals is also regulated by humoral substances, e.g. catecholamines. We studied whether humoral factors released during exercise regulate AMPK activity in contracting and resting muscles as well as in abdominal subcutaneous adipose tissue in humans. In resting leg muscle and adipose tissue the AMPK activity was not up-regulated by humoral factors during one-legged knee extensor exercise even when arm cranking exercise, inducing a approximately 20-fold increase in plasma catecholamine level, was added simultaneously. In exercising leg muscle the AMPK activity was increased by one-legged knee extensor exercise eliciting a whole body respiratory load of only 30% .VO(2,peak) but was not further increased by adding arm cranking exercise. In conclusion, during exercise with combined leg kicking and arm cranking, the AMPK activity in human skeletal muscle is restricted to contracting muscle without influence of marked increased catecholamine levels. Also, with this type of exercise the catecholamines or other humoral factors do not seem to be physiological regulators of AMPK in the subcutaneous adipose tissue. PMID:17962330

  10. Matrine-induced autophagy regulated by p53 through AMP-activated protein kinase in human hepatoma cells.

    PubMed

    Xie, Shan-Bu; He, Xing-Xing; Yao, Shu-Kun

    2015-08-01

    Matrine, one of the main extract components of Sophora flavescens, has been shown to exhibit inhibitory effects on some tumors through autophagy. However, the mechanism underlying the effect of matrine remains unclear. The cultured human hepatocellular carcinoma cell line HepG2 and SMMC‑7721 were treated with matrine. Signal transduction and gene expression profile were determined. Matrine stimulated autophagy in SMMC‑7721 cells in a mammalian target of rapamycin (mTOR)-dependent manner, but in an mTOR-independent manner in HepG2 cells. Next, in HepG2 cells, autophagy induced by matrine was regulated by p53 inactivation through AMP-activated protein kinase (AMPK) signaling transduction, then AMPK suppression switched autophagy to apoptosis. Furthermore, the interferon (IFN)-inducible genes, including interferon α-inducible protein 27 (IFI27) and interferon induced transmembrane protein 1 (IFITM1), which are downstream effector of p53, might be modulated by matrine-induced autophagy. In addition, we found that the p53 protein isoforms, p53β, p53γ, ∆133p53, and ∆133p53γ, due to alternative splicing of intron 9, might be regulated by the p53-mediated autophagy. These results show that matrine induces autophagy in human hepatoma cells through a novel mechanism, which is p53/AMPK signaling pathway involvement in matrine-promoted autophagy. PMID:26034977

  11. Dibenzoylmethane exerts metabolic activity through regulation of AMP-activated protein kinase (AMPK)-mediated glucose uptake and adipogenesis pathways.

    PubMed

    Kim, Nami; Kim, Hong Min; Lee, Eun Soo; Lee, Jung Ok; Lee, Hye Jeong; Lee, Soo Kyung; Moon, Ji Wook; Kim, Ji Hae; Kim, Joong Kwan; Kim, Su Jin; Park, Sun Hwa; Chung, Choon Hee; Kim, Hyeon Soo

    2015-01-01

    Dibenzoylmethane (DBM) has been shown to exert a variety of beneficial effects on human health. However, the mechanism of action is poorly understood. In this study, DBM increased phosphorylation of AMP-activated protein kinase (AMPK) and stimulated glucose uptake in a skeletal muscle cell line. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor blocked DBM-induced glucose uptake. DBM increased the concentration of intracellular calcium and glucose uptake due to DBM was abolished by STO-609 (a calcium/calmodulin-dependent protein kinase inhibitor). DBM stimulated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which was blocked by pretreatment with compound C, an AMPK inhibitor. The expression of glucose transporter type 4 (GLUT4) was increased by DBM. The translocation of GLUT4 to the plasma membrane was also increased by DBM in AMPK dependently. In addition, DBM suppressed weight gain and prevented fat accumulation in the liver and abdomen in mice fed a high-fat diet. In pre-adipocyte cells, DBM decreased the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis. Expression of the adipogenic gene, fatty acid synthase (FAS), was suppressed by DBM in an AMPK-dependent manner. These results showed that the beneficial metabolic effects of DBM might be due to regulation of glucose uptake via AMPK in skeletal muscle and inhibition of adipogenesis in pre-adipocytes. PMID:25756788

  12. Salicylate acutely stimulates 5'-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscles.

    PubMed

    Serizawa, Yasuhiro; Oshima, Rieko; Yoshida, Mitsuki; Sakon, Ichika; Kitani, Kazuto; Goto, Ayumi; Tsuda, Satoshi; Hayashi, Tatsuya

    2014-10-10

    Salicylate (SAL) has been recently implicated in the antidiabetic effect in humans. We assessed whether 5'-AMP-activated protein kinase (AMPK) in skeletal muscle is involved in the effect of SAL on glucose homeostasis. Rat fast-twitch epitrochlearis and slow-twitch soleus muscles were incubated in buffer containing SAL. Intracellular concentrations of SAL increased rapidly (<5 min) in both skeletal muscles, and the Thr(172) phosphorylation of the α subunit of AMPK increased in a dose- and time-dependent manner. SAL increased both AMPKα1 and AMPKα2 activities. These increases in enzyme activity were accompanied by an increase in the activity of 3-O-methyl-D-glucose transport, and decreases in ATP, phosphocreatine, and glycogen contents. SAL did not change the phosphorylation of insulin receptor signaling including insulin receptor substrate 1, Akt, and p70 ribosomal protein S6 kinase. These results suggest that SAL may be transported into skeletal muscle and may stimulate AMPK and glucose transport via energy deprivation in multiple muscle types. Skeletal muscle AMPK might be part of the mechanism responsible for the metabolic improvement induced by SAL. PMID:25256746

  13. Inhibition of AMP-Activated Protein Kinase at the Allosteric Drug-Binding Site Promotes Islet Insulin Release.

    PubMed

    Scott, John W; Galic, Sandra; Graham, Kate L; Foitzik, Richard; Ling, Naomi X Y; Dite, Toby A; Issa, Samah M A; Langendorf, Chris G; Weng, Qing Ping; Thomas, Helen E; Kay, Thomas W; Birnberg, Neal C; Steinberg, Gregory R; Kemp, Bruce E; Oakhill, Jonathan S

    2015-06-18

    The AMP-activated protein kinase (AMPK) is a metabolic stress-sensing αβγ heterotrimer responsible for energy homeostasis. Pharmacological inhibition of AMPK is regarded as a therapeutic strategy in some disease settings including obesity and cancer; however, the broadly used direct AMPK inhibitor compound C suffers from poor selectivity. We have discovered a dihydroxyquinoline drug (MT47-100) with novel AMPK regulatory properties, being simultaneously a direct activator and inhibitor of AMPK complexes containing the β1 or β2 isoform, respectively. Allosteric inhibition by MT47-100 was dependent on the β2 carbohydrate-binding module (CBM) and determined by three non-conserved CBM residues (Ile81, Phe91, Ile92), but was independent of β2-Ser108 phosphorylation. Whereas MT47-100 regulation of total cellular AMPK activity was determined by β1/β2 expression ratio, MT47-100 augmented glucose-stimulated insulin secretion from isolated mouse pancreatic islets via a β2-dependent mechanism. Our findings highlight the therapeutic potential of isoform-specific AMPK allosteric inhibitors. PMID:26091167

  14. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells

    PubMed Central

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin. PMID:27348124

  15. Novel epigallocatechin gallate (EGCG) analogs activate AMP-activated protein kinase pathway and target cancer stem cells

    PubMed Central

    Chen, Di; Pamu, Sreedhar; Cui, Qiuzhi; Chan, Tak Hang; Dou, Q. Ping

    2012-01-01

    AMP-activated protein kinase (AMPK) is a critical monitor of cellular energy status and also controls processes related to tumor development, including cell cycle progression, protein synthesis, cell growth and survival. Therefore AMPK as an anti-cancer target has received intensive attention recently. It has been reported that the anti-diabetic drug metformin and some natural compounds, such as quercetin, genistein, capsaicin and green tea polyphenol epigallocatechin gallate (EGCG), can activate AMPK and inhibit cancer cell growth. Indeed, natural products have been the most productive source of leads for the development of anti-cancer drugs but perceived disadvantages, such as low bioavailability and week potency, have limited their development and use in the clinic. In this study we demonstrated that synthetic EGCG analogs 4 and 6 were more potent AMPK activators than metformin and EGCG. Activation of AMPK by these EGCG analogs resulted in inhibition of cell proliferation, up-regulation of the cyclin-dependent kinase inhibitor p21, down-regulation of mTOR pathway, and suppression of stem cell population in human breast cancer cells. Our findings suggest that novel potent and specific AMPK activators can be discovered from natural and synthetic sources that have potential to be used for anti-cancer therapy in the clinic. PMID:22459208

  16. Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine deaminase.

    PubMed

    Green, H; Chan, T

    1973-11-23

    In the presence of 10(-4) to 10(-5) molar adenosine, established cell lines of fibroblastic or lymphoid origin die of pyrimidine starvation. Less than lethal concentrations inhibit cell growth. Over a broad concentration range, the effects of adenosine are prevented by providing a suitable pyrimidine source. We suggest that the recently described immune deficiency disease associated with absence of adenosine deaminase may be the result of pyrimidine starvation induced by adenosine nucleotides in cells of the lymphoid system. PMID:4795749

  17. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

    SciTech Connect

    Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H. )

    1990-04-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 3}H)NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the ({sup 3}H)NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

  18. PRODUCTION OF EXTRACELLULAR GUANOSINE-5'-MONOPHOSPHATE BY BACILLUS SUBTILIS

    PubMed Central

    Demain, A. L.; Miller, I. M.; Hendlin, D.

    1964-01-01

    Demain, A. L. (Merck Sharp & Dohme Research Laboratories, Rahway, N.J.), I. M. Miller, and D. Hendlin. Production of extracellular guanosine-5'-monophosphate by Bacillus subtilis. J. Bacteriol. 88:991–995. 1964.—Wild-type Bacillus subtilis colonies were found to feed purineless mutants. A strain with high feeding capacity was selected for study, with a guanineless mutant of B. subtilis used as the assay organism. The factor was excreted during its growth phase in a complex medium containing starch and soybean meal extract. Nutritional studies led to the development of a defined medium to be used for biochemical studies and to aid in the isolation of the factor. Starch was replaced by maltose and the soybean meal extract by Mn++. Production of the factor was sensitive to the pH of the medium during growth. Practically its entire extracellular accumulation occurred before visible lysis. The factor was identified as guanosine-5'-monophosphate derived by extracellular enzymatic hydrolysis of excreted ribonucleic acid. PMID:14219064

  19. Avian renal proximal tubule urate secretion is inhibited by cellular stress-induced AMP-activated protein kinase.

    PubMed

    Bataille, Amy M; Maffeo, Carla L; Renfro, J Larry

    2011-06-01

    Urate is a potent antioxidant at high concentrations but it has also been associated with a wide variety of health risks. Plasma urate concentration is determined by ingestion, production, and urinary excretion; however, factors that regulate urate excretion remain uncertain. The objective of this study was to determine whether cellular stress, which has been shown to affect other renal transport properties, modulates urate secretion in the avian renal proximal tubule. Chick kidney proximal tubule epithelial cell primary culture monolayers were used to study the transepithelial transport of radiolabeled urate. This model allowed examination of the processes, such as multidrug resistance protein 4 (Mrp4, Abcc4), which subserve urate secretion in a functional, intact, homologous system. Our results show that the recently implicated urate efflux transporter, breast cancer resistance protein (ABCG2), does not significantly contribute to urate secretion in this system. Exposure to a high concentration of zinc for 6 h induced a cellular stress response and a striking decrease in transepithelial urate secretion. Acute exposure to zinc had no effect on transepithelial urate secretion or isolated membrane vesicle urate transport, suggesting involvement of a cellular stress adaptation. Activation of AMP-activated protein kinase (AMPK), a candidate modulator of ATP-dependent urate efflux, by 5'-aminoimidazole-4-carboxamide 1-β-d-ribo-furanoside caused a decrease in urate secretion similar to that seen with zinc-induced cellular stress. This effect was prevented with the AMPK inhibitor compound C. Notably, the decrease in urate secretion seen with zinc-induced cellular stress was also prevented by compound C, implicating AMPK in regulation of renal uric acid excretion. PMID:21429974

  20. Energy-Dependent Modulation of Glucagon-Like Signaling in Drosophila via the AMP-Activated Protein Kinase

    PubMed Central

    Braco, Jason T.; Gillespie, Emily L.; Alberto, Gregory E.; Brenman, Jay E.; Johnson, Erik C.

    2012-01-01

    Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks. PMID:22798489

  1. Low-intensity contraction activates the alpha1-isoform of 5'-AMP-activated protein kinase in rat skeletal muscle.

    PubMed

    Toyoda, Taro; Tanaka, Satsuki; Ebihara, Ken; Masuzaki, Hiroaki; Hosoda, Kiminori; Sato, Kenji; Fushiki, Tohru; Nakao, Kazuwa; Hayashi, Tatsuya

    2006-03-01

    Skeletal muscle expresses two catalytic subunits, alpha1 and alpha2, of the 5'-AMP-activated protein kinase (AMPK), which has been implicated in contraction-stimulated glucose transport and fatty acid oxidation. Muscle contraction activates the alpha2-containing AMPK complex (AMPKalpha2), but this activation may occur with or without activation of the alpha1-containing AMPK complex (AMPKalpha1), suggesting that AMPKalpha2 is the major isoform responsible for contraction-induced metabolic events in skeletal muscle. We report for the first time that AMPKalpha1, but not AMPKalpha2, can be activated in contracting skeletal muscle. Rat epitrochlearis muscles were isolated and incubated in Krebs-Ringer bicarbonate buffer containing pyruvate. In muscles stimulated to contract at a frequency of 1 and 2 Hz during the last 2 min of incubation, AMPKalpha1 activity increased twofold and AMPKalpha2 activity remained unchanged. Muscle stimulation did not change the muscle AMP concentration or the AMP-to-ATP ratio. AMPK activation was associated with increased phosphorylation of Thr(172) of the alpha-subunit, the primary activation site. Muscle stimulation increased the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, and the rate of 3-O-methyl-d-glucose transport. In contrast, increasing the frequency (>or=5 Hz) or duration (>or=5 min) of contraction activated AMPKalpha1 and AMPKalpha2 and increased AMP concentration and the AMP/ATP ratio. These results suggest that 1) AMPKalpha1 is the predominant isoform activated by AMP-independent phosphorylation in low-intensity contracting muscle, 2) AMPKalpha2 is activated by an AMP-dependent mechanism in high-intensity contracting muscle, and 3) activation of each isoform enhances glucose transport and ACC phosphorylation in skeletal muscle. PMID:16249251

  2. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells.

    PubMed

    Jin, Benjamin Y; Sartoretto, Juliano L; Gladyshev, Vadim N; Michel, Thomas

    2009-10-13

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H(2)O(2) is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H(2)O(2) treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC(50) for H(2)O(2)-promoted phosphorylation of AMPK is 65 + or - 15 microM, within the physiological range of cellular H(2)O(2) concentrations. The Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) inhibitor STO-609 abolishes H(2)O(2)-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKbeta abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H(2)O(2) biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H(2)O(2) generation, which is blocked by PEG-catalase. eNOS(-/-) mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS(-/-) mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKbeta is critically involved in mediating the phosphorylation of AMPK promoted by H(2)O(2) in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H(2)O(2) generation in endothelial cells. PMID:19805165

  3. Opposing Activity Changes in AMP Deaminase and AMP-Activated Protein Kinase in the Hibernating Ground Squirrel

    PubMed Central

    Cicerchi, Christina; Garcia, Gabriela E.; Roncal-Jimenez, Carlos A.; Trostel, Jessica; Jain, Swati; Mant, Colin T.; Rivard, Christopher J.; Ishimoto, Takuji; Shimada, Michiko; Sanchez-Lozada, Laura Gabriela; Nakagawa, Takahiko; Jani, Alkesh; Stenvinkel, Peter; Martin, Sandra L.; Johnson, Richard J.

    2015-01-01

    Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel. PMID:25856396

  4. Stress-induced activation of the AMP-activated protein kinase in the freeze-tolerant frog Rana sylvatica.

    PubMed

    Rider, Mark H; Hussain, Nusrat; Horman, Sandrine; Dilworth, Stephen M; Storey, Kenneth B

    2006-12-01

    Survival in the frozen state depends on biochemical adaptations that deal with multiple stresses on cells including long-term ischaemia and tissue dehydration. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the metabolic re-sculpting that occurs during freezing. AMPK activity and the phosphorylation state of translation factors were measured in liver and skeletal muscle of wood frogs (Rana sylvatica) subjected to anoxia, dehydration, freezing, and thawing after freezing. AMPK activity was increased 2-fold in livers of frozen frogs compared with the controls whereas in skeletal muscle, AMPK activity increased 2.5-, 4.5- and 3-fold in dehydrated, frozen and frozen/thawed animals, respectively. Immunoblotting with phospho-specific antibodies revealed an increase in the phosphorylation state of eukaryotic elongation factor-2 at the inactivating Thr56 site in livers from frozen frogs and in skeletal muscles of anoxic frogs. No change in phosphorylation state of eukaryotic initiation factor-2alpha at the inactivating Ser51 site was seen in the tissues under any of the stress conditions. Surprisingly, ribosomal protein S6 phosphorylation was increased 2-fold in livers from frozen frogs and 10-fold in skeletal muscle from frozen/thawed animals. However, no change in translation capacity was detected in cell-free translation assays with skeletal muscle extracts under any of the experimental conditions. The changes in phosphorylation state of translation factors are discussed in relation to the control of protein synthesis and stress-induced AMPK activation. PMID:16973146

  5. Expression of Phosphorylated AMP-Activated Protein Kinase Predicts Response to Transarterial Chemoembolization in Postoperative Cases of Hepatocellular Carcinoma

    PubMed Central

    Zheng, Long-Yi; Wu, Lu; Lu, Jin; Zou, Da-Jin; Huang, Qin

    2016-01-01

    Abstract Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in the world. Transcatheter arterial chemoembolization (TACE) was commonly used for HCC patients postoperatively. However, the survival benefits of adjuvant TACE were controversial due to the extensive heterogeneity of HCC. Hence, there is a critical need to explore potential biomarkers that can predict the clinical response to TACE. The AMP-activated protein kinase (AMPK) is a highly conserved heterotrimeric serine/threonine kinase that plays a central role in linking metabolism and cancer development. In this study, we aimed at evaluating the association of pAMPKα (Thr172) status with clinical outcomes in HCC patients treated with or without postoperative adjuvant TACE. pAMPKα (Thr172) expression was assessed using immunohistochemical analysis in a cohort of 378 Chinese HCC patients who had undergone tumor resection. Kaplan–Meier analysis and multivariate Cox proportional hazards models were used to study the impact on clinical outcomes. High pAMPKα (Thr172) expression was associated with improved disease-free and overall survival and was an independent prognostic factor for overall survival by multivariate analysis. Furthermore, low pAMPKα (Thr172) expression level was correlated with high percentage of OV6+ tumor-initiating cells (T-ICs) in HCC specimens. To our knowledge, it can be demonstrated for the first time that pAMPKα (Thr172) status is associated with response to postoperative adjuvant TACE. High pAMPKα (Thr172) level in HCC may serve as a positive predictor of survival in HCC patients undergoing TACE. PMID:26986101

  6. T3-induced liver AMP-activated protein kinase signaling: Redox dependency and upregulation of downstream targets

    PubMed Central

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Morales, Paula; Ceballo, Juan; Fischer, Alvaro; Escudero, Nicolás; Escobar, Oscar

    2014-01-01

    AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum β-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca2+-calmodulin-dependent protein kinase kinase-β and transforming growth factor-β-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of β-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury. PMID:25516653

  7. Energy-dependent modulation of glucagon-like signaling in Drosophila via the AMP-activated protein kinase.

    PubMed

    Braco, Jason T; Gillespie, Emily L; Alberto, Gregory E; Brenman, Jay E; Johnson, Erik C

    2012-10-01

    Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks. PMID:22798489

  8. Possible involvement of AMP-activated protein kinase in PGE1-induced synthesis of osteoprotegerin in osteoblasts

    PubMed Central

    KAINUMA, SHINGO; OTSUKA, TAKANOBU; KUROYANAGI, GEN; YAMAMOTO, NAOHIRO; MATSUSHIMA-NISHIWAKI, RIE; KOZAWA, OSAMU; TOKUDA, HARUHIKO

    2016-01-01

    AMP-activated protein kinase (AMPK) is firmly established as a central regulator of cellular energy homeostasis. We have previously reported that prostaglandin E1 (PGE1) stimulates the synthesis of osteoprotegerin through p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. The present study investigated the involvement of AMPK in PGE1-induced osteoprotegerin synthesis in MC3T3-E1 cells. The levels of osteoprotegerin were measured using an enzyme-linked immunosorbent assay, while the phosphorylation of AMPK, acetyl-CoA carboxylase, p38 MAP kinase and SAPK/JNK were analyzed by western blotting. In addition, the mRNA expression levels of osteoprotegerin were determined by a reverse transcription-quantitative polymerase chain reaction. It was revealed that PGE1 significantly induced the phosphorylation of the α and β subunits of AMPK in a time-dependent manner (P<0.05). In addition, acetyl-CoA carboxylase, a direct substrate of AMPK, was significantly phosphorylated by PGE1 (P<0.05). Compound C, an AMPK inhibitor, was revealed to suppress the phosphorylation of acetyl-CoA carboxylase, which significantly reduced the release and mRNA expression levels of PGE1-stimulated osteoprotegerin (P<0.05). However, the PGE1-induced phosphorylation of p38 MAP kinase and SAPK/JNK were not affected by compound C. The results of the present study indicated that AMPK may positively regulate PGE1-stimulated osteoprotegerin synthesis in osteoblasts; thus providing novel insight into the regulatory mechanisms underlying bone metabolism. PMID:27168848

  9. Opposing activity changes in AMP deaminase and AMP-activated protein kinase in the hibernating ground squirrel.

    PubMed

    Lanaspa, Miguel A; Epperson, L Elaine; Li, Nanxing; Cicerchi, Christina; Garcia, Gabriela E; Roncal-Jimenez, Carlos A; Trostel, Jessica; Jain, Swati; Mant, Colin T; Rivard, Christopher J; Ishimoto, Takuji; Shimada, Michiko; Sanchez-Lozada, Laura Gabriela; Nakagawa, Takahiko; Jani, Alkesh; Stenvinkel, Peter; Martin, Sandra L; Johnson, Richard J

    2015-01-01

    Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel. PMID:25856396

  10. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

    PubMed Central

    Jin, Benjamin Y.; Sartoretto, Juliano L.; Gladyshev, Vadim N.; Michel, Thomas

    2009-01-01

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H2O2 is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H2O2 treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC50 for H2O2-promoted phosphorylation of AMPK is 65 ± 15 μM, within the physiological range of cellular H2O2 concentrations. The Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) inhibitor STO-609 abolishes H2O2-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKβ abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H2O2 biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H2O2 generation, which is blocked by PEG-catalase. eNOS−/− mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS−/− mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKβ is critically involved in mediating the phosphorylation of AMPK promoted by H2O2 in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H2O2 generation in endothelial cells. PMID:19805165

  11. RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer.

    PubMed

    Guo, Chengcheng; Hao, Chuncheng; Shao, RuPing; Fang, Bingliang; Correa, Arlene M; Hofstetter, Wayne L; Roth, Jack A; Behrens, Carmen; Kalhor, Neda; Wistuba, Ignacio I; Swisher, Stephen G; Pataer, Apar

    2015-05-10

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expression of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinase(T172) [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  12. Glucose Availability and AMP-Activated Protein Kinase Link Energy Metabolism and Innate Immunity in the Bovine Endometrium

    PubMed Central

    Turner, Matthew L.; Cronin, James G.; Noleto, Pablo G.; Sheldon, I. Martin

    2016-01-01

    Defences against the bacteria that usually infect the endometrium of postpartum cattle are impaired when there is metabolic energy stress, leading to endometritis and infertility. The endometrial response to bacteria depends on innate immunity, with recognition of pathogen-associated molecular patterns stimulating inflammation, characterised by secretion of interleukin (IL)-1β, IL-6 and IL-8. How metabolic stress impacts tissue responses to pathogens is unclear, but integration of energy metabolism and innate immunity means that stressing one system might affect the other. Here we tested the hypothesis that homeostatic pathways integrate energy metabolism and innate immunity in bovine endometrial tissue. Glucose deprivation reduced the secretion of IL-1β, IL-6 and IL-8 from ex vivo organ cultures of bovine endometrium challenged with the pathogen-associated molecular patterns lipopolysaccharide and bacterial lipopeptide. Endometrial inflammatory responses to lipopolysaccharide were also reduced by small molecules that activate or inhibit the intracellular sensor of energy, AMP-activated protein kinase (AMPK). However, inhibition of mammalian target of rapamycin, which is a more global metabolic sensor than AMPK, had little effect on inflammation. Similarly, endometrial inflammatory responses to lipopolysaccharide were not affected by insulin-like growth factor-1, which is an endocrine regulator of metabolism. Interestingly, the inflammatory responses to lipopolysaccharide increased endometrial glucose consumption and induced the Warburg effect, which could exacerbate deficits in glucose availability in the tissue. In conclusion, metabolic energy stress perturbed inflammatory responses to pathogen-associated molecular patterns in bovine endometrial tissue, and the most fundamental regulators of cellular energy, glucose availability and AMPK, had the greatest impact on innate immunity. PMID:26974839

  13. Perturbing microtubule integrity blocks AMP-activated protein kinase-induced meiotic resumption in cultured mouse oocytes.

    PubMed

    Ya, Ru; Downs, Stephen M

    2014-02-01

    The oocyte meiotic spindle is comprised of microtubules (MT) that bind chromatin and regulate both metaphase plate formation and karyokinesis during meiotic maturation; however, little information is known about their role in meiosis reinitiation. This study was conducted to determine if microtubule integrity is required for meiotic induction and to ascertain how it affects activation of AMP-activated protein kinase (AMPK), an important participant in the meiotic induction process. Treatment with microtubule-disrupting agents nocodazole and vinblastine suppressed meiotic resumption in a dose-dependent manner in both arrested cumulus cell-enclosed oocytes (CEO) stimulated with follicle-stimulating hormone (FSH) and arrested denuded oocytes (DO) stimulated with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR). This effect coincided with suppression of AMPK activation as determined by western blotting and germinal vesicle immunostaining. Treatment with the MT stabilizer paclitaxel also suppressed meiotic induction. Targeting actin filament polymerization had only a marginal effect on meiotic induction. Immunolocalization experiments revealed that active AMPK colocalized with γ-tubulin during metaphase I and II stages, while it localized at the spindle midzone during anaphase. This discrete localization pattern was dependent on MT integrity. Treatment with nocodazole led to disruption of proper spindle pole localization of active AMPK, while paclitaxel induced excessive polymerization of spindle MT and formation of ectopic asters with accentuated AMPK colocalization. Although stimulation of AMPK increased the rate of germinal vesicle breakdown (GVB), spindle formation and polar body (PB) extrusion, the kinase had no effect on peripheral movement of the spindle. These data suggest that the meiosis-inducing action and localization of AMPK are regulated by MT spindle integrity during mouse oocyte maturation. PMID:23199370

  14. Glucose Availability and AMP-Activated Protein Kinase Link Energy Metabolism and Innate Immunity in the Bovine Endometrium.

    PubMed

    Turner, Matthew L; Cronin, James G; Noleto, Pablo G; Sheldon, I Martin

    2016-01-01

    Defences against the bacteria that usually infect the endometrium of postpartum cattle are impaired when there is metabolic energy stress, leading to endometritis and infertility. The endometrial response to bacteria depends on innate immunity, with recognition of pathogen-associated molecular patterns stimulating inflammation, characterised by secretion of interleukin (IL)-1β, IL-6 and IL-8. How metabolic stress impacts tissue responses to pathogens is unclear, but integration of energy metabolism and innate immunity means that stressing one system might affect the other. Here we tested the hypothesis that homeostatic pathways integrate energy metabolism and innate immunity in bovine endometrial tissue. Glucose deprivation reduced the secretion of IL-1β, IL-6 and IL-8 from ex vivo organ cultures of bovine endometrium challenged with the pathogen-associated molecular patterns lipopolysaccharide and bacterial lipopeptide. Endometrial inflammatory responses to lipopolysaccharide were also reduced by small molecules that activate or inhibit the intracellular sensor of energy, AMP-activated protein kinase (AMPK). However, inhibition of mammalian target of rapamycin, which is a more global metabolic sensor than AMPK, had little effect on inflammation. Similarly, endometrial inflammatory responses to lipopolysaccharide were not affected by insulin-like growth factor-1, which is an endocrine regulator of metabolism. Interestingly, the inflammatory responses to lipopolysaccharide increased endometrial glucose consumption and induced the Warburg effect, which could exacerbate deficits in glucose availability in the tissue. In conclusion, metabolic energy stress perturbed inflammatory responses to pathogen-associated molecular patterns in bovine endometrial tissue, and the most fundamental regulators of cellular energy, glucose availability and AMPK, had the greatest impact on innate immunity. PMID:26974839

  15. AMP-activated protein kinase: a key regulator of energy balance with many roles in human disease.

    PubMed

    Grahame Hardie, D

    2014-12-01

    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that regulates cellular and whole-body energy balance. A recently reported crystal structure has illuminated the complex regulatory mechanisms by which AMP and ADP cause activation of AMPK, involving phosphorylation by the upstream kinase LKB1. Once activated by falling cellular energy status, AMPK activates catabolic pathways that generate ATP whilst inhibiting anabolic pathways and other cellular processes that consume ATP. A role of AMPK is implicated in many human diseases. Mutations in the γ2 subunit cause heart disease due to excessive glycogen storage in cardiac myocytes, leading to ventricular pre-excitation. AMPK-activating drugs reverse many of the metabolic defects associated with insulin resistance, and recent findings suggest that the insulin-sensitizing effects of the widely used antidiabetic drug metformin are mediated by AMPK. The upstream kinase LKB1 is a tumour suppressor, and AMPK may exert many of its antitumour effects. AMPK activation promotes the oxidative metabolism typical of quiescent cells, rather than the aerobic glycolysis observed in tumour cells and cells involved in inflammation, explaining in part why AMPK activators have both antitumour and anti-inflammatory effects. Salicylate (the major in vivo metabolite of aspirin) activates AMPK, and this could be responsible for at least some of the anticancer and anti-inflammatory effects of aspirin. In addition to metformin and salicylates, novel drugs that modulate AMPK are likely to enter clinical trials soon. Finally, AMPK may be involved in viral infection: downregulation of AMPK during hepatitis C virus infection appears to be essential for efficient viral replication. PMID:24824502

  16. Regulation of 5'AMP-activated protein kinase activity and substrate utilization in exercising human skeletal muscle.

    PubMed

    Wojtaszewski, Jorgen F P; MacDonald, Christopher; Nielsen, Jakob N; Hellsten, Ylva; Hardie, D Grahame; Kemp, Bruce E; Kiens, Bente; Richter, Erik A

    2003-04-01

    The metabolic role of 5'AMP-activated protein kinase (AMPK) in regulation of skeletal muscle metabolism in humans is unresolved. We measured isoform-specific AMPK activity and beta-acetyl-CoA carboxylase (ACCbeta) Ser(221) phosphorylation and substrate balance in skeletal muscle of eight athletes at rest, during cycling exercise for 1 h at 70% peak oxygen consumption, and 1 h into recovery. The experiment was performed twice, once in a glycogen-loaded (glycogen concentration approximately 900 mmol/kg dry wt) and once in a glycogen-depleted (glycogen concentration approximately 160 mmol/kg dry wt) state. At rest, plasma long-chain fatty acids (FA) were twofold higher in the glycogen-depleted than in the loaded state, and muscle alpha1 AMPK (160%) and alpha2 AMPK (145%) activities and ACCbeta Ser(221) phosphorylation (137%) were also significantly higher in the glycogen-depleted state. During exercise, alpha2 AMPK activity, ACCbeta Ser(221) phosphorylation, plasma catecholamines, and leg glucose and net FA uptake were significantly higher in the glycogen-depleted than in the glycogen-loaded state without apparent differences in muscle high-energy phosphates. Thus exercise in the glycogen-depleted state elicits an enhanced uptake of circulating fuels that might be associated with elevated muscle AMPK activation. It is concluded that muscle AMPK activity and ACCbeta Ser(221) phosphorylation at rest and during exercise are sensitive to the fuel status of the muscle. During exercise, this dependence may in part be mediated by humoral factors. PMID:12488245

  17. Altered content of AMP-activated protein kinase isoforms in skeletal muscle from spinal cord injured subjects.

    PubMed

    Kostovski, Emil; Boon, Hanneke; Hjeltnes, Nils; Lundell, Leonidas S; Ahlsén, Maria; Chibalin, Alexander V; Krook, Anna; Iversen, Per Ole; Widegren, Ulrika

    2013-11-01

    AMP-activated protein kinase (AMPK) is a pivotal regulator of energy homeostasis. Although downstream targets of AMPK are widely characterized, the physiological factors governing isoform expression of this protein kinase are largely unknown. Nerve/contractile activity has a major impact on the metabolic phenotype of skeletal muscle, therefore likely to influence AMPK isoform expression. Spinal cord injury represents an extreme form of physical inactivity, with concomitant changes in skeletal muscle metabolism. We assessed the influence of longstanding and recent spinal cord injury on protein abundance of AMPK isoforms in human skeletal muscle. We also determined muscle fiber type as a marker of glycolytic or oxidative metabolism. In subjects with longstanding complete injury, protein abundance of the AMPKγ3 subunit, as well as myosin heavy chain (MHC) IIa and IIx, were increased, whereas abundance of the AMPKγ1 subunit and MHC I were decreased. Similarly, abundance of AMPKγ3 and MHC IIa proteins were increased, whereas AMPKα2, -β1, and -γ1 subunits and MHC I abundance was decreased during the first year following injury, reflecting a more glycolytic phenotype of the skeletal muscle. However, in incomplete cervical lesions, partial recovery of muscle function attenuated the changes in the isoform profile of AMPK and MHC. Furthermore, exercise training (electrically stimulated leg cycling) partly normalized mRNA expression of AMPK isoforms. Thus, physical activity affects the relative expression of AMPK isoforms. In conclusion, skeletal muscle abundance of AMPK isoforms is related to physical activity and/or muscle fiber type. Thus, physical/neuromuscular activity is an important determinant of isoform abundance of AMPK and MCH. This further underscores the need for physical activity as part of a treatment regimen after spinal cord injury to maintain skeletal muscle metabolism. PMID:24022865

  18. Hepatic Glycogen Supercompensation Activates AMP-Activated Protein Kinase, Impairs Insulin Signaling, and Reduces Glycogen Deposition in the Liver

    PubMed Central

    Winnick, Jason J.; An, Zhibo; Ramnanan, Christopher J.; Smith, Marta; Irimia, Jose M.; Neal, Doss W.; Moore, Mary Courtney; Roach, Peter J.; Cherrington, Alan D.

    2011-01-01

    OBJECTIVE The objective of this study was to determine how increasing the hepatic glycogen content would affect the liver’s ability to take up and metabolize glucose. RESEARCH DESIGN AND METHODS During the first 4 h of the study, liver glycogen deposition was stimulated by intraportal fructose infusion in the presence of hyperglycemic-normoinsulinemia. This was followed by a 2-h hyperglycemic-normoinsulinemic control period, during which the fructose infusion was stopped, and a 2-h experimental period in which net hepatic glucose uptake (NHGU) and disposition (glycogen, lactate, and CO2) were measured in the absence of fructose but in the presence of a hyperglycemic-hyperinsulinemic challenge including portal vein glucose infusion. RESULTS Fructose infusion increased net hepatic glycogen synthesis (0.7 ± 0.5 vs. 6.4 ± 0.4 mg/kg/min; P < 0.001), causing a large difference in hepatic glycogen content (62 ± 9 vs. 100 ± 3 mg/g; P < 0.001). Hepatic glycogen supercompensation (fructose infusion group) did not alter NHGU, but it reduced the percent of NHGU directed to glycogen (79 ± 4 vs. 55 ± 6; P < 0.01) and increased the percent directed to lactate (12 ± 3 vs. 29 ± 5; P = 0.01) and oxidation (9 ± 3 vs. 16 ± 3; P = NS). This change was associated with increased AMP-activated protein kinase phosphorylation, diminished insulin signaling, and a shift in glycogenic enzyme activity toward a state discouraging glycogen accumulation. CONCLUSIONS These data indicate that increases in hepatic glycogen can generate a state of hepatic insulin resistance, which is characterized by impaired glycogen synthesis despite preserved NHGU. PMID:21270252

  19. Effects of adenosine on intrarenal oxygenation.

    PubMed

    Dinour, D; Brezis, M

    1991-11-01

    Although generally a vasodilator, adenosine vasoconstricts cortical vessels in the kidney, reduces glomerular filtration rate (GFR), and increases medullary blood flow, effects likely to improve the medullary O2 deficiency characteristic of mammalian kidneys. To evaluate a possible role of adenosine in medullary O2 balance, we investigated the effect of adenosine upon cortical and medullary tissue PO2. Adenosine was infused into renal interstitium through chronically implanted capsules. Cortical and medullary PO2 were measured using sensitive Clark-type O2 microelectrodes inserted into kidneys of anesthetized rats at the respective depths of 1.8 and 3.7 mm. Infusion of adenosine (0.1-0.5 mumol/min) increased medullary PO2 from 17 +/- 3 (SE) to 40 +/- 5 mmHG (P less than 0.001) and decreased cortical PO2 from 64 +/- 4 to 47 +/- 3 mmHg (P less than 0.001). After the infusion was stopped, PO2 returned to baseline at both sites. Coadministration of adenosine receptor antagonist 8-phenyltheophylline (0.01 mumol/min) prevented both cortical and medullary effects of adenosine. We concluded that adenosine could play an important protective and regulatory role in renal medullary O2 balance. PMID:1951710

  20. Adenosine Neuromodulation and Traumatic Brain Injury

    PubMed Central

    Lusardi, T.A

    2009-01-01

    Adenosine is a ubiquitous signaling molecule, with widespread activity across all organ systems. There is evidence that adenosine regulation is a significant factor in traumatic brain injury (TBI) onset, recovery, and outcome, and a growing body of experimental work examining the therapeutic potential of adenosine neuromodulation in the treatment of TBI. In the central nervous system (CNS), adenosine (dys)regulation has been demonstrated following TBI, and correlated to several TBI pathologies, including impaired cerebral hemodynamics, anaerobic metabolism, and inflammation. In addition to acute pathologies, adenosine function has been implicated in TBI comorbidities, such as cognitive deficits, psychiatric function, and post-traumatic epilepsy. This review presents studies in TBI as well as adenosine-related mechanisms in co-morbidities of and unfavorable outcomes resulting from TBI. While the exact role of the adenosine system following TBI remains unclear, there is increasing evidence that a thorough understanding of adenosine signaling will be critical to the development of diagnostic and therapeutic tools for the treatment of TBI. PMID:20190964

  1. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  2. Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut

    PubMed Central

    Burnstock, G; Campbell, G; Satchell, D; Smythe, ANNE

    1997-01-01

    Stimulation of the vagal non-adrenergic inhibitory innervation caused the release of adenosine and inosine into vascular perfusates from the stomachs of guinea-pigs and toads. Stimulation of portions of Auerbach's plexus isolated from turkey gizzard caused the release of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). ATP, added to solutions perfused through the toad stomach vasculature, was broken down to adenosine, inosine and adenine. Of a series of purine and pyrimidine derivatives tested for inhibitory activity on the guinea-pig isolated taenia coli, ATP and ADP were the most potent. ATP caused inhibition of twelve other gut preparations previously shown to contain non-adrenergic inhibitory nerves. The inhibitory action of ATP was not prevented by tetrodotoxin. Quinidine antagonized relaxations of the guinea-pig taenia coli caused by catecholamines or adrenergic nerve stimulation. Higher concentrations of quinidine antagonized relaxations caused by ATP or non-adrenergic inhibitory nerve stimulation. When tachyphylaxis to ATP had been produced in the rabbit ileum, there was a consistent depression of the responses to non-adrenergic inhibitory nerve stimulation but not of responses to adrenergic nerve stimulation. It is suggested that ATP or a related nucleotide is the transmitter substance released by the non-adrenergic inhibitory innervation of the gut. PMID:9142414

  3. An improved method for the enzymatic transformation of nucleosides into 5'-monophosphates.

    PubMed

    Barai, Vladimir N; Kvach, Sergei V; Zinchenko, Anatoli I; Mikhailopulo, Igor A

    2004-12-01

    An improved method to transform nucleosides into 5'-monophosphates using nucleoside phosphotransferase from Erwinia herbicola is reported. The method is based on the shift in the equilibrium state of the reaction to the formation of desired product due to its precipitation by Zn2+. Under optimal conditions, the extent of nucleoside transformations into nucleoside-5'-monophosphates were 41-91% (mol). PMID:15672226

  4. Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Kristjansson, Hordur; Sadler, Martha H.; Hochstein, Lawrence I.

    1986-01-01

    Membranes prepared from various members of the genus Halobacterium contained a Triton X-l00 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90 percent of total protein. The 60-kDa subunit reacted with dicyclohexyl-carbodiimide when inhibition was carried out in an acidic medium. The enzyme from H. saccharovorum, possesses properties of an F(1)F(0) as well as an E(1)E(2) ATPase.

  5. AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD.

    PubMed

    Brandauer, Josef; Andersen, Marianne A; Kellezi, Holti; Risis, Steve; Frøsig, Christian; Vienberg, Sara G; Treebak, Jonas T

    2015-01-01

    The mitochondrial protein deacetylase sirtuin (SIRT) 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS) handling. We determined the requirement of AMP-activated protein kinase (AMPK) for exercise training-induced increases in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p < 0.01) and superoxide dismutase 2 (MnSOD; p < 0.05) protein abundance in quadriceps muscle of wild-type (WT; n = 13-15), but not AMPK α2 kinase dead (KD; n = 12-13) mice. We also observed a strong trend for increased MnSOD abundance in exercise-trained skeletal muscle of healthy humans (p = 0.051; n = 6). To further elucidate a role for AMPK in mediating these effects, we treated WT (n = 7-8) and AMPK α2 KD (n = 7-9) mice with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). Four weeks of daily AICAR injections (500 mg/kg) resulted in AMPK-dependent increases in SIRT3 (p < 0.05) and MnSOD (p < 0.01) in WT, but not AMPK α2 KD mice. We also tested the effect of repeated AICAR treatment on mitochondrial protein levels in mice lacking the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α KO; n = 9-10). Skeletal muscle SIRT3 and MnSOD protein abundance was reduced in sedentary PGC-1α KO mice (p < 0.01) and AICAR-induced increases in SIRT3 and MnSOD protein abundance was only observed in WT mice (p < 0.05). Finally, the acetylation status of SIRT3 target lysine residues on MnSOD (K122) or oligomycin-sensitivity conferring protein (OSCP; K139) was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling in skeletal muscle in response to exercise training. PMID:25852572

  6. AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD

    PubMed Central

    Brandauer, Josef; Andersen, Marianne A.; Kellezi, Holti; Risis, Steve; Frøsig, Christian; Vienberg, Sara G.; Treebak, Jonas T.

    2015-01-01

    The mitochondrial protein deacetylase sirtuin (SIRT) 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS) handling. We determined the requirement of AMP-activated protein kinase (AMPK) for exercise training-induced increases in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p < 0.01) and superoxide dismutase 2 (MnSOD; p < 0.05) protein abundance in quadriceps muscle of wild-type (WT; n = 13–15), but not AMPK α2 kinase dead (KD; n = 12–13) mice. We also observed a strong trend for increased MnSOD abundance in exercise-trained skeletal muscle of healthy humans (p = 0.051; n = 6). To further elucidate a role for AMPK in mediating these effects, we treated WT (n = 7–8) and AMPK α2 KD (n = 7–9) mice with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). Four weeks of daily AICAR injections (500 mg/kg) resulted in AMPK-dependent increases in SIRT3 (p < 0.05) and MnSOD (p < 0.01) in WT, but not AMPK α2 KD mice. We also tested the effect of repeated AICAR treatment on mitochondrial protein levels in mice lacking the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α KO; n = 9–10). Skeletal muscle SIRT3 and MnSOD protein abundance was reduced in sedentary PGC-1α KO mice (p < 0.01) and AICAR-induced increases in SIRT3 and MnSOD protein abundance was only observed in WT mice (p < 0.05). Finally, the acetylation status of SIRT3 target lysine residues on MnSOD (K122) or oligomycin-sensitivity conferring protein (OSCP; K139) was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling in skeletal muscle in response to exercise training. PMID:25852572

  7. [AMP-activated protein kinase activation regulates adhesion of monocytes to vascular endothelial cells and the underlying mechanism].

    PubMed

    Bai, Hong-Bo; Wang, Yun; Zhang, Yu-Hua; Zhang, Yuan

    2016-02-25

    The present study was aimed to explore the effect of AMP-activated protein kinase (AMPK) on monocyte adhesion to vascular endothelial cells and underlying molecular mechanism. Tumor necrosis factor α (TNFα)-activated human aortic endothelial cells (HAECs) were treated with different concentrations of AMPK agonist 5-Aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) or AMPK inhibitor compound C. And other HAECs were overexpressed with constitutive active or dominant negative AMPK protein and then treated with TNFα. The rates of monocytes adhering to endothelial cells were detected by fluorescent staining. Intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels and protein secretions were detected by quantitative PCR and ELISA, respectively. Acetylation of NF-κB p65 at lysine 221 site was assessed by Western blot. NF-κB p65 DNA binding activity was analyzed by an ELISA-based method. By using small interfering RNA based strategy, p300 expression in HAECs was down-regulated and then cells were incubated with TNFα. NF-κB p65 DNA binding activity, ICAM-1 and VCAM-1 expressions and adhesion rates were detected, respectively. The activity of p300 was also detected by ELISA. The results showed that AICAR treatment significantly reduced monocyte-endothelial adhesion rate, as well as ICAM-1 and VCAM-1 mRNA levels and protein secretions, in TNFα-activated HAECs. Moreover, transfection of constitutive active AMPKα but not dominant negative AMPKα strongly diminished TNFα-induced upregulation of ICAM-1 and VCAM-1 mRNA expressions and secretions, as well as monocyte-endothelial adhesion. Furthermore, AMPK activation decreased TNFα-mediated acetylation of NF-κB p65 at Lys221 site and reduced NF-κB p65 DNA binding activity. Silencing p300 by siRNA significantly abolished the effect of TNFα- induced adhesion molecules expression and monocyte-endothelial adhesion. Blocking AMPK activation by compound C almost

  8. Female mice are more susceptible to nonalcoholic fatty liver disease: sex-specific regulation of the hepatic AMP-activated protein kinase-plasminogen activator inhibitor 1 cascade, but not the hepatic endotoxin response.

    PubMed

    Spruss, Astrid; Henkel, Janin; Kanuri, Giridhar; Blank, Daniela; Püschel, Gerhard P; Bischoff, Stephan C; Bergheim, Ina

    2012-01-01

    As significant differences between sexes were found in the susceptibility to alcoholic liver disease in human and animal models, it was the aim of the present study to investigate whether female mice also are more susceptible to the development of non-alcoholic fatty liver disease (NAFLD). Male and female C57BL/6J mice were fed either water or 30% fructose solution ad libitum for 16 wks. Liver damage was evaluated by histological scoring. Portal endotoxin levels and markers of Kupffer cell activation and insulin resistance, plasminogen activator inhibitor 1 (PAI-1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK ) were measured in the liver. Adiponectin mRNA expression was determined in adipose tissue. Hepatic steatosis was almost similar between male and female mice; however, inflammation was markedly more pronounced in livers of female mice. Portal endotoxin levels, hepatic levels of myeloid differentiation primary response gene (88) (MyD88) protein and of 4-hydroxynonenal protein adducts were elevated in animals with NAFLD regardless of sex. Expression of insulin receptor substrate 1 and 2 was decreased to a similar extent in livers of male and female mice with NAFLD. The less pronounced susceptibility to liver damage in male mice was associated with a superinduction of hepatic pAMPK in these mice whereas, in livers of female mice with NAFLD, PAI-1 was markedly induced. Expression of adiponectin in visceral fat was significantly lower in female mice with NAFLD but unchanged in male mice compared with respective controls. In conclusion, our data suggest that the sex-specific differences in the susceptibility to NAFLD are associated with differences in the regulation of the adiponectin-AMPK-PAI-1 signaling cascade. PMID:22952059

  9. Optical Aptasensors for Adenosine Triphosphate

    PubMed Central

    Ng, Stella; Lim, Hui Si; Ma, Qian; Gao, Zhiqiang

    2016-01-01

    Nucleic acids are among the most researched and applied biomolecules. Their diverse two- and three-dimensional structures in conjunction with their robust chemistry and ease of manipulation provide a rare opportunity for sensor applications. Moreover, their high biocompatibility has seen them being used in the construction of in vivo assays. Various nucleic acid-based devices have been extensively studied as either the principal element in discrete molecule-like sensors or as the main component in the fabrication of sensing devices. The use of aptamers in sensors - aptasensors, in particular, has led to improvements in sensitivity, selectivity, and multiplexing capacity for a wide verity of analytes like proteins, nucleic acids, as well as small biomolecules such as glucose and adenosine triphosphate (ATP). This article reviews the progress in the use of aptamers as the principal component in sensors for optical detection of ATP with an emphasis on sensing mechanism, performance, and applications with some discussion on challenges and perspectives. PMID:27446501

  10. Gas-phase protonation thermochemistry of adenosine.

    PubMed

    Touboul, David; Bouchoux, Guy; Zenobi, Renato

    2008-09-18

    The goal of this work was to obtain a detailed insight on the gas-phase protonation energetic of adenosine using both mass spectrometric experiments and quantum chemical calculations. The experimental approach used the extended kinetic method with nanoelectrospray ionization and collision-induced dissociation tandem mass spectrometry. This method provides experimental values for proton affinity, PA(adenosine) = 979 +/- 1 kJ.mol (-1), and for the "protonation entropy", Delta p S degrees (adenosine) = S degrees (adenosineH +) - S degrees (adenosine) = -5 +/- 5 J.mol (-1).K (-1). The corresponding gas-phase basicity is consequently equal to: GB(adenosine) = 945 +/- 2 kJ.mol (-1) at 298K. Theoretical calculations conducted at the B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G(d,p) level, including 298 K enthalpy correction, predict a proton affinity value of 974 kJ.mol (-1) after consideration of isodesmic proton transfer reactions with pyridine as the reference base. Moreover, computations clearly showed that N3 is the most favorable protonation site for adenosine, due to a strong internal hydrogen bond involving the hydroxyl group at the 2' position of the ribose sugar moiety, unlike observations for adenine and 2'-deoxyadenosine, where protonation occurs on N1. The existence of negligible protonation entropy is confirmed by calculations (theoretical Delta p S degrees (adenosine) approximately -2/-3 J.mol (-1).K (-1)) including conformational analysis and entropy of hindered rotations. Thus, the calculated protonation thermochemical properties are in good agreement with our experimental measurements. It may be noted that the new PA value is approximately 10 kJ.mol (-1) lower than the one reported in the National Institute of Standards and Technology (NIST) database, thus pointing to a correction of the tabulated protonation thermochemistry of adenosine. PMID:18720985

  11. Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase.

    PubMed

    Floyd, E E; Jones, M E

    1985-08-01

    The multifunctional protein uridine 5'-monophosphate (UMP) synthase catalyzes the final two reactions of the de novo biosynthesis of UMP in mammalian cells by the sequential action of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23). This protein is composed of one or two identical subunits; the monomer weighs of 51,500 daltons. UMP synthase from mouse Ehrlich ascites cells can exist as three distinct species as determined by sucrose density gradient centrifugation: a 3.6 S monomer, a 5.1 S dimer, and a 5.6 S conformationally altered dimer. Limited digestion of each of these three species with trypsin produced a 28,500-dalton peptide that was relatively resistant to further proteolysis. The peptide appears to be one of the two enzyme domains of UMP synthase for it retained only OMP decarboxylase activity. Similar results were obtained when UMP synthase was digested with elastase. OMP decarboxylase activity was less stable for the domain than for UMP synthase; the domain can rapidly lose activity upon storage or upon dilution. The size of the mammalian OMP decarboxylase domain is similar to that of yeast OMP decarboxylase. If the polypeptides which are cleaved from UMP synthase by trypsin are derived exclusively from either the amino or the carboxyl end of UMP synthase, then the size of a fragment possessing the orotate phosphoribosyltransferase domain could be as large as 23,000 daltons which is similar in size to the orotate phosphoribosyltransferase of yeast and of Escherichia coli. PMID:3839509

  12. Orally Active Adenosine A1 Receptor Agonists with Antinociceptive Effects in Mice

    PubMed Central

    Korboukh, Ilia; Hull-Ryde, Emily A.; Rittiner, Joseph E.; Randhawa, Amarjit S.; Coleman, Jennifer; Fitzpatrick, Brendan J.; Setola, Vincent; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.; Jin, Jian

    2012-01-01

    Adenosine A1 receptor (A1AR) agonists have antinociceptive effects in multiple preclinical models of acute and chronic pain. Although numerous A1AR agonists have been developed, clinical applications of these agents have been hampered by their cardiovascular side effects. Herein we report a series of novel A1AR agonists, some of which are structurally related to adenosine 5′-monophosphate (5′-AMP), a naturally occurring nucleotide that itself activates A1AR. These novel compounds potently activate A1AR in several orthogonal in vitro assays and are subtype selective for A1AR over A2AAR, A2BAR, and A3AR. Among them, UNC32A (3a) is orally active and has dose-dependent antinociceptive effects in wild-type mice. The antinociceptive effects of 3a were completely abolished in A1AR knockout mice, revealing a strict dependence on A1AR for activity. The apparent lack of cardiovascular side effects when administered orally and high affinity (Ki of 36 nM for the human A1AR) make this compound potentially suitable as a therapeutic. PMID:22738238

  13. Adenosine triphosphate inhibition of yeast trehalase.

    PubMed

    Panek, A D

    1969-09-01

    Yeast trehalase has been found to be inhibited non-competitively by adenosine triphosphate. Such a biological control could explain the accumulation of trehalose during the stationary phase of the growth curve. PMID:5370287

  14. Role of adenosine receptors in caffeine tolerance

    SciTech Connect

    Holtzman, S.G.; Mante, S.; Minneman, K.P. )

    1991-01-01

    Caffeine is a competitive antagonist at adenosine receptors. Receptor up-regulation during chronic drug treatment has been proposed to be the mechanism of tolerance to the behavioral stimulant effects of caffeine. This study reassessed the role of adenosine receptors in caffeine tolerance. Separate groups of rats were given scheduled access to drinking bottles containing plain tap water or a 0.1% solution of caffeine. Daily drug intake averaged 60-75 mg/kg and resulted in complete tolerance to caffeine-induced stimulation of locomotor activity, which could not be surmounted by increasing the dose of caffeine. 5'-N-ethylcarboxamidoadenosine (0.001-1.0 mg/kg) dose dependently decreased the locomotor activity of caffeine-tolerant rats and their water-treated controls but was 8-fold more potent in the latter group. Caffeine (1.0-10 mg/kg) injected concurrently with 5-N-ethylcarboxamidoadenosine antagonized the decreases in locomotor activity comparably in both groups. Apparent pA2 values for tolerant and control rats also were comparable: 5.05 and 5.11. Thus, the adenosine-antagonist activity of caffeine was undiminished in tolerant rats. The effects of chronic caffeine administration on parameters of adenosine receptor binding and function were measured in cerebral cortex. There were no differences between brain tissue from control and caffeine-treated rats in number and affinity of adenosine binding sites or in receptor-mediated increases (A2 adenosine receptor) and decreases (A1 adenosine receptor) in cAMP accumulation. These results are consistent with theoretical arguments that changes in receptor density should not affect the potency of a competitive antagonist. Experimental evidence and theoretical considerations indicate that up-regulation of adenosine receptors is not the mechanism of tolerance to caffeine-induced stimulation of locomotor activity.

  15. Recent improvements in the development of A2B adenosine receptor agonists

    PubMed Central

    Tabrizi, Mojgan Aghazadeh; Fruttarolo, Francesca; Romagnoli, Romeo; Preti, Delia

    2009-01-01

    Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A1, A2A, A2B and A3 (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N6-, C2-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA1Ki = 1050 nM, hA2AKi = 1550 nM, hA2B EC50 = 82 nM, hA3Ki > 5 μM) and its 2-chloro analogue 23 (hA1Ki = 3500 nM, hA2AKi = 4950 nM, hA2B EC50 = 210 nM, hA3Ki > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA1, hA2A, hA3 EC50 > 10 μM; hA2B EC50 = 3 nM) is currently under preclinical-phase investigation for treating coronary

  16. Recent improvements in the development of A2B adenosine receptor agonists

    PubMed Central

    Tabrizi, Mojgan Aghazadeh; Fruttarolo, Francesca; Romagnoli, Romeo; Preti, Delia

    2008-01-01

    Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A1, A2A, A2B and A3 (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N6-, C2-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA1Ki = 1050 nM, hA2AKi = 1550 nM, hA2B EC50 = 82 nM, hA3Ki > 5 μM) and its 2-chloro analogue 23 (hA1Ki = 3500 nM, hA2AKi = 4950 nM, hA2B EC50 = 210 nM, hA3Ki > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA1, hA2A, hA3 EC50 > 10 μM; hA2B EC50 = 3 nM) is currently under preclinical-phase investigation for treating coronary

  17. Regulation of Lymphocyte Function by Adenosine

    PubMed Central

    Linden, Joel; Cekic, Caglar

    2014-01-01

    Adenosine regulates the interaction between lymphocytes and the vasculature and is important for controlling lymphocyte trafficking in response to tissue injury or infection. Adenosine can blunt the effects of T cell receptor (TCR) activation primarily by activating adenosine A2A receptors (A2AR) and signaling via cyclic AMP and protein kinase A (PKA). PKA reduces proximal TCR signaling by phosphorylation of C-terminal Src kinase (Csk), nuclear factor of activated T cells (NF-AT) and cyclic AMP response element binding protein (CREB). PKA activation can either enhance or inhibit the survival of T cells depending on the strength and duration of signaling. Inducible enzymes such as CD73 and CD39 regulate adenosine formation and degradation in vivo. The extravasation of lymphocytes through blood vessels is influenced by A2AR-mediated suppression of Intercellular Adhesion Molecule 1 (ICAM) expression on lymphocytes and diminished production of IFNγ and IFNγ-inducible chemokines that are chemotactic to activated lymphocytes. Adenosine also decreases the barrier function of vascular endothelium by activating A2BRs. In sum, adenosine signaling is influenced by tissue inflammation and injury through induction of receptors and enzymes and has generally inhibitory effects on lymphocyte migration into inflamed tissues due to PKA-mediated effects on adhesion molecules, IFNγ production and endothelial barrier function. PMID:22772752

  18. Adenosine receptor agonists attenuate and adenosine receptor antagonists exacerbate opiate withdrawal signs.

    PubMed

    Kaplan, G B; Sears, M T

    1996-01-01

    Previous studies have demonstrated a role for adenosine in mediating opiate effects. Adenosine receptors and their functions have been shown to be regulated by chronic opiate treatment. This study examines the role of adenosine receptors in the expression of opiate withdrawal behaviors. The effects of single doses of parenterally administered adenosine receptor subtype-selective agonists and antagonists on opiate withdrawal signs in morphine-dependent mice were measured. Mice received subcutaneous morphine pellet treatment for 72 h and then underwent naloxone-precipitated withdrawal after pretreatment with adenosinergic agents. Adenosine agonists attenuated different opiate withdrawal signs. The A1 agonist R-N6(phenylisopropyl)adenosine (0, 0.01, 0.02 mg/kg, IP) significantly reduced wet dog shakes and withdrawal diarrhea, while the A2a-selective agonist 2-p-(2-carboxethyl)phenylethylamino-5'-N-ethylcarboxamido adenosine or CGS 21680 (0, 0.01, 0.05 mg/kg, IP) significantly inhibited teeth chattering and forepaw treads. Adenosine receptor antagonists enhanced different opiate withdrawal signs. The adenosine A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (0, 1, 10 mg/kg, IP) significantly increased weight loss and the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (0, 1 and 10 mg/kg, IP) enhanced wet dog shakes and withdrawal diarrhea. Treatment effects of adenosinergic agents were not due to nonspecific motor effects, as demonstrated by activity monitoring studies. These results support a role for adenosine receptors in the expression of opiate withdrawal and suggest the potential utility of adenosine agonists in its treatment. PMID:8741956

  19. Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency

    PubMed Central

    Garone, Caterina; Garcia-Diaz, Beatriz; Emmanuele, Valentina; Lopez, Luis C; Tadesse, Saba; Akman, Hasan O; Tanji, Kurenai; Quinzii, Catarina M; Hirano, Michio

    2014-01-01

    Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2−/−) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2−/− mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2−/−200dCMP/dTMP) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency. Subject Categories Genetics, Gene Therapy & Genetic Disease; Metabolism PMID:24968719

  20. Nitric oxide and cyclic guanosine monophosphate signaling in the eye.

    PubMed

    Murad, Ferid

    2008-06-01

    This brief review describes the components and pathways utilized in nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) signaling. Since the discovery of the effects of NO and cGMP on smooth muscle relaxation about 30 years ago, the field has expanded in many directions such that many, but not all, biochemical and biological effects seem to be regulated by these unique signaling molecules. While many of the effects of NO are due to activation of soluble guanylyl cyclase (sGC) that can be considered the receptor for NO, cGMP, in turn, can activate a cGMP-dependent protein kinase (PKG) to phosphorylate an array of proteins. Some of the effects of cGMP can be independent of PKG and are due to effects on ion channels or cyclic nucleotide phosphodiesterases. Also, some of the effects of NO can be independent of sGC activation. The isoenzymes and macromolecules that participate in these signaling pathways can serve as molecular targets to identify compounds that increase or decrease their activation and thus serve as chemical leads for discovering novel drugs for a variety of diseases. Some examples are given. However, with about 90,000 publications in the field since our first reports in 1977, this brief review can only give the readers a sample of the excitement and opportunities we have found in this cell signaling system. PMID:18443613

  1. Phosphatidylinositol 3-Monophosphate Is Involved in Toxoplasma Apicoplast Biogenesis

    PubMed Central

    Tawk, Lina; Dubremetz, Jean-François; Montcourrier, Philippe; Chicanne, Gaëtan; Merezegue, Fabrice; Richard, Véronique; Payrastre, Bernard; Meissner, Markus; Vial, Henri J.; Roy, Christian

    2011-01-01

    Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs. PMID:21379336

  2. Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis.

    PubMed

    Tawk, Lina; Dubremetz, Jean-François; Montcourrier, Philippe; Chicanne, Gaëtan; Merezegue, Fabrice; Richard, Véronique; Payrastre, Bernard; Meissner, Markus; Vial, Henri J; Roy, Christian; Wengelnik, Kai; Lebrun, Maryse

    2011-02-01

    Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs. PMID:21379336

  3. Bioconjugation of zirconium uridine monophosphate: application to myoglobin direct electrochemistry.

    PubMed

    Qiao, Yuanbiao; Jian, Fangfang; Bai, Qian

    2008-03-14

    Porous nano-granule of zirconium uridine monophosphate, Zr(UMP)2.H2O is, for the first time, synthesized under mild experimental conditions and applied to the bioconjugation of myoglobin (Mb) to realize its direct electron transfer. UV-vis and resonance Raman spectroscopies prove that Mb in the Zr(UMP)2.H2O film maintains its secondary structure similar to the native state. The conjugation film of the Mb-Zr(UMP)2.H2O on the glassy carbon (GC) electrode gives a well-defined and quasi-reversible cyclic voltammogram, which reflects the direct electron transfer of the heme Fe III/Fe II couple of Mb. On the basis of the satisfying bioelectrocatalysis of the nano-conjugation of Mb and genetic substrate, a kind of mediator-free biosensor for H2O2 is developed. The linear range for H2O2 detection is estimated to be 3.92-180.14 microM. The apparent Michaelis-Menten constant (Km) and the detection limit based on the signal-to-noise ratio of 3 are found to be 196.1 microM and 1.52 microM, respectively. Both the apparent Michaelis-Menten constant and the detection limit herein are much lower than currently reported values from other Mb films. This kind of sensor possesses excellent stability, long-term life (more than 20 days) and good reproducibility. PMID:18180152

  4. Ag(+)-mediated assembly of 5'-guanosine monophosphate.

    PubMed

    Loo, Kristine; Degtyareva, Natalya; Park, Jihae; Sengupta, Bidisha; Reddish, Michaeal; Rogers, Christopher C; Bryant, Andrea; Petty, Jeffrey T

    2010-04-01

    Polymorphic forms of nucleic acids provide platforms for new nanomaterials, and transition metal cations give access to alternative arrangements of nucleobases by coordinating with electron-rich functional groups. Interaction of Ag(+) with 5'-guanosine monophosphate (5'-GMP) is considered in this work. Ag(+) promotes nucleotide stacking and aggregation, as indicated by the increased viscosity of 5'-GMP solutions with Ag(+), magnification of the circular dichroism response of guanine by Ag(+), and exothermic reactions between Ag(+) and guanine derivatives. Isothermal titration calorimetry studies show that the reaction is favored starting at 10 microM 5'-GMP. Utilizing the exothermic heat change associated with reaction of Ag(+) with 5'-GMP, local structure within the aggregate was assessed. On the basis of the salt dependence of the reaction and comparison with the corresponding nucleoside, the dianionic phosphate of 5'-GMP is one binding site for Ag(+), although this electrostatic interaction is not a dominant contribution to the overall heat change. Another binding site is the N7 on the nucleobase, as determined via studies with 7-deazaguanosine. Besides this binding site, Ag(+) also associates with the O6, as earlier studies deduced from the shift in the carbonyl stretching frequency associated with adduct formation. With these two binding sites on the nucleobase, the empirical stoichiometry of approximately 1 Ag(+):nucleobase derived from the calorimetry studies indicates that Ag(+) coordinates two nucleobases. The proposed structural model is a Ag(+)-mediated guanine dimer within a base stacked aggregate. PMID:20205377

  5. Photoaffinity labeling of A1-adenosine receptors

    SciTech Connect

    Klotz, K.N.; Cristalli, G.; Grifantini, M.; Vittori, S.; Lohse, M.J.

    1985-11-25

    The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6-phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A1-subtype. It competes for (TH)N6-phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of (TH)N6-phenylisopropyladenosine binding after extensive washing; the Ki value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity label of high specific radioactivity ( SVI-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for SVI-AHPIA binding to rat brain membranes with an order of potency characteristic for A1-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of Mr = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A1-subtype. The results indicate that SVI-AHPIA identifies the ligand-binding subunit of the A1-adenosine receptor, which is a peptide with Mr = 35,000.

  6. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis

    SciTech Connect

    Polekhina, Galina Feil, Susanne C.; Gupta, Abhilasha; O’Donnell, Paul; Stapleton, David; Parker, Michael W.

    2005-01-01

    The glycogen-binding domain of the AMP-activated kinase β subunit has been crystallized in the presence of β-cyclodextrin. The structure has been determined by single isomorphous replacement and threefold averaging using in-house X-ray data collected from selenomethionine-substituted protein. AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic α subunit and two regulatory subunits, β and γ. Mutations in the γ subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the β subunit. Here, the crystallization of GBD in the presence of β-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein.

  7. Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods

    NASA Astrophysics Data System (ADS)

    Liao, Yu-Ju; Shiang, Yen-Chun; Chen, Li-Yi; Hsu, Chia-Lun; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-11-01

    We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin-Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ˜56 nm and diameter ˜12 nm), G-actin-Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin-Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin-Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin-Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin-luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

  8. Effects of different concentrations of metal ions on degradation of adenosine triphosphate in common carp (Cyprinus carpio) fillets stored at 4°C: An in vivo study.

    PubMed

    Li, Dapeng; Qin, Na; Zhang, Longteng; Lv, Jian; Li, Qingzheng; Luo, Yongkang

    2016-11-15

    The impact of different concentrations of Na(+), K(+), Ca(2+), Mg(2+), Fe(2+), and Zn(2+) on the degradation of adenosine triphosphate (ATP) and the influence of these ions on the activity of adenosine monophosphate deaminase (AMP-deaminase) and acid phosphatase (ACP) in common carp fillets (in vivo) during 4°C storage was examined. The content of ATP, inosine monophosphate (IMP), and hypoxanthine (Hx), and the activity of AMP-deaminase and ACP were determined. Results indicated that the effects of different concentrations of six kinds of metal ions on AMP-deaminase and ACP were not the same. Na(+), K(+), Fe(2+), and Zn(2+) enhanced AMP-deaminase activity, which led to the rapid degradation of ATP and to the generation of a large quantity of IMP within a short time. Ca(2+) and Mg(2+) delayed the change in AMP-deaminase and ACP activity in carp and caused a further delay in the degradation of ATP. Fe(2+) and Zn(2+) inhibited ACP activity, which reduced the decomposition of IMP and the formation of Hx. PMID:27283700

  9. Characterization of a monoclonal antibody to thymidine glycol monophosphate

    SciTech Connect

    Chen, B.X.; Hubbard, K.; Ide, H.; Wallace, S.S.; Erlanger, B.F. )

    1990-11-01

    A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions.

  10. Nucleoside transporter expression and adenosine uptake in the rat cochlea.

    PubMed

    Khan, Abdul F; Thorne, Peter R; Muñoz, David J B; Wang, Carol J H; Housley, Gary D; Vlajkovic, Srdjan M

    2007-02-12

    Even though extracellular adenosine plays multiple roles in the cochlea, the mechanisms that control extracellular adenosine concentrations in this organ are unclear. This study investigated the expression of nucleoside transporters and adenosine uptake in the rat cochlea. Reverse transcription-polymerase chain reaction revealed the expression of mRNA transcripts for two equilibrative (ENT1 and ENT2) and two concentrative (CNT1 and CNT2) nucleoside transporters. Exogenous adenosine perfused through the cochlear perilymphatic compartment was taken up by cells lining the compartment. Adenosine uptake was sensitive to changes in extracellular Na concentrations and inhibited by nitrobenzylthioinosine (an adenosine uptake blocker). The study suggests that the bi-directional nucleoside transport supports the uptake and recycling of purines and regulates the activation of adenosine receptors by altering adenosine concentrations in cochlear fluid spaces. PMID:17314663

  11. Novel adenosine receptors in rat hippocampus identification and characterization

    SciTech Connect

    Chin, J.H.; Mashman, W.E.; DeLorenzo, R.J.

    1985-05-06

    2-chloro(/sup 3/H)adenosine, a stable analog of adenosine, was used to investigate the presence of adenosine receptors in rat hippocampal membranes that may mediate the depressant effects of adenosine on synaptic transmission in this tissue. Equilibrium binding studies reveal the presence of a previously undescribed class of receptors with a K/sub D/ of 4.7 ..mu..M and a Bmax of 130 pmol/mg of protein. Binding is sensitive to alkylxanthines and to a number of adenosine-related compounds. The pharmacological properties of this binding site are distinct from those of the A1 and A2 adenosine receptors associated with adenylate cyclase. The results suggest that this adenosine binding site is a novel central purinergic receptor through which adenosine may regulate hippocampal excitability. 50 references, 2 figures, 1 table.

  12. Pioglitazone stimulates AMP-activated protein kinase signalling and increases the expression of genes involved in adiponectin signalling, mitochondrial function and fat oxidation in human skeletal muscle in vivo: a randomised trial

    PubMed Central

    Sriwijitkamol, A.; Wajcberg, E.; Tantiwong, P.; Li, M.; Prentki, M.; Madiraju, M.; Jenkinson, C. P.; Cersosimo, E.; Musi, N.; DeFronzo, R. A.

    2016-01-01

    Aims/hypothesis The molecular mechanisms by which thiazolidinediones improve insulin sensitivity in type 2 diabetes are not fully understood. We hypothesised that pioglitazone would activate the adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway and increase the expression of genes involved in adiponectin signalling, NEFA oxidation and mitochondrial function in human skeletal muscle. Methods A randomised, double-blind, parallel study was performed in 26 drug-naive type 2 diabetes patients treated with: (1) pioglitazone (n=14) or (2) aggressive nutritional therapy (n=12) to reduce HbA1c to levels observed in the pioglitazone-treated group. Participants were assigned randomly to treatment using a table of random numbers. Before and after 6 months, patients reported to the Clinical Research Center of the Texas Diabetes Institute for a vastus lateralis muscle biopsy followed by a 180 min euglycaemic–hyperinsulinaemic (80 mU m−2 min−1) clamp. Results All patients in the pioglitazone (n=14) or nutritional therapy (n=12) group were included in the analysis. Pioglitazone significantly increased plasma adiponectin concentration by 79% and reduced fasting plasma NEFA by 35% (both p<0.01). Following pioglitazone, insulin-stimulated glucose disposal increased by 30% (p<0.01), and muscle AMPK and acetyl-CoA carboxylase (ACC) phosphorylation increased by 38% and 53%, respectively (p<0.05). Pioglitazone increased mRNA levels for adiponectin receptor 1 and 2 genes (ADIPOR1, ADIPOR2), peroxisome proliferator-activated receptor gamma, coactivator 1 gene (PPARGC1) and multiple genes involved in mitochondrial function and fat oxidation. Despite a similar reduction in HbA1c and similar improvement in insulin sensitivity with nutritional therapy, there were no significant changes in muscle AMPK and ACC phosphorylation, or the expression of ADIPOR1, ADIPOR2, PPARGC1 and genes involved in mitochondrial function and fat oxidation. No adverse (or unexpected) effects

  13. Kinetics and mechanism of the acid-catalyzed hydrolysis of a hypermodified nucleoside wyosine and its 5'-monophosphate.

    PubMed Central

    Golankiewicz, B; Zielonacka-Lis, E; Folkman, W

    1985-01-01

    The rates of acid-catalyzed hydrolysis of a hypermodified nucleoside, wyosine and its 5'-monophosphate were determined at various pH, temperature and buffer concentrations. The results show that despite distinct differences in structure and the glycosyl bond stability, the hydrolysis of wyosine proceeds via cleavage of the C-N bond by A-1 mechanism, analogously to simple nucleosides. Unlike majority of other monophosphates studied so far, wyosine 5'-monophosphate is not more stable than respective nucleoside. PMID:4000960

  14. Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

    PubMed Central

    Snyder, F F; Mendelsohn, J; Seegmiller, J E

    1976-01-01

    The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 muM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes. Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemagglutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 muM adenosine, and deamination at concentrations greater than 5 muM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine

  15. Extracellular formation and uptake of adenosine during skeletal muscle contraction in the rat: role of adenosine transporters.

    PubMed

    Lynge, J; Juel, C; Hellsten, Y

    2001-12-01

    1. The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. 2. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km = 177 +/- 36 microM and Vmax = 1.9 +/- 0.2 nmol x ml(-1) x s(-1) (0.7 nmol (mg protein)(-1) x s(-1)). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72% inhibition) or dipyridamol (64% inhibition; P < 0.05). 3. In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57% larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. 4. Inhibition of ecto-5'-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70% lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. 5. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the

  16. Body weight management effect of burdock (Arctium lappa L.) root is associated with the activation of AMP-activated protein kinase in human HepG2 cells.

    PubMed

    Kuo, Daih-Huang; Hung, Ming-Chi; Hung, Chao-Ming; Liu, Li-Min; Chen, Fu-An; Shieh, Po-Chuen; Ho, Chi-Tang; Way, Tzong-Der

    2012-10-01

    Burdock (Arcticum lappa L.) root is used in folk medicine and also as a vegetable in Asian countries. In the present study, burdock root treatment significantly reduced body weight in rats. To evaluate the bioactive compounds, we successively extracted the burdock root with ethanol (AL-1), and fractionated it with n-hexane (AL-2), ethyl acetate (AL-3), n-butanol (AL-4), and water (AL-5). Among these fractions, AL-2 contained components with the most effective hypolipidemic potential in human hepatoma HepG2 cells. AL-2 decreased the expression of fatty acid synthase (FASN) and inhibited the activity of acetyl-coenzyme A carboxylase (ACC) by stimulating AMP-activated protein kinase (AMPK) through the LKB1 pathway. Three active compounds were identified from the AL-2, namely α-linolenic acid, methyl α-linolenate, and methyl oleate. These results suggest that burdock root is expected to be useful for body weight management. PMID:25005949

  17. 5-aminoimidazole-4-carboxamide Riboside Induces Apoptosis Through AMP-activated Protein Kinase-independent and NADPH Oxidase-dependent Pathways

    PubMed Central

    Wi, Sae Mi

    2014-01-01

    It is debatable whether AMP-activated protein kinase (AMPK) activation is involved in anti-apoptotic or pro-apoptotic signaling. AICAR treatment increases AMPK-α1 phosphorylation, decreases intracellular reactive oxygen species (ROS) levels, and significantly increases Annexin V-positive cells, DNA laddering, and caspase activity in human myeloid cell. AMPK activation is therefore implicated in apoptosis. However, AMPK-α1-knockdown THP-1 cells are more sensitive to apoptosis than control THP-1 cells are, suggesting that the apoptosis is AMPK-independent. Low doses of AICAR induce cell proliferation, whereas high doses of AICAR suppress cell proliferation. Moreover, these effects are significantly correlated with the downregulation of intracellular ROS, strongly suggesting that AICAR-induced apoptosis is critically associated with the inhibition of NADPH oxidase by AICAR. Collectively, our results demonstrate that in AICAR-induced apoptosis, intracellular ROS levels are far more relevant than AMPK activation. PMID:25360075

  18. Adenosine reagent-free detection by co-immobilization of adenosine deaminase and phenol red on an optical biostrip.

    PubMed

    Bartzoka, Foteini; Venetsanou, Katerina; Clonis, Yannis

    2015-01-01

    Adenosine detection in human serum is important because this ribonucleoside has established clinical applications, modulating many physiological processes. Furthermore, a simple and cheap detection method is useful in adenosine production processes. Adenosine can be determined enzymatically using either S-adenosyl-homocysteine hydrolase and (3) [H]-adenosine, or adenosine kinase combined with GTP and luciferase, or an amperometric biosensor carrying adenosine deaminase (ADA), purine nucleoside phosphorylase, and xanthine oxidase. We developed a simple and cheap method relying on a transparent biostrip bearing ADA and the indicator phenol red (PR), co-immobilized to polyacrylamide, itself chemically adhered to a derivatized glass strip. The ADA-catalyzed conversion of adenosine to inosine and ammonia leads to a local pH alteration, changing the absorbance maximum of PR (from 425 to 567 nm), which is measured optically. The biostrip shows an analytical range 0.05-1.5 mM adenosine and is reusable when stored at 4 °C. When the biostrip was tested with serum, spiked with adenosine (70 and 100 μM), and filtered for protein and adenosine phosphates depletion, it showed good adenosine recovery. In summary, we show the proof-of-concept that adenosine can be determined reagent-free, at moderate sensitivity on an easy to construct, cheap, and reusable biostrip, based on commercially available molecular entities. PMID:25293641

  19. Emodin protects against high-fat diet-induced obesity via regulation of AMP-activated protein kinase pathways in white adipose tissue.

    PubMed

    Tzeng, Thing-Fong; Lu, Hung-Jen; Liou, Shorong-Shii; Chang, Chia Ju; Liu, I-Min

    2012-06-01

    Emodin is an active herbal component traditionally used in China for treating a variety of diseases. The aim of this study was to examine the effect of emodin on the reducing lipid accumulation in white adipose tissue of high-fat diet-fed rats, and on the regulation of the expression of the genes involved in lipid metabolism to elucidate the mechanisms. After being fed a high-fat diet for two weeks, rats were dosed orally with emodin (20, 40, 80 mg/kg/day) or pioglitazone (20 mg/kg/day), once daily for eight weeks. Changes in body weight, feeding pattern, serum lipids, coronary artery risk index, and atherogenic index were investigated. Subcutaneous white adipose tissues were isolated for pathology histology and Western blot analyses. Changes of triglyceride accumulation in differentiated 3 T3-L1 adipocytes were also investigated. Emodin exhibited a significant concentration-dependent decrease in the intracellular accumulation of triglyceride in 3 T3-L1 adipocytes. Emodin (80 mg/kg/day) displayed similar characteristics to pioglitazone (20 mg/kg/day) in reducing body weight gain and plasma lipid levels as well as the coronary artery risk and atherogenic indices of high-fat diet-fed rats. Emodin also caused dose related reductions in epididymal white adipose tissue sizes in high-fat diet-fed rats. Emodin and pioglitazone enhanced the phosphorylation of AMP-activated protein kinase and its primary downstream targeting enzyme, acetyl-CoA carboxylase, upregulated gene expression of carnitine palmitoyl transferase 1, and downregulated sterol regulatory element binding protein 1 and fatty acid synthase protein levels in the epididymal white adipose tissue of high-fat diet-fed rats. Our findings suggest that emodin could attenuate lipid accumulation in white adipose tissue through AMP-activated protein kinase activation. PMID:22673833

  20. DNA polymorphisms and transcript abundance of PRKAG2 and phosphorylated AMP-activated protein kinase in the rumen are associated with gain and feed intake in beef steers.

    PubMed

    Lindholm-Perry, A K; Kuehn, L A; Oliver, W T; Kern, R J; Cushman, R A; Miles, J R; McNeel, A K; Freetly, H C

    2014-08-01

    Beef steers with variation in feed efficiency phenotypes were evaluated previously on a high-density SNP panel. Ten markers from rs110125325-rs41652818 on bovine chromosome 4 were associated with average daily gain (ADG). To identify the gene(s) in this 1.2-Mb region responsible for variation in ADG, genotyping with 157 additional markers was performed. Several markers (n = 41) were nominally associated with ADG, and three of these, including the only marker to withstand Bonferroni correction, were located within the protein kinase, AMP-activated, gamma 2 non-catalytic subunit (PRKAG2) gene. An additional population of cross-bred steers (n = 406) was genotyped for validation. One marker located within the PRKAG2 loci approached a significant association with gain. To evaluate PRKAG2 for differences in transcript abundance, we measured expression in the liver, muscle, rumen and intestine from steers (n = 32) with extreme feed efficiency phenotypes collected over two seasons. No differences in PRKAG2 transcript abundance were detected in small intestine, liver or muscle. Correlation between gene expression level of PRKAG2 in rumen and average daily feed intake (ADFI) was detected in both seasons (P < 0.05); however, the direction differed by season. Lastly, we evaluated AMP-activated protein kinase (AMPK), of which PRKAG2 is a subunit, for differences among ADG and ADFI and found that the phosphorylated form of AMPK was associated with ADFI in the rumen. These data suggest that PRKAG2 and its mature protein, AMPK, are involved in feed efficiency traits in beef steers. This is the first evidence to suggest that rumen AMPK may be contributing to ADFI in cattle. PMID:24730749

  1. Internalization and desensitization of adenosine receptors

    PubMed Central

    Klaasse, Elisabeth C.; de Grip, Willem J.; Beukers, Margot W.

    2007-01-01

    Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A1, A2A, A2B and A3 receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A1Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A2AR and A2BR undergo much faster downregulation, usually shorter than 1 h. The A3R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A3R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed. PMID:18368531

  2. Changes in histone phosphorylation and associated early metabolic events in pig lymphocyte cultures transformed by phytohaemagglutinin or 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate

    PubMed Central

    Cross, Margaret E.; Ord, Margery G.

    1971-01-01

    1. Pig lymphocytes were transformed by dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) at concentrations of 0.01–0.1μm. The pattern of incorporation of label from [5-3H]uridine and [6-3H]thymidine into RNA and DNA respectively was identical with that obtained with unpurified phytohaemagglutinin. 2. Chlorpromazine (0.1μm) prevented the stimulation of [5-3H]uridine incorporation into RNA by phytohaemagglutinin, but only slightly lowered the lymphocyte response to dibutyryl cyclic AMP. 3. An increase in the size and specific radioactivity of the intracellular Pi pool was found immediately after stimulation by both phytohaemagglutinin and dibutyryl cyclic AMP. This was followed after some 30min by a rise in the specific radioactivity and concentration of ATP. 4. There was an immediate increase in the specific radioactivity of phosphate groups of histones; by about 45min after stimulation only the histones remaining after extraction of histone fraction F1 continued to incorporate 32P from [32P]Pi. 5. Histone kinase activity increased in the first 30min after stimulation; subsequently histone F1 kinase activity decreased, but activity with the other histones as substrate continued to increase for a further 30min. Kinase activation was effected by cyclic AMP (adenosine 3′:5′-cyclic monophosphate). 6. Histone phosphatase activity behaved similarly to that of the kinase. PMID:4331256

  3. Neuroprotective effects of adenosine deaminase in the striatum.

    PubMed

    Tamura, Risa; Ohta, Hiroyuki; Satoh, Yasushi; Nonoyama, Shigeaki; Nishida, Yasuhiro; Nibuya, Masashi

    2016-04-01

    Adenosine deaminase (ADA) is a ubiquitous enzyme that catabolizes adenosine and deoxyadenosine. During cerebral ischemia, extracellular adenosine levels increase acutely and adenosine deaminase catabolizes the increased levels of adenosine. Since adenosine is a known neuroprotective agent, adenosine deaminase was thought to have a negative effect during ischemia. In this study, however, we demonstrate that adenosine deaminase has substantial neuroprotective effects in the striatum, which is especially vulnerable during cerebral ischemia. We used temporary oxygen/glucose deprivation (OGD) to simulate ischemia in rat corticostriatal brain slices. We used field potentials as the primary measure of neuronal damage. For stable and efficient electrophysiological assessment, we used transgenic rats expressing channelrhodopsin-2, which depolarizes neurons in response to blue light. Time courses of electrically evoked striatal field potential (eFP) and optogenetically evoked striatal field potential (optFP) were recorded during and after oxygen/glucose deprivation. The levels of both eFP and optFP decreased after 10 min of oxygen/glucose deprivation. Bath-application of 10 µg/ml adenosine deaminase during oxygen/glucose deprivation significantly attenuated the oxygen/glucose deprivation-induced reduction in levels of eFP and optFP. The number of injured cells decreased significantly, and western blot analysis indicated a significant decrease of autophagic signaling in the adenosine deaminase-treated oxygen/glucose deprivation slices. These results indicate that adenosine deaminase has protective effects in the striatum. PMID:26746865

  4. 2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors.

    PubMed

    Gao, Zhan-Guo; Mamedova, Liaman K; Chen, Peiran; Jacobson, Kenneth A

    2004-11-15

    The affinity and efficacy at four subtypes (A(1), A(2A), A(2B) and A(3)) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N(6)-position, several 2-substituents were found to be critical structural determinants for the A(3)AR activation. The following adenosine 2-ethers were moderately potent partial agonists (K(i), nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A(3)AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)adenosine as an A(3)AR antagonist right-shifted the concentration-response curve for the inhibition by NECA of cyclic AMP accumulation with a K(B) value of 212 nM, which is similar to its binding affinity (K(i) = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A(1)AR in comparison to the A(3)AR, but fully efficacious, with binding K(i) values over 100 nM. The 2-phenylethyl moiety resulted in higher A(3)AR affinity (K(i) in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (K(i) = 3.8 nM) was found to be the most potent and selective (>50-fold) A(2A) agonist in this series. Mixed A(2A)/A(3)AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A(2B)AR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC(50) = 1.4 microM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC(50) = 1.8 microM) were found to be relatively potent A(2B) agonists, although less potent than NECA (EC(50) = 140 nM). PMID:15476669

  5. Differences in responsiveness of intrapulmonary artery and vein to arachidonic acid: mechanism of arterial relaxation involves cyclic guanosine 3':5'-monophosphate and cyclic adenosine 3':5'-monophosphate

    SciTech Connect

    Ignarro, L.J.; Harbison, R.G.; Wood, K.S.; Wolin, M.S.; McNamara, D.B.; Hyman, A.L.; Kadowitz, P.J.

    1985-06-01

    The objective of this study was to examine the relationship between responses of bovine intrapulmonary artery and vein to arachidonic acid and cyclic nucleotide levels in order to better understand the mechanism of relaxation elicited by arachidonic acid and acetylcholine. Arachidonic acid relaxed phenylephrine-precontracted arterial rings and elevated both cyclic GMP and cyclic AMP levels in arteries with intact endothelium. In contrast, endothelium-damaged arterial rings contracted to arachidonic acid without demonstrating significant changes in cyclic nucleotide levels. Indomethacin partially inhibited endothelium-dependent relaxation and abolished cyclic AMP accumulation whereas methylene blue, a guanylate cyclase inhibitor, partially inhibited relaxation and abolished cyclic GMP accumulation in response to arachidonic acid. All vessel responses were blocked by a combination of the two inhibitors. Prostaglandin (PG) I2 relaxed arterial rings and elevated cyclic AMP levels whereas PGE2 and PGF2 alpha caused contraction, suggesting that the indomethacin-sensitive component of arachidonic acid-elicited relaxation is due to PGI2 formation and cyclic AMP accumulation. The methylene blue-sensitive component is attributed to an endothelium-dependent but cyclooxygenase-independent generation of a substance causing cyclic GMP accumulation. Intrapulmonary veins contracted to arachidonic acid with no changes in cyclic nucleotide levels and PGI2 was without effect. Homogenates of intrapulmonary artery and vein formed 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from (/sup 14/C)arachidonic acid, which was inhibited by indomethacin. Thus, bovine intrapulmonary vein may not possess receptors for PGI2.

  6. 2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors

    PubMed Central

    Gao, Zhan-Guo; Mamedova, Liaman K.; Chen, Peiran; Jacobson, Kenneth A.

    2012-01-01

    The affinity and efficacy at four subtypes (A1, A2A, A2B and A3) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N6-position, several 2-substituents were found to be critical structural determinants for the A3AR activation. The following adenosine 2-ethers were moderately potent partial agonists (Ki, nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A3AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)a-denosine as an A3AR antagonist right-shifted the concentration–response curve for the inhibition by NECA of cyclic AMP accumulation with a KB value of 212 nM, which is similar to its binding affinity (Ki = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A1AR in comparison to the A3AR, but fully efficacious, with binding Ki values over 100 nM. The 2-phenylethyl moiety resulted in higher A3AR affinity (Ki in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (Ki = 3.8 nM) was found to be the most potent and selective (>50-fold) A2A agonist in this series. Mixed A2A/A3AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A2BAR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC50 = 1.4 µM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC50 = 1.8 (M) were found to be relatively potent A2B agonists, although less potent than NECA (EC50 = 140 nM). PMID:15476669

  7. Silk polymer-based adenosine release: therapeutic potential for epilepsy.

    PubMed

    Wilz, Andrew; Pritchard, Eleanor M; Li, Tianfu; Lan, Jing-Quan; Kaplan, David L; Boison, Detlev

    2008-09-01

    Adenosine augmentation therapies (AAT) make rational use of the brain's own adenosine-based seizure control system and hold promise for the therapy of refractory epilepsy. In an effort to develop an AAT compatible with future clinical application, we developed a novel silk protein-based release system for adenosine. Adenosine releasing brain implants with target release doses of 0, 40, 200, and 1000ng adenosine per day were prepared by embedding adenosine containing microspheres into nanofilm-coated silk fibroin scaffolds. In vitro, the respective polymers released 0, 33.4, 170.5, and 819.0ng adenosine per day over 14 days. The therapeutic potential of the implants was validated in a dose-response study in the rat model of kindling epileptogenesis. Four days prior to the onset of kindling, adenosine releasing polymers were implanted into the infrahippocampal cleft and progressive acquisition of kindled seizures was monitored over a total of 48 stimulations. We document a dose-dependent retardation of seizure acquisition. In recipients of polymers releasing 819ng adenosine per day, kindling epileptogenesis was delayed by one week corresponding to 18 kindling stimulations. Histological analysis of brain samples confirmed the correct location of implants and electrodes. We conclude that silk-based delivery of around 1000ng adenosine per day is a safe and efficient strategy to suppress seizures. PMID:18514814

  8. Adenosine Signaling During Acute and Chronic Disease States

    PubMed Central

    Karmouty-Quintana, Harry; Xia, Yang; Blackburn, Michael R.

    2013-01-01

    Adenosine is a signaling nucleoside that is produced following tissue injury, particularly injury involving ischemia and hypoxia. The production of extracellular adenosine and its subsequent signaling through adenosine receptors plays an important role in orchestrating injury responses in multiple organs. There are four adenosine receptors that are widely distributed on immune, epithelial, endothelial, neuronal and stromal cells throughout the body. Interestingly, these receptors are subject to altered regulation following injury. Studies in mouse models and human cells and tissues have identified that the production of adenosine and its subsequent signaling through its receptors plays largely beneficial roles in acute disease states, with the exception of brain injury. In contrast, if elevated adenosine levels are sustained beyond the acute injury phase, adenosine responses can become detrimental by activating pathways that promote tissue injury and fibrosis. Understanding when during the course of disease adenosine signaling is beneficial as opposed to detrimental and defining the mechanisms involved will be critical for the advancement of adenosine based therapies for acute and chronic diseases. The purpose of this review is to discuss key observations that define the beneficial and detrimental aspects of adenosine signaling during acute and chronic disease states with an emphasis on cellular processes such as inflammatory cell regulation, vascular barrier function and tissue fibrosis. PMID:23340998

  9. Adenosine diphosphate-degrading activity in placenta.

    PubMed

    Barradas, M; Khokher, M; Hutton, R; Craft, I L; Dandona, P

    1983-02-01

    1. The degradation of ADP by the placenta and umbilical artery was investigated. 2. Supernatants from incubations of finely chopped placental and umbilical arterial tissue were incubated with [14C]ADP for various durations from 0 to 30 min. 3. Products of ADP degradation were separated by thin-layer chromatography and radioactivity incorporated into each product was measured. 4. Placental supernatants induced a more rapid degradation of ADP than the umbilical artery supernatants. The main product of ADP degradation by placental supernatants at 30 min was adenosine, whereas that of umbilical artery was AMP. 5. This conversion by placenta of ADP, a potent platelet aggregator and vasoconstrictor, into adenosine, a potent platelet anti-aggregator and vasodilator, may be important in the maintenance of perfusion of the foetoplacental unit. PMID:6822058

  10. Adenosine thallium 201 myocardial perfusion scintigraphy

    SciTech Connect

    Verani, M.S. )

    1991-07-01

    Pharmacologic coronary vasodilation as an adjunct to myocardial perfusion imaging has become increasingly important in the evaluation of patients with coronary artery disease, in view of the large number of patients who cannot perform an adequate exercise test or in whom contraindications render exercise inappropriate. Adenosine is a very potent coronary vasodilator and when combined with thallium 201 scintigraphy produces images of high quality, with the added advantages of a very short half-life (less than 10 seconds) and the ability to adjust the dose during the infusion, which may enhance safety and curtail the duration of side effects. The reported sensitivity and specificity of adenosine thallium 201 scintigraphy for the detection of coronary artery disease are high and at least comparable with imaging after exercise or dipyridamole administration. 23 refs.

  11. Chemoelectrical energy conversion of adenosine triphosphate

    NASA Astrophysics Data System (ADS)

    Sundaresan, Vishnu Baba; Sarles, Stephen Andrew; Leo, Donald J.

    2007-04-01

    Plant and animal cell membranes transport charged species, neutral molecules and water through ion pumps and channels. The energy required for moving species against established concentration and charge gradients is provided by the biological fuel - adenosine triphosphate (ATP) -synthesized within the cell. The adenosine triphosphatase (ATPases) in a plant cell membrane hydrolyze ATP in the cell cytoplasm to pump protons across the cell membrane. This establishes a proton gradient across the membrane from the cell exterior into the cell cytoplasm. This proton motive force stimulates ion channels that transport nutrients and other species into the cell. This article discusses a device that converts the chemical energy stored in adenosine triphosphate into electrical power using a transporter protein, ATPase. The V-type ATPase proteins used in our prototype are extracted from red beet(Beta vulgaris) tonoplast membranes and reconstituted in a bilayer lipid membrane or BLM formed from POPC and POPS lipids. A pH7 medium that can support ATP hydrolysis is provided on both sides of the membrane and ATP is dissolved in the pH7 buffer on one side of the membrane. Hydrolysis of ATP results in the formation of a phosphate ion and adenosine diphosphate. The energy from the reaction activates ATPase in the BLM and moves a proton across the membrane. The charge gradient established across the BLM due to the reaction and ion transport is converted into electrical current by half-cell reference electrodes. The prototype ATPase cell with an effective BLM area of 4.15 mm2 carrying 15 μl of ATPase proteins was observed to develop a steady state peak power output of 70 nW, which corresponds to a specific power of 1.69 μW/cm2 and a current density of 43.4 μA/cm2 of membrane area.

  12. Prevalence of unidirectional Na+-dependent adenosine transport and altered potential for adenosine generation in diabetic cardiac myocytes.

    PubMed

    Podgorska, M; Kocbuch, K; Grden, M; Szutowicz, A; Pawelczyk, T

    2006-05-01

    Adenosine is an important physiological regulator of the cardiovascular system. The goal of our study was to assess the expression level of nucleoside transporters (NT) in diabetic rat cardiomyocytes and to examine the activities of adenosine metabolizing enzymes. Isolated rat cardiomyocytes displayed the presence of detectable amounts of mRNA for ENT1, ENT2, CNT1, and CNT2. Overall adenosine (10 microM) transport in cardiomyocytes isolated from normal rat was 36 pmol/mg/min. The expression level of equilibrative transporters (ENT1, ENT2) decreased and of concentrative transporters (CNT1, CNT2) increased in myocytes isolated from diabetic rat. Consequently, overall adenosine transport decreased by 30%, whereas Na(+)-dependent adenosine uptake increased 2-fold, and equilibrative transport decreased by 60%. The activity ratio of AMP deaminase/5'-nucleotidase in cytosol of normal cardiomyocytes was 11 and increased to 15 in diabetic cells. The activity of ecto-5'-nucleotidase increased 2-fold in diabetic cells resulting in a rise of the activity ratio of ecto-5'-nucleotidase/adenosine deaminase from 28 to 56.These results indicate that in rat cardiomyocytes diabetes alters activities of adenosine metabolizing enzymes in such a way that conversion of AMP to IMP is favored in the cytosolic compartment, whereas the capability to produce adenosine extracellularly is increased. This is accompanied by an increased unidirectional Na(+)-dependent uptake of adenosine and significantly reduced bidirectional adenosine transport. PMID:16369729

  13. ATP- and adenosine-mediated signaling in the central nervous system: adenosine stimulates glutamate release from astrocytes via A2a adenosine receptors.

    PubMed

    Nishizaki, Tomoyuki

    2004-02-01

    Adenosine enhanced intracellular Ca(2+) concentrations in astrocytes via A(2a) adenosine receptors involving protein kinase A (PKA) activation. The Ca(2+) rise is inhibited by brefeldin A, an inhibitor of vesicular transport; but not by neomycin and U73122, phospholipase C inhibitors; xestospongin, an IP(3)-receptor inhibitor; ryanodine, a ryanodine-receptor inhibitor; TMB-8, an endoplasmic reticulum calcium-release blocker; octanol, a gap-junction inhibitor; or cadmium, a non-selective, calcium-channel blocker. Adenosine stimulates astrocytic glutamate release via an A(2a) adenosine receptors/PKA pathway, and the release is inhibited by the vesicular transport inhibitors brefeldin A and bafilomycin A1. A(2a) adenosine receptors and the ensuing PKA events, thus, are endowed with vesicular Ca(2+) release from an unknown intracellular calcium store and vesicular glutamate release from astrocytes. PMID:14978344

  14. Use of adenosine echocardiography for diagnosis of coronary artery disease

    SciTech Connect

    Zoghbi, W.A. )

    1991-07-01

    Two-dimensional echocardiography combined with exercise is sensitive and specific in the detection of coronary artery disease (CAD) by demonstrating transient abnormalities in wall motion. Frequently, however, patients cannot achieve maximal exercise because of various factors. Pharmacologic stress testing with intravenous adenosine was evaluated as a means of detecting CAD in a noninvasive manner. Patients with suspected CAD underwent echocardiographic imaging and simultaneous thallium 201 single-photon emission computed tomography during the intravenous administration of 140 micrograms/kg/min of adenosine. An increase in heart rate, decrease in blood pressure, and increase in double product were observed during adenosine administration. Initial observations revealed that wall motion abnormalities were induced by adenosine in areas of perfusion defects. The adenosine infusion was well tolerated, and symptoms disappeared within 1 to 2 minutes after termination of the infusion. Therefore preliminary observations suggest that adenosine echocardiography appears to be useful in the assessment of CAD.

  15. AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism.

    PubMed

    Pinkosky, Stephen L; Filippov, Sergey; Srivastava, Rai Ajit K; Hanselman, Jeffrey C; Bradshaw, Cheryl D; Hurley, Timothy R; Cramer, Clay T; Spahr, Mark A; Brant, Ashley F; Houghton, Jacob L; Baker, Chris; Naples, Mark; Adeli, Khosrow; Newton, Roger S

    2013-01-01

    ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease. PMID:23118444

  16. Fermented Rhus verniciflua Stokes Extract Exerts an Antihepatic Lipogenic Effect in Oleic-Acid-Induced HepG2 Cells via Upregulation of AMP-Activated Protein Kinase.

    PubMed

    Lee, Myoung-Sun; Kim, Joo-Seok; Cho, Sun-Mi; Lee, Seon Ok; Kim, Sung-Hoon; Lee, Hyo-Jeong

    2015-08-19

    Rhus verniciflua Stokes has been used as a traditional medicine and food supplement in Korea. In the present study, fermented R. verniciflua Stokes extract (FRVE), an allergen-free extract of R. verniciflua Stokes fermented with the yeast Saccharomyces carlsbergensis, was assessed for its lipid-lowering potential in an in vitro non-alcoholic fatty liver disease model. FRVE markedly suppressed lipid accumulation and intracellular triglycerides (TGs) in the presence of oleic acid (OA). Additionally, FRVE decreased both mRNA and protein levels of lipid-synthesis- and cholesterol-metabolism-related factors, such as sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), glycerol-3-phosphate acyltransferase (GPAT), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), in OA-induced HepG2 cells. Moreover, FRVE activated low-density lipoprotein receptor (LDLR), AMP-activated protein kinase (AMPK), and fatty acid oxidation-related factors peroxisome proliferator activated receptor α (PPARα) and carnitine palmitoyltransferase 1 (CPT-1). Further, the AMPK inhibitor compound C suppressed the increased expression of AMPK phosphorylation induced by FRVE. Phenolics and cosanols in FRVE increased the phosphorylation of AMPK and decreased that of SREBP-1. Taken together, our findings suggest that FRVE has antilipogenic potential in non-alcoholic fatty livers via AMPK upregulation. PMID:26176317

  17. Endogenous Ligand for GPR120, Docosahexaenoic Acid, Exerts Benign Metabolic Effects on the Skeletal Muscles via AMP-activated Protein Kinase Pathway*

    PubMed Central

    Kim, Nami; Lee, Jung Ok; Lee, Hye Jeong; Kim, Hyung Ip; Kim, Joong Kwan; Lee, Yong Woo; Lee, Soo Kyung; Kim, Su Jin; Park, Sun Hwa; Kim, Hyeon Soo

    2015-01-01

    Docosahexaenoic acid (DHA) is an endogenous ligand of G protein-coupled receptor 120 (GPR120). However, the mechanisms underlying DHA action are poorly understood. In this study, DHA stimulated glucose uptake in the skeletal muscles in an AMP-activated protein kinase (AMPK)-dependent manner. GPR120-mediated increase in intracellular Ca2+ was critical for DHA-mediated AMPK phosphorylation and glucose uptake. In addition, DHA stimulated GLUT4 translocation AMPK-dependently. Inhibition of AMPK and Ca2+/calmodulin-dependent protein kinase kinase blocked DHA-induced glucose uptake. DHA and GW9508, a GPR120 agonist, increased GPR120 expression. DHA-mediated glucose uptake was not observed in GPR120 knockdown conditions. DHA increased AMPK phosphorylation, glucose uptake, and intracellular Ca2+ concentration in primary cultured myoblasts. Taken together, these results indicated that the beneficial metabolic role of DHA was attributed to its ability to regulate glucose via the GPR120-mediated AMPK pathway in the skeletal muscles. PMID:26134561

  18. Stearoyl lysophosphatidylcholine prevents lipopolysaccharide-induced extracellular release of high mobility group box-1 through AMP-activated protein kinase activation.

    PubMed

    Kim, Joung-Min; Han, Hui-Jing; Hur, Young-Hoe; Quan, Hui; Kwak, Sang-Hyun; Choi, Jeong-Il; Bae, Hong-Beom

    2015-09-01

    Previous studies have suggested that stearoyl lysophosphatidlycholine (LPC) protects against lethal experimental sepsis by inhibiting lipopolysaccharide (LPS)-induced extracellular release of high-mobility group box 1 (HMGB1). However, limited information exists on the mechanism by which stearoyl-LPC suppresses the extracellular release of HMGB1 in monocyte/macrophages stimulated with LPS. In this study, we found that stearoyl-LPC increased the phosphorylation of AMP-activated protein kinase (AMPK) in macrophages. Exposure of LPS-stimulated macrophages to stearoyl-LPC decreased the extracellular release of HMGB1 in peritoneal macrophages, which were inhibited by the AMPK inhibitor, compound C. In addition, stearoyl-LPC-mediated suppression of HMGB1 release was abolished by siRNA-mediated knock-down of AMPKα1. Stearoyl-LPC increased the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of activated AMPK, in mice lungs and decreased HMGB1 levels in bronchoalveolar lavage fluids in mice administered LPS. These results reveal a novel mechanism by which stearoyl-LPC regulates LPS-mediated cellular translocation of HMGB1. PMID:26218280

  19. Hypolipidemic activity of Taraxacum mongolicum associated with the activation of AMP-activated protein kinase in human HepG2 cells.

    PubMed

    Liu, Yan-Jin; Shieh, Po-Chuen; Lee, Jang-Chang; Chen, Fu-An; Lee, Chih-Hung; Kuo, Sheng-Chu; Ho, Chi-Tang; Kuo, Daih-Huang; Huang, Li-Jiau; Way, Tzong-Der

    2014-08-01

    This study investigated the hypolipidemic effect and potential mechanisms of T. mongolicum extracts. T. mongolicum was extracted by refluxing three times with water (TM-1), 50% ethanol (TM-2) and 95% ethanol (TM-3). TM-2 contained components with the most effective hypolipidemic potentials in HepG2 cells. Extended administration of TM-2 stimulated a significant reduction in body weight and levels of serum triglyceride LDL-C and total cholesterol in rats. To evaluate the bioactive compounds, we successively fractionated TM-2 with n-hexane (TM-4), dichloromethane (TM-5), ethyl acetate (TM-6), and water (TM-7). TM-4 fraction had the most effective hypolipidemic potential in HepG2 cells, and it decreased the expression of fatty acid synthase (FASN) and inhibited the activity of acetyl-coenzyme A carboxylase (ACC) through the phosphorylation of AMP-activated protein kinase (AMPK). Linoleic acid, phytol and tetracosanol are bioactive compounds identified from TM-4. These results suggest that T. mongolicum is expected to be useful for hypolipidemic effects. PMID:24903219

  20. AMP-Activated Protein Kinase Attenuates High Salt-Induced Activation of Epithelial Sodium Channels (ENaC) in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Li, Xin-Yuan; Hu, Qing-Qing; Ma, He-Ping

    2016-01-01

    Recent studies suggest that the epithelial sodium channel (ENaC) is expressed in the endothelial cells. To test whether high salt affects the NO production via regulation of endothelial ENaC, human umbilical vein endothelial cells (HUVECs) were incubated in solutions containing either normal or high sodium (additional 20 mM NaCl). Our data showed that high sodium treatment significantly increased α-, β-, and γ-ENaC expression levels in HUVECs. Using the cell-attached patch-clamp technique, we demonstrated that high sodium treatment significantly increased ENaC open probability (PO). Moreover, nitric oxide synthase (eNOS) phosphorylation (Ser 1177) levels and NO production were significantly decreased by high sodium in HUVECs; the effects of high sodium on eNOS phosphorylation and NO production were inhibited by a specific ENaC blocker, amiloride. Our results showed that high sodium decreased AMP-activated kinase (AMPK) phosphorylation in endothelial cells. On the other hand, metformin, an AMPK activator, prevented high sodium-induced upregulation of ENaC expression and PO. Moreover, metformin prevented high salt-induced decrease in NO production and eNOS phosphorylation. These results suggest that high sodium stimulates ENaC activation by negatively modulating AMPK activity, thereby leading to reduction in eNOS activity and NO production in endothelial cells.

  1. Bai-Hu-Jia-Ren-Shen-Tang Decoction Reduces Fatty Liver by Activating AMP-Activated Protein Kinase In Vitro and In Vivo

    PubMed Central

    Liu, Hui-Kang; Hung, Tzu-Min; Huang, Hsiu-Chen; Lee, I-Jung; Chang, Chia-Chuan; Cheng, Jing-Jy; Lin, Lie-Chwen; Huang, Cheng

    2015-01-01

    Obesity and associated conditions, such as type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are currently a worldwide health problem. In Asian traditional medicine, Bai-Hu-Jia-Ren-Shen-Tang (BHJRST) is widely used in diabetes patients to reduce thirst. However, whether it has a therapeutic effect on T2DM or NAFLD is not known. The aim of this study was to examine whether BHJRST had a lipid-lowering effect using a HuS-E/2 cell model of fatty liver induced by palmitate and in a db/db mouse model of dyslipidemia. Incubation of HuS-E/2 cells with palmitate markedly increased lipid accumulation and expression of adipose triglyceride lipase (ATGL), which is involved in lipolysis. BHJRST significantly decreased lipid accumulation and increased ATGL levels and phosphorylation of AMP-activated protein kinase (AMPK) and its primary downstream target, acetyl-CoA carboxylase (ACC), which are involved in fatty acid oxidation. Furthermore, after twice daily oral administration for six weeks, BHJRST significantly reduced hepatic fat accumulation in db/db mice, as demonstrated by increased hepatic AMPK and ACC phosphorylation, reduced serum triglyceride levels, and reduced hepatic total lipid content. The results show that BHJRST has a lipid-lowering effect in the liver that is mediated by activation of the AMPK signaling pathway. PMID:26508982

  2. Chronic Glutathione Depletion Confers Protection against Alcohol-induced Steatosis: Implication for Redox Activation of AMP-activated Protein Kinase Pathway

    PubMed Central

    Chen, Ying; Singh, Surendra; Matsumoto, Akiko; Manna, Soumen K.; Abdelmegeed, Mohamed A.; Golla, Srujana; Murphy, Robert C.; Dong, Hongbin; Song, Byoung-Joon; Gonzalez, Frank J.; Thompson, David C.; Vasiliou, Vasilis

    2016-01-01

    The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2–related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD. PMID:27403993

  3. Inactivation of MARK4, an AMP-activated protein kinase (AMPK)-related kinase, leads to insulin hypersensitivity and resistance to diet-induced obesity.

    PubMed

    Sun, Chao; Tian, Liang; Nie, Jia; Zhang, Hai; Han, Xiao; Shi, Yuguang

    2012-11-01

    MARK4, also known as Par-1d/MarkL1, is a member of the AMP-activated protein kinase (AMPK)-related family of kinases, which are implicated in the regulation of dynamic biological functions, including glucose and energy homeostasis. However, the physiological function of MARK4 in mammals remains elusive. Here, we investigated a role for MARK4 in regulating energy homeostasis by generating mice with targeted inactivation of the mark4 gene. We show that MARK4 deficiency in mice caused hyperphagia, hyperactivity, and hypermetabolism, leading to protection from diet-induced obesity and its related metabolic complications through up-regulation of brown fat activity. Consequently, MARK4 deficiency mitigated insulin resistance associated with diet-induced obesity by dramatically enhancing insulin-stimulated AKT phosphorylation in major metabolic tissues. Ablation of MARK4 also significantly improved glucose homeostasis by up-regulating the activity and expression of AMPK kinase in key metabolic tissues. Taken together, these data identify a key role of MARK4 in energy metabolism, implicating the kinase as a novel drug target for the treatment of obesity and type 2 diabetes. PMID:22992738

  4. When phosphorylated at Thr148, the β2-subunit of AMP-activated kinase does not associate with glycogen in skeletal muscle.

    PubMed

    Xu, Hongyang; Frankenberg, Noni T; Lamb, Graham D; Gooley, Paul R; Stapleton, David I; Murphy, Robyn M

    2016-07-01

    The 5'-AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of α- and β-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, ∼60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an ∼80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total β2-AMPK that was phosphorylated in whole muscle homogenates measured by immunoprecipitation. These findings suggest that, in rat skeletal muscle, β2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate β2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of β2-AMPK in skeletal muscle. PMID:27099349

  5. AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages.

    PubMed

    Kemmerer, Marina; Finkernagel, Florian; Cavalcante, Marcela Frota; Abdalla, Dulcineia Saes Parra; Müller, Rolf; Brüne, Bernhard; Namgaladze, Dmitry

    2015-01-01

    AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload. PMID:26098914

  6. Combined Treatment of MCF-7 Cells with AICAR and Methotrexate, Arrests Cell Cycle and Reverses Warburg Metabolism through AMP-Activated Protein Kinase (AMPK) and FOXO1

    PubMed Central

    Fodor, Tamás; Szántó, Magdolna; Abdul-Rahman, Omar; Nagy, Lilla; Dér, Ádám; Kiss, Borbála; Bai, Peter

    2016-01-01

    Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK) jointly with methotrexate (MTX), a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1β or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer. PMID:26919657

  7. AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages

    PubMed Central

    Kemmerer, Marina; Finkernagel, Florian; Cavalcante, Marcela Frota; Abdalla, Dulcineia Saes Parra; Müller, Rolf; Brüne, Bernhard; Namgaladze, Dmitry

    2015-01-01

    AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload. PMID:26098914

  8. AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of {beta}-catenin at Ser 552

    SciTech Connect

    Zhao, Junxing; Yue, Wanfu; Zhu, Mei J.; Sreejayan, Nair; Du, Min

    2010-04-23

    AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; its activity is regulated by a plethora of physiological conditions, exercises and many anti-diabetic drugs. Recent studies show that AMPK involves in cell differentiation but the underlying mechanism remains undefined. Wingless Int-1 (Wnt)/{beta}-catenin signaling pathway regulates the differentiation of mesenchymal stem cells through enhancing {beta}-catenin/T-cell transcription factor 1 (TCF) mediated transcription. The objective of this study was to determine whether AMPK cross-talks with Wnt/{beta}-catenin signaling through phosphorylation of {beta}-catenin. C3H10T1/2 mesenchymal cells were used. Chemical inhibition of AMPK and the expression of a dominant negative AMPK decreased phosphorylation of {beta}-catenin at Ser 552. The {beta}-catenin/TCF mediated transcription was correlated with AMPK activity. In vitro, pure AMPK phosphorylated {beta}-catenin at Ser 552 and the mutation of Ser 552 to Ala prevented such phosphorylation, which was further confirmed using [{gamma}-{sup 32}P]ATP autoradiography. In conclusion, AMPK phosphorylates {beta}-catenin at Ser 552, which stabilizes {beta}-catenin, enhances {beta}-catenin/TCF mediated transcription, expanding AMPK from regulation of energy metabolism to cell differentiation and development via cross-talking with the Wnt/{beta}-catenin signaling pathway.

  9. HL156A, a novel AMP-activated protein kinase activator, is protective against peritoneal fibrosis in an in vivo and in vitro model of peritoneal fibrosis.

    PubMed

    Ju, Kyung Don; Kim, Hyo Jin; Tsogbadrakh, Bodokhsuren; Lee, Jinho; Ryu, Hyunjin; Cho, Eun Jin; Hwang, Young-Hwan; Kim, Kiwon; Yang, Jaeseok; Ahn, Curie; Oh, Kook-Hwan

    2016-03-01

    HL156A is a novel AMP-activated protein kinase (AMPK) activator. We aimed to investigate the protective mechanism of HL156A against peritoneal fibrosis (PF) in in vivo and in vitro models. The rat PF model was induced by daily intraperitoneally injection of chlorhexidine (CHX) solution containing 0.1% CHX gluconate and 15% ethanol for 4 wk. The rats in the treatment group were treated with HL156A (1 mg·kg(-1)·day(-1)). Control rats were injected with vehicle alone. In vitro, cultured rat peritoneal mesothelial cells (RPMCs) were treated with either high glucose (HG; 50 mM), normal glucose (NG; 5 mM), NG+HL156A, or HG+HL156A. HL156A in supplemented rats ameliorated peritoneal calcification, cocoon formation, bowel obstruction, and PF. Immunohistochemistry showed reduced fibronectin accumulation in the peritoneum of HL156A-treated rats compared with those injected with CHX alone. HL156A treatment of RPMCs inhibited HG-induced myofibroblast transdifferentiation and markers of epithelial-mesenchymal transition (EMT). Moreover, HL156A ameliorated HG-induced transforming growth factor-β1, Smad3, Snail, and fibronectin expression in the RPMCs via AMPK upregulation. These results suggest that HL156A exhibits a protective effect in PF progression. Further research is warranted to seek the therapeutic potential of HL156A as an antifibrotic agent in peritoneal dialysis patients. PMID:26661649

  10. Combined Treatment of MCF-7 Cells with AICAR and Methotrexate, Arrests Cell Cycle and Reverses Warburg Metabolism through AMP-Activated Protein Kinase (AMPK) and FOXO1.

    PubMed

    Fodor, Tamás; Szántó, Magdolna; Abdul-Rahman, Omar; Nagy, Lilla; Dér, Ádám; Kiss, Borbála; Bai, Peter

    2016-01-01

    Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK) jointly with methotrexate (MTX), a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1β or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer. PMID:26919657

  11. Snf1/AMP-activated protein kinase activates Arf3p to promote invasive yeast growth via a non-canonical GEF domain

    PubMed Central

    Hsu, Jia-Wei; Chen, Kuan-Jung; Lee, Fang-Jen S.

    2015-01-01

    Active GTP-bound Arf GTPases promote eukaryotic cell membrane trafficking and cytoskeletal remodelling. Arf activation is accelerated by guanine nucleotide-exchange factors (GEFs) using the critical catalytic glutamate in all known Sec7 domain sequences. Yeast Arf3p, a homologue of mammalian Arf6, is required for yeast invasive responses to glucose depletion. Here we identify Snf1p as a GEF that activates Arf3p when energy is limited. SNF1 is the yeast homologue of AMP-activated protein kinase (AMPK), which is a key regulator of cellular energy homeostasis. As activation of Arf3p does not depend on the Snf1p kinase domain, assay of regulatory domain fragments yield evidence that the C-terminal hydrophobic α-helix core of Snf1p is a non-canonical GEF for Arf3p activation. Thus, our study reveals a novel mechanism for regulating cellular responses to energy deprivation, in particular invasive cell growth, through direct Arf activation by Snf1/AMPK. PMID:26198097

  12. Ketogenic diet delays the phase of circadian rhythms and does not affect AMP-activated protein kinase (AMPK) in mouse liver.

    PubMed

    Genzer, Yoni; Dadon, Maayan; Burg, Chen; Chapnik, Nava; Froy, Oren

    2015-12-01

    Ketogenic diet (KD) is used for weight loss or to treat epilepsy. KD leads to liver AMP-activated protein kinase (AMPK) activation, which would be expected to inhibit gluconeogenesis. However, KD leads to increased hepatic glucose output. As AMPK and its active phosphorylated form (pAMPK) show circadian oscillation, this discrepancy could stem from wrong-time-of-day sampling. The effect of KD was tested on mouse clock gene expression, AMPK, mTOR, SIRT1 and locomotor activity for 2 months and compared to low-fat diet (LFD). KD led to 1.5-fold increased levels of blood glucose and insulin. Brain pAMPK/AMPK ratio was 40% higher under KD, whereas that in liver was not affected. KD led to 40% and 20% down-regulation of the ratio of pP70S6K/P70S6K, the downstream target of mTOR, in the brain and liver, respectively. SIRT1 levels were 40% higher in the brain, but 40% lower in the liver of KD-fed mice. Clock genes showed delayed rhythms under KD. In the brain of KD-fed mice, amplitudes of clock genes were down-regulated, whereas 6-fold up-regulation was found in the liver. The metabolic state under KD indicates reduced satiety in the brain and reduced anabolism alongside increased gluconeogenesis in the liver. PMID:26408964

  13. Dieckol, a phlorotannin isolated from a brown seaweed, Ecklonia cava, inhibits adipogenesis through AMP-activated protein kinase (AMPK) activation in 3T3-L1 preadipocytes.

    PubMed

    Ko, Seok-Chun; Lee, Myoungsook; Lee, Ji-Hyeok; Lee, Seung-Hong; Lim, Yunsook; Jeon, You-Jin

    2013-11-01

    In this study, we assessed the potential inhibitory effect of 5 species of brown seaweeds on adipogenesis the differentiation of 3T3-L1 preadipocytes into mature adipocytes by measuring Oil-Red O staining. The Ecklonia cava extract tested herein evidenced profound adipogenesis inhibitory effect, compared to that exhibited by the other four brown seaweed extracts. Thus, E. cava was selected for isolation of active compounds and finally the three polyphenol compounds of phlorotannins were obtained and their inhibitory effect on adipogenesis was observed. Among the phlorotannins, dieckol exhibited greatest potential adipogenesis inhibition and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding proteins (C/EBPα), sterol regulatory element-binding protein 1 (SREBP1) and fatty acid binding protein 4 (FABP4) in a dose-dependent manner. The specific mechanism mediating the effects of dieckol was confirmed by AMP-activated protein kinase (AMPK) activation. These results demonstrate inhibitory effect of dieckol compound on adipogenesis through the activation of the AMPK signal pathway. PMID:24211593

  14. Chronic Glutathione Depletion Confers Protection against Alcohol-induced Steatosis: Implication for Redox Activation of AMP-activated Protein Kinase Pathway.

    PubMed

    Chen, Ying; Singh, Surendra; Matsumoto, Akiko; Manna, Soumen K; Abdelmegeed, Mohamed A; Golla, Srujana; Murphy, Robert C; Dong, Hongbin; Song, Byoung-Joon; Gonzalez, Frank J; Thompson, David C; Vasiliou, Vasilis

    2016-01-01

    The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2-related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD. PMID:27403993

  15. Non-esterified fatty acids activate the AMP-activated protein kinase signaling pathway to regulate lipid metabolism in bovine hepatocytes.

    PubMed

    Li, Xinwei; Li, Xiaobing; Chen, Hui; Lei, Liancheng; Liu, Juxiong; Guan, Yuan; Liu, Zhaoxi; Zhang, Liang; Yang, Wentao; Zhao, Chenxu; Fu, Shixin; Li, Peng; Liu, Guowen; Wang, Zhe

    2013-01-01

    Non-esterified fatty acids (NEFAs) act as signaling molecules involved in regulating genes expression to modulate lipid metabolism. However, the regulation mechanism of NEFAs on lipid metabolism in dairy cows is unclear. The AMP-activated protein kinase (AMPK) signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of NEFAs and AMPKα inhibitors (BML-275). NEFAs increased AMPKα phosphorylation through up-regulating the protein levels of liver kinase B1. Activated AMPKα increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α (PPARα). NEFAs also directly activate the PPARα independent of AMPKα. Activated PPARα increased the lipolytic genes expression to increase lipid oxidation. Furthermore, activated AMPKα inhibited the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid synthesis. Activated AMPKα phosphorylated and inhibited acetyl-CoA carboxylase and increased carnitine palmitoyltransferase-1 activity, which increased lipid oxidation. Consequently, the triglyceride content in the NEFAs-treated hepatocytes was significantly decreased. These results indicate that NEFAs activate the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in hepatocytes, which in turn, generates more ATP to relieve the negative energy balance in transition dairy cows. PMID:23690240

  16. Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

    PubMed

    Cheng, Yi-Fang; Young, Guang-Huar; Lin, Jiun-Tsai; Jang, Hyun-Hwa; Chen, Chin-Chen; Nong, Jing-Yi; Chen, Po-Ku; Kuo, Cheng-Yi; Kao, Shao-Hsuan; Liang, Yao-Jen; Chen, Han-Min

    2015-01-01

    The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK. PMID:26544976

  17. Distinct Time Course of the Decrease in Hepatic AMP-Activated Protein Kinase and Akt Phosphorylation in Mice Fed a High Fat Diet

    PubMed Central

    Shiwa, Mami; Yoneda, Masayasu; Okubo, Hirofumi; Ohno, Haruya; Kobuke, Kazuhiro; Monzen, Yuko; Kishimoto, Rui; Nakatsu, Yusuke; Asano, Tomoichiro; Kohno, Nobuoki

    2015-01-01

    AMP-activated protein kinase (AMPK) plays an important role in insulin resistance, which is characterized by the impairment of the insulin-Akt signaling pathway. However, the time course of the decrease in AMPK and Akt phosphorylation in the liver during the development of obesity and insulin resistance caused by feeding a high fat diet (HFD) remains controversial. Moreover, it is unclear whether the impairment of AMPK and Akt signaling pathways is reversible when changing from a HFD to a standard diet (SD). Male ddY mice were fed the SD or HFD for 3 to 28 days, or fed the HFD for 14 days, followed by the SD for 14 days. We examined the time course of the expression and phosphorylation levels of AMPK and Akt in the liver by immunoblotting. After 3 days of feeding on the HFD, mice gained body weight, resulting in an increased oil red O staining, indicative of hepatic lipid accumulation, and significantly decreased AMPK phosphorylation, in comparison with mice fed the SD. After 14 days on the HFD, systemic insulin resistance occurred and Akt phosphorylation significantly decreased. Subsequently, a change from the HFD to SD for 3 days, after 14 days on the HFD, ameliorated the impairment of AMPK and Akt phosphorylation and systemic insulin resistance. Our findings indicate that AMPK phosphorylation decreases early upon feeding a HFD and emphasizes the importance of prompt lifestyle modification for decreasing the risk of developing diabetes. PMID:26266809

  18. AMP-activated protein kinase α2 protects against liver injury from metastasized tumors via reduced glucose deprivation-induced oxidative stress.

    PubMed

    Qiu, Shu-Lan; Xiao, Zhi-Cheng; Piao, Chun-Mei; Xian, Ying-Lin; Jia, Li-Xin; Qi, Yong-Fen; Han, Jia-Huai; Zhang, You-Yi; Du, Jie

    2014-03-28

    It is well known that tumors damage affected tissues; however, the specific mechanism underlying such damage remains elusive. AMP-activated protein kinase (AMPK) senses energetic changes and regulates glucose metabolism. In this study, we examined the mechanisms by which AMPK promotes metabolic adaptation in the tumor-bearing liver using a murine model of colon cancer liver metastasis. Knock-out of AMPK α2 significantly enhanced tumor-induced glucose deprivation in the liver and increased the extent of liver injury and hepatocyte death. Mechanistically, we observed that AMPK α2 deficiency resulted in elevated reactive oxygen species, reduced mitophagy, and increased cell death in response to tumors or glucose deprivation in vitro. These results imply that AMPK α2 is essential for attenuation of liver injury during tumor metastasis via hepatic glucose deprivation and mitophagy-mediated inhibition of reactive oxygen species production. Therefore, AMPK α2 might represent an important therapeutic target for colon cancer metastasis-induced liver injury. PMID:24515110

  19. Prolyl isomerase Pin1 negatively regulates AMP-activated protein kinase (AMPK) by associating with the CBS domain in the γ subunit.

    PubMed

    Nakatsu, Yusuke; Iwashita, Misaki; Sakoda, Hideyuki; Ono, Hiraku; Nagata, Kengo; Matsunaga, Yasuka; Fukushima, Toshiaki; Fujishiro, Midori; Kushiyama, Akifumi; Kamata, Hideaki; Takahashi, Shin-Ichiro; Katagiri, Hideki; Honda, Hiroaki; Kiyonari, Hiroshi; Uchida, Takafumi; Asano, Tomoichiro

    2015-10-01

    AMP-activated protein kinase (AMPK) plays a critical role in metabolic regulation. In this study, first, it was revealed that Pin1 associates with any isoform of γ, but not with either the α or the β subunit, of AMPK. The association between Pin1 and the AMPK γ1 subunit is mediated by the WW domain of Pin1 and the Thr(211)-Pro-containing motif located in the CBS domain of the γ1 subunit. Importantly, overexpression of Pin1 suppressed AMPK phosphorylation in response to either 2-deoxyglucose or biguanide stimulation, whereas Pin1 knockdown by siRNAs or treatment with Pin1 inhibitors enhanced it. The experiments using recombinant Pin1, AMPK, LKB1, and PP2C proteins revealed that the protective effect of AMP against PP2C-induced AMPKα subunit dephosphorylation was markedly suppressed by the addition of Pin1. In good agreement with the in vitro data, the level of AMPK phosphorylation as well as the expressions of mitochondria-related genes, such as PGC-1α, which are known to be positively regulated by AMPK, were markedly higher with reduced triglyceride accumulation in the muscles of Pin1 KO mice as compared with controls. These findings suggest that Pin1 plays an important role in the pathogenic mechanisms underlying impaired glucose and lipid metabolism, functioning as a negative regulator of AMPK. PMID:26276391

  20. Leptin Receptor Signaling in the Hypothalamus Regulates Hepatic Autonomic Nerve Activity via Phosphatidylinositol 3-Kinase and AMP-Activated Protein Kinase

    PubMed Central

    Yamamoto, Naoki; Morgan, Donald A.; Kurata, Yasutaka; Shibamoto, Toshishige

    2015-01-01

    Leptin action in the brain has emerged as an important regulator of liver function independently from its effects on food intake and body weight. The autonomic nervous system plays a key role in the regulation of physiological processes by leptin. Here, we used direct recording of nerve activity from sympathetic or vagal nerves subserving the liver to investigate how brain action of leptin controls hepatic autonomic nerve activity. Intracerebroventricular (ICV) administration of leptin activated hepatic sympathetic traffic in rats and mice in dose- and receptor-dependent manners. The hepatic sympatho-excitatory effects of leptin were also observed when leptin was microinjected directly into the arcuate nucleus (ARC), but not into the ventromedial hypothalamus (VMH). Moreover, using pharmacological and genetic approaches, we show that leptin-induced increase in hepatic sympathetic outflow depends on PI3K but not AMP-activated protein kinase (AMPK), STAT3, or ERK1/2. Interestingly, ICV leptin also increased hepatic vagal nerve activity in rats. We show that this response is reproduced by intra-ARC, but not intra-VMH, leptin administration and requires PI3K and AMPK. We conclude that central leptin signaling conveys the information to the liver through the sympathetic and parasympathetic branches of the autonomic nervous system. Our data also provide important insight into the molecular events underlying leptin's control of hepatic autonomic nerve activity by implicating PI3K and AMPK pathways. PMID:25589743

  1. Adverse effects of AMP-activated protein kinase alpha2-subunit deletion and high-fat diet on heart function and ischemic tolerance in aged female mice.

    PubMed

    Slámová, K; Papoušek, F; Janovská, P; Kopecký, J; Kolář, F

    2016-03-14

    AMP-activated protein kinase (AMPK) plays a role in metabolic regulation under stress conditions, and inadequate AMPK signaling may be also involved in aging process. The aim was to find out whether AMPK alpha2-subunit deletion affects heart function and ischemic tolerance of adult and aged mice. AMPK alpha2(-/-) (KO) and wild type (WT) female mice were compared at the age of 6 and 18 months. KO mice exhibited subtle myocardial AMPK alpha2-subunit protein level, but no difference in AMPK alpha1-subunit was detected between the strains. Both alpha1- and alpha2-subunits of AMPK and their phosphorylation decreased with advanced age. Left ventricular fractional shortening was lower in KO than in WT mice of both age groups and this difference was maintained after high-fat feeding. Infarct size induced by global ischemia/reperfusion of isolated hearts was similar in both strains at 6 months of age. Aged WT but not KO mice exhibited improved ischemic tolerance compared with the younger group. High-fat feeding for 6 months during aging abolished the infarct size-reduction in WT without affecting KO animals; nevertheless, the extent of injury remained larger in KO mice. The results demonstrate that adverse effects of AMPK alpha2-subunit deletion and high-fat feeding on heart function and myocardial ischemic tolerance in aged female mice are not additive. PMID:26596312

  2. Stearoyl lysophosphatidylcholine enhances the phagocytic ability of macrophages through the AMP-activated protein kinase/p38 mitogen activated protein kinase pathway.

    PubMed

    Quan, Hui; Hur, Young-Hoe; Xin, Chun; Kim, Joung-Min; Choi, Jeong-Il; Kim, Man-Young; Bae, Hong-Beom

    2016-10-01

    A previous study showed that stearoyl lysophosphatidylcholine (sLPC) suppressed extracellular high mobility group box 1 translocation in macrophages stimulated with lipopolysaccharide through AMP-activated protein kinase (AMPK) activation. In the present study, we investigated whether sLPC-induced AMPK activation could enhance macrophages phagocytosis of bacteria. We found that sLPC increased phosphorylation of AMPK and acetyl-CoA carboxylase, a downstream target of AMPK, in a time- and dose-dependent manner in macrophages. Furthermore, sLPC increased the uptake of FITC-conjugated Escherichia coli by macrophages in a dose-dependent manner, and treatment with an AMPK inhibitor (compound C) or siRNA to AMPKα1 reversed this uptake. sLPC increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK), but inhibition of AMPK activity with compound C or siRNA to AMPKα1 prevented the sLPC-induced increase in p38 MAPK phosphorylation. SB203580, a p38 MAPK inhibitor, decreased sLPC-induced phagocytosis. In vivo, systemic administration of sLPC to mice led to increased AMPK and p38 MAPK activity in the lung and to increased phagocytosis of fluorescent E. coli in bronchoalveolar lavage cells. These results suggest that sLPC increases macrophages phagocytosis through activation of the AMPK/p38 MAPK pathway. Therefore, sLPC is a candidate pharmacological agent for the treatment of bacterial infections in clinically relevant conditions. PMID:27517519

  3. Antidiabetic Effect of Methanolic Extract from Berberis julianae Schneid. via Activation of AMP-Activated Protein Kinase in Type 2 Diabetic Mice

    PubMed Central

    Yang, Jing; Zhao, Ping; Wan, Dingrong; Zhou, Qi; Wang, Chao; Shu, Guangwen; Mei, Zhinan; Yang, Xinzhou

    2014-01-01

    We have investigated the antidiabetic effect and mechanism of methanolic extract of Berberis julianae Schneid. (BJSME) in STZ induced Type 2 diabetes mellitus mice. T2DM mice were induced by high fat diet and low dose streptozotocin (STZ). BJSME was orally administrated at the doses of 60, 120, and 240 mg/kg/d, for 21 days. Metformin was used as positive control drug. Food intake, body weight, plasma glucose, oral glucose tolerance test, insulin tolerance test, insulin, and blood-lipid content were measured. The effects of BJSME on the glucose transporter 4 (GLUT4) translocation in L6 myotubes and the GLUT4 protein expression in skeletal muscle as well as phosphorylation of the AMP-activated protein kinase (AMPK) in liver and muscle were examined. In vitro and in vivo results indicate that BJSME increased GLUT4 translocation by 1.8-fold and BJSME significantly improved the oral glucose tolerance and low density lipoprotein cholesterol (LDL-C) of serum and reduced body weight, glucose, and other related blood-lipid contents. The BJSME treatment also stimulated the phosphorylation of AMPK. Thus, BJSME seems to possess promising beneficial effects for the treatment of T2DM with the possible mechanism via stimulating AMPK activity. PMID:25258641

  4. Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation.

    PubMed

    Jacobs, René L; Lingrell, Susanne; Dyck, Jason R B; Vance, Dennis E

    2007-02-16

    5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK. PMID:17179149

  5. Polymerization of actin in RBL-2H3 cells can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used.

    PubMed Central

    Apgar, J R

    1994-01-01

    Crosslinking of the IgE receptor on rat basophilic leukemia (RBL) cells using the multivalent antigen DNP-BSA leads to a rapid and sustained increase in the filamentous actin content of the cells. Stimulation of RBL cells through the adenosine receptor also induces a very rapid polymerization of actin, which peaks in 45-60 s and is equivalent in magnitude to the F-actin response elicited through stimulation of the IgE receptor. However, in contrast to the IgE mediated response, which remains elevated for over 30 min, the F-actin increase induced by the adenosine analogue 5'-(N-ethylcarboxamido)-adenosine (NECA) is relatively transient and returns to baseline values within 5-10 min. While previous work has shown that the polymerization of actin in RBL cells stimulated through the IgE receptor is mediated by protein kinase C (PKC), protein kinase inhibitors have no effect on the F-actin response activated through the adenosine receptor. In contrast, pretreatment of the cells with pertussis toxin completely inhibits the F-actin response to NECA but has relatively little effect on the response induced through the IgE receptor. Stimulation of RBL cells through either receptor causes increased production of phosphatidylinositol mono-phosphate (PIP) and phosphatidylinositol bis-phosphate (PIP2), which correlates with the F-actin response. Production of PIP and PIP2 may be important downstream signals since these polyphosphoinositides are able to regulate the interaction of gelsolin and profilin with actin. Thus the polymerization of actin can be triggered through either the adenosine receptor or the IgE receptor, but different upstream signaling pathways are being used. The IgE mediated response requires the activation of PKC while stimulation through the adenosine receptor is PKC independent but involves a G protein. PMID:8049523

  6. A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment

    PubMed Central

    Ohta, Akio

    2016-01-01

    Within tumors, some areas are less oxygenated than others. Since their home ground is under chronic hypoxia, tumor cells adapt to this condition by activating aerobic glycolysis; however, this hypoxic environment is very harsh for incoming immune cells. Deprivation of oxygen limits availability of energy sources and induces accumulation of extracellular adenosine in tumors. Extracellular adenosine, upon binding with adenosine receptors on the surface of various immune cells, suppresses pro-inflammatory activities. In addition, signaling through adenosine receptors upregulates a number of anti-inflammatory molecules and immunoregulatory cells, leading to the establishment of a long-lasting immunosuppressive environment. Thus, due to hypoxia and adenosine, tumors can discourage antitumor immune responses no matter how the response was induced, whether it was spontaneous or artificially introduced with a therapeutic intention. Preclinical studies have shown the significance of adenosine in tumor survival strategy by demonstrating tumor regression after inactivation of adenosine receptors, inhibition of adenosine-producing enzymes, or reversal of tissue hypoxia. These promising results indicate a potential use of the inhibitors of the hypoxia–adenosine pathway for cancer immunotherapy. PMID:27066002

  7. Effects of adenosine, adenosine triphosphate and structural analogues on glucagon secretion from the perfused pancreas of rat in vitro.

    PubMed Central

    Chapal, J.; Loubatières-Mariani, M. M.; Roye, M.; Zerbib, A.

    1984-01-01

    The effects of adenosine, adenosine triphosphate (ATP) and structural analogues have been studied on glucagon secretion from the isolated perfused pancreas of the rat in the presence of glucose (2.8 mM). Adenosine induced a transient increase of glucagon secretion. This effect was concentration-dependent in the range of 0.165 to 165 microM. ATP also induced an increase, but the effect was no greater at 165 microM than at 16.5 microM. 2-Chloroadenosine, an analogue more resistant to metabolism or uptake systems than adenosine, was more effective. Among the three structural analogues of ATP or ADP studied, beta, gamma-methylene ATP which can be hydrolyzed into AMP and adenosine had an effect similar to adenosine or ATP at the same concentrations (1.65 and 16.5 microM); in contrast alpha, beta-methylene ATP and alpha, beta-methylene ADP (resistant to hydrolysis into AMP and adenosine) were ineffective. Theophylline (50 microM) a specific blocker of the adenosine receptor, suppressed the glucagon peak induced by adenosine, 2-chloroadenosine, ATP and beta, gamma-methylene ATP (1.65 microM). An inhibitor of 5' nucleotidase, alpha, beta-methylene ADP (16.5 microM), reduced the glucagon increase induced by ATP and did not affect the response to adenosine (1.65 microM). These results support the hypothesis of adenosine receptors (P1-purinoceptors) on the pancreatic glucagon secretory cells and indicate that ATP acts after hydrolysis to adenosine. PMID:6097328

  8. Adenosine: Tipping the balance towards hepatic steatosis and fibrosis

    PubMed Central

    Robson, Simon C.; Schuppan, Detlef

    2010-01-01

    Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the histochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5′-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver. PMID:20395005

  9. Granulosa cell apoptosis, aromatase activity, cyclic adenosine 3',5'-monophosphate response to gonadotropins, and follicular fluid steroid levels during spontaneous and induced follicular atresia in ewes.

    PubMed

    Jolly, P D; Tisdall, D J; De'ath, G; Heath, D A; Lun, S; Hudson, N L; McNatty, K P

    1997-04-01

    The aims of the present study in ewes were 1) to test the hypothesis that apoptosis in granulosa cells is one of the processes involved in the structural demise of follicles and 2) to define the temporal relationships among the occurrence and degree of apoptosis in granulosa cells, aromatase activity, production of cyclic AMP (cAMP) by granulosa cells in response to FSH or LH, concentrations of estradiol 17 beta (E2) and progesterone in follicular fluid, and the characteristic morphometric changes associated with the process of follicular atresia. To address these aims, ewes were treated with either saline or steroid-free bovine follicular fluid (bFF) at 60 h after estrus, and ovarian follicles > or = 3 mm diameter were recovered at 0, 12, 18, or 24 h later. Apoptotic granulosa cells were identified by the presence of oligonucleosomes after 3'-end labeling of extracted DNA with [32P]alpha dideoxy ATP (ddATP). The degree of oligonucleosome formation, based on the intensity of radiolabeling, was given an apoptosis score (AP) of 0 (nondetectable), 1 (slight), 2 (moderate), or 3 (marked). Moreover, a labeling index (LI) was calculated from the amount of radiolabeled ddATP incorporated into low-molecular weight (< 4.2 kb) DNA fragments. On the basis of morphometric criteria, 73% (141 of 194) of the follicles classified as healthy had apoptotic granulosa cells compared to 86% (18 of 21) of the follicles classified as atretic. In the bFF-but not saline-treated ewes, the concentrations of plasma FSH had declined to basal values at 12 h after treatment. At the beginning of the treatment period, the degree of granulosa cell apoptosis was either undetectable (AP = 0, 47% of follicles) or slight (AP = 1, 44% of follicles) in the majority of follicles. After 12 h from the bFF but not the saline injection, there was a significant increase in the proportion of follicles (> or = 3 mm diameter) per ewe containing apoptotic granulosa cells (p < 0.001) and a significant decrease in the number of follicles per ewe with aromatase activity (p < 0.05) and with follicular fluid E2 > 20 ng/ml (p < 0.05). By 24 h after bFF treatment, apoptosis was evident in all follicles (> or = 3 mm diameter), fewer follicles contained FSH-responsive granulosa cells in terms of cAMP production (p < 0.05), and none were LH-responsive. A significant negative relationship was found between the degree of granulosa cell death as measured by L1 and follicular fluid E2 concentrations. In summary, the presence of apoptotic granulosa cells in an appreciable number of follicles considered to be healthy by morphometric criteria and before their commitment to preovulatory enlargement and ovulation suggests that apoptosis may be a physiological process in developing follicles and/or a very early event in atresia. Collectively, these data provide strong evidence that granulosa cells may die by apoptosis before there is an appreciable decrease in the capacity of the granulosa cell layer as a whole to respond to gonadotropins or to produce E2. PMID:9096862

  10. Inhibition of Cyclic Adenosine Monophosphate (cAMP)-response Element-binding Protein (CREB)-binding Protein (CBP)/β-Catenin Reduces Liver Fibrosis in Mice

    PubMed Central

    Osawa, Yosuke; Oboki, Keisuke; Imamura, Jun; Kojika, Ekumi; Hayashi, Yukiko; Hishima, Tsunekazu; Saibara, Toshiji; Shibasaki, Futoshi; Kohara, Michinori; Kimura, Kiminori

    2015-01-01

    Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP)/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4)- or bile duct ligation (BDL)-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs) and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80+ CD11b+ and Ly6Clow CD11b+ macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP)-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis. PMID:26870800

  11. Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis

    NASA Technical Reports Server (NTRS)

    Scott, D. K.; Brakenhoff, K. D.; Clohisy, J. C.; Quinn, C. O.; Partridge, N. C.

    1992-01-01

    Collagenase is synthesized and secreted by rat osteoblastic cells in response to PTH. We have previously demonstrated that this effect involves a substantial increase in collagenase mRNA via transcription. Northern blots and nuclear run-on assays were performed to further investigate the induction of collagenase by PTH in the rat osteoblastic cell line UMR 106-01. Detectable amounts of collagenase mRNA were not apparent until 2 h of PTH treatment, showed the greatest abundance at 4 h, and declined to approximately 30% of maximum by 8 h. The changes in the rate of transcription of the collagenase gene in response to PTH paralleled and preceded the changes in the steady state mRNA levels. After an initial lag period of about 1 h, collagenase transcription rates increased from very low levels to a maximal response at 2 h, returning to about 50% of maximum by 10 h. The increased transcriptional rate of the collagenase gene was found to be dependent on the concentration of PTH, with a half-maximal response at approximately 7 x 10(-10) M rat PTH-(1-34) and a maximal effect with a dose of 10(-8) M. The PTH-mediated induction of collagenase transcriptional activity was completely abolished by cycloheximide, while transcription of the beta-actin gene was unaffected by the translation inhibitor. These data suggest that a protein factor(s) is required for PTH-mediated transcriptional induction of collagenase. Since PTH increases intracellular levels of several potential second messengers, agents that mimic these substances were employed to determine which signal transduction pathway is predominant in the PTH-mediated stimulation of collagenase transcription.(ABSTRACT TRUNCATED AT 250 WORDS).

  12. Bleomycin-induced pulmonary fibrosis in hamsters. An alveolar macrophage product increases fibroblast prostaglandin E2 and cyclic adenosine monophosphate and suppresses fibroblast proliferation and collagen production.

    PubMed Central

    Clark, J G; Kostal, K M; Marino, B A

    1983-01-01

    Bleomycin-induced pulmonary fibrosis in hamsters is associated with collagen accumulation that results from increased lung collagen synthesis rates. However, 1-2 wk after intratracheal instillation of bleomycin, lung collagen synthesis rates decline toward control values. To evaluate the potential role of the bronchoalveolar macrophage in regulating lung collagen production, we studied the effects of macrophages from normal and bleomycin-treated hamsters upon fibroblasts in vitro. We observed: (a) Medium from macrophage cultures decreased fibroblast [3H]thymidine incorporation and nondialyzable [3H]hydroxyproline production in a dose-dependent manner. Fibroblast cell counts were decreased in exposed cultures, and fibroblast viability was unchanged. Procollagen prolyl hydroxylation and prolyl-transfer RNA-specific activity were not altered by macrophage medium; this indicates that [3H]hydroxyproline reflects collagen production rate under the experimental conditions. (b) The suppressive effect of macrophage medium was selective for collagen since collagen production decreased more than noncollagen protein production. (c) Medium from bleomycin-treated hamster macrophages suppressed fibroblast proliferation and collagen production to a greater degree than medium from normal hamster macrophages. (d) Fibroblast suppression by macrophage medium was associated with increased fibroblast endogenous prostaglandin E2 production and intracellular cyclic AMP (cAMP). (e) Incubation of fibroblasts with indomethacin before exposure completely inhibited prostaglandin E2 production and increases in cAMP, and eliminated suppression of fibroblast proliferation and collagen production. The macrophage-derived suppressive factor has an apparent molecular weight of 20,000-30,000 and is heat stable. It does not appear to be species restricted since both hamster and human lung fibroblasts are similarly suppressed. It is at least in part preformed in macrophages obtained by lavage, but its production can also be stimulated in vitro. We concluded that alveolar macrophages release a product that stimulates endogenous fibroblast prostaglandin E2 production and cAMP formation with resultant suppression of fibroblast proliferation and collagen production. Enhanced release of suppressive factor by macrophages during a time when lung collagen production is declining in bleomycin-induced pulmonary fibrosis suggests that macrophages may limit collagen accumulation in pulmonary fibrosis. PMID:6196378

  13. Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system

    SciTech Connect

    Dave, J.R.; Eiden, L.E.; Eskay, R.L.

    1985-06-01

    Specific binding sites for rat corticotropin-releasing factor (rCRF) are present in rat adrenal medulla, ventral prostate, spleen, liver, kidney, and testis and bovine chromaffin cells in culture. Maximal binding of (/sup 125/I)rCRF occurred within 25 min at 4 C and was saturable. Scatchard analysis of rCRF binding to rat adrenal membranes and bovine chromaffin cells revealed the existence of two classes of binding sites. One class had a relatively higher apparent affinity and lower number of binding sites, whereas the other class had a relatively lower affinity and higher number of binding sites. CRF induced a dose-related increase in rat adrenal membrane adenylate cyclase activity and cAMP levels in bovine chromaffin cells. Nanomolar concentrations of rCRF maximally stimulated adenylate cyclase activity in rat adrenal membranes and maximally increased cAMP levels in bovine chromaffin cells to 86% and 130% above control values, respectively. The demonstration of specific CRF-binding sites in a variety of peripheral tissues and the finding that activation of specific CRF-binding sites in adrenal tissue stimulates the adenylate cyclase-cAMP system suggest that CRF may have an important regulatory role in various peripheral tissues.

  14. Conservation and divergence of the cyclic adenosine monophosphate-protein kinase A (cAMP–PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F. verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cyclic AMP (cAMP)-PKA pathway is a central signaling cascade that transmits extracellular stimuli and governs cell responses through the second messenger cAMP. The importance of cAMP signaling in fungal biology has been well documented. Two key conserved components, adenylate cyclase (AC) and ca...

  15. Regulation of insulin release from isolated islets of Langerhans of the rat in pregnancy. The role of adenosine 3′:5′-cyclic monophosphate

    PubMed Central

    Green, I. C.; Howell, S. L.; Montague, W.; Taylor, K. W.

    1973-01-01

    1. The concentrations of cyclic AMP were compared in islets of Langerhans isolated from the pancreases of normal female and pregnant rats and were higher in islets in pregnancy. 2. There was also a significant increase in adenylate cyclase activity in homogenates of islets from pregnant rats compared with those from normal rats. 3. Increased cyclic AMP concentration in islets from pregnant rats was reflected in increased protein kinase activity. When the cyclic AMP-dependent protein kinase activity was increased by 3-isobutyl-1-methylxanthine this stimulated activity was significantly greater in pregnancy. 4. Insulin-secretion studies with islets from normal and pregnant rats showed that theophylline or 3-isobutyl-1-methylxanthine, which raise intracellular cyclic AMP concentrations, caused a significantly greater insulin secretion in pregnancy. 5. It was also found that in the presence of a glucose concentration too low to stimulate insulin secretion, the latter could be induced if the cyclic AMP concentrations were raised sufficiently with 3-isobutyl-1-methylxanthine. 6. It is suggested that the higher cyclic AMP concentrations observed in islets in pregnancy mediate the greater insulin-secretory capacity, as well as the greater sensitivity of these islets to low glucose concentrations. PMID:16742808

  16. Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Jiang, Keyong; Sun, Shujuan; Liu, Mei; Wang, Baojie; Meng, Xiaolin; Wang, Lei

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) ( P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

  17. A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium.

    PubMed

    Vijayaraghavan, S; Trautman, K D; Goueli, S A; Carr, D W

    1997-06-01

    Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1gamma2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or alpha-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways. PMID:9166697

  18. Comparative involvement of cyclic nucleotide phosphodiesterases and adenylyl cyclase on adrenocorticotropin-induced increase of cyclic adenosine monophosphate in rat and human glomerulosa cells.

    PubMed

    Côté, M; Payet, M D; Rousseau, E; Guillon, G; Gallo-Payet, N

    1999-08-01

    The present study investigated the role and identity of cyclic nucleotide phosphodiesterases (PDEs) in the regulation of basal and ACTH-stimulated levels of intracellular cAMP in human and rat adrenal glomerulosa cells. Comparative dose-response curves indicated that maximal hormone-stimulated cAMP accumulation was 11- and 24-fold higher in human and rat cells, compared with cAMP production obtained in corresponding membranes, respectively. Similarly to 3-isobutyl-1-methyl-xanthine, 25 microM erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, a specific PDE2 inhibitor), caused a large increase in ACTH-stimulated cAMP accumulation; by contrast, it did not change cAMP production in membranes. Moreover, in membrane fractions, addition of 10 microM cGMP inhibited ACTH-induced cAMP production, an effect completely reversed by addition of 25 microM EHNA. These results indicate that PDE2 activity is involved in the regulation of cAMP accumulation induced by ACTH, and suggest that ACTH inhibits this activity. Indeed, time-course studies indicated that ACTH induced a rapid decrease in cGMP production, resulting in PDE2 inhibition, which in turn, contributed [with adenylyl cyclase (AC) activation] to an accumulation in cAMP for 15 min. Thereafter, cAMP content decreased, because of cAMP-stimulated PDE2, as confirmed by measurement of PDE activity that was activated by ACTH, but only after a 10-min incubation. Hence, we demonstrate that the ACTH-induced increase in intracellular cAMP is the result of a balance between activation of AC and direct modulation of PDE2 activity, an effect mediated by cGMP content. Although similar results were observed in both models, PDE2 involvement is more important in rat than in human adrenal glomerulosa cells, whereas AC is more stimulated in human than in rat glomerulosa cells. PMID:10433216

  19. Control of the Synthesis of Fatty-Acid Synthetase in Rat Liver by Insulin, Glucagon, and Adenosine 3′:5′ Cyclic Monophosphate

    PubMed Central

    Lakshmanan, M. R.; Nepokroeff, Carl M.; Porter, John W.

    1972-01-01

    The usual increase in the activity of liver fatty-acid synthetase that occurs on refeeding of a fat-free diet to previously fasted rats is abolished in diabetic animals. Insulin specifically restores this increase by enhancement of the rate of synthesis of fatty-acid synthetase. However, glucagon and cyclic AMP inhibit the increase in the activity of fatty-acid synthetase. Therefore, the concentration of fatty-acid synthetase in rat liver is under the control of the relative concentrations of insulin and glucagon. PMID:4345502

  20. Effectors of cyclic adenosine 5'-monophosphate up-regulating-oxytocin receptors in rabbit amnion cells: isoproterenol, parathyroid hormone-related protein, and potentiation by cortisol.

    PubMed

    Jeng, Y J; Hinko, A; Soloff, M S

    1995-11-01

    Forskolin (FSK; an activator of adenylyl cyclase) and cortisol synergistically increase the concentration of oxytocin receptors (OTRs) in rabbit amnion cells. The aims of this study were to characterize potential physiological regulators of OTR concentrations acting through adenylyl cyclase and to clarify the mechanisms of potentiation by cAMP and cortisol. Both isoproterenol (ISO) and parathyroid hormone-related protein (PTHrP) elevated amnion cell cAMP levels and OTR concentrations. The effects of ISO and PTHrP on OTR were potentiated by cortisol. Cortisol had no effect on the ability of ISO or PTHrP to stimulate adenylyl cyclase activity, and cAMP did not affect the number or affinity of glucocorticoid receptors in whole cells or in cytosol. Adenylyl cyclase activation, however, caused conversion of mifepristone (RU486) from a glucocorticoid antagonist to agonist. Thus, mifepristone elevated OTR receptor concentrations in the presence of FSK. In contrast, a structurally related glucocorticoid antagonist, onapristone (ZK98 299), was unaffected by cAMP. Because glucocorticoid receptors bound to mifepristone are capable of interacting with DNA, whereas onapristone-occupied receptors are not, we conclude that cAMP affects glucocoticoid receptor-DNA interactions, accounting for the synergistic effects of cAMP and cortisol on OTRs. PMID:8527507

  1. Dual specificity and novel structural folding of yeast phosphodiesterase-1 for hydrolysis of second messengers cyclic adenosine and guanosine 3',5'-Monophosphate

    DOE PAGESBeta

    Tian, Yuanyuan; Cui, Wenjun; Huang, Manna; Robinson, Howard; Wan, Yiqian; Wang, Yousheng; Ke, Hengming

    2014-08-05

    Cyclic nucleotide phosphodiesterases (PDEs) decompose second messengers cAMP and cGMP that play critical roles in many physiological processes. PDE1 of Saccharomyces cerevisiae has been subcloned and expressed in Escherichia coli. Recombinant yPDE1 has a KM of 110 μM and a kcat of 16.9 s⁻¹ for cAMP and a KM of 105 μM and a kcat of 11.8 s₅⁻¹ for cGMP. Thus, the specificity constant (kcat/KMcAMP)/(kcat/KMcGMP) of 1.4 indicates a dual specificity of yPDE1 for hydrolysis of both cAMP and cGMP. The crystal structures of unliganded yPDE1 and its complex with GMP at 1.31 Å resolution reveal a new structural foldingmore » that is different from those of human PDEs but is partially similar to that of some other metalloenzymes such as metallo-β-lactamase. In spite of their different structures and divalent metals, yPDE1 and human PDEs may share a common mechanism for hydrolysis of cAMP and cGMP.« less

  2. Parathyroid hormone induces E4bp4 messenger ribonucleic acid expression primarily through cyclic adenosine 3',5'-monophosphate signaling in osteoblasts.

    PubMed

    Ozkurt, Ibrahim C; Pirih, Flavia Q; Tetradis, Sotirios

    2004-08-01

    PTH binding to its receptor activates protein kinase A (PKA), protein kinase C (PKC), and calcium signaling to induce transcription of primary response genes in osteoblasts. Adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4BP4/NFIL3), a transcriptional repressor, is a PTH-induced primary response gene in primary mouse osteoblasts (MOBs). Here we investigate the signaling pathway(s) that lead to PTH induction of E4bp4 mRNA expression. Ten and 100 nm PTH induced maximum E4bp4 expression in MOBs. Forskolin (FSK), an adenylate cyclase inducer, 8-bromo-cAMP, a cAMP analog, and phorbol myristate acetate, a PKC activator, increased E4bp4 mRNA levels, whereas ionomycin, a calcium ionophore, had no effect. Pretreatment of cells with 30 microm H89, a PKA inhibitor, strongly inhibited PTH- and FSK-induced E4bp4 expression. In contrast, overnight pretreatment with 1 microm phorbol myristate acetate to down-regulate PKC signaling did not alter PTH and FSK effects. Moreover, PTH (3-34) that does not activate cAMP signaling did not increase E4bp4 expression. Prostaglandin E(2), which signals through cAMP, increased E4bp4 mRNA at all doses, whereas prostaglandin F(2alpha) that primarily activates PKC and calcium signaling, induced E4bp4 only at high doses and fluprostenol that only activates PKC and calcium signaling, had no effect. Finally, 80 microg/kg PTH (1-34) ip injection induced E4bp4 mRNA expression at 1 h in mice. In contrast, 80 microg/kg PTH (3-34) had no effect. Our data suggest that PTH-induced E4bp4 mRNA expression is mediated primarily through cAMP-PKA signaling in vitro and in vivo. In conjunction with our previous report, we hypothesize that E4bp4 attenuates transcription of osteoblastic genes possessing E4bp4 promoter binding sites. PMID:15087429

  3. Inhibition of Cyclic Adenosine Monophosphate (cAMP)-response Element-binding Protein (CREB)-binding Protein (CBP)/β-Catenin Reduces Liver Fibrosis in Mice.

    PubMed

    Osawa, Yosuke; Oboki, Keisuke; Imamura, Jun; Kojika, Ekumi; Hayashi, Yukiko; Hishima, Tsunekazu; Saibara, Toshiji; Shibasaki, Futoshi; Kohara, Michinori; Kimura, Kiminori

    2015-11-01

    Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP)/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4)- or bile duct ligation (BDL)-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs) and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80(+) CD11b(+) and Ly6C(low) CD11b(+) macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP)-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis. PMID:26870800

  4. Heterooligomerization between vasotocin and corticotropin-releasing hormone (CRH) receptors augments CRH-stimulated 3',5'-cyclic adenosine monophosphate production.

    PubMed

    Mikhailova, Marina V; Mayeux, Philip R; Jurkevich, Alexander; Kuenzel, Wayne J; Madison, Farrah; Periasamy, Ammasi; Chen, Ye; Cornett, Lawrence E

    2007-09-01

    In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15-18%, and the distance between receptors was 5-6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction. PMID:17536010

  5. The role of bicarbonate ions and of adenosine 3',5'-monophosphate (cAMP) in chloride transport by epithelial cells of bullfrog small intestine.

    PubMed

    Armstrong, W M; Youmans, S J

    1980-01-01

    In an HCO3-free medium, isolated segments of bullfrog small intestine, stripped of their external muscle layers, displayed a small, serosal positive PD that did not, on the average, differ significantly from zero. Similarly, in this medium, the mean values of Isc and of net Na+ and Cl- absorption under short-circuit conditions did not differ significantly from zero. External HCO3- (25 mM) induced a highly significant serosal negative PD and Isc and a large net absorption of Cl-. Net Cl- absorption exceeded Isc, i.e., there was a significant net flux, JR, which was consistent with a net secretion of HCO3-. The ratio of the internal Cl-activity of the absorptive cells (alpha Cli) to its equilibrium value was larger in the presence than in the absence of HCO3-. In the presence of HCO3-, cAMP, added to the serosal medium, reversed the serosal negative PD and Isc, and inhibited, though it did not completely abolish, net Cl- absorption. JR was unchanged; tissue Cl- and alpha Cli were reduced, and tissue Na+ decreased and tissue K+ increased. When HCO3- and Cl- were removed from the bathing medium, the electrical response of the tissue to cAMP, though greatly attenuated, was not completely abolished. Under these conditions, cAMP induced a significant net Na+ absorption. A model for ion transport in the absorptive cells of the small intestine is proposed that is consistent with these findings. PMID:6249145

  6. Effects of limited exposure of rabbit chondrocyte cultures to parathyroid hormone and dibutyryl adenosine 3',5'-monophosphate on cartilage-characteristic proteoglycan synthesis

    SciTech Connect

    Kato, Y.; Koike, T.; Iwamoto, M.; Kinoshita, M.; Sato, K.; Hiraki, Y.; Suzuki, F.

    1988-05-01

    Treatment of rabbit chondrocyte cultures with PTH or (Bu)2cAMP for 30 h increased by 2- to 3-fold the incorporation of (35S)sulfate and 3H radioactivity with glucosamine as the precursor into large chondroitin sulfate proteoglycans characteristically found in cartilage matrix. However, PTH and (Bu)2cAMP did not increase either (35S)sulfate incorporation into small proteoglycans or the incorporation of 3H radioactivity into hyaluronic acid and other glycosaminoglycans. PTH and (Bu)2cAMP also increased the incorporation of (3H) serine into both proteoglycans and total protein. In all cultures described above, the stimulation of (3H)serine incorporation into proteoglycans exceeded that of (3H)serine incorporation into total protein. These data indicate that PTH and (Bu)2cAMP selectively stimulate cartilage proteoglycan synthesis while they increase total protein synthesis. Since cAMP seems to play a mediatory role in the action of PTH, we elected to examine the effects of a limited exposure of chondrocytes to PTH or (Bu)2cAMP on the synthesis of proteoglycans. Treatment with PTH or (Bu)2cAMP for only the initial 2-7 h did not increase the rates of incorporation of (35S)sulfate, the 3H radioactivity with glucosamine, and (3H)serine into proteoglycans, as measured at 30 h, despite the fact that this treatment brought about a rapid and transient rise in the cAMP level. Furthermore, the application of prostaglandin I2 at concentrations that increased cAMP levels in a similar fashion as did PTH did not affect (35S) sulfate incorporation into proteoglycans.

  7. Pretreatment of rats with increased bioavailable berberine attenuates cerebral ischemia-reperfusion injury via down regulation of adenosine-5'monophosphate kinase activity.

    PubMed

    Chen, Weijia; Wei, Shengnan; Yu, Yang; Xue, Huan; Yao, Fan; Zhang, Ming; Xiao, Jun; Hatch, Grant M; Chen, Li

    2016-05-15

    Berberine (BBR) exhibits multiple beneficial biological effects. However, poor bioavailability of BBR has limited its clinical application. We previously demonstrated that solid dispersion of BBR with sodium caprate (HGSD) remarkably improves its bioavailability. We examined whether this increased bioavailability of BBR could protect the brain from ischemia-reperfusion (IR) induced injury. Rats treated with HGSD, SC and saline for 7 days then subjected to cerebral ischemia reperfusion by middle cerebral artery occlusion for 2h followed 12h reperfusion. Neurological deficit scores, infarct size, SOD, MDA and NO levels were examined. P-AMPK, Bax, cleaved-Caspase-3 in brain was determined. To further probe for the mechanism of beneficial effect of HGSD, PC12 cells were incubated with serum from control or HGSD pretreated animals, incubated with 300μM H2O2 to induce apoptosis. Caspase-3 activity and cell apoptosis was evaluated. HGSD pretreatment significantly attenuated neurological deficit scores, reduced infarct size, increased SOD and decreased MDA and NO after cerebral IR injury compared to controls. Meanwhile, HGSD pretreatment significantly reduced expression of p-AMPK, Bax, cleaved-Caspase-3 after cerebral IR injury. Sodium caprate (100mg/kg/d) pretreatment alone did not exhibit any of these beneficial effects. PC12 cell apoptosis was attenuated when cells were cultured with HGSD serum compared to control. The presence of AMPK activator (AICAR) attenuated whereas AMPK inhibitor (Compound C) augmented the protective effect of HGSD serum on PC12 cell apoptosis.The results indicate that HGSD-pretreatment of rats protects the brain from ischemia-reperfusion injury and the mechanism is due to its anti-apoptotic effect mediated by decreased activation of AMPK. PMID:26957053

  8. Arginine vasopressin increases cellular free calcium concentration and adenosine 3',5'-monophosphate production in rat renal papillary collecting tubule cells in culture

    SciTech Connect

    Ishikawa, S.; Okada, K.; Saito, T.

    1988-09-01

    The role of calcium (Ca) in the cellular action of arginine vasopressin (AVP) was examined in rat renal papillary collecting tubule cells in culture. AVP increased both the cellular free Ca concentration ((Ca2+)i) using fura-2, and cAMP production in a dose-dependent manner. AVP-induced cellular Ca mobilization was totally blocked by the antagonist to the antidiuretic action of AVP, and somewhat weakened by the antagonist to the vascular action of AVP. 1-Deamino-8-D-AVP (dDAVP). an antidiuretic analog of AVP, also increased (Ca2+) significantly. Cellular Ca mobilization was not obtained with cAMP, forskolin (a diterpene activator of adenylate cyclase), or phorbol-12-myristate-13-acetate. The early phase of (Ca2+)i depended on the intracellular Ca pool, since an AVP-induced rise in (Ca2+)i was obtained in cells pretreated with Ca-free medium containing 1 mM EGTA, verapamil, or cobalt, which blocked cellular Ca uptake. Also, AVP increased /sup 45/Ca2+ influx during the initial 10 min, which initiated the sustained phase of cellular Ca mobilization. However, cellular cAMP production induced by AVP during the 10-min observation period was diminished in the cells pretreated with Ca-free medium, verapamil, or cobalt, but was still significantly higher than the basal level. This was also diminished by a high Ca concentration in medium. These results indicate that 1) AVP concomitantly regulates cellular free Ca as well as its second messenger cAMP production; 2) AVP-induced elevation of cellular free Ca is dependent on both the cellular Ca pool and extracellular Ca; and 3) there is an optimal level of extracellular Ca to modulate the AVP action in renal papillary collecting tubule cells.

  9. A Biphasic and Brain-Region Selective Down-Regulation of Cyclic Adenosine Monophosphate Concentrations Supports Object Recognition in the Rat

    PubMed Central

    Hotte, Maïte; Dauphin, François; Freret, Thomas; Boulouard, Michel; Levallet, Guenaëlle

    2012-01-01

    Background We aimed to further understand the relationship between cAMP concentration and mnesic performance. Methods and Findings Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p.), rolipram (PDE4 inhibitor, 0.3 mg/kg, i.p.) and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p.) before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices) before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold), prefrontal (1.3 fold) and perirhinal (1.3 fold) cortices from controls rat while decreased in prefrontal cortex (∼0.83 to 0.62 fold) from drug-treated rats (except for milrinone+RS 67333 treatment). After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold) and the perirhinal cortex (2.1 fold) from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold) and milrinone (respectively 1.46 fold and 1.56 fold)-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h). Conclusions Our results strongly suggest that a “pre-sample” early increase in cAMP levels followed by a specific lowering of cAMP concentrations in each brain sub-region linked to the object recognition paradigm support learning efficacy after a middle-term delay. PMID:22359674

  10. Glial restricted precursor cell transplant with cyclic adenosine monophosphate improved some autonomic functions but resulted in a reduced graft size after spinal cord contusion injury in rats.

    PubMed

    Nout, Yvette S; Culp, Esther; Schmidt, Markus H; Tovar, C Amy; Pröschel, Christoph; Mayer-Pröschel, Margot; Noble, Mark D; Beattie, Michael S; Bresnahan, Jacqueline C

    2011-01-01

    Transplantation of glial restricted precursor (GRP) cells has been shown to reduce glial scarring after spinal cord injury (SCI) and, in combination with neuronal restricted precursor (NRP) cells or enhanced expression of neurotrophins, to improve recovery of function after SCI. We hypothesized that combining GRP transplants with rolipram and cAMP would improve functional recovery, similar to that seen after combining Schwann cell transplants with increasing cAMP. A short term study, (1) uninjured control, (2) SCI+vehicle, and (3) SCI+cAMP, showed that spinal cord [cAMP] was increased 14days after SCI. We used 51 male rats subjected to a thoracic SCI for a 12-week survival study: (1) SCI+vehicle, (2) SCI+GRP, (3) SCI+cAMP, (4) SCI+GRP+cAMP, and (5) uninjured endpoint age-matched control (AM). Rolipram was administered for 2weeks after SCI. At 9days after SCI, GRP transplantation and injection of dibutyryl-cAMP into the spinal cord were performed. GRP cells survived, differentiated, and formed extensive transplants that were well integrated with host tissue. Presence of GRP cells increased the amount of tissue in the lesion; however, cAMP reduced the graft size. White matter sparing at the lesion epicenter was not affected. Serotonergic input to the lumbosacral spinal cord was not affected by treatment, but the amount of serotonin immediately caudal to the lesion was reduced in the cAMP groups. Using telemetric monitoring of corpus spongiosum penis pressure we show that the cAMP groups regained the same number of micturitions per 24hours when compared to the AM group, however, the frequency of peak pressures was increased in these groups compared to the AM group. In contrast, the GRP groups had similar frequency of peak pressures compared to baseline and the AM group. Animals that received GRP cells regained the same number of erectile events per 24hours compared to baseline and the AM group. Since cAMP reduced the GRP transplant graft, and some modest positive effects were seen that could be attributable to both GRP or cAMP, future research is required to determine how cAMP affects survival, proliferation, and/or function of progenitor cells and how this is related to function. cAMP may not always be a desirable addition to a progenitor cell transplantation strategy after SCI. PMID:21040723

  11. Adenosine augments interleukin-10 production by microglial cells through an A2B adenosine receptor-mediated process

    PubMed Central

    Koscsó, Balázs; Csóka, Balázs; Selmeczy, Zsolt; Himer, Leonóra; Pacher, Pál; Virág, László; Haskó, György

    2011-01-01

    Microglia are activated by pathogen-associated molecular patterns and produce pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and is a ligand of four G protein-coupled adenosine receptors (ARs), which are the A1AR, A2AAR, A2BAR and A3AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. Here, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of pro-inflammatory cytokines. Since the order of potency of selective AR agonists in inducing IL-10 production was 5′-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) ≥ 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS21680), and the A2BAR antagonist MRS-1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A2BAR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered shRNA blocked the enhancing effect of adenosine on IL-10 production confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A2BARs augment IL-10 production by activated murine microglia. PMID:22116830

  12. A(3) adenosine receptor ligands: history and perspectives.

    PubMed

    Baraldi, P G; Cacciari, B; Romagnoli, R; Merighi, S; Varani, K; Borea, P A; Spalluto, G

    2000-03-01

    Adenosine regulates many physiological functions through specific cell membrane receptors. On the basis of pharmacological studies and molecular cloning, four different adenosine receptors have been identified and classified as A(1), A(2A), A(2B), and A(3). These adenosine receptors are members of the G-protein-coupled receptor family. While adenosine A(1) and A(2A) receptor subtypes have been pharmacologically characterized through the use of selective ligands, the A(3) adenosine receptor subtype is presently under study in order to better understand its physio-pathological functions. Activation of adenosine A(3) receptors has been shown to stimulate phospholipase C and D and to inhibit adenylate cyclase. Activation of A(3) adenosine receptors also causes the release of inflammatory mediators such as histamine from mast cells. These mediators are responsible for processes such as inflammation and hypotension. It has also been suggested that the A(3) receptor plays an important role in brain ischemia, immunosuppression, and bronchospasm in several animal models. Based on these results, highly selective A(3) adenosine receptor agonists and/or antagonists have been indicated as potential drugs for the treatment of asthma and inflammation, while highly selective agonists have been shown to possess cardioprotective effects. The updated material related to this field of research has been rationalized and arranged in order to offer an overview of the topic. PMID:10723024

  13. Comorbidities in Neurology: Is adenosine the common link?

    PubMed

    Boison, Detlev; Aronica, Eleonora

    2015-10-01

    Comorbidities in Neurology represent a major conceptual and therapeutic challenge. For example, temporal lobe epilepsy (TLE) is a syndrome comprised of epileptic seizures and comorbid symptoms including memory and psychiatric impairment, depression, and sleep dysfunction. Similarly, Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic Lateral Sclerosis (ALS) are accompanied by various degrees of memory dysfunction. Patients with AD have an increased likelihood for seizures, whereas all four conditions share certain aspects of psychosis, depression, and sleep dysfunction. This remarkable overlap suggests common pathophysiological mechanisms, which include synaptic dysfunction and synaptotoxicity, as well as glial activation and astrogliosis. Astrogliosis is linked to synapse function via the tripartite synapse, but astrocytes also control the availability of gliotransmitters and adenosine. Here we will specifically focus on the 'adenosine hypothesis of comorbidities' implying that astrocyte activation, via overexpression of adenosine kinase (ADK), induces a deficiency in the homeostatic tone of adenosine. We present evidence from patient-derived samples showing astrogliosis and overexpression of ADK as common pathological hallmark of epilepsy, AD, PD, and ALS. We discuss a transgenic 'comorbidity model', in which brain-wide overexpression of ADK and resulting adenosine deficiency produces a comorbid spectrum of seizures, altered dopaminergic function, attentional impairment, and deficits in cognitive domains and sleep regulation. We conclude that dysfunction of adenosine signaling is common in neurological conditions, that adenosine dysfunction can explain co-morbid phenotypes, and that therapeutic adenosine augmentation might be effective for the treatment of comorbid symptoms in multiple neurological conditions. PMID:25979489

  14. Adenosine: Essential for life but licensed to kill

    PubMed Central

    Gama, Vivian; Deshmukh, Mohanish

    2016-01-01

    In this issue of Molecular Cell, Long et al. (Long et al., 2013) report a cell death priming mechanism activated by p53 that senses extracellular adenosine accumulated following chemotherapy or hypoxia, providing a novel connection between adenosine signaling and apoptosis. PMID:25884366

  15. Targeting of Adenosine Receptors in Ischemia-Reperfusion Injury

    PubMed Central

    Laubach, Victor E.; French, Brent A.; Okusa, Mark D.

    2010-01-01

    Importance of the field Ischemia-reperfusion (IR) injury is a common clinical problem after transplantation as well as myocardial infarction and stroke. IR initiates an inflammatory response leading to rapid tissue damage. Adenosine, produced in response to IR, is generally considered as a protective signaling molecule and elicits its physiological responses through four distinct adenosine receptors. The short half-life, lack of specificity, and rapid metabolism limits the use of adenosine as a therapeutic agent. Thus intense research efforts have focused on the synthesis and implementation of specific adenosine receptor agonists and antagonists as potential therapeutic agents for a variety of inflammatory conditions including IR injury. Areas covered by this review This review summarizes current knowledge on IR injury with a focus on lung, heart, and kidney, and examines studies that have advanced our understanding of the role of adenosine receptors and the therapeutic potential of adenosine receptor agonists and antagonists for the prevention of IR injury. What the reader will gain The reader will gain insight into the role of adenosine receptor signaling in IR injury. Take home message No clinical therapies are currently available that specifically target IR injury; however, targeting of specific adenosine receptors may offer therapeutic strategies in this regard. PMID:21110787

  16. Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis

    PubMed Central

    Song, Zhenyuan; Deaciuc, Ion; Zhou, Zhanxiang; Song, Ming; Chen, Theresa; Hill, Daniell; McClain, Craig J.

    2014-01-01

    Although simple steatosis was originally thought to be a pathologically inert histological change, fat accumulation in the liver may play a critical role not only in disease initiation, but also in the progression to nonalcoholic steatohepatitis and cirrhosis. Therefore, prevention of fat accumulation in the liver may be an effective therapy for multiple stages of nonalcoholic fatty liver disease (NAFLD). Promising beneficial effects of betaine supplementation on human NAFLD have been reported in some pilot clinical studies; however, data related to betaine therapy in NAFLD are limited. In this study, we examined the effects of betaine on fat accumulation in the liver induced by high-sucrose diet and evaluated mechanisms by which betaine could attenuate or prevent hepatic steatosis in this model. Male C57BL/6 mice weighing 20 ± 0.5 g (means ± SE) were divided into four groups (8 mice per group) and started on one of four treatments: standard diet (SD), SD+betaine, high-sucrose diet (HS), and HS + betaine. Betaine was supplemented in the drinking water at a concentration of 1% (wt/vol) (anhydrous). Long-term feeding of high-sucrose diet to mice caused significant hepatic steatosis accompanied by markedly increased lipogenic activity. Betaine significantly attenuated hepatic steatosis in this animal model, and this change was associated with increased activation of hepatic AMP-activated protein kinase (AMPK) and attenuated lipogenic capability (enzyme activities and gene expression) in the liver. Our findings are the first to suggest that betaine might serve as a therapeutic tool to attenuate hepatic steatosis by targeting the hepatic AMPK system. PMID:17702954

  17. AMP-activated Protein Kinase α2 Subunit Is Required for the Preservation of Hepatic Insulin Sensitivity by n-3 Polyunsaturated Fatty Acids

    PubMed Central

    Jelenik, Tomas; Rossmeisl, Martin; Kuda, Ondrej; Jilkova, Zuzana Macek; Medrikova, Dasa; Kus, Vladimir; Hensler, Michal; Janovska, Petra; Miksik, Ivan; Baranowski, Marcin; Gorski, Jan; Hébrard, Sophie; Jensen, Thomas E.; Flachs, Pavel; Hawley, Simon; Viollet, Benoit; Kopecky, Jan

    2010-01-01

    OBJECTIVE The induction of obesity, dyslipidemia, and insulin resistance by high-fat diet in rodents can be prevented by n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). We tested a hypothesis whether AMP-activated protein kinase (AMPK) has a role in the beneficial effects of n-3 LC-PUFAs. RESEARCH DESIGN AND METHODS Mice with a whole-body deletion of the α2 catalytic subunit of AMPK (AMPKα2−/−) and their wild-type littermates were fed on either a low-fat chow, or a corn oil-based high-fat diet (cHF), or a cHF diet with 15% lipids replaced by n-3 LC-PUFA concentrate (cHF+F). RESULTS Feeding a cHF diet induced obesity, dyslipidemia, hepatic steatosis, and whole-body insulin resistance in mice of both genotypes. Although cHF+F feeding increased hepatic AMPKα2 activity, the body weight gain, dyslipidemia, and the accumulation of hepatic triglycerides were prevented by the cHF+F diet to a similar degree in both AMPKα2−/− and wild-type mice in ad libitum-fed state. However, preservation of hepatic insulin sensitivity by n-3 LC-PUFAs required functional AMPKα2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content. Under hyperinsulinemic-euglycemic conditions, AMPKα2 was essential for preserving low levels of both hepatic and plasma triglycerides, as well as plasma free fatty acids, in response to the n-3 LC-PUFA treatment. CONCLUSIONS Our results show that n-3 LC-PUFAs prevent hepatic insulin resistance in an AMPKα2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity. AMPKα2 is also essential for hypolipidemic and antisteatotic effects of n-3 LC-PUFA under insulin-stimulated conditions. PMID:20693347

  18. Nordihydroguaiaretic acid protects against high-fat diet-induced fatty liver by activating AMP-activated protein kinase in obese mice

    SciTech Connect

    Lee, Myoung-Su; Kim, Daeyoung; Jo, Keunae; Hwang, Jae-Kwan

    2010-10-08

    Research highlights: {yields} NDGA decreases high-fat diet-induced body weight gain and adiposity. {yields} NDGA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} NDGA improves lipid storage in vitro through altering lipid regulatory proteins. {yields} Inhibition of lipid storage in vivo and in vitro is mediated by AMPK activation. -- Abstract: Nonalcoholic fatty liver disease, one of the most common causes of chronic liver disease, is strongly associated with metabolic syndrome. Nordihydroguaiaretic acid (NDGA) has been reported to inhibit lipoprotein lipase; however, the effect of NDGA on hepatic lipid metabolism remains unclear. We evaluated body weight, adiposity, liver histology, and hepatic triglyceride content in high-fat diet (HFD)-fed C57BL/6J mice treated with NDGA. In addition, we characterized the underlying mechanism of NDGA's effects in HepG2 hepatocytes by Western blot and RT-PCR analysis. NDGA (100 or 200 mg/kg/day) reduced weight gain, fat pad mass, and hepatic triglyceride accumulation, and improved serum lipid parameters in mice fed a HFD for 8 weeks. NDGA significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the liver and in HepG2 hepatocytes. NDGA downregulated the level of mature SREBP-1 and its target genes (acetyl-CoA carboxylase and fatty acid synthase), but, it upregulated expression of genes involved in fatty acid oxidation, such as peroxisome proliferator-activated receptor (PPAR){alpha}, PPAR{gamma} coactivator-1, carnitine palmitoyl transferase-1, and uncoupling protein-2. The specific AMPK inhibitor compound C attenuated the effects of NDGA on expression of lipid metabolism-related proteins in HepG2 hepatocytes. The beneficial effects of NDGA on HFD-induced hepatic triglyceride accumulation are mediated through AMPK signaling pathways, suggesting a potential target for preventing NAFLD.

  19. Docosahexaenoic acid inhibits proteolytic processing of sterol regulatory element-binding protein-1c (SREBP-1c) via activation of AMP-activated kinase.

    PubMed

    Deng, Xiong; Dong, Qingming; Bridges, Dave; Raghow, Rajendra; Park, Edwards A; Elam, Marshall B

    2015-12-01

    In hyperinsulinemic states including obesity and T2DM, overproduction of fatty acid and triglyceride contributes to steatosis of the liver, hyperlipidemia and hepatic insulin resistance. This effect is mediated in part by the transcriptional regulator sterol responsive element binding protein-1c (SREBP-1c), which stimulates the expression of genes involved in hepatic fatty acid and triglyceride synthesis. SREBP-1c is up regulated by insulin both via increased transcription of nascent full-length SREBP-1c and by enhanced proteolytic processing of the endoplasmic reticulum (ER)-bound precursor to yield the transcriptionally active n-terminal form, nSREBP-1c. Polyunsaturated fatty acids of marine origin (n-3 PUFA) prevent induction of SREBP-1c by insulin thereby reducing plasma and hepatic triglycerides. Despite widespread use of n-3 PUFA supplements to reduce triglycerides in clinical practice, the exact mechanisms underlying their hypotriglyceridemic effect remain elusive. Here we demonstrate that the n-3 PUFA docosahexaenoic acid (DHA; 22:5 n-3) reduces nSREBP-1c by inhibiting regulated intramembrane proteolysis (RIP) of the nascent SREBP-1c. We further show that this effect of DHA is mediated both via activation of AMP-activated protein kinase (AMPK) and by inhibition of mechanistic target of rapamycin complex 1 (mTORC1). The inhibitory effect of AMPK on SREBP-1c processing is linked to phosphorylation of serine 365 of SREBP-1c in the rat. We have defined a novel regulatory mechanism by which n-3 PUFA inhibit induction of SREBP-1c by insulin. These findings identify AMPK as an important negative regulator of hepatic lipid synthesis and as a potential therapeutic target for hyperlipidemia in obesity and T2DM. PMID:26327595

  20. Up-regulation of lipolysis genes and increased production of AMP-activated protein kinase protein in the skeletal muscle of rats after resistance training

    PubMed Central

    An, Jae-Heung; Yoon, Jin-Hwan; Suk, Min-Hwa; Shin, Yun-A

    2016-01-01

    The purpose of this study was to investigate the expression of lipogenesis- and lipolysis-related genes and proteins in skeletal muscles after 12 weeks of resistance training. Sprague-Dawley rats (n=12) were randomly divided into control (resting) and resistance training groups. A tower-climbing exercise, in which rats climbed to the top of their cage with a weight applied to their tails, used for resistance training. After 12 weeks, rats from the resistance training group had lower body weights (411.66±14.71 g vs. 478.33±24.63 g in the control), there was no significant difference between the two groups in the concentrations of total cholesterol, and high or low density lipoprotein cholesterol. However, the concentration of triglyceride was lower in resistance-trained rats (59.83±14.05 μg/mL vs 93.33±33.89 μg/mL in the control). The mRNA expression levels of the lipogenesis-related genes sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase were not significantly different between the resistance-trained and control rats; however, mRNA expression of the lipolysis-related carnitine palmitoyl transferase 1 and malonyl-CoA decarboxylase increased significantly with resistance training. AMP-activated protein kinase protein levels also significantly increased in resistance training group compared with in the control group. These results suggested that resistance exercise training contributing to reduced weight gain may be in part be due to increase the lipolysis metabolism and energy expenditure in response to resistance training. PMID:27419110

  1. The 5’-AMP-Activated Protein Kinase (AMPK) Is Involved in the Augmentation of Antioxidant Defenses in Cryopreserved Chicken Sperm

    PubMed Central

    Nguyen, Thi Mong Diep; Seigneurin, François; Froment, Pascal; Combarnous, Yves; Blesbois, Elisabeth

    2015-01-01

    Semen cryopreservation is a unique tool for the management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations which make difficult the restoration of sperm energy-dependent functions needed for fertilization. 5’-AMP activated protein kinase (AMPK) is a key sensor and regulator of intracellular energy metabolism. Mitochondria functions are known to be severely affected during sperm cryopreservation with deleterious oxidative and peroxidative effects leading to cell integrity and functions damages. The aim of this study was thus to examine the role of AMPK on the peroxidation/antioxidant enzymes defense system in frozen-thawed sperm and its consequences on sperm functions. Chicken semen was diluted in media supplemented with or without AMPK activators (AICAR or Metformin [MET]) or inhibitor (Compound C [CC]) and then cryopreserved. AMPKα phosphorylation, antioxidant enzymes activities, mitochondrial potential, ATP, citrate, viability, acrosome reaction ability (AR) and various motility parameters were negatively affected by the freeze-thaw process while reactive oxygen species (ROS) production, lipid peroxidation (LPO) and lactate concentration were dramatically increased. AICAR partially restored superoxide dismutase (SOD), Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), increased ATP, citrate, and lactate concentration and subsequently decreased the ROS and LPO (malondialdehyde) in frozen-thawed semen. Motility parameters were increased (i.e., + 23% for motility, + 34% for rapid sperm) as well as AR (+ 100%). MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased. CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters. Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation, thus

  2. Antcin K, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects via Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in Muscles

    PubMed Central

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching; Yang, Chang-Syun

    2016-01-01

    The purpose of this study was to screen firstly the potential effects of antcin K (AnK), the main constituent of the fruiting body of Antrodia camphorata, in vitro and further evaluate the activities and mechanisms in high-fat-diet- (HFD-) induced mice. Following 8-week HFD-induction, mice were treated with AnK, fenofibrate (Feno), metformin (Metf), or vehicle for 4 weeks afterward. In C2C12 myotube cells, the membrane GLUT4 and phospho-Akt expressions were higher in insulin and AnK-treated groups than in the control group. It was observed that AnK-treated mice significantly lowered blood glucose, triglyceride, total cholesterol, and leptin levels in AnK-treated groups. Of interest, AnK at 40 mg/kg/day dosage displayed both antihyperglycemic effect comparable to Metf (300 mg/kg/day) and antihypertriglyceridemic effect comparable to Feno (250 mg/kg/day). The combination of significantly increased skeletal muscular membrane expression levels of glucose transporter 4 (GLUT4) but decreased hepatic glucose-6-phosphatase (G6 Pase) mRNA levels by AnK thus contributed to a decrease in blood glucose levels. Furthermore, AnK enhanced phosphorylation of AMP-activated protein kinase (phospho-AMPK) expressions in the muscle and liver. Moreover, AnK treatment exhibited inhibition of hepatic fatty acid synthase (FAS) but enhancement of fatty acid oxidation peroxisome proliferator-activated receptor α (PPARα) expression coincident with reduced sterol response element binding protein-1c (SREBP-1c) mRNA levels in the liver may contribute to decreased plasma triglycerides, hepatic steatosis, and total cholesterol levels. The present findings indicate that AnK displays an advantageous therapeutic potential for the management of type 2 diabetes and hyperlipidemia. PMID:27242912

  3. AMP-activated protein kinase regulates the vacuolar H+-ATPase via direct phosphorylation of the A subunit (ATP6V1A) in the kidney

    PubMed Central

    Alzamora, Rodrigo; Al-Bataineh, Mohammad M.; Liu, Wen; Gong, Fan; Li, Hui; Thali, Ramon F.; Joho-Auchli, Yolanda; Brunisholz, René A.; Satlin, Lisa M.; Neumann, Dietbert; Pastor-Soler, Núria M.

    2013-01-01

    The vacuolar H+-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibited V-ATPase-dependent H+ secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3−-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues. PMID:23863464

  4. Insulin down-regulates the expression of ubiquitin E3 ligases partially by inhibiting the activity and expression of AMP-activated protein kinase in L6 myotubes

    PubMed Central

    Deng, Hu-Ping; Chai, Jia-Ke; Shen, Chuan-An; Zhang, Xi-Bo; Ma, Li; Sun, Tian-Jun; Hu, Qing-Gang; Chi, Yun-Fei; Dong, Ning

    2015-01-01

    While insulin is an anabolic hormone, AMP-activated protein kinase (AMPK) is not only a key energy regulator, but it can also control substrate metabolism directly by inducing skeletal muscle protein degradation. The hypothesis of the present study was that insulin inhibits AMPK and thus down-regulates the expression of the ubiquitin E3 ligases, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in skeletal muscle cells. Differentiated L6 myotubes were treated with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and/or compound C to stimulate and/or block AMPK respectively. These treatments were also conducted in the presence or absence of insulin and the cells were analysed by western blot and quantitative real-time PCR. In addition, nuleotide levels were determined using HPLC. The activation of AMPK with AICAR enhanced the mRNA levels of MAFbx and MuRF1. Insulin reduced the phosphorylation and activity AMPK, which was accompanied by reduced MAFbx and MuRF1 mRNA levels. Using a protein kinase B (PKB/Akt) inhibitor, we found that insulin regulates AMPK through the activation of Akt. Furthermore, insulin down-regulated AMPK α2 mRNA. We conclude that insulin inhibits AMPK through Akt phosphorylation in L6 myotubes, which may serve as a possible signalling pathway for the down-regulation of protein degradation. In addition, decreased expression of AMPK α2 may partially participate in inhibiting the activity of AMPK. PMID:26193886

  5. Activation of AMP-activated protein kinase regulates hippocampal neuronal pH by recruiting Na(+)/H(+) exchanger NHE5 to the cell surface.

    PubMed

    Jinadasa, Tushare; Szabó, Elöd Z; Numat, Masayuki; Orlowski, John

    2014-07-25

    Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H(+)-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na(+)/H(+) exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress. PMID:24936055

  6. Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

    SciTech Connect

    Sung, Jin Young; Choi, Hyoung Chul

    2011-05-06

    Highlights: {yields} Aspirin-induced AMPK phosphorylation was greater in VSMC from SHR than WKY. {yields} Aspirin-induced AMPK phosphorylation inhibited proliferation of VSMC from SHR. {yields} Low basal AMPK phosphorylation in SHR elicits increased VSMC proliferation. {yields} Inhibition of AMPK restored decreased VSMC proliferation by aspirin in SHR. {yields} Aspirin exerts anti-proliferative effect through AMPK activation in VSMC from SHR. -- Abstract: Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.

  7. Molecular characterization and expression of AMP-activated protein kinase in response to low-salinity stress in the Pacific white shrimp Litopenaeus vannamei.

    PubMed

    Xu, Chang; Li, Erchao; Xu, Zhixin; Wang, Shifeng; Chen, Ke; Wang, Xiaodan; Li, Tongyu; Qin, Jian G; Chen, Liqiao

    2016-08-01

    AMP-activated protein kinase (AMPK) serves as a major regulator of cellular energy metabolism by activating ATP production pathways and blocking ATP consumption. However, information on AMPK genes in aquatic animals is limited. In this study, three subunits of AMPK were cloned from the Pacific white shrimp Litopenaeus vannamei. The full-length cDNAs of the α, β and γ subunits were 1617, 1243 and 3467bp long, respectively, with open reading frames of 1566, 873 and 2988bp encoding for 521, 290 and 996 amino acids, respectively. Amino acid sequence alignments of the three subunits showed that the functional domains in the L. vannamei proteins retained the highest similarity with those of other animals, at 89%, 58%, and 75%, respectively. The expression levels of the three subunits were higher in the muscle and gills than in the eyestalk and hepatopancreas. The mRNA levels of AMPK-α and AMPK-β were up-regulated in the hepatopancreas and muscle after acute low-salinity stress at 3psu for 6h compared with control salinity at 20psu. After 8-week salinity stress at 3psu, AMPK-α and AMPK-β mRNA levels in the hepatopancreas were significantly higher than those of the control at 30psu. However, in the muscle only AMPK-γ mRNA was significantly up-regulated at low salinity relative to controls. Muscle and hepatopancreas showed increases in AMPK protein after 6h exposure to low salinity, but there were no differences seen after long term acclimation. The change patterns of protein were slightly differing from the mRNA patterns due to the distinguishing function of individual subunits of AMPK. These findings confirm that three AMPK subunits are present in L. vannamei and that all encode proteins with conserved functional domains. The three AMPK subunits are all regulated at the transcriptional and protein levels to manage excess energy expenditure during salinity stress. PMID:27095693

  8. Regulation of Pancreatic β Cell Mass by Cross-Interaction between CCAAT Enhancer Binding Protein β Induced by Endoplasmic Reticulum Stress and AMP-Activated Protein Kinase Activity

    PubMed Central

    Matsuda, Tomokazu; Takahashi, Hiroaki; Mieda, Yusuke; Shimizu, Shinobu; Kawamoto, Takeshi; Matsuura, Yuki; Takai, Tomoko; Suzuki, Emi; Kanno, Ayumi; Koyanagi-Kimura, Maki; Asahara, Shun-ichiro; Bartolome, Alberto; Yokoi, Norihide; Inoue, Hiroshi; Ogawa, Wataru; Seino, Susumu; Kido, Yoshiaki

    2015-01-01

    During the development of type 2 diabetes, endoplasmic reticulum (ER) stress leads to not only insulin resistance but also to pancreatic beta cell failure. Conversely, cell function under various stressed conditions can be restored by reducing ER stress by activating AMP-activated protein kinase (AMPK). However, the details of this mechanism are still obscure. Therefore, the current study aims to elucidate the role of AMPK activity during ER stress-associated pancreatic beta cell failure. MIN6 cells were loaded with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) and metformin to assess the relationship between AMPK activity and CCAAT enhancer binding protein β (C/EBPβ) expression levels. The effect of C/EBPβ phosphorylation on expression levels was also investigated. Vildagliptin and metformin were administered to pancreatic beta cell-specific C/EBPβ transgenic mice to investigate the relationship between C/EBPβ expression levels and AMPK activity in the pancreatic islets. When pancreatic beta cells are exposed to ER stress, the accumulation of the transcription factor C/EBPβ lowers the AMP/ATP ratio, thereby decreasing AMPK activity. In an opposite manner, incubation of MIN6 cells with AICAR or metformin activated AMPK, which suppressed C/EBPβ expression. In addition, administration of the dipeptidyl peptidase-4 inhibitor vildagliptin and metformin to pancreatic beta cell-specific C/EBPβ transgenic mice decreased C/EBPβ expression levels and enhanced pancreatic beta cell mass in proportion to the recovery of AMPK activity. Enhanced C/EBPβ expression and decreased AMPK activity act synergistically to induce ER stress-associated pancreatic beta cell failure. PMID:26091000

  9. Nectandrin B, a lignan isolated from nutmeg, inhibits liver X receptor-α-induced hepatic lipogenesis through AMP-activated protein kinase activation.

    PubMed

    Choi, Du Gon; Kim, Eun Kyung; Yang, Jin Won; Song, Jae Sook; Kim, Young-Mi

    2015-11-01

    Nonalcoholic fatty liver disease is recognized as the most commonly occurring chronic liver disease. Liver X receptor α (LXRα) and sterol regulatory element-binding protein (SREBP)-1c play a central role in de novo fatty acid synthesis. This study investigated pharmacological effects of nectandrin B, a lignan isolated from nutmeg extract, on hepatic lipogenesis stimulated by LXRα-SREBP-1c-mediated pathway and the possible molecular basis. The reporter gene assay revealed that nectandrin B completely represses LXRα activity enhanced by a synthetic LXRα ligand (T0901317) in HepG2 cells. The inhibitory effect was further supported by the suppression of mRNA expression of LXRα target genes, SREBP-1c and LXRα itself. Nectandrin B also inhibited the increase in SREBP-1c expression promoted by insulin plus high glucose, major contributors to hepatic lipid accumulation. LXRα-SREBP-1c-mediated induction of acetyl-CoA carboxylase 1 and fatty acid synthase, major genes for de novo lipogenesis, was suppressed by nectandrin B. Moreover, Oil Red O staining showed that nectandrin B notably attenuates LXRα-induced lipid accumulation. AMP-activated protein kinase (AMPK) inhibits the activities of LXRα and SREBP-1c. Nectandrin B strongly activated AMPK signaling in HepG2 cells. Taken together, the suppressive effects of nectandrin B on lipogenic gene expression and lipid accumulation in hepatocytes may be due to its inhibitory effect on the LXRα-SREBP-1c pathway presumably via AMPK activation. These results suggest the potential of nectandrin B as a therapeutic candidate for fatty liver disease. PMID:26790190

  10. An ent-kaurane diterpenoid from Croton tonkinensis induces apoptosis by regulating AMP-activated protein kinase in SK-HEP1 human hepatocellular carcinoma cells.

    PubMed

    Sul, Young Hoon; Lee, Myung Sun; Cha, Eun Young; Thuong, Phuong Thien; Khoi, Nguyen Minh; Song, In Sang

    2013-01-01

    Hepatocellular carcinoma (HCC) is the most common type of liver cancer with high mortality worldwide. Traditional chemotherapy for HCC is not widely accepted by clinical practitioners because of its toxic side effects. Thus, there is a need to identify chemotherapeutic drugs against HCC. AMP-activated protein kinase (AMPK) is a biologic sensor for cellular energy status that acts a tumor suppressor and a potential cancer therapeutic target. The traditional Vietnamese medicinal plant Croton tonkinensis shows cytotoxicity in various cancer cells; however, its anticancer mechanism remains unclear. In this study, we determined whether the ent-kaurane diterpenoid ent-18-acetoxy-7β-hydroxy kaur-15-oxo-16-ene (CrT1) isolated from this plant plays a role as a chemotherapeutic drug targeting AMPK. CrT1 blocked proliferation in dose- and time-dependent manners in human hepatocellular carcinoma SK-HEP1 cells. CrT1 induced sub-G(1) arrest and caspase-dependent apoptosis. CrT1 activated caspase-3, -7, -8, -9, and poly(ADP-ribose) polymerase, and its effect was inhibited by z-VAD-fmk suppressing caspase-3 cleavage. CrT1 induced increases in p53 and Bax levels but decreased Bcl(2) levels. In addition, CrT1 resulted in increased translocation of cytochrome c into the cytoplasm. We showed that CrT1-activated AMPK activation was followed by modulating the mammalian target of rapamycin/p70S6K pathway and was inactivated by treating cells with compound C. Treatment with CrT1 and aminoimidazole carboxamide ribonucleotide (AICAR) synergistically activated AMPK. CrT1-induced AMPK activation regulated cell viability and apoptosis. These results suggest that CrT1 is a novel AMPK activator and that AMPK activation in SK-HEP1 cells is responsible for CrT1-induced anticancer activity including apoptosis. PMID:23302650

  11. δ-Opioid receptors stimulate the metabolic sensor AMP-activated protein kinase through coincident signaling with G(q/11)-coupled receptors.

    PubMed

    Olianas, Maria C; Dedoni, Simona; Olianas, Alessandra; Onali, Pierluigi

    2012-02-01

    AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-D-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G(q/11)-coupled receptors, potentiating Ca(2+) release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake. PMID:22031472

  12. A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion.

    PubMed Central

    Pan, David A; Hardie, D Grahame

    2002-01-01

    We have identified single genes encoding homologues of the alpha, beta and gamma subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila melanogaster. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative alpha, beta or gamma subunits ablated this activity, and abolished expression of the alpha subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, we showed that activation was associated with phosphorylation of a threonine residue (Thr-184) within the 'activation loop' of the alpha subunit. We also identified a homologue of acetyl-CoA carboxylase (DmACC) in Drosophila and, using a phosphospecific antibody, showed that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK alpha subunit was mainly detected in the nucleus. Our results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs. PMID:12093363

  13. Sodium Tanshinone IIA Silate Inhibits High Glucose-Induced Vascular Smooth Muscle Cell Proliferation and Migration through Activation of AMP-Activated Protein Kinase

    PubMed Central

    Wu, Wen-yu; Yan, Hong; Wang, Xin-bo; Gui, Yu-zhou; Gao, Fei; Tang, Xi-lan; Qin, Yin-lin; Su, Mei; Chen, Tao; Wang, Yi-ping

    2014-01-01

    The proliferation of vascular smooth muscle cells may perform a crucial role in the pathogenesis of diabetic vascular disease. AMPK additionally exerts several salutary effects on vascular function and improves vascular abnormalities. The current study sought to determine whether sodium tanshinone IIA silate (STS) has an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation and migration under high glucose conditions mimicking diabetes without dyslipidemia, and establish the underlying mechanism. In this study, STS promoted the phosphorylation of AMP-activated protein kinase (AMPK) at T172 in VSMCs. VSMC proliferation was enhanced under high glucose (25 mM glucose, HG) versus normal glucose conditions (5.5 mM glucose, NG), and this increase was inhibited significantly by STS treatment. We utilized western blotting analysis to evaluate the effects of STS on cell-cycle regulatory proteins and found that STS increased the expression of p53 and the Cdk inhibitor, p21, subsequent decreased the expression of cell cycle-associated protein, cyclin D1. We further observed that STS arrested cell cycle progression at the G0/G1 phase. Additionally, expression and enzymatic activity of MMP-2, translocation of NF-κB, as well as VSMC migration were suppressed in the presence of STS. Notably, Compound C (CC), a specific inhibitor of AMPK, as well as AMPK siRNA blocked STS-mediated inhibition of VSMC proliferation and migration. We further evaluated its potential for activating AMPK in aortas in animal models of type 2 diabetes and found that Oral administration of STS for 10 days resulted in activation of AMPK in aortas from ob/ob or db/db mice. In conclusion, STS inhibits high glucose-induced VSMC proliferation and migration, possibly through AMPK activation. The growth suppression effect may be attributable to activation of AMPK-p53-p21 signaling, and the inhibitory effect on migration to the AMPK/NF-κB signaling axis. PMID:24739942

  14. AMP-Activated Protein Kinase Suppresses Autoimmune Central Nervous System Disease by Regulating M1-Type Macrophage-Th17 Axis.

    PubMed

    Mangalam, Ashutosh K; Rattan, Ramandeep; Suhail, Hamid; Singh, Jaspreet; Hoda, Md Nasrul; Deshpande, Mandar; Fulzele, Sadanand; Denic, Alexander; Shridhar, Viji; Kumar, Ashok; Viollet, Benoit; Rodriguez, Moses; Giri, Shailendra

    2016-08-01

    The AMP-activated protein kinase, AMPK, is an energy-sensing, metabolic switch implicated in various metabolic disorders; however, its role in inflammation is not well defined. We have previously shown that loss of AMPK exacerbates experimental autoimmune encephalomyelitis (EAE) disease severity. In this study, we investigated the mechanism through which AMPK modulates inflammatory disease like EAE. AMPKα1 knockout (α1KO) mice with EAE showed severe demyelination and inflammation in the brain and spinal cord compared with wild-type due to higher expression of proinflammatory Th17 cytokines, including IL-17, IL-23, and IL-1β, impaired blood-brain barrier integrity, and increased infiltration of inflammatory cells in the CNS. Infiltrated CD4 cells in the brains and spinal cords of α1KO with EAE were significantly higher compared with wild-type EAE and were characterized as IL-17 (IL-17 and GM-CSF double-positive) CD4 cells. Increased inflammatory response in α1KO mice was due to polarization of macrophages (Mϕ) to proinflammatory M1 type phenotype (IL-10(low)IL-23/IL-1β/IL-6(high)), and these M1 Mϕ showed stronger capacity to induce allogenic as well as Ag-specific (myelin oligodendrocyte glycoprotein [MOG]35-55) T cell response. Mϕ from α1KO mice also enhanced the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. The increased encephalitogenic MOG-restricted CD4(+) T cells were due to an autocrine effect of IL-1β/IL-23-mediated induction of IL-6 production in α1KO Mϕ, which in turn induce IL-17 and GM-CSF production in CD4 cells. Collectively, our data indicate that AMPK controls the inflammatory disease by regulating the M1 phenotype-Th17 axis in an animal model of multiple sclerosis. PMID:27354217

  15. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    SciTech Connect

    Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  16. Specific Deletion of AMP-Activated Protein Kinase (α1AMPK) in Murine Oocytes Alters Junctional Protein Expression and Mitochondrial Physiology

    PubMed Central

    Bertoldo, Michael J.; Guibert, Edith; Faure, Melanie; Ramé, Christelle; Foretz, Marc; Viollet, Benoit; Dupont, Joëlle; Froment, Pascal

    2015-01-01

    Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed. PMID:25767884

  17. AMP-activated protein kinase mediates apoptosis in response to bioenergetic stress through activation of the pro-apoptotic Bcl-2 homology domain-3-only protein BMF.

    PubMed

    Kilbride, Seán M; Farrelly, Angela M; Bonner, Caroline; Ward, Manus W; Nyhan, Kristine C; Concannon, Caoimhín G; Wollheim, Claes B; Byrne, Maria M; Prehn, Jochen H M

    2010-11-12

    Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation. PMID:20841353

  18. Antiaging Gene Klotho Deficiency Promoted High-Fat Diet-Induced Arterial Stiffening via Inactivation of AMP-Activated Protein Kinase.

    PubMed

    Lin, Yi; Chen, Jianglei; Sun, Zhongjie

    2016-03-01

    Klotho was originally discovered as an aging-suppressor gene. The objective of this study is to investigate whether klotho gene deficiency affects high-fat diet (HFD)-induced arterial stiffening. Heterozygous Klotho-deficient (KL(+/-)) mice and WT littermates were fed on HFD or normal diet. HFD increased pulse wave velocity within 5 weeks in KL(+/-) mice but not in wild-type mice, indicating that klotho deficiency accelerates and exacerbates HFD-induced arterial stiffening. A greater increase in blood pressure was found in KL(+/-) mice fed on HFD. Protein expressions of phosphorylated AMP-activated protein kinase-α (AMPKα), phosphorylated endothelial nitric oxide synthase (eNOS), and manganese-dependent superoxide dismutase (Mn-SOD) were decreased, whereas protein expressions of collagen I, transforming growth factor-β1, and Runx2 were increased in aortas of KL(+/-) mice fed on HFD. Interestingly, daily injections of an AMPKα activator, 5-aminoimidazole-4-carboxamide-3-ribonucleoside, abolished the increases in pulse wave velocity, blood pressure, and blood glucose in KL(+/-) mice fed on HFD. Treatment with 5-aminoimidazole-4-carboxamide-3-ribonucleoside for 2 weeks not only abolished the downregulation of phosphorylated AMPKα, phosphorylated eNOS, and Mn-SOD levels but also attenuated the increased levels of collagen I, transforming growth factor-β1, Runx2, superoxide, elastic lamellae breaks, and calcification in aortas of KL(+/-) mice fed on HFD. In cultured mouse aortic smooth muscle cells, cholesterol plus KL-deficient serum decreased phosphorylation levels of AMPKα and LKB1 (an important upstream regulator of AMPKα activity) but increased collagen I synthesis, which can be eliminated by activation of AMPKα by 5-aminoimidazole-4-carboxamide-3-ribonucleoside. In conclusions, Klotho deficiency promoted HFD-induced arterial stiffening and hypertension via downregulation of AMPKα activity. PMID:26781278

  19. Effects of Bofu-Tsusho-San on Diabetes and Hyperlipidemia Associated with AMP-Activated Protein Kinase and Glucose Transporter 4 in High-Fat-Fed Mice

    PubMed Central

    Lin, Cheng-Hsiu; Kuo, Yueh-Hsiung; Shih, Chun-Ching

    2014-01-01

    This study was undertaken to examine the effect and mechanism of Bofu-tsusho-san formula (BO) on hyperglycemia and hyperlipidemia and in mice fed with a high-fat (HF) diet. The C57BL/6J mice were received control/HF diet for 12 weeks, and oral administration of BO (at three doses) or rosiglitazone (Rosi) or vehicle for the last 4 weeks. Blood, skeletal muscle and tissues were examined by means of measuring glycaemia and dyslipidaemia-associated events. BO treatment effectively prevented HF diet-induced increases in the levels of triglyceride (TG), free fatty acid (FFA) and leptin (p < 0.01, p < 0.01, p < 0.01, respectively). BO treatment exhibited reduced both visceral fat mass and hepatic triacylglycerol content; moreover, BO treatment displayed significantly decreased both the average area of the cut of adipocytes and ballooning of hepatocytes. BO treatment exerted increased the protein contents of glucose transporter 4 (GLUT4) in skeletal muscle, and caused lowered blood glucose levels. BO treatment displayed increased levels of phosphorylated AMP-activated protein kinase (AMPK) in both skeletal muscle and liver tissue. Furthermore, BO reduced the hepatic expression of glucose-6-phosphatase (G6Pase) and phosphenolpyruvate carboxykinase (PEPCK) and glucose production. Therefore, it is possible that the activation of AMPK by BO leads to diminished gluconeogenesis in liver tissue. BO increased hepatic expressions of peroxisome proliferator-activated receptor α (PPARα), whereas down-regulating decreasing expressions of fatty acid synthesis, including sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FAS), resulting in a decrease in circulating triglycerides. This study originally provides the evidence that amelioration of dyslipidemic and diabetic state by BO in HF-fed mice occurred by regulation of GLUT4, SREBP1c, FAS, PPARα, adiponectin and AMPK phosphorylation. PMID:25375187

  20. Piperidine alkaloids from Piper retrofractum Vahl. protect against high-fat diet-induced obesity by regulating lipid metabolism and activating AMP-activated protein kinase.

    PubMed

    Kim, Kyung Jin; Lee, Myoung-Su; Jo, Keunae; Hwang, Jae-Kwan

    2011-07-22

    The fruits of Piper retrofractum Vahl. have been used for their anti-flatulent, expectorant, antitussive, antifungal, and appetizing properties in traditional medicine, and they are reported to possess gastroprotective and cholesterol-lowering properties. However, their anti-obesity activity remains unexplored. The present study was conducted to isolate the anti-obesity constituents from P. retrofractum Vahl. and evaluate their effects in high-fat diet (HFD)-induced obese mice. Piperidine alkaloids from P. retrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, were isolated as the anti-obesity constituents through a peroxisome proliferator-activated receptor δ (PPARδ) transactivation assay. The molecular mechanism was investigated in 3T3-L1 adipocytes and L6 myocytes. PRPA treatment activated AMP-activated protein kinase (AMPK) signaling and PPARδ protein and also regulated the expression of lipid metabolism-related proteins. In the animal model, oral PRPA administration (50, 100, or 300mg/kg/day for 8weeks) significantly reduced HFD-induced body weight gain without altering the amount of food intake. Fat pad mass was reduced in the PRPA treatment groups, as evidenced by reduced adipocyte size. In addition, elevated serum levels of total cholesterol, low-density lipoprotein cholesterol, total lipid, leptin, and lipase were suppressed by PRPA treatment. PRPA also protected against the development of nonalcoholic fatty liver by decreasing hepatic triglyceride accumulation. Consistent with the in vitro results, PRPA activated AMPK signaling and altered the expression of lipid metabolism-related proteins in liver and skeletal muscle. Taken together, these findings demonstrate that PRPAs attenuate HFD-induced obesity by activating AMPK and PPARδ, and regulate lipid metabolism, suggesting their potential anti-obesity effects. PMID:21741367

  1. Ptc1 protein phosphatase 2C contributes to glucose regulation of SNF1/AMP-activated protein kinase (AMPK) in Saccharomyces cerevisiae.

    PubMed

    Ruiz, Amparo; Xu, Xinjing; Carlson, Marian

    2013-10-25

    The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αβγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability. PMID:24019512

  2. Up-regulation of lipolysis genes and increased production of AMP-activated protein kinase protein in the skeletal muscle of rats after resistance training.

    PubMed

    An, Jae-Heung; Yoon, Jin-Hwan; Suk, Min-Hwa; Shin, Yun-A

    2016-06-01

    The purpose of this study was to investigate the expression of lipogenesis- and lipolysis-related genes and proteins in skeletal muscles after 12 weeks of resistance training. Sprague-Dawley rats (n=12) were randomly divided into control (resting) and resistance training groups. A tower-climbing exercise, in which rats climbed to the top of their cage with a weight applied to their tails, used for resistance training. After 12 weeks, rats from the resistance training group had lower body weights (411.66±14.71 g vs. 478.33±24.63 g in the control), there was no significant difference between the two groups in the concentrations of total cholesterol, and high or low density lipoprotein cholesterol. However, the concentration of triglyceride was lower in resistance-trained rats (59.83±14.05 μg/mL vs 93.33±33.89 μg/mL in the control). The mRNA expression levels of the lipogenesis-related genes sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase were not significantly different between the resistance-trained and control rats; however, mRNA expression of the lipolysis-related carnitine palmitoyl transferase 1 and malonyl-CoA decarboxylase increased significantly with resistance training. AMP-activated protein kinase protein levels also significantly increased in resistance training group compared with in the control group. These results suggested that resistance exercise training contributing to reduced weight gain may be in part be due to increase the lipolysis metabolism and energy expenditure in response to resistance training. PMID:27419110

  3. Different mechanisms of extracellular adenosine accumulation by reduction of the external Ca(2+) concentration and inhibition of adenosine metabolism in spinal astrocytes.

    PubMed

    Eguchi, Ryota; Akao, Sanae; Otsuguro, Ken-ichi; Yamaguchi, Soichiro; Ito, Shigeo

    2015-05-01

    Extracellular adenosine is a neuromodulator in the central nervous system. Astrocytes mainly participate in adenosine production, and extracellular adenosine accumulates under physiological and pathophysiological conditions. Inhibition of intracellular adenosine metabolism and reduction of the external Ca(2+) concentration ([Ca(2+)]e) participate in adenosine accumulation, but the precise mechanisms remain unclear. This study investigated the mechanisms underlying extracellular adenosine accumulation in cultured rat spinal astrocytes. The combination of adenosine kinase and deaminase (ADK/ADA) inhibition and a reduced [Ca(2+)]e increased the extracellular adenosine level. ADK/ADA inhibitors increased the level of extracellular adenosine but not of adenine nucleotides, which was suppressed by inhibition of equilibrative nucleoside transporter (ENT) 2. Unlike ADK/ADA inhibition, a reduced [Ca(2+)]e increased the extracellular level not only of adenosine but also of ATP. This adenosine increase was enhanced by ENT2 inhibition, and suppressed by sodium polyoxotungstate (ecto-nucleoside triphosphate diphosphohydrolase inhibitor). Gap junction inhibitors suppressed the increases in adenosine and adenine nucleotide levels by reduction of [Ca(2+)]e. These results indicate that extracellular adenosine accumulation by ADK/ADA inhibition is due to the adenosine release via ENT2, while that by reduction of [Ca(2+)]e is due to breakdown of ATP released via gap junction hemichannels, after which ENT2 incorporates adenosine into the cells. PMID:26003082

  4. Adenosine receptors and asthma in humans.

    PubMed

    Wilson, C N

    2008-10-01

    According to an executive summary of the GINA dissemination committee report, it is now estimated that approximately 300 million people (5% of the global population or 1 in 20 persons) have asthma. Despite the scientific progress made over the past several decades toward improving our understanding of the pathophysiology of asthma, there is still a great need for improved therapies, particularly oral therapies that enhance patient compliance and that target new mechanisms of action. Adenosine is an important signalling molecule in human asthma. By acting on extracellular G-protein-coupled ARs on a number of different cell types important in the pathophysiology of human asthma, adenosine affects bronchial reactivity, inflammation and airway remodelling. Four AR subtypes (A(1), A(2a), A(2b) and A(3)) have been cloned in humans, are expressed in the lung, and are all targets for drug development for human asthma. This review summarizes what is known about these AR subtypes and their function in human asthma as well as the pros and cons of therapeutic approaches to these AR targets. A number of molecules with high affinity and high selectivity for the human AR subtypes have entered clinical trials or are poised to enter clinical trials as anti-asthma treatments. With the availability of these molecules for testing in humans, the function of ARs in human asthma, as well as the safety and efficacy of approaches to the different AR targets, can now be determined. PMID:18852693

  5. L-theanine elicits an umami taste with inosine 5'-monophosphate.

    PubMed

    Narukawa, Masataka; Morita, Kanako; Hayashi, Yukako

    2008-11-01

    We investigated the taste synergy between L-theanine and the flavour enhancer, inosine 5'-monophosphate (IMP), by using a human sensory evaluation. When L-theanine was added to IMP, only the umami taste was enhanced. We then investigated this synergistic effect of L-theanine in mice by gustatory nerve recording. We confirmed the synergism between L-theanine and IMP for the umami taste. PMID:18997398

  6. Novel Characteristics of Trypanosoma brucei Guanosine 5'-monophosphate Reductase Distinct from Host Animals

    PubMed Central

    Kimura, Chihiro; Shinohara, Takahiro; Tomiyama, Ai; Imamura, Akira; Kuwamura, Mitsuru; Nishimura, Kazuhiko; Fujimori, Ko; Shuto, Satoshi; Ishibashi, Osamu; Kubata, Bruno Kilunga; Inui, Takashi

    2016-01-01

    The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5’-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5’-monophosphate (GMP) to inosine 5’-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine β-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K+ and NH4+ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR’s was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis. PMID:26731263

  7. The Role of Adenosine Signaling in Sickle Cell Therapeutics

    PubMed Central

    Field, Joshua J.; Nathan, David G.; Linden, Joel

    2014-01-01

    Recent data suggest a role for adenosine signaling in the pathogenesis of sickle cell disease (SCD). Signaling through the adenosine A2A receptor (A2AR) has demonstrated beneficial effects in SCD. Activation of A2ARs decreases inflammation in mice and patients with SCD largely by blocking activation of invariant NKT cells. Decreased inflammation may reduce the severity of vaso-occlusive crises. In contrast, adenosine signaling through the A2B receptor (A2BR) may be detrimental for patients with SCD. Priapism and the formation of sickle erythrocytes may be a consequence of A2BR activation on corpus cavernosal cells and erythrocytes, respectively. Whether adenosine signaling predominantly occurs through A2ARs or A2BRs may depend on differing levels of adenosine and disease state (steady state versus crisis). There may be opportunities to develop novel therapeutic approaches targeting A2ARs and/or A2BRs for patients with SCD. PMID:24589267

  8. Chronic benzodiazepine treatment and cortical responses to adenosine and GABA.

    PubMed

    Mally, J; Connick, J H; Stone, T W

    1990-10-22

    The effects of chronic treatment of mice with clonazepam have been examined on the responses of neocortical slices to adenosine, 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA). Responses to these agonists were measured as changes in the depolarisation induced by N-methyl-D-aspartate (NMDA). Added to the superfusion medium diazepam blocked responses to adenosine but not 5-HT; this effect was not observed with 2-chloroadenosine or in the presence of 2-hydroxynitrobenzylthioguanosine. GABA was inactive in control slices but chronic treatment with clonazepam induced responses to GABA and enhanced responses to adenosine but not 5-HT. It is suggested that the induction of GABA responses may reflect the up-regulation of GABA receptors, but the increase of adenosine responses by clonazepam implies that there is no simple relationship between adenosine receptor binding and functional responses. PMID:1979931

  9. Recipient pretransplant inosine monophosphate dehydrogenase activity in nonmyeloablative hematopoietic cell transplantation.

    PubMed

    Bemer, Meagan J; Risler, Linda J; Phillips, Brian R; Wang, Joanne; Storer, Barry E; Sandmaier, Brenda M; Duan, Haichuan; Raccor, Brianne S; Boeckh, Michael J; McCune, Jeannine S

    2014-10-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5'-monophosphate to xanthosine 5'-monophosphate (XMP). We developed a highly sensitive liquid chromatography-mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNCs) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation but not with chronic GVHD, relapse, nonrelapse mortality, or overall mortality. We conclude that quantitation of the recipient's pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient's sensitivity to MMF. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

  10. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    SciTech Connect

    M Cassera; M Ho; E Merino; E Burgos; A Rinaldo-Matthis; S Almo; V Schramm

    2011-12-31

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

  11. An adenosine kinase inhibitor, ABT-702, inhibits spinal nociceptive transmission by adenosine release via equilibrative nucleoside transporters in rat.

    PubMed

    Otsuguro, Ken-ichi; Tomonari, Yuki; Otsuka, Saori; Yamaguchi, Soichiro; Kon, Yasuhiro; Ito, Shigeo

    2015-10-01

    Adenosine kinase (AK) inhibitor is a potential candidate for controlling pain, but some AK inhibitors have problems of adverse effects such as motor impairment. ABT-702, a non-nucleoside AK inhibitor, shows analgesic effect in animal models of pain. Here, we investigated the effects of ABT-702 on synaptic transmission via nociceptive and motor reflex pathways in the isolated spinal cord of neonatal rats. The release of adenosine from the spinal cord was measured by HPLC. ABT-702 inhibited slow ventral root potentials (sVRPs) in the nociceptive pathway more potently than monosynaptic reflex potentials (MSRs) in the motor reflex pathway. The inhibitory effects of ABT-702 were mimicked by exogenously applied adenosine, blocked by 8CPT (8-cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist, and augmented by EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), an adenosine deaminase (ADA) inhibitor. Equilibrative nucleoside transporter (ENT) inhibitors reversed the effects of ABT-702, but not those of adenosine. ABT-702 released adenosine from the spinal cord, an effect that was also reversed by ENT inhibitors. The ABT-702-facilitated release of adenosine by way of ENTs inhibits nociceptive pathways more potently than motor reflex pathways in the spinal cord via activation of A1 receptors. This feature is expected to lead to good analgesic effects, but, caution may be required for the use of AK inhibitors in the case of ADA dysfunction or a combination with ENT inhibitors. PMID:26066576

  12. Simultaneous interaction with base and phosphate moieties modulates the phosphodiester cleavage of dinucleoside 3',5'-monophosphates by dinuclear Zn2+ complexes of di(azacrown) ligands.

    PubMed

    Wang, Qi; Lönnberg, Harri

    2006-08-23

    Five dinucleating ligands (1-5) and one trinucleating ligand (6) incorporating 1,5,9-triazacyclododecan-3-yloxy groups attached to an aromatic scaffold have been synthesized. The ability of the Zn(2+) complexes of these ligands to promote the transesterification of dinucleoside 3',5'-monophosphates to a 2',3'-cyclic phosphate derived from the 3'-linked nucleoside by release of the 5'-linked nucleoside has been studied over a narrow pH range, from pH 5.8 to 7.2, at 90 degrees C. The dinuclear complexes show marked base moiety selectivity. Among the four dinucleotide 3',5'-phosphates studied, viz. adenylyl-3',5'-adenosine (ApA), adenylyl-3',5'-uridine (ApU), uridylyl-3',5'-adenosine (UpA), and uridylyl-3',5'-uridine (UpU), the dimers containing one uracil base (ApU and UpA) are cleaved up to 2 orders of magnitude more readily than those containing either two uracil bases (UpU) or two adenine bases (ApA). The trinuclear complex (6), however, cleaves UpU as readily as ApU and UpA, while the cleavage of ApA remains slow. UV spectrophotometric and (1)H NMR spectroscopic studies with one of the dinucleating ligands (3) verify binding to the bases of UpU and ApU at less than millimolar concentrations, while no interaction with the base moieties of ApA is observed. With ApU and UpA, one of the Zn(2+)-azacrown moieties in all likelihood anchors the cleaving agent to the uracil base of the substrate, while the other azacrown moiety serves as a catalyst for the phosphodiester transesterification. With UpU, two azacrown moieties are engaged in the base moiety binding. The catalytic activity is, hence, lost, but it can be restored by addition of a third azacrown group on the cleaving agent. PMID:16910666

  13. Chaperoning of the A1-adenosine receptor by endogenous adenosine - an extension of the retaliatory metabolite concept.

    PubMed

    Kusek, Justyna; Yang, Qiong; Witek, Martin; Gruber, Christian W; Nanoff, Christian; Freissmuth, Michael

    2015-01-01

    Cell-permeable orthosteric ligands can assist folding of G protein-coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A1-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A1-receptor-Y(288)A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A1 antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A1-selective antagonist with an affinity indistinguishable from the wild-type A1 receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V2-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O2) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A1-receptor-Y(288)A within 1 hour. These findings were recapitulated for the wild-type A1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A1 receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A1 receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress. PMID:25354767

  14. Ethanol Tolerance Affects Endogenous Adenosine Signaling in Mouse Hippocampus.

    PubMed

    Zhang, Dali; Xiong, Wei; Jackson, Michael F; Parkinson, Fiona E

    2016-07-01

    Ethanol has many pharmacological effects, including increases in endogenous adenosine levels and adenosine receptor activity in brain. Ethanol consumption is associated with both positive and negative health outcomes, but tolerance to the behavioral effects of ethanol can lead to increased consumption, which increases the risk of negative health outcomes. The present study was performed to test whether a 7-day treatment with ethanol is linked to reduced adenosine signaling and whether this is a consequence of reduced ecto-5'-nucleotidase activity. Wild-type (CD73(+/+)) and ecto-5'-nucleotidase-deficient (CD73(-/-)) mice were treated with ethanol (2 g/kg) or saline for 7 days. In CD73(+/+) mice, repeated ethanol treatment reduced the hypothermic and ataxic effects of acute ethanol, indicating the development of tolerance to the acute effects of ethanol. In CD73(+/+) mice, this 7-day ethanol treatment led to increased hippocampal synaptic activity and reduced adenosine A1 receptor activity under both basal and low Mg(2+) conditions. These effects of ethanol tolerance were associated with an 18% decrease in activity of ecto-5'-nucleotidase activity in hippocampal cell membranes. In contrast, ethanol treatment was not associated with changes in synaptic activity or adenosine signaling in hippocampus from CD73(-/-) mice. These data indicate that ethanol treatment is associated with a reduction in adenosine signaling through adenosine<