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Sample records for activate amp-activated protein

  1. The regulation of AMP-activated protein kinase by phosphorylation.

    PubMed Central

    Stein, S C; Woods, A; Jones, N A; Davison, M D; Carling, D

    2000-01-01

    The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP. PMID:10642499

  2. 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

    2011-02-08

    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.

  3. Functional modulation of AMP-activated protein kinase by cereblon.

    PubMed

    Lee, Kwang Min; Jo, Sooyeon; Kim, Hyunyoung; Lee, Jongwon; Park, Chul-Seung

    2011-03-01

    Mutations in cereblon (CRBN), a substrate binding component of the E3 ubiquitin ligase complex, cause a form of mental retardation in humans. However, the cellular proteins that interact with CRBN remain largely unknown. Here, we report that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK α1) and inhibits the activation of AMPK activation. The ectopic expression of CRBN reduces phosphorylation of AMPK α1 and, thus, inhibits the enzyme in a nutrient-independent manner. Moreover, AMPK α1 can be potently activated by suppressing endogenous CRBN using CRBN-specific small hairpin RNAs. Thus, CRBN may act as a negative modulator of the AMPK signaling pathway in vivo.

  4. AMP-activated protein kinase and metabolic control

    PubMed Central

    Viollet, Benoit; Andreelli, Fabrizio

    2011-01-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is a major regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. It is now recognized that pharmacological activation of AMPK improves blood glucose homeostasis, lipid profile and blood pressure in insulin-resistant rodents. Indeed, AMPK activation mimics the beneficial effects of physical activity or those of calorie restriction by acting on multiple cellular targets. In addition it is now demonstrated that AMPK is one of the probable (albeit indirect) targets of major antidiabetic drugs including, the biguanides (metformin) and thiazolidinediones, as well as of insulin sensitizing adipokines (e.g., adiponectin). Taken together, such findings highlight the logic underlying the concept of targeting the AMPK pathway for the treatment of metabolic syndrome and type 2 diabetes. PMID:21484577

  5. 5'-AMP-activated protein kinase signaling in Caenorhabditis elegans.

    PubMed

    Beale, Elmus G

    2008-01-01

    5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.

  6. Effects of AMP-activated protein kinase in cerebral ischemia.

    PubMed

    Li, Jun; McCullough, Louise D

    2010-03-01

    AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK is found in most mammalian tissues including the brain. As a key metabolic and stress sensor/effector, AMPK is activated under conditions of nutrient deprivation, vigorous exercise, or heat shock. However, it is becoming increasingly recognized that changes in AMPK activation not only signal unmet metabolic needs, but also are involved in sensing and responding to 'cell stress', including ischemia. The downstream effect of AMPK activation is dependent on many factors, including the severity of the stressor as well as the tissue examined. This review discusses recent in vitro and in vivo studies performed in the brain/neuronal cells and vasculature that have contributed to our understanding of AMPK in stroke. Recent data on the potential role of AMPK in angiogenesis and neurogenesis and the interaction of AMPK with 3-hydroxy-3-methy-glutaryl-CoA reductase inhibitors (statins) agents are highlighted. The interaction between AMPK and nitric oxide signaling is also discussed.

  7. Effects of AMP-activated protein kinase in cerebral ischemia

    PubMed Central

    Li, Jun; McCullough, Louise D

    2010-01-01

    AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK is found in most mammalian tissues including the brain. As a key metabolic and stress sensor/effector, AMPK is activated under conditions of nutrient deprivation, vigorous exercise, or heat shock. However, it is becoming increasingly recognized that changes in AMPK activation not only signal unmet metabolic needs, but also are involved in sensing and responding to ‘cell stress', including ischemia. The downstream effect of AMPK activation is dependent on many factors, including the severity of the stressor as well as the tissue examined. This review discusses recent in vitro and in vivo studies performed in the brain/neuronal cells and vasculature that have contributed to our understanding of AMPK in stroke. Recent data on the potential role of AMPK in angiogenesis and neurogenesis and the interaction of AMPK with 3-hydroxy-3-methy-glutaryl-CoA reductase inhibitors (statins) agents are highlighted. The interaction between AMPK and nitric oxide signaling is also discussed. PMID:20010958

  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. AMP-activated protein kinase--an archetypal protein kinase cascade?

    PubMed

    Hardie, D G; MacKintosh, R W

    1992-10-01

    Mammalian AMP-activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events.

  10. Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase.

    PubMed

    Henin, N; Vincent, M F; Gruber, H E; Van den Berghe, G

    1995-04-01

    AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 microM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 microM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half-maximal inhibition was obtained at approximately 100 microM, and near-complete inhibition at 500 microM AICAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP-activated protein kinase.

  11. 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.

  12. Activation of AMP-activated protein kinase revealed by hydrogen/deuterium exchange Mass Spectrometry

    PubMed Central

    Landgraf, Rachelle R.; Goswami, Devrishi; Rajamohan, Francis; Harris, Melissa S.; Calabrese, Matthew; Hoth, Lise R.; Magyar, Rachelle; Pascal, Bruce D.; Chalmers, Michael J.; Busby, Scott A.; Kurumbail, Ravi; Griffin, Patrick R.

    2013-01-01

    Summary AMP-Activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme by hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and β subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the β subunit and in the kinase domain of the α subunit suggesting that the molecular binding site of latter resides between the α and β subunits. The distinct short and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands. PMID:24076403

  13. AMP-activated protein kinase—an energy sensor that regulates all aspects of cell function

    PubMed Central

    Hardie, D. Grahame

    2011-01-01

    AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains cellular energy homeostasis. It arose very early during eukaryotic evolution, and its ancestral role may have been in the response to starvation. Recent work shows that the kinase is activated by increases not only in AMP, but also in ADP. Although best known for its effects on metabolism, AMPK has many other functions, including regulation of mitochondrial biogenesis and disposal, autophagy, cell polarity, and cell growth and proliferation. Both tumor cells and viruses establish mechanisms to down-regulate AMPK, allowing them to escape its restraining influences on growth. PMID:21937710

  14. Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

    PubMed

    Funahashi, Koji; Cao, Xia; Yamauchi, Masako; Kozaki, Yasuko; Ishiguro, Naoki; Kambe, Fukushi

    2009-01-01

    We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

  15. 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...

  16. 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

  17. Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

    PubMed Central

    Heathcote, Helen R.; Mancini, Sarah J.; Strembitska, Anastasiya; Jamal, Kunzah; Reihill, James A.; Palmer, Timothy M.; Gould, Gwyn W.; Salt, Ian P.

    2016-01-01

    The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues. PMID:27784766

  18. Berberine promotes glucose consumption independently of AMP-activated protein kinase activation.

    PubMed

    Xu, Miao; Xiao, Yuanyuan; Yin, Jun; Hou, Wolin; Yu, Xueying; Shen, Li; Liu, Fang; Wei, Li; Jia, Weiping

    2014-01-01

    Berberine is a plant alkaloid with anti-diabetic action. Activation of AMP-activated protein kinase (AMPK) pathway has been proposed as mechanism for berberine's action. This study aimed to examine whether AMPK activation was necessary for berberine's glucose-lowering effect. We found that in HepG2 hepatocytes and C2C12 myotubes, berberine significantly increased glucose consumption and lactate release in a dose-dependent manner. AMPK and acetyl coenzyme A synthetase (ACC) phosphorylation were stimulated by 20 µmol/L berberine. Nevertheless, berberine was still effective on stimulating glucose utilization and lactate production, when the AMPK activation was blocked by (1) inhibition of AMPK activity by Compound C, (2) suppression of AMPKα expression by siRNA, and (3) blockade of AMPK pathway by adenoviruses containing dominant-negative forms of AMPKα1/α2. To test the effect of berberine on oxygen consumption, extracellular flux analysis was performed in Seahorse XF24 analyzer. The activity of respiratory chain complex I was almost fully blocked in C2C12 myotubes by berberine. Metformin, as a positive control, showed similar effects as berberine. These results suggest that berberine and metformin promote glucose metabolism by stimulating glycolysis, which probably results from inhibition of mitochondrial respiratory chain complex I, independent of AMPK activation.

  19. 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

  20. 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

  1. AMP-activated protein kinase: a target for drugs both ancient and modern.

    PubMed

    Hardie, D Grahame; Ross, Fiona A; Hawley, Simon A

    2012-10-26

    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. It is activated, by a mechanism requiring the tumor suppressor LKB1, by metabolic stresses that increase cellular ADP:ATP and/or AMP:ATP ratios. Once activated, it switches on catabolic pathways that generate ATP, while switching off biosynthetic pathways and cell-cycle progress. These effects suggest that AMPK activators might be useful for treatment and/or prevention of type 2 diabetes and cancer. Indeed, AMPK is activated by the drugs metformin and salicylate, the latter being the major breakdown product of aspirin. Metformin is widely used to treat diabetes, while there is epidemiological evidence that both metformin and aspirin provide protection against cancer. We review the mechanisms of AMPK activation by these and other drugs, and by natural products derived from traditional herbal medicines.

  2. A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated protein kinase.

    PubMed

    Xu, Z-X; Liang, J; Haridas, V; Gaikwad, A; Connolly, F P; Mills, G B; Gutterman, J U

    2007-11-01

    Avicins, a family of plant triterpene electrophiles, can trigger apoptosis-associated tumor cell death, and suppress chemical-induced carcinogenesis by its anti-inflammatory, anti-mutagenic, and antioxidant properties. Here, we show that tumor cells treated with benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone, an apoptosis inhibitor, and Bax(-/-)Bak(-/-) apoptosis-resistant cells can still undergo cell death in response to avicin D treatment. We demonstrate that this non-apoptotic cell death is mediated by autophagy, which can be suppressed by chloroquine, an autophagy inhibitor, and by specific knockdown of autophagy-related gene-5 (Atg5) and Atg7. Avicin D decreases cellular ATP levels, stimulates the activation of AMP-activated protein kinase (AMPK), and inhibits mammalian target of rapamycin (mTOR) and S6 kinase activity. Suppression of AMPK by compound C and dominant-negative AMPK decreases avicin D-induced autophagic cell death. Furthermore, avicin D-induced autophagic cell death can be abrogated by knockdown of tuberous sclerosis complex 2 (TSC2), a key mediator linking AMPK to mTOR inhibition, suggesting that AMPK activation is a crucial event targeted by avicin D. These findings indicate the therapeutic potential of avicins by triggering autophagic cell death.

  3. 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.

  4. Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice

    PubMed Central

    Huang, Jingyu; Simcox, Judith; Mitchell, T. Creighton; Jones, Deborah; Cox, James; Luo, Bai; Cooksey, Robert C.; Boros, Laszlo G.; McClain, Donald A.

    2013-01-01

    Excess iron is associated with hepatic damage and diabetes in humans, although the detailed molecular mechanisms are not known. To investigate how iron regulates glucose homeostasis, we fed C57BL/6J male mice with high-iron (HI) diets (2 or 20 g Fe/kg chow). Mice fed an HI diet exhibited elevated AMP-activated protein kinase (AMPK) activity and impaired insulin signaling in skeletal muscle and liver. Consistent with the increased AMPK activity, glucose uptake was enhanced in mice fed an HI diet. The effects of improved glucose tolerance induced by HI feeding were abolished in transgenic mice with expression of muscle specific dominant-negative AMPK. Glucose output was suppressed in the liver of wild-type mice fed an HI diet, due to decreased expression of gluconeogenic genes and decreased substrate (lactate) from peripheral glycolysis. Iron activated AMPK by increasing deacetylase and decreasing LKB1 acetylation, in turn stimulating the phosphorylation of LKB1 and AMPK. The effects of HI diet were abrogated by treatment of the mice with N-acetyl cysteine, suggesting a redox-dependent mechanism for increasing deacetylase activity. In addition, tissue from iron-fed mice exhibited an elevated AMP/ATP ratio, further contributing to AMPK activation. In summary, a diet high in iron improves glucose tolerance by activating AMPK through mechanisms that include deacetylation.—Huang J., Simcox, J., Mitchell, T. C., Jones, D., Cox, J., Luo, B., Cooksey, R. C., Boros, L. G., McClain, D. A. Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice. PMID:23515442

  5. Apelin-13 protects against apoptosis by activating AMP-activated protein kinase pathway in ischemia stroke.

    PubMed

    Yang, Yi; Zhang, Xiang-Jian; Li, Li-Tao; Cui, Hai-Ying; Zhang, Cong; Zhu, Chun-Hua; Miao, Jiang-Yong

    2016-01-01

    Apelin has been proved to be protective against apoptosis induced by ischemic reperfusion. However, mechanisms whereby apelin produces neuroprotection remain to be elucidated. AMP-activated protein kinase (AMPK) is a master energy sensor that monitors levels of key energy metabolites. It is activated via AMPKαThr172 phosphorylation during cerebral ischemia and appears to be neuroprotective. In this study, we investigated the effect of apelin on AMPKα and tested whether apelin protecting against apoptosis was associated with AMPK signals. Focal transient cerebral ischemia/reperfusion (I/R) model in male ICR mice was induced by 60 min of ischemia followed by reperfusion. Apelin-13 was injected intracerebroventricularly 15 min before reperfusion. AMPK inhibitor, compound C, was injected to mice intraperitoneally at the onset of ischemia. In experiment 1, the effect of apelin-13 on AMPKα was measured. In experiment 2, the relevance of AMPKα and apelin-13' effect on apoptosis was measured. Data showed that apelin-13 significantly increased AMPKα phosphorylation level after cerebral I/R. Apelin-13, with the co-administration of saline, reduced apoptosis cells, down-regulated Bax and cleaved-caspase3 and up-regulated Bcl2. However, with the co-administration of compound C, apelin-13 was inefficient in affecting apoptosis and Bax, Bcl2 and cleaved-caspase3. The study provided the evidence that apelin-13 up-regulated AMPKα phosphorylation level in cerebral ischemia insults and AMPK signals participated in the mechanism of apelin-mediated neuroprotection.

  6. 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.

  7. 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

  8. Activation of AMP-activated protein kinase inhibits ER stress and renal fibrosis.

    PubMed

    Kim, Hyosang; Moon, Soo Young; Kim, Joon-Seok; Baek, Chung Hee; Kim, Miyeon; Min, Ji Yeon; Lee, Sang Koo

    2015-02-01

    It has been suggested that endoplasmic reticulum (ER) stress facilitates fibrotic remodeling. Therefore, modulation of ER stress may serve as one of the possible therapeutic approaches to renal fibrosis. We examined whether and how activation of AMP-activated protein kinase (AMPK) suppressed ER stress induced by chemical ER stress inducers [tunicamycin (TM) and thapsigargin (TG)] and also nonchemical inducers in tubular HK-2 cells. We further investigated the in vivo effects of AMPK on ER stress and renal fibrosis. Western blot analysis, immunofluorescence, small interfering (si)RNA experiments, and immunohistochemical staining were performed. Metformin (the best known clinical activator of AMPK) suppressed TM- or TG-induced ER stress, as shown by the inhibition of TM- or TG-induced upregulation of glucose-related protein (GRP)78 and phosphorylated eukaryotic initiation factor-2α through induction of heme oxygenase-1. Metformin inhibited TM- or TG-induced epithelial-mesenchymal transitions as well. Compound C (AMPK inhibitor) blocked the effect of metformin, and 5-aminoimidazole-4-carboxamide-1β riboside (another AMPK activator) exerted the same effects as metformin. Transfection with siRNA targeting AMPK blocked the effect of metformin. Consistent with the results of cell culture experiments, metformin reduced renal cortical GRP78 expression and increased heme oxygenase-1 expression in a mouse model of ER stress-induced acute kidney injury by TM. Activation of AMPK also suppressed ER stress by transforming growth factor-β, ANG II, aldosterone, and high glucose. Furthermore, metformin reduced GRP78 expression and renal fibrosis in a mouse model of unilateral ureteral obstruction. In conclusion, AMPK may serve as a promising therapeutic target through reducing ER stress and renal fibrosis.

  9. Low salt concentrations activate AMP-activated protein kinase in mouse macula densa cells.

    PubMed

    Cook, Natasha; Fraser, Scott A; Katerelos, Marina; Katsis, Frosa; Gleich, Kurt; Mount, Peter F; Steinberg, Gregory R; Levidiotis, Vicki; Kemp, Bruce E; Power, David A

    2009-04-01

    The energy-sensing kinase AMP-activated protein kinase (AMPK) is associated with the sodium-potassium-chloride cotransporter NKCC2 in the kidney and phosphorylates it on a regulatory site in vitro. To identify a potential role for AMPK in salt sensing at the macula densa, we have used the murine macula densa cell line MMDD1. In this cell line, AMPK was rapidly activated by isosmolar low-salt conditions. In contrast to the known salt-sensing pathway in the macula densa, AMPK activation occurred in the presence of either low sodium or low chloride and was unaffected by inhibition of NKCC2 with bumetanide. Assays using recombinant AMPK demonstrated activation of an upstream kinase by isosmolar low salt. The specific calcium/calmodulin-dependent kinase kinase inhibitor STO-609 failed to suppress AMPK activation, suggesting that it was not part of the signal pathway. AMPK activation was associated with increased phosphorylation of the specific substrate acetyl-CoA carboxylase (ACC) at Ser(79), as well as increased NKCC2 phosphorylation at Ser(126). AMPK activation due to low salt concentrations was inhibited by an adenovirus construct encoding a kinase dead mutant of AMPK, leading to reduced ACC Ser(79) and NKCC2 Ser(126) phosphorylation. This work demonstrates that AMPK activation in macula densa-like cells occurs via isosmolar changes in sodium or chloride concentration, leading to phosphorylation of ACC and NKCC2. Phosphorylation of these substrates in vivo is predicted to increase intracellular chloride and so reduce the effect of salt restriction on tubuloglomerular feedback and renin secretion.

  10. Impact of 5'-amp-activated Protein Kinase on Male Gonad and Spermatozoa Functions

    PubMed Central

    Nguyen, Thi Mong Diep

    2017-01-01

    As we already know, the male reproductive system requires less energetic investment than the female one. Nevertheless, energy balance is an important feature for spermatozoa production in the testis and for spermatozoa properties after ejaculation. The 5'-AMP-activated protein kinase, AMPK, is a sensor of cell energy, that regulates many metabolic pathways and that has been recently shown to control spermatozoa quality and functions. It is indeed involved in the regulation of spermatozoa quality through its action on the proliferation of testicular somatic cells (Sertoli and Leydig), on spermatozoa motility and acrosome reaction. It also favors spermatozoa quality through the management of lipid peroxidation and antioxidant enzymes. I review here the most recent data available on the roles of AMPK in vertebrate spermatozoa functions. PMID:28386541

  11. Metformin reduces airway inflammation and remodeling via activation of AMP-activated protein kinase.

    PubMed

    Park, Chan Sun; Bang, Bo-Ram; Kwon, Hyouk-Soo; Moon, Keun-Ai; Kim, Tae-Bum; Lee, Ki-Young; Moon, Hee-Bom; Cho, You Sook

    2012-12-15

    Recent reports have suggested that metformin has anti-inflammatory and anti-tissue remodeling properties. We investigated the potential effect of metformin on airway inflammation and remodeling in asthma. The effect of metformin treatment on airway inflammation and pivotal characteristics of airway remodeling were examined in a murine model of chronic asthma generated by repetitive challenges with ovalbumin and fungal-associated allergenic protease. To investigate the underlying mechanism of metformin, oxidative stress levels and AMP-activated protein kinase (AMPK) activation were assessed. To further elucidate the role of AMPK, we examined the effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) as a specific activator of AMPK and employed AMPKα1-deficient mice as an asthma model. The role of metformin and AMPK in tissue fibrosis was evaluated using a bleomycin-induced acute lung injury model and in vitro experiments with cultured fibroblasts. Metformin suppressed eosinophilic inflammation and significantly reduced peribronchial fibrosis, smooth muscle layer thickness, and mucin secretion. Enhanced AMPK activation and decreased oxidative stress in lungs was found in metformin-treated asthmatic mice. Similar results were observed in the AICAR-treated group. In addition, the enhanced airway inflammation and fibrosis in heterozygous AMPKα1-deficient mice were induced by both allergen and bleomycin challenges. Fibronectin and collagen expression was diminished by metformin through AMPKα1 activation in cultured fibroblasts. Therefore metformin reduced both airway inflammation and remodeling at least partially through the induction of AMPK activation and decreased oxidative stress. These data provide insight into the beneficial role of metformin as a novel therapeutic drug for chronic asthma.

  12. Involvement of hypothalamic AMP-activated protein kinase in leptin-induced sympathetic nerve activation.

    PubMed

    Tanida, Mamoru; Yamamoto, Naoki; Shibamoto, Toshishige; Rahmouni, Kamal

    2013-01-01

    In mammals, leptin released from the white adipose tissue acts on the central nervous system to control feeding behavior, cardiovascular function, and energy metabolism. Central leptin activates sympathetic nerves that innervate the kidney, adipose tissue, and some abdominal organs in rats. AMP-activated protein kinase (AMPK) is essential in the intracellular signaling pathway involving the activation of leptin receptors (ObRb). We investigated the potential of AMPKα2 in the sympathetic effects of leptin using in vivo siRNA injection to knockdown AMPKα2 in rats, to produce reduced hypothalamic AMPKα2 expression. Leptin effects on body weight, food intake, and blood FFA levels were eliminated in AMPKα2 siRNA-treated rats. Leptin-evoked enhancements of the sympathetic nerve outflows to the kidney, brown and white adipose tissues were attenuated in AMPKα2 siRNA-treated rats. To check whether AMPKα2 was specific to sympathetic changes induced by leptin, we examined the effects of injecting MT-II, a melanocortin-3 and -4 receptor agonist, on the sympathetic nerve outflows to the kidney and adipose tissue. MT-II-induced sympatho-excitation in the kidney was unchanged in AMPKα2 siRNA-treated rats. However, responses of neural activities involving adipose tissue to MT-II were attenuated in AMPKα2 siRNA-treated rats. These results suggest that hypothalamic AMPKα2 is involved not only in appetite and body weight regulation but also in the regulation of sympathetic nerve discharges to the kidney and adipose tissue. Thus, AMPK might function not only as an energy sensor, but as a key molecule in the cardiovascular, thermogenic, and lipolytic effects of leptin through the sympathetic nervous system.

  13. 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.

  14. AMP-activated protein kinase α1-sensitive activation of AP-1 in cardiomyocytes.

    PubMed

    Voelkl, Jakob; Alesutan, Ioana; Primessnig, Uwe; Feger, Martina; Mia, Sobuj; Jungmann, Andreas; Castor, Tatsiana; Viereck, Robert; Stöckigt, Florian; Borst, Oliver; Gawaz, Meinrad; Schrickel, Jan Wilko; Metzler, Bernhard; Katus, Hugo A; Müller, Oliver J; Pieske, Burkert; Heinzel, Frank R; Lang, Florian

    2016-08-01

    AMP-activated protein kinase (Ampk) regulates myocardial energy metabolism and plays a crucial role in the response to cell stress. In the failing heart, an isoform shift of the predominant Ampkα2 to the Ampkα1 was observed. The present study explored possible isoform specific effects of Ampkα1 in cardiomyocytes. To this end, experiments were performed in HL-1 cardiomyocytes, as well as in Ampkα1-deficient and corresponding wild-type mice and mice following AAV9-mediated cardiac overexpression of constitutively active Ampkα1. As a result, in HL-1 cardiomyocytes, overexpression of constitutively active Ampkα1 increased the phosphorylation of Pkcζ. Constitutively active Ampkα1 further increased AP-1-dependent transcriptional activity and mRNA expression of the AP-1 target genes c-Fos, Il6 and Ncx1, effects blunted by Pkcζ silencing. In HL-1 cardiomyocytes, angiotensin-II activated AP-1, an effect blunted by silencing of Ampkα1 and Pkcζ, but not of Ampkα2. In wild-type mice, angiotensin-II infusion increased cardiac Ampkα1 and cardiac Pkcζ protein levels, as well as c-Fos, Il6 and Ncx1 mRNA expression, effects blunted in Ampkα1-deficient mice. Pressure overload by transverse aortic constriction (TAC) similarly increased cardiac Ampkα1 and Pkcζ abundance as well as c-Fos, Il6 and Ncx1 mRNA expression, effects again blunted in Ampkα1-deficient mice. AAV9-mediated cardiac overexpression of constitutively active Ampkα1 increased Pkcζ protein abundance and the mRNA expression of c-Fos, Il6 and Ncx1 in cardiac tissue. In conclusion, Ampkα1 promotes myocardial AP-1 activation in a Pkcζ-dependent manner and thus contributes to cardiac stress signaling.

  15. The Interplay of AMP-activated Protein Kinase and Androgen Receptor in Prostate Cancer Cells†

    PubMed Central

    Shen, Min; Zhang, Zhen; Ratnam, Manohar; Dou, Q. Ping

    2013-01-01

    AMP-activated protein kinase (AMPK) has recently emerged as a potential target for cancer therapy due to the observation that activation of AMPK inhibits tumor cell growth. It is well-known that androgen receptor (AR) signaling is a major driver for the development and progression of prostate cancer and that downregulation of AR is a critical step in the induction of apoptosis in prostate cancer cells. However, little is known about the potential interaction between AMPK and AR signaling pathways. In the current study, we showed that activation of AMPK by metformin caused decrease of AR protein level through suppression of AR mRNA expression and promotion of AR protein degradation, demonstrating that AMPK activation is upstream of AR downregulation. We also showed that inhibition of AR function by an anti-androgen or its siRNA enhanced AMPK activation and growth inhibition whereas overexpression of AR delayed AMPK activation and increased prostate cancer cellular resistance to metformin treatment, suggesting that AR suppresses AMPK signaling-mediated growth inhibition in a feedback mechanism. Our findings thus reveal a novel AMPK-AR regulatory loop in prostate cancer cells and should have a potential clinical significance. PMID:24129850

  16. Sensing of energy and nutrients by AMP-activated protein kinase.

    PubMed

    Hardie, D Grahame

    2011-04-01

    AMP-activated protein kinase (AMPK) is a cellular energy sensor that exists in almost all eukaryotes. Genetic studies in lower eukaryotes suggest that the ancestral role of AMPK was in response to starvation for a carbon source and that AMPK is involved in life-span extension in response to caloric restriction. In mammals, AMPK is activated by an increasing cellular AMP:ATP ratio (which signifies a decrease in energy) caused by metabolic stresses that interfere with ATP production (eg, hypoxia) or that accelerate ATP consumption (eg, muscle contraction). Because glucose deprivation can increase the AMP:ATP ratio, AMPK can also act as a glucose sensor. AMPK activation occurs by a dual mechanism that involves allosteric activation and phosphorylation by upstream kinases. Once activated, AMPK switches on catabolic pathways that generate ATP (eg, the uptake and oxidation of glucose and fatty acids and mitochondrial biogenesis) while switching off ATP-consuming, anabolic pathways (eg, the synthesis of lipids, glucose, glycogen, and proteins). In addition to the acute effects via direct phosphorylation of metabolic enzymes, AMPK has longer-term effects by regulating transcription. These features make AMPK an ideal drug target in the treatment of metabolic disorders such as insulin resistance and type 2 diabetes. The antidiabetic drug metformin (which is derived from an herbal remedy) works in part by activating AMPK, whereas many xenobiotics or "nutraceuticals," including resveratrol, quercetin, and berberine, are also AMPK activators. Most of these agents activate AMPK because they inhibit mitochondrial function.

  17. Protective benefits of AMP-activated protein kinase in hepatic ischemia-reperfusion injury

    PubMed Central

    Zhang, Min; Yang, Dan; Gong, Xianqiong; Ge, Pu; Dai, Jie; Lin, Ling; Zhang, Li

    2017-01-01

    Hepatic ischemia-reperfusion injury (HIRI) is a major cause of hepatic failure and death after liver trauma, haemorrhagic shock, resection surgery and liver transplantation. AMP-activated protein kinase (AMPK) is an energy sensitive kinase that plays crucial roles in the regulation of metabolic homeostasis. In HIRI, ischemia induces the decline of ATP and the increased ratio of AMP/ATP, which promotes the phosphorylation and activation of AMPK. Three AMPK kinases, liver kinase B1 (LKB1), Ca2+/calmodulin-depedent protein kinase kinase β (CaMKKβ) and TGF-β-activated kinase-1 (TAK1), are main upstream kinases for the phosphorylation of AMPK. In addition to the changed AMP/ATP ratio, the activated CaMKKβ by increased intracelluar Ca2+ and the overproduction of reactive oxygen species (ROS) are also involved in the activation of AMPK during HIRI. The activated AMPK might provide protective benefits in HIRI via prevention of energy decline, inhibition of inflammatory response, suppression of hepatocyte apoptosis and attenuation of oxidative stress. Thus, AMPK might become a novel target for the pharmacological intervention of HIRI. PMID:28386315

  18. 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

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

    PubMed

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnès; 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(2+)-dependent AMPK activation via calmodulin-dependent protein kinase kinase-beta(CaMKKbeta), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKbeta inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  20. AMP-activated protein kinase and energy balance in breast cancer

    PubMed Central

    Zhao, Hong; Orhan, Yelda C; Zha, Xiaoming; Esencan, Ecem; Chatterton, Robert T; Bulun, Serdar E

    2017-01-01

    Cancer growth and metastasis depends on the availability of energy. Energy-sensing systems are critical in maintaining a balance between the energy supply and utilization of energy for tumor growth. A central regulator in this process is AMP-activated protein kinase (AMPK). In times of energy deficit, AMPK is allosterically modified by the binding of increased levels of AMP and ADP, making it a target of specific AMPK kinases (AMPKKs). AMPK signaling prompts cells to produce energy at the expense of growth and motility, opposing the actions of insulin and growth factors. Increasing AMPK activity may thus prevent the proliferation and metastasis of tumor cells. Activated AMPK also suppresses aromatase, which lowers estrogen formation and prevents breast cancer growth. Biguanides can be used to activate AMPK, but AMPK activity is modified by many different interacting factors; understanding these factors is important in order to control the abnormal growth processes that lead to breast cancer neoplasia. Fatty acids, estrogens, androgens, adipokines, and another energy sensor, sirtuin-1, alter the phosphorylation and activation of AMPK. Isoforms of AMPK differ among tissues and may serve specific functions. Targeting AMPK regulatory processes at points other than the upstream AMPKKs may provide additional approaches for prevention of breast cancer neoplasia, growth, and metastasis. PMID:28337254

  1. Berberine regulates AMP-activated protein kinase signaling pathways and inhibits colon tumorigenesis in mice.

    PubMed

    Li, Weidong; Hua, Baojin; Saud, Shakir M; Lin, Hongsheng; Hou, Wei; Matter, Matthias S; Jia, Libin; Colburn, Nancy H; Young, Matthew R

    2015-10-01

    Colorectal cancer, a leading cause of cancer death, has been linked to inflammation and obesity. Berberine, an isoquinoline alkaloid, possesses anti-inflammatory, anti-diabetes and anti-tumor properties. In the azoxymethane initiated and dextran sulfate sodium (AOM/DSS) promoted colorectal carcinogenesis mouse model, berberine treated mice showed a 60% reduction in tumor number (P = 0.009), a 48% reduction in tumors <2 mm, (P = 0.05); 94% reduction in tumors 2-4 mm, (P = 0.001), and 100% reduction in tumors >4 mm (P = 0.02) compared to vehicle treated mice. Berberine also decreased AOM/DSS induced Ki-67 and COX-2 expression. In vitro analysis showed that in addition to its anti-proliferation activity, berberine also induced apoptosis in colorectal cancer cell lines. Berberine activated AMP-activated protein kinase (AMPK), a major regulator of metabolic pathways, and inhibited mammalian target of rapamycin (mTOR), a downstream target of AMPK. Furthermore, 4E-binding protein-1 and p70 ribosomal S6 kinases, downstream targets of mTOR, were down regulated by berberine treatment. Berberine did not affect Liver kinase B1 (LKB1) activity or the mitogen-activated protein kinase pathway. Berberine inhibited Nuclear Factor kappa-B (NF-κB) activity, reduced the expression of cyclin D1 and survivin, induced phosphorylation of p53 and increased caspase-3 cleavage in vitro. Berberine inhibition of mTOR activity and p53 phosphorylation was found to be AMPK dependent, while inhibition NF-κB was AMPK independent. In vivo, berberine also activated AMPK, inhibited mTOR and p65 phosphorylation and activated caspase-3 cleavage. Our data suggests that berberine suppresses colon epithelial proliferation and tumorigenesis via AMPK dependent inhibition of mTOR activity and AMPK independent inhibition of NF-κB.

  2. 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.

  3. Comprehensive Characterization of AMP-activated Protein Kinase Catalytic Domain by Top-down Mass Spectrometry

    PubMed Central

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

    2015-01-01

    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 has 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. PMID:26489410

  4. AMP-activated protein kinase enhances the phagocytic ability of macrophages and neutrophils

    PubMed Central

    Bae, Hong-Beom; Zmijewski, Jaroslaw W.; Deshane, Jessy S.; Tadie, Jean-Marc; Chaplin, David D.; Takashima, Seiji; Abraham, Edward

    2011-01-01

    Although AMPK plays well-established roles in the modulation of energy balance, recent studies have shown that AMPK activation has potent anti-inflammatory effects. In the present experiments, we examined the role of AMPK in phagocytosis. We found that ingestion of Escherichia coli or apoptotic cells by macrophages increased AMPK activity. AMPK activation increased the ability of neutrophils or macrophages to ingest bacteria (by 46±7.8 or 85±26%, respectively, compared to control, P<0.05) and the ability of macrophages to ingest apoptotic cells (by 21±1.4%, P<0.05 compared to control). AMPK activation resulted in cytoskeletal reorganization, including enhanced formation of actin and microtubule networks. Activation of PAK1/2 and WAVE2, which are downstream effectors of Rac1, accompanied AMPK activation. AMPK activation also induced phosphorylation of CLIP-170, a protein that participates in microtubule synthesis. The increase in phagocytosis was reversible by the specific AMPK inhibitor compound C, siRNA to AMPKα1, Rac1 inhibitors, or agents that disrupt actin or microtubule networks. In vivo, AMPK activation resulted in enhanced phagocytosis of bacteria in the lungs by 75 ± 5% vs. control (P<0.05). These results demonstrate a novel function for AMPK in enhancing the phagocytic activity of neutrophils and macrophages.—Bae, H. -B., Zmijewski, J. W., Deshane, J. S., Tadie, J. -M., Chaplin, D. D., Takashima, S., Abraham, E. AMP-activated protein kinase enhances the phagocytic ability of macrophages and neutrophils. PMID:21885655

  5. AMP-activated protein kinase (AMPK) α2 subunit mediates glycolysis in postmortem skeletal muscle.

    PubMed

    Liang, Junfang; Yang, Qiyuan; Zhu, Mei-Jun; Jin, Ye; Du, Min

    2013-11-01

    Postmortem glycolysis is directly linked to the incidences of PSE (pale, soft and exudative) and DFD (dark, firm and dry) meats which cause significant loss to meat industry. AMP-activated protein kinase (AMPK) is a major regulator of postmortem glycolysis. However, there are two isoforms of the AMPKα catalytic subunit, and their roles in glycolysis of postmortem muscle remain unclear. The objective was to identify the isoform specific roles of AMPK in postmortem glycolysis. Wild type, AMPKα1, and AMPKα2 knockout (KO) mice were used in the current study. AMPK in Longissimus muscle was activated shortly after death. AMPKα2 but not AMPKα1 KO abolished the activity of AMPK in postmortem muscle. In addition, AMPKα2 KO reduced postmortem pH decline and the generation of lactate, while AMPKα1 KO had no significant effect. Finally, the glycogen content of skeletal muscle was reduced in AMPKα2 KO but not AMPKα1 KO mice. Data clearly demonstrate that AMPKα2 catalytic subunit mainly regulates postmortem glycolysis in muscle.

  6. Neuroprotective Effects of AMP-Activated Protein Kinase on Scopolamine Induced Memory Impairment

    PubMed Central

    Kim, Soo-Jeong; Lee, Jun-Ho; Chung, Hwan-Suck; Song, Joo-Hyun; Ha, Joohun

    2013-01-01

    AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, is activated in response to cellular stress when intracellular levels of AMP increase. We investigated the neuroprotective effects of AMPK against scopolamine-induced memory impairment in vivo and glutamate-induced cytotoxicity in vitro. An adenovirus expressing AMPK wild type alpha subunit (WT) or a dominant negative form (DN) was injected into the hippocampus of rats using a stereotaxic apparatus. The AMPK WT-injected rats showed significant reversal of the scopolamine induced cognitive deficit as evaluated by escape latency in the Morris water maze. In addition, they showed enhanced acetylcholinesterase (AChE)-reactive neurons in the hippocampus, implying increased cholinergic activity in response to AMPK. We also studied the cellular mechanism by which AMPK protects against glutamate-induced cell death in primary cultured rat hippocampal neurons. We further demonstrated that AMPK WT-infected cells increased cell viability and reduced Annexin V positive hippocampal neurons. Western blot analysis indicated that AMPK WT-infected cells reduced the expression of Bax and had no effects on Bcl-2, which resulted in a decreased Bax/Bcl-2 ratio. These data suggest that AMPK is a useful cognitive impairment treatment target, and that its beneficial effects are mediated via the protective capacity of hippocampal neurons. PMID:23946693

  7. Nicotine induces negative energy balance through hypothalamic AMP-activated protein kinase.

    PubMed

    Martínez de Morentin, Pablo B; Whittle, Andrew J; Fernø, Johan; Nogueiras, Rubén; Diéguez, Carlos; Vidal-Puig, Antonio; López, Miguel

    2012-04-01

    Smokers around the world commonly report increased body weight after smoking cessation as a major factor that interferes with their attempts to quit. Numerous controlled studies in both humans and rodents have reported that nicotine exerts a marked anorectic action. The effects of nicotine on energy homeostasis have been mostly pinpointed in the central nervous system, but the molecular mechanisms controlling its action are still not fully understood. The aim of this study was to investigate the effect of nicotine on hypothalamic AMP-activated protein kinase (AMPK) and its effect on energy balance. Here we demonstrate that nicotine-induced weight loss is associated with inactivation of hypothalamic AMPK, decreased orexigenic signaling in the hypothalamus, increased energy expenditure as a result of increased locomotor activity, increased thermogenesis in brown adipose tissue (BAT), and alterations in fuel substrate utilization. Conversely, nicotine withdrawal or genetic activation of hypothalamic AMPK in the ventromedial nucleus of the hypothalamus reversed nicotine-induced negative energy balance. Overall these data demonstrate that the effects of nicotine on energy balance involve specific modulation of the hypothalamic AMPK-BAT axis. These targets may be relevant for the development of new therapies for human obesity.

  8. Functional characterization of AMP-activated protein kinase signaling in tumorigenesis.

    PubMed

    Cheng, Ji; Zhang, Tao; Ji, Hongbin; Tao, Kaixiong; Guo, Jianping; Wei, Wenyi

    2016-12-01

    AMP-activated protein kinase (AMPK) is a ubiquitously expressed metabolic sensor among various species. Specifically, cellular AMPK is phosphorylated and activated under certain stressful conditions, such as energy deprivation, in turn to activate diversified downstream substrates to modulate the adaptive changes and maintain metabolic homeostasis. Recently, emerging evidences have implicated the potential roles of AMPK signaling in tumor initiation and progression. Nevertheless, a comprehensive description on such topic is still in scarcity, especially in combination of its biochemical features with mouse modeling results to elucidate the physiological role of AMPK signaling in tumorigenesis. Hence, we performed this thorough review by summarizing the tumorigenic role of each component along the AMPK signaling, comprising of both its upstream and downstream effectors. Moreover, their functional interplay with the AMPK heterotrimer and exclusive efficacies in carcinogenesis were chiefly explained among genetically altered mice models. Importantly, the pharmaceutical investigations of AMPK relevant medications have also been highlighted. In summary, in this review, we not only elucidate the potential functions of AMPK signaling pathway in governing tumorigenesis, but also potentiate the future targeted strategy aiming for better treatment of aberrant metabolism-associated diseases, including cancer.

  9. Protective effects of AMP-activated protein kinase in the cardiovascular system.

    PubMed

    Xu, Qiang; Si, Liang-Yi

    2010-11-01

    Cardiovascular diseases remain the leading cause of mortality worldwide. Recent studies of AMP-activated protein kinase (AMPK), a highly conserved sensor of cellular energy status, suggest that there might be therapeutic value in targeting the AMPK signaling pathway. AMPK is found in most mammalian tissues, including those of the cardiovascular system. As cardiovascular diseases are typically associated with blood flow occlusion and blood occlusion may induce rapid energy deficit, AMPK activation may occur during the early phase upon nutrient deprivation in cardiovascular organs. Therefore, investigation of AMPK in cardiovascular organs may help us to understand the pathophysiology of defence mechanisms in these organs. Recent studies have provided proof of concept for the idea that AMPK is protective in heart as well as in vascular endothelial and smooth muscle cells. Moreover, dysfunction of the AMPK signalling pathway is involved in the genesis and development of various cardiovascular diseases, including atherosclerosis, hypertension and stroke. The roles of AMPK in the cardiovascular system, as they are currently understood, will be presented in this review. The interaction between AMPK and other cardiovascular signalling pathways such as nitric oxide signalling is also discussed.

  10. The AMP-activated protein kinase α2 catalytic subunit controls whole-body insulin sensitivity

    PubMed Central

    Viollet, Benoit; Andreelli, Fabrizio; Jørgensen, Sebastian B.; Perrin, Christophe; Geloen, Alain; Flamez, Daisy; Mu, James; Lenzner, Claudia; Baud, Olivier; Bennoun, Myriam; Gomas, Emmanuel; Nicolas, Gaël; Wojtaszewski, Jørgen F.P.; Kahn, Axel; Carling, David; Schuit, Frans C.; Birnbaum, Morris J.; Richter, Erik A.; Burcelin, Rémy; Vaulont, Sophie

    2003-01-01

    AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To better understand the physiological role of AMPK, we generated a knockout mouse model in which the AMPKα2 catalytic subunit gene was inactivated. AMPKα2–/– mice presented high glucose levels in the fed period and during an oral glucose challenge associated with low insulin plasma levels. However, in isolated AMPKα2–/– pancreatic islets, glucose- and L-arginine–stimulated insulin secretion were not affected. AMPKα2–/– mice have reduced insulin-stimulated whole-body glucose utilization and muscle glycogen synthesis rates assessed in vivo by the hyperinsulinemic euglycemic clamp technique. Surprisingly, both parameters were not altered in mice expressing a dominant-negative mutant of AMPK in skeletal muscle. Furthermore, glucose transport was normal in incubated isolated AMPKα2–/– muscles. These data indicate that AMPKα2 in tissues other than skeletal muscles regulates insulin action. Concordantly, we found an increased daily urinary catecholamine excretion in AMPKα2–/– mice, suggesting altered function of the autonomic nervous system that could explain both the impaired insulin secretion and insulin sensitivity observed in vivo. Therefore, extramuscular AMPKα2 catalytic subunit is important for whole-body insulin action in vivo, probably through modulation of sympathetic nervous activity. PMID:12511592

  11. The AMP-activated protein kinase alpha2 catalytic subunit controls whole-body insulin sensitivity.

    PubMed

    Viollet, Benoit; Andreelli, Fabrizio; Jørgensen, Sebastian B; Perrin, Christophe; Geloen, Alain; Flamez, Daisy; Mu, James; Lenzner, Claudia; Baud, Olivier; Bennoun, Myriam; Gomas, Emmanuel; Nicolas, Gaël; Wojtaszewski, Jørgen F P; Kahn, Axel; Carling, David; Schuit, Frans C; Birnbaum, Morris J; Richter, Erik A; Burcelin, Rémy; Vaulont, Sophie

    2003-01-01

    AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To better understand the physiological role of AMPK, we generated a knockout mouse model in which the AMPKalpha2 catalytic subunit gene was inactivated. AMPKalpha2(-/-) mice presented high glucose levels in the fed period and during an oral glucose challenge associated with low insulin plasma levels. However, in isolated AMPKalpha2(-/-) pancreatic islets, glucose- and L-arginine-stimulated insulin secretion were not affected. AMPKalpha2(-/-) mice have reduced insulin-stimulated whole-body glucose utilization and muscle glycogen synthesis rates assessed in vivo by the hyperinsulinemic euglycemic clamp technique. Surprisingly, both parameters were not altered in mice expressing a dominant-negative mutant of AMPK in skeletal muscle. Furthermore, glucose transport was normal in incubated isolated AMPKalpha2(-/-) muscles. These data indicate that AMPKalpha2 in tissues other than skeletal muscles regulates insulin action. Concordantly, we found an increased daily urinary catecholamine excretion in AMPKalpha2(-/-) mice, suggesting altered function of the autonomic nervous system that could explain both the impaired insulin secretion and insulin sensitivity observed in vivo. Therefore, extramuscular AMPKalpha2 catalytic subunit is important for whole-body insulin action in vivo, probably through modulation of sympathetic nervous activity.

  12. Structural basis for glycogen recognition by AMP-activated protein kinase.

    PubMed

    Polekhina, Galina; Gupta, Abhilasha; van Denderen, Bryce J W; Feil, Susanne C; Kemp, Bruce E; Stapleton, David; Parker, Michael W

    2005-10-01

    AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK beta subunit acts as a scaffold for the alpha catalytic and gamma regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK beta glycogen binding domain in complex with beta-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. beta-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two beta-cyclodextrin glucose units and a leucine residue piercing the beta-cyclodextrin ring. Mutation of key beta-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate's helical surface.

  13. Stimulation of Brain AMP-Activated Protein Kinase Attenuates Inflammation and Acute Lung Injury in Sepsis

    PubMed Central

    Mulchandani, Nikhil; Yang, Weng-Lang; Khan, Mohammad Moshahid; Zhang, Fangming; Marambaud, Philippe; Nicastro, Jeffrey; Coppa, Gene F; Wang, Ping

    2015-01-01

    Sepsis and septic shock are enormous public health problems with astronomical financial repercussions on health systems worldwide. The central nervous system (CNS) is closely intertwined in the septic process but the underlying mechanism is still obscure. AMP-activated protein kinase (AMPK) is a ubiquitous energy sensor enzyme and plays a key role in regulation of energy homeostasis and cell survival. In this study, we hypothesized that activation of AMPK in the brain would attenuate inflammatory responses in sepsis, particularly in the lungs. Adult C57BL/6 male mice were treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, 20 ng), an AMPK activator, or vehicle (normal saline) by intracerebroventricular (ICV) injection, followed by cecal ligation and puncture (CLP) at 30 min post-ICV. The septic mice treated with AICAR exhibited elevated phosphorylation of AMPKα in the brain along with reduced serum levels of aspartate aminotransferase, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), compared with the vehicle. Similarly, the expressions of TNF-α, IL-1β, keratinocyte-derived chemokine and macrophage inflammatory protein-2 as well as myeloperoxidase activity in the lungs of AICAR-treated mice were significantly reduced. Moreover, histological findings in the lungs showed improvement of morphologic features and reduction of apoptosis with AICAR treatment. We further found that the beneficial effects of AICAR on septic mice were diminished in AMPKα2 deficient mice, showing that AMPK mediates these effects. In conclusion, our findings reveal a new functional role of activating AMPK in the CNS to attenuate inflammatory responses and acute lung injury in sepsis. PMID:26252187

  14. AMP-activated protein kinase has diet-dependent and -independent roles in Drosophila oogenesis.

    PubMed

    Laws, Kaitlin M; Drummond-Barbosa, Daniela

    2016-12-01

    Multiple aspects of organismal physiology influence the number and activity of stem cells and their progeny, including nutritional status. Previous studies demonstrated that Drosophila germline stem cells (GSCs), follicle stem cells (FSCs), and their progeny sense and respond to diet via complex mechanisms involving many systemic and local signals. AMP-activated protein kinase, or AMPK, is a highly conserved regulator of energy homeostasis known to be activated under low cellular energy conditions; however, its role in the ovarian response to diet has not been investigated. Here, we describe nutrient-dependent and -independent requirements for AMPK in Drosophila oogenesis. We found that AMPK is cell autonomously required for the slow down in GSC and follicle cell proliferation that occurs on a poor diet. Similarly, AMPK activity is necessary in the germline for the degeneration of vitellogenic stages in response to nutrient deprivation. In contrast, AMPK activity is not required within the germline to modulate its growth. Instead, AMPK acts in follicle cells to negatively regulate their growth and proliferation, thereby indirectly limiting the size of the underlying germline cyst within developing follicles. Paradoxically, AMPK is required for GSC maintenance in well-fed flies (when AMPK activity is presumably at its lowest), suggesting potentially important roles for basal AMPK activity in specific cell types. Finally, we identified a nutrient-independent, developmental role for AMPK in cyst encapsulation by follicle cells. These results uncover specific AMPK requirements in multiple cell types in the ovary and suggest that AMPK can function outside of its canonical nutrient-sensing role in specific developmental contexts.

  15. Role of the energy sensor AMP-activated protein kinase in renal physiology and disease.

    PubMed

    Hallows, Kenneth R; Mount, Peter F; Pastor-Soler, Núria M; Power, David A

    2010-05-01

    The ultrasensitive energy sensor AMP-activated protein kinase (AMPK) orchestrates the regulation of energy-generating and energy-consuming pathways. AMPK is highly expressed in the kidney where it is reported to be involved in a variety of physiological and pathological processes including ion transport, podocyte function, and diabetic renal hypertrophy. Sodium transport is the major energy-consuming process in the kidney, and AMPK has been proposed to contribute to the coupling of ion transport with cellular energy metabolism. Specifically, AMPK has been identified as a regulator of several ion transporters of significance in renal physiology, including the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial sodium channel (ENaC), the Na(+)-K(+)-2Cl(-) cotransporter (NKCC), and the vacuolar H(+)-ATPase (V-ATPase). Identified regulators of AMPK in the kidney include dietary salt, diabetes, adiponectin, and ischemia. Activation of AMPK in response to adiponectin is described in podocytes, where it reduces albuminuria, and in tubular cells, where it reduces glycogen accumulation. Reduced AMPK activity in the diabetic kidney is associated with renal accumulation of triglyceride and glycogen and the pathogenesis of diabetic renal hypertrophy. Acute renal ischemia causes a rapid and powerful activation of AMPK, but the functional significance of this observation remains unclear. Despite the recent advances, there remain significant gaps in the present understanding of both the upstream regulating pathways and the downstream substrates for AMPK in the kidney. A more complete understanding of the AMPK pathway in the kidney offers potential for improved therapies for several renal diseases including diabetic nephropathy, polycystic kidney disease, and ischemia-reperfusion injury.

  16. AMP-activated protein kinase counteracted the inhibitory effect of glucose on the phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes.

    PubMed

    Hubert, A; Husson, A; Chédeville, A; Lavoinne, A

    2000-09-22

    The effect of AMP-activated protein kinase (AMPK) in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression was studied in isolated rat hepatocytes. Activation of AMPK by AICAR counteracted the inhibitory effect of glucose on the PEPCK gene expression, both at the mRNA and the transcriptional levels. It is proposed that a target for AMPK is involved in the inhibitory effect of glucose on PEPCK gene transcription.

  17. 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.

  18. 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

  19. 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

  20. Unpredictable chronic mild stress induces anxiety and depression-like behaviors and inactivates AMP-activated protein kinase in mice.

    PubMed

    Zhu, Shenghua; Wang, Junhui; Zhang, Yanbo; Li, Victor; Kong, Jiming; He, Jue; Li, Xin-Min

    2014-08-12

    The unpredictable chronic mild stress (UCMS) model was developed based upon the stress-diathesis hypothesis of depression. Most effects of UCMS can be reversed by antidepressants, demonstrating a strong predictive validity of this model for depression. However, the mechanisms underlying the effects induced by UCMS remain incompletely understood. Increasing evidence has shown that AMP-activated protein kinase (AMPK) regulates intracellular energy metabolism and is especially important for neurons because neurons are known to have small energy reserves. Abnormalities in the AMPK pathway disturb normal brain functions and synaptic integrity. In the present study, we first investigated the effects of UCMS on a battery of different tests measuring anxiety and depression-like behaviors in female C57BL/6N mice after 4 weeks of UCMS exposure. Stressed mice showed suppressed body weight gain, heightened anxiety, and increased immobility in the forced swim and tail suspension tests. These results are representative of some of the core symptoms of depression. Simultaneously, we observed decrease of synaptic proteins in the cortex of mice subjected to UCMS, which is associated with decreased levels of phosphorylated AMP-activated protein kinase α (AMPKα) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). Our findings suggest that AMPKα inactivation might be a mechanism by which UCMS causes anxiety/depression-like behaviors in mice.

  1. AMP-activated protein kinase supports the NGF-induced viability of human HeLa cells to glucose starvation.

    PubMed

    Ting, Luo; Bo, Wan; Li, Ruwei; Chen, Xinya; Wang, Yingli; Jun, Zhou; Yu, Long

    2010-07-01

    As an important cellular energy regulation kinase, AMP-activated protein kinase (AMPK) has been demonstrated as a key molecule in the development of tolerance to nutrient starvation. Activation of AMPK includes the phosphorylation of Thr172 of the alpha-subunit. Nerve growth factor (NGF) was originally isolated for its ability to stimulate both survival and differentiation in peripheral neurons, but many investigations have shown that the NGF also plays an important role in survival, growth and invasion of many human cancers. In this study, we used CCK-8 cell viability assay to find that NGF could facilitate the viability of HeLa cells following glucose deprivation while not in glucose-normal control groups. This effect of NGF-induced viability promotion to glucose starvation can be suppressed by Compound C, a specific inhibitor of AMPK. Meanwhile, western blot analysis showed that AMPKalpha1/alpha2 Thr172 phosphorylation level in HeLa cells was up-regulated after NGF treatment under glucose starvation, and Compound C was able to reduce the AMPKalpha1/alpha2 Thr172 phosphorylation level which was up-regulated by NGF in HeLa cells. Taken together, these results indicate that AMP-activated protein kinase supports the NGF-induced viability of human HeLa cells to glucose starvation.

  2. ATP-Induced Inflammasome Activation and Pyroptosis Is Regulated by AMP-Activated Protein Kinase in Macrophages

    PubMed Central

    Zha, Qing-Bing; Wei, Hong-Xia; Li, Chen-Guang; Liang, Yi-Dan; Xu, Li-Hui; Bai, Wen-Jing; Pan, Hao; He, Xian-Hui; Ouyang, Dong-Yun

    2016-01-01

    Adenosine triphosphate (ATP) is released by bacteria and host cells during bacterial infection as well as sterile tissue injury, acting as an inducer of inflammasome activation. Previous studies have shown that ATP treatment leads to AMP-activated protein kinase (AMPK) activation. However, it is unclear whether AMPK signaling has been involved in the regulation of ATP-induced inflammasome activation and subsequent pyroptosis. In this study, we aimed to investigate this issue in lipopolysaccharide-activated murine macrophages. Our results showed that AMPK signaling was activated in murine macrophages upon ATP treatment, which was accompanied by inflammasome activation and pyroptosis as evidenced by rapid cell membrane rupture as well as mature interleukin (IL)-1β and active caspase-1p10 release. The ATP-induced inflammasome activation and pyroptosis were markedly suppressed by an AMPK inhibitor compound C or small-interfering RNA-mediated knockdown of AMPKα, but could be greatly enhanced by metformin (a well-known AMPK agonist). Importantly, metformin administration increased the mortality of mice with bacterial sepsis, which was likely because metformin treatment enhanced the systemic inflammasome activation as indicated by elevated serum and hepatic IL-1β levels. Collectively, these data indicated that the AMPK signaling positively regulated ATP-induced inflammasome activation and pyroptosis in macrophages, highlighting the possibility of AMPK-targeting therapies for inflammatory diseases involving inflammasome activation. PMID:28018360

  3. Yeast AMP-activated Protein Kinase Monitors Glucose Concentration Changes and Absolute Glucose Levels*

    PubMed Central

    Bendrioua, Loubna; Smedh, Maria; Almquist, Joachim; Cvijovic, Marija; Jirstrand, Mats; Goksör, Mattias; Adiels, Caroline B.; Hohmann, Stefan

    2014-01-01

    Analysis of the time-dependent behavior of a signaling system can provide insight into its dynamic properties. We employed the nucleocytoplasmic shuttling of the transcriptional repressor Mig1 as readout to characterize Snf1-Mig1 dynamics in single yeast cells. Mig1 binds to promoters of target genes and mediates glucose repression. Mig1 is predominantly located in the nucleus when glucose is abundant. Upon glucose depletion, Mig1 is phosphorylated by the yeast AMP-activated kinase Snf1 and exported into the cytoplasm. We used a three-channel microfluidic device to establish a high degree of control over the glucose concentration exposed to cells. Following regimes of glucose up- and downshifts, we observed a very rapid response reaching a new steady state within less than 1 min, different glucose threshold concentrations depending on glucose up- or downshifts, a graded profile with increased cell-to-cell variation at threshold glucose concentrations, and biphasic behavior with a transient translocation of Mig1 upon the shift from high to intermediate glucose concentrations. Fluorescence loss in photobleaching and fluorescence recovery after photobleaching data demonstrate that Mig1 shuttles constantly between the nucleus and cytoplasm, although with different rates, depending on the presence of glucose. Taken together, our data suggest that the Snf1-Mig1 system has the ability to monitor glucose concentration changes as well as absolute glucose levels. The sensitivity over a wide range of glucose levels and different glucose concentration-dependent response profiles are likely determined by the close integration of signaling with the metabolism and may provide for a highly flexible and fast adaptation to an altered nutritional status. PMID:24627493

  4. Development of Novel Alkene Oxindole Derivatives As Orally Efficacious AMP-Activated Protein Kinase Activators

    PubMed Central

    2013-01-01

    Adenosine 5′-monophosphate-activated protein kinase (AMPK) is emerging as a promising drug target for its regulatory function in both glucose and lipid metabolism. Compound PT1 (5) was originally identified from high throughput screening as a small molecule activator of AMPK through the antagonization of the autoinhibition in α subunits. In order to enhance its potency at AMPK and bioavailability, structure–activity relationship studies have been performed and resulted in a novel series of AMPK activators based on an alkene oxindole scaffold. Following their evaluation in pharmacological AMPK activation assays, lead compound 24 was identified to possess improved potency as well as favorable pharmacokinetic profile. In the diet-induced obesity (DIO) mouse model, compound 24 was found to improve glucose tolerance and alleviate insulin resistance. The in vitro and in vivo data for these alkene oxindoles warrant further studies for their potential therapeutic medications in metabolic associated diseases. PMID:24900695

  5. 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.

  6. 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

  7. 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

  8. Antidiabetic activities of extract from Malva verticillata seed via the activation of AMP-activated protein kinase.

    PubMed

    Jeong, Yong-Tae; Song, Chi-Hyun

    2011-09-01

    Stimulation of AMP-activated protein kinase (AMPK) signaling followed by increase of glucose uptake in L6 myotubes were studied with organic solvent extract of Malva verticillata (MV) seeds. Ethanol extract of M. verticillata seeds (MVE) significantly increased the phosphorylation level of AMPK, acetyl-CoA carboxylase (ACC), and glucose uptake in L6 myotube cells. The MVE was fractionated with n-hexane (MVE-H), chloroform (MVE-C), ethylacetate (MVE-E), n-butanol (MVE-B), and water (MVE-W). MVE-H (150 microgram/ml) showed the highest phosphorylating activity and increased glucose uptake by 2.3-fold. Oral administration of MVE-H (40 mg/kg) for 4 weeks to type 2 diabetic (db/db) mice reduced non-fasting and fasting blood glucose levels by 17.1% and 23.3%, respectively. Phosphorylation levels of AMPK and ACC in the soleus muscle and liver tissue of db/db mice were significantly increased by the administration of MVE-H. MVE-H was further fractionated using preparative HPLC to identify the AMPK-activating compounds. The NMR and GC-MS analyses revealed that β-sitosterol was a major effective compound in MVE-H. Phosphorylation levels of AMPK and ACC, and glucose uptake were significantly increased by the treatment of MVE-S (β-sitosterol) isolated from M. verticillata to L6 cells, and these effects were attenuated by an AMPK inhibitor (Compound C) pretreatment. These results, taken together, demonstrate that increased glucose uptake in L6 myotubes by MVE-H treatment is mainly accomplished through the activation of AMPK. Our finding suggests that the extract isolated from M. verticillata seed would be beneficial for the treatment of metabolic disease including type 2 diabetes and hyperlipidemia.

  9. Oolong, black and pu-erh tea suppresses adiposity in mice via activation of AMP-activated protein kinase.

    PubMed

    Yamashita, Yoko; Wang, Liuqing; Wang, Lihua; Tanaka, Yuki; Zhang, Tianshun; Ashida, Hitoshi

    2014-10-01

    It is well known that tea has a variety of beneficial impacts on human health, including anti-obesity effects. It is well documented that green tea and its constituent catechins suppress obesity, but the effects of other types of tea on obesity and the potential mechanisms involved are not yet fully understood. In this study, we investigated the suppression of adiposity by oolong, black and pu-erh tea and characterized the underlying molecular mechanism in vivo. We found that the consumption of oolong, black or pu-erh tea for a period of one week significantly decreased visceral fat without affecting body weight in male ICR mice. On a mechanistic level, the consumption of tea enhanced the phosphorylation of AMP-activated protein kinase (AMPK) in white adipose tissue (WAT). This was accompanied by the induction of WAT protein levels of uncoupling protein 1 and insulin-like growth factor binding protein 1. Our results indicate that oolong, black and pu-erh tea, and in particular, black tea, suppresses adiposity via phosphorylation of the key metabolic regulator AMPK and increases browning of WAT.

  10. Metformin-induced AMP-activated protein kinase activation regulates phenylephrine-mediated contraction of rat aorta.

    PubMed

    Sung, Jin Young; Choi, Hyoung Chul

    2012-05-11

    The aim of the present study is to determine the effects and molecular mechanisms by which activation of LKB1-AMP-activated protein kinase (AMPK) by metformin regulates vascular smooth muscle contraction. The essential ability of vascular smooth muscle cells (VSMCs) to contract and relax in response to an elevation and reduction in intravascular pressure is necessary for appropriate blood flow regulation. Thus, vessel contraction is a critical mechanism for systemic blood flow regulation. In cultured rat VSMCs, AMPK activation through LKB1 by metformin-inhibited phenylephrine-mediated myosin light chain kinase (MLCK) and myosin light chain phosphorylation (p-MLC). Conversely, inhibition of AMPK and LKB1 reversed phenylephrine-induced MLCK and p-MLC phosphorylation. Measurement of the tension trace in rat aortic rings also showed that the effect of AMPK activation by metformin decreased phenylephrine-induced contraction. Metformin inhibited PE-induced p-MLC and α-smooth muscle actin co-localization. Our results suggest that activation of AMPK by LKB1 decreases VSMC contraction by inhibiting MLCK and p-MLC, indicating that induction by the AMPK-LKB1 pathway may be a new therapeutic target to lower high blood pressure.

  11. AMP-activated protein kinase (AMPK) activators from Myristica fragrans (nutmeg) and their anti-obesity effect.

    PubMed

    Nguyen, Phi Hung; Le, Thi Van Thu; Kang, Hu Won; Chae, Jooyoung; Kim, Sang Kyum; Kwon, Kwang-iI; Seo, Dae Bang; Lee, Sang Jun; Oh, Won Keun

    2010-07-15

    AMP-activated protein kinase (AMPK) is a potential therapeutic target for the treatment of metabolic syndrome including obesity and type-2 diabetes. As part of an ongoing search for new AMPK activators from plants, this study found that the total extract of Myristica fragrans (nutmeg) activated the AMPK enzyme in differentiated C2C12 cells. As active constituents, seven 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignans, tetrahydrofuroguaiacin B (1), saucernetindiol (2), verrucosin (3), nectandrin B (4), nectandrin A (5), fragransin C(1) (6), and galbacin (7) were isolated from this extract. Among the isolates, compounds 1, 4, and 5 at 5 microM produced strong AMPK stimulation in differentiated C2C12 cells. In addition, the preventive effect of a tetrahydrofuran mixture (THF) on weight gain in a diet-induced animal model was further examined. These results suggest that nutmeg and its active constituents can be used not only for the development of agents to treat obesity and possibly type-2 diabetes but may also be beneficial for other metabolic disorders.

  12. Silibinin activates AMP-activated protein kinase to protect neuronal cells from oxygen and glucose deprivation-re-oxygenation.

    PubMed

    Xie, Zhi; Ding, Sheng-quan; Shen, Ya-fang

    2014-11-14

    In this study, we explored the cytoprotective potential of silibinin against oxygen-glucose deprivation (OGD)-induced neuronal cell damages, and studied underling mechanisms. In vitro model of ischemic stroke was created by keeping neuronal cells (SH-SY5Y cells and primary mouse cortical neurons) in an OGD condition followed by re-oxygenation. Pre-treatment of silibinin significantly inhibited OGD/re-oxygenation-induced necrosis and apoptosis of neuronal cells. OGD/re-oxygenation-induced reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) reduction were also inhibited by silibinin. At the molecular level, silibinin treatment in SH-SY5Y cells and primary cortical neurons led to significant AMP-activated protein kinase (AMPK) signaling activation, detected by phosphorylations of AMPKα1, its upstream kinase liver kinase B1 (LKB1) and the downstream target acetyl-CoA Carboxylase (ACC). Pharmacological inhibition or genetic depletion of AMPK alleviated the neuroprotective ability of silibinin against OGD/re-oxygenation. Further, ROS scavenging ability by silibinin was abolished with AMPK inhibition or silencing. While A-769662, the AMPK activator, mimicked silibinin actions and suppressed ROS production and neuronal cell death following OGD/re-oxygenation. Together, these results show that silibinin-mediated neuroprotection requires activation of AMPK signaling.

  13. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

    PubMed

    Fleming, Ingrid; Schulz, Christian; Fichtlscherer, Birgit; Kemp, Bruce E; Fisslthaler, Beate; Busse, Rudi

    2003-11-01

    Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

  14. The Extract of Herbal Medicines Activates AMP-Activated Protein Kinase in Diet-Induced Obese Rats

    PubMed Central

    Shin, Hye-Yeon; Chung, SaeYeon; Kim, Soon Re; Lee, Ji-Hye; Seo, Hye-Sook; Shin, Yong-Cheol; Ko, Seong-Gyu

    2013-01-01

    Our study investigated whether the extract of six herbal medicines (OB-1) has an inhibitory effect on obesity. High-fat diet-(HFD-) induced rats and controls were treated with 40 mg/100 g body weight of OB-1 or saline once a day for 5 weeks. After significant changes in body weight were induced, OB-1 and saline were administered to each subgroup of HFD and control groups for additional 5 weeks. No statistically significant decrease of body weight in OB-1-treated rats was found compared to controls. However, OB-1-treated rats were found to be more active in an open-field test and have a reduction in the size of adipocytes compared to controls. We observed no changes in the mRNA expressions of leptin and adiponectin from adipocytes between OB-1- and saline-treated rats with HFD-induced obesity group. However, OB-1 treatments were shown to be inversely correlated with accumulation of lipid droplets in liver tissue, suggesting that OB-1 could inhibit a lipid accumulation by blocking the pathway related to lipid metabolism. Moreover, the phosphorylation of AMP-activated protein kinase (AMPK) was significantly increased in OB-1-treated rats with HFD compared to controls. These results suggest that OB-1 has no direct antiobesity effect and, however, could be a regulator of cellular metabolism. PMID:23533517

  15. 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

    2014-06-05

    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.

  16. 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.

  17. Chrysophanic Acid Suppresses Adipogenesis and Induces Thermogenesis by Activating AMP-Activated Protein Kinase Alpha In vivo and In vitro

    PubMed Central

    Lim, Hara; Park, Jinbong; Kim, Hye-Lin; Kang, JongWook; Jeong, Mi-Young; Youn, Dong-Hyun; Jung, Yunu; Kim, Yong-Il; Kim, Hyun-Ju; Ahn, Kwang Seok; Kim, Su-Jin; Choe, Seong-Kyu; Hong, Seung-Heon; Um, Jae-Young

    2016-01-01

    Chrysophanic acid (CA) is a member of the anthraquinone family abundant in rhubarb, a widely used herb for obesity treatment in Traditional Korean Medicine. Though several studies have indicated numerous features of CA, no study has yet reported the effect of CA on obesity. In this study, we tried to identify the anti-obesity effects of CA. By using 3T3-L1 adipocytes and primary cultured brown adipocytes as in vitro models, high-fat diet (HFD)-induced obese mice, and zebrafish as in vivo models, we determined the anti-obesity effects of CA. CA reduced weight gain in HFD-induced obese mice. They also decreased lipid accumulation and the expressions of adipogenesis factors including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in 3T3-L1 adipocytes. In addition, uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), the brown fat specific thermogenic genes, were up-regulated in brown adipocytes by CA treatment. Furthermore, when co-treated with Compound C, the AMP-activated protein kinase (AMPK) inhibitor, the action of CA on AMPKα was nullified in both types of adipocytes, indicating the multi-controlling effect of CA was partially via the AMPKα pathway. Given all together, these results indicate that CA can ameliorate obesity by controlling the adipogenic and thermogenic pathway at the same time. On these bases, we suggest the new potential of CA as an anti-obese pharmacotherapy. PMID:28008317

  18. Chrysophanic Acid Suppresses Adipogenesis and Induces Thermogenesis by Activating AMP-Activated Protein Kinase Alpha In vivo and In vitro.

    PubMed

    Lim, Hara; Park, Jinbong; Kim, Hye-Lin; Kang, JongWook; Jeong, Mi-Young; Youn, Dong-Hyun; Jung, Yunu; Kim, Yong-Il; Kim, Hyun-Ju; Ahn, Kwang Seok; Kim, Su-Jin; Choe, Seong-Kyu; Hong, Seung-Heon; Um, Jae-Young

    2016-01-01

    Chrysophanic acid (CA) is a member of the anthraquinone family abundant in rhubarb, a widely used herb for obesity treatment in Traditional Korean Medicine. Though several studies have indicated numerous features of CA, no study has yet reported the effect of CA on obesity. In this study, we tried to identify the anti-obesity effects of CA. By using 3T3-L1 adipocytes and primary cultured brown adipocytes as in vitro models, high-fat diet (HFD)-induced obese mice, and zebrafish as in vivo models, we determined the anti-obesity effects of CA. CA reduced weight gain in HFD-induced obese mice. They also decreased lipid accumulation and the expressions of adipogenesis factors including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in 3T3-L1 adipocytes. In addition, uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), the brown fat specific thermogenic genes, were up-regulated in brown adipocytes by CA treatment. Furthermore, when co-treated with Compound C, the AMP-activated protein kinase (AMPK) inhibitor, the action of CA on AMPKα was nullified in both types of adipocytes, indicating the multi-controlling effect of CA was partially via the AMPKα pathway. Given all together, these results indicate that CA can ameliorate obesity by controlling the adipogenic and thermogenic pathway at the same time. On these bases, we suggest the new potential of CA as an anti-obese pharmacotherapy.

  19. Fasting alters protein expression of AMP-activated protein kinase in the hypothalamus of broiler chicks (Gallus gallus domesticus).

    PubMed

    Song, Zhigang; Liu, Lei; Yue, Yunshuang; Jiao, Hongchao; Lin, Hai; Sheikhahmadi, Ardashir; Everaert, Nadia; Decuypere, Eddy; Buyse, Johan

    2012-09-15

    An experiment was conducted to investigate the effects of fasting and re-feeding on hypothalamic 5'-AMP-activated protein kinase (AMPK) levels and (an)orexigenic neuropeptides. Male Arbor Acres chicks (7-day-old, n=160) were allocated to four equal treatment groups: control chicks (fed ad libitum for 48 h, C48), chicks that were fasted for 48 h (F48), chicks that were first fasted for 48 h and then re-fed for 24h (F48C24), and chicks that were fed ad libitum for 72h (C72). Fasting for 48 h significantly (P<0.05) increased the ratio of phosphorylated AMPKα to total AMPKα and phosphorylated LKB1 to total LKB1, whereas re-feeding for 24h reduced these ratios to that of the ad libitum fed C72 chicks. The gene expressions of agouti-related peptide (AgRP), neuropeptide Y (NPY), melanocortin receptor 4, melanin-concentrating hormone, prepro-orexins and carnitine palmitoyltransferase-1 were significantly (P<0.05) increased in the fasted chicks relative to the ad libitum fed C48 group. The gene expression of pro-opiomelanocortin (POMC), as well as cocaine- and amphetamine-regulated transcript (CART) was not affected by the nutritional status. Fasting significantly (P<0.05) decreased the mRNA levels of fatty acid synthase (FAS) and sterol regulatory element binding protein-1 (SREBP-1). The results suggest that the LKB1/AMPK signal pathway is involved in the energy homeostasis of fasted chicks, and its possible role in feed intake regulation might be mediated by the AgRP/NPY rather than the POMC/CART pathway.

  20. Bavachalcone-induced manganese superoxide dismutase expression through the AMP-activated protein kinase pathway in human endothelial cells.

    PubMed

    Dang, Yanqi; Ling, Shuang; Duan, Ju; Ma, Jing; Ni, Rongzhen; Xu, Jin-Wen

    2015-01-01

    Mitochondrial oxidative stress has been suggested as a major etiological factor in cardiovascular diseases. Manganese superoxide dismutase (MnSOD) is an essential antioxidant mitochondrial enzyme. Although polyphenols can induce MnSOD expression, their mechanism of action remains unclear. We examined the effect of bavachalcone, a bioactive compound isolated from Psoralea corylifolia, on MnSOD protein expression and explored whether this effect is mediated through the AMP-activated protein kinase (AMPK) signaling pathway. Our data showed that bavachalcone enhanced the luciferase activity of the MnSOD promoter and increased MnSOD mRNA and protein expressions. Moreover, bavachalcone suppressed the mitochondrial superoxide production in endothelial cells. Conversely, bavachalcone stimulated liver kinase B1 and AMPKα phosphorylation. mRNA interference by using short hairpin RNA (shRNA) of AMPK inhibited bavachalcone-induced MnSOD expression. A-769662, an AMPK activator, also stimulated AMPK activity and increased MnSOD expression. Furthermore, AMPK knockdown by shRNA-AMPK reversed the inhibitory effects of bavachalcone on mitochondrial superoxide production in endothelial cells. These findings indicate that bavachalcone can protect the endothelial function by increasing AMPK activity and MnSOD expression and reducing mitochondrial oxidative stress. .

  1. 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.

  2. 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.

  3. Nonsteroidal anti-inflammatory drug flufenamic acid is a potent activator of AMP-activated protein kinase.

    PubMed

    Chi, Yuan; Li, Kai; Yan, Qiaojing; Koizumi, Schuichi; Shi, Liye; Takahashi, Shuhei; Zhu, Ying; Matsue, Hiroyuki; Takeda, Masayuki; Kitamura, Masanori; Yao, Jian

    2011-10-01

    Flufenamic acid (FFA) is a nonsteroidal anti-inflammatory drug (NSAID). It has anti-inflammatory and antipyretic properties. In addition, it modulates multiple channel activities. The mechanisms underlying the pharmacological actions of FFA are presently unclear. Given that AMP-activated protein kinase (AMPK) has both anti-inflammatory and channel-regulating functions, we examined whether FFA induces AMPK activation. 1) Exposure of several different types of cells to FFA resulted in an elevation of AMPKα phosphorylation at Thr172. This effect of FFA was reproduced by functionally and structurally similar mefenamic acid, tolfenamic acid, niflumic acid, and meclofenamic acid. 2) FFA-induced activation of AMPK was largely abolished by the treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (an intracellular Ca(2+) chelator) or depletion of extracellular Ca(2+), whereas it was mimicked by stimulation of cells with the Ca(2+) ionophore 5-(methylamino)-2-({(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl}methyl)-1,3-benzoxazole-4-carboxylic acid (A23187) or ionomycin. 3) FFA triggered a rise in intracellular Ca(2+), which was abolished by cyclosporine, a blocker of mitochondrial permeability transition pore. Cyclosporine also abolished FFA-induced activation of AMPK. 4) Inhibition of Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ) with 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609) or down-regulation of CaMKKβ with short interfering RNA largely abrogated FFA-induced activation of AMPK. 5) FFA significantly suppressed nuclear factor-κB activity and inducible nitric-oxide synthase expression triggered by interleukin-1β and tumor necrosis factor α. This suppression was also largely abrogated by STO-609. Taken together, we conclude that FFA induces AMPK activation through the Ca(2+)-CaMKKβ pathway

  4. 14-Deoxyandrographolide alleviates ethanol-induced hepatosteatosis through stimulation of AMP-activated protein kinase activity in rats.

    PubMed

    Mandal, Samir; Mukhopadhyay, Sibabrata; Bandhopadhyay, Sukdeb; Sen, Gargi; Biswas, Tuli

    2014-03-01

    Andrographis paniculata (AP) is a traditional medicinal plant of Ayurveda. It grows widely in Asia and is prescribed in the treatment of liver diseases. Here we have investigated the beneficial role of 14-deoxyandrographolide (14-DAG), a bioactive diterpenoid from AP, against alcoholic steatosis in rats. 14-DAG was extracted from aerial parts (leaves and stems) of AP. Rats were fed with ethanol for 8 weeks. Animals were treated with 14-DAG during the last 4 weeks of ethanol treatment. In vitro studies were undertaken in a human hepatocellular liver carcinoma cell line culture. Hepatosteatosis was assessed from histopathological studies of liver sections. Acetyl-CoA, malonyl-CoA, and triglyceride contents were determined using commercially available kits. Fatty acid synthesis was evaluated from incorporation of 1-(14)C acetate. Regulation of fatty acid oxidation and lipogenesis were monitored with immunoblotting and immunoprecipitation studies. Ethanol exposure led to hepatotoxicity, as evident from the marked enhancement in the levels of AST and ALT. The values decreased almost to control levels in response to 14-DAG treatment. Results showed that ethanol feeding induced deactivation of AMP-activated protein kinase (AMPK) that led to enhanced lipid synthesis and decreased fatty acid oxidation, culminating in hepatic fat accumulation. Treatment with 14-DAG activated AMPK through induction of cyclic AMP-protein kinase A pathway. Activation of AMPK was followed by down-regulation of sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, leading to suppression of lipogenesis. This was associated with up-regulation of sirtuin 1 and depletion of malonyl-CoA, in favor of increased fatty acid oxidation. 14-DAG controlled ethanol-induced hepatosteatosis by interfering with dysregulation of lipid metabolism. In conclusion, our results indicated that 14-DAG was capable of preventing the development of fatty liver through AMPK

  5. Crystal Structures of the Adenylate Sensor from Fission Yeast AMP-Activated Protein Kinase

    SciTech Connect

    Townley,R.; Shapiro, L.

    2007-01-01

    The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular adenosine triphosphate (ATP) and AMP levels. Here we report crystal structures at 2.6 and 2.9 Angstrom resolution for ATP- and AMP-bound forms of a core {alpha}{beta}{gamma} adenylate-binding domain from the fission yeast AMPK homologue. ATP and AMP bind competitively to a single site in the {gamma} subunit, with their respective phosphate groups positioned near function-impairing mutants. Surprisingly, ATP binds without counter ions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

  6. Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway.

    PubMed

    Solaz-Fuster, Maria Carmen; Gimeno-Alcañiz, José Vicente; Ros, Susana; Fernandez-Sanchez, Maria Elena; Garcia-Fojeda, Belen; Criado Garcia, Olga; Vilchez, David; Dominguez, Jorge; Garcia-Rocha, Mar; Sanchez-Piris, Maribel; Aguado, Carmen; Knecht, Erwin; Serratosa, Jose; Guinovart, Joan Josep; Sanz, Pascual; Rodriguez de Córdoba, Santiago

    2008-03-01

    Lafora progressive myoclonus epilepsy (LD) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. LD is caused by mutations in two genes, EPM2A and EPM2B, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. Previously, we and others have suggested that the interactions between laforin and PTG (a regulatory subunit of type 1 protein phosphatase) and between laforin and malin are critical in the pathogenesis of LD. Here, we show that the laforin-malin complex downregulates PTG-induced glycogen synthesis in FTO2B hepatoma cells through a mechanism involving ubiquitination and degradation of PTG. Furthermore, we demonstrate that the interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK). These findings provide further insights into the critical role of the laforin-malin complex in the control of glycogen metabolism and unravel a novel link between the energy sensor AMPK and glycogen metabolism. These data advance our understanding of the functional role of laforin and malin, which hopefully will facilitate the development of appropriate LD therapies.

  7. Metformin attenuates ventricular hypertrophy by activating the AMP-activated protein kinase-endothelial nitric oxide synthase pathway in rats.

    PubMed

    Zhang, Cheng-Xi; Pan, Si-Nian; Meng, Rong-Sen; Peng, Chao-Quan; Xiong, Zhao-Jun; Chen, Bao-Lin; Chen, Guang-Qin; Yao, Feng-Juan; Chen, Yi-Li; Ma, Yue-Dong; Dong, Yu-Gang

    2011-01-01

    1. Metformin is an activator of AMP-activated protein kinase (AMPK). Recent studies suggest that pharmacological activation of AMPK inhibits cardiac hypertrophy. In the present study, we examined whether long-term treatment with metformin could attenuate ventricular hypertrophy in a rat model. The potential involvement of nitric oxide (NO) in the effects of metformin was also investigated. 2. Ventricular hypertrophy was established in rats by transaortic constriction (TAC). Starting 1 week after the TAC procedure, rats were treated with metformin (300 mg/kg per day, p.o.), N(G)-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg per day, p.o.) or both for 8 weeks prior to the assessment of haemodynamic function and cardiac hypertrophy. 3. Cultured cardiomyocytes were used to examine the effects of metformin on the AMPK-endothelial NO synthase (eNOS) pathway. Cells were exposed to angiotensin (Ang) II (10⁻⁶ mol/L) for 24 h under serum-free conditions in the presence or absence of metformin (10⁻³ mol/L), compound C (10⁻⁶ mol/L), L-NAME (10⁻⁶ mol/L) or their combination. The rate of incorporation of [³H]-leucine was determined, western blotting analyses of AMPK-eNOS, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) were undertaken and the concentration of NO in culture media was determined. 4. Transaortic constriction resulted in significant haemodynamic dysfunction and ventricular hypertrophy. Myocardial fibrosis was also evident. Treatment with metformin improved haemodynamic function and significantly attenuated ventricular hypertrophy. Most of the effects of metformin were abolished by concomitant L-NAME treatment. L-NAME on its own had no effect on haemodynamic function and ventricular hypertrophy in TAC rats. 5. In cardiomyocytes, metformin inhibited AngII-induced protein synthesis, an effect that was suppressed by the AMPK inhibitor compound C or the eNOS inhibitor L-NAME. The improvement in cardiac structure and

  8. 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

  9. β-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase

    PubMed Central

    Ali, Nada; Ling, Naomi; Krishnamurthy, Srinath; Oakhill, Jonathan S.; Scott, John W.; Stapleton, David I.; Kemp, Bruce E.; Anand, Ganesh Srinivasan; Gooley, Paul R.

    2016-01-01

    The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the βCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high. PMID:28000716

  10. 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

  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.

  12. Human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by AMP-activated protein kinase.

    PubMed

    Kim, Eung-Kyun; Lim, Seyoung; Park, Ji-Man; Seo, Jeong Kon; Kim, Jae Ho; Kim, Kyong Tai; Ryu, Sung Ho; Suh, Pann-Ghill

    2012-04-01

    AMP-activated protein kinase (AMPK) is an energy-sensing kinase that has recently been shown to regulate the differentiation of preadipocytes and osteoblasts. However, the role of AMPK in stem cell differentiation is largely unknown. Using in vitro culture models, the present study demonstrates that AMPK is a critical regulatory factor for osteogenic differentiation. We observed that expression and phosphorylation of AMPK were increased during osteogenesis in human adipose tissue-derived mesenchymal stem cells (hAMSC). To elucidate the role of AMPK in osteogenic differentiation, we investigated the effect of AMPK inhibition or knockdown on mineralization of hAMSC. Compound C, an AMPK inhibitor, reduced mineralized matrix deposition and suppressed the expression of osteoblast-specific genes, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). Knockdown of AMPK by shRNA-lentivirus infection also reduced osteogenesis. In addition, inhibition or knockdown of AMPK during osteogenesis inhibited ERK phosphorylation, which is required for osteogenesis. Interestingly, inhibition of AMPK induced adipogenic differentiation of hAMSC, even in osteogenic induction medium (OIM). These results provide a potential mechanism involving AMPK activation in osteogenic differentiation of hAMSC and suggest that commitment of hAMSC to osteogenic or adipogenic lineage is governed by activation or inhibition of AMPK, respectively.

  13. 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

  14. Adenosine Monophosphate (AMP)-Activated Protein Kinase: A New Target for Nutraceutical Compounds

    PubMed Central

    Marín-Aguilar, Fabiola; Pavillard, Luis E.; Giampieri, Francesca; Bullón, Pedro; Cordero, Mario D.

    2017-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inflammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identified as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases. PMID:28146060

  15. 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

  16. Adiponectin protects the rats liver against chronic intermittent hypoxia induced injury through AMP-activated protein kinase pathway

    PubMed Central

    Ding, Wenxiao; Zhang, Qiang; Dong, Yanbin; Ding, Ning; Huang, Hanpeng; Zhu, Xianji; Hutchinson, Sean; Gao, Xingya; Zhang, Xilong

    2016-01-01

    This study was performed to assess the effect of chronic intermittent hypoxia (CIH) on the liver, the associated mechanisms and the potential therapeutic roles of adiponectin (Ad). Sixty rats were randomly assigned to four groups: the normal control (NC), NC and Ad supplement (NC + Ad), CIH, and CIH and Ad supplement (CIH + Ad) groups. The rats in the CIH and CIH + Ad groups were exposed to a hypoxic environment for 4 months. Rats in the NC + Ad and CIH + Ad groups were also treated with an intravenous injection of Ad (10 ug), twice a week. The plasma levels of hepatic enzymes, serum triglyceride, liver triglyceride, fasting blood glucose and hepatic cell apoptosis in hepatic tissue, were higher in the CIH group than in the NC and NC + Ad groups. However, the Ad supplementation in the CIH + Ad group rescued the hepatic tissue insult by activating the AMP-activated protein kinase (AMPK) pathway. In conclusion, Ad could protect against CIH-induced hepatic injury partly through the AMPK pathway. PMID:27678302

  17. Low Concentrations of Metformin Suppress Glucose Production in Hepatocytes through AMP-activated Protein Kinase (AMPK)*♦

    PubMed Central

    Cao, Jia; Meng, Shumei; Chang, Evan; Beckwith-Fickas, Katherine; Xiong, Lishou; Cole, Robert N.; Radovick, Sally; Wondisford, Fredric E.; He, Ling

    2014-01-01

    Metformin is a first-line antidiabetic agent taken by 150 million people across the world every year, yet its mechanism remains only partially understood and controversial. It was proposed that suppression of glucose production in hepatocytes by metformin is AMPK-independent; however, unachievably high concentrations of metformin were employed in these studies. In the current study, we find that metformin, via an AMP-activated protein kinase (AMPK)-dependent mechanism, suppresses glucose production and gluconeogenic gene expression in primary hepatocytes at concentrations found in the portal vein of animals (60–80 μm). Metformin also inhibits gluconeogenic gene expression in the liver of mice administered orally with metformin. Furthermore, the cAMP-PKA pathway negatively regulates AMPK activity through phosphorylation at Ser-485/497 on the α subunit, which in turn reduces net phosphorylation at Thr-172. Because diabetic patients often have hyperglucagonemia, AMPKα phosphorylation at Ser-485/497 is a therapeutic target to improve metformin efficacy. PMID:24928508

  18. Glabridin induces glucose uptake via the AMP-activated protein kinase pathway in muscle cells.

    PubMed

    Sawada, Keisuke; Yamashita, Yoko; Zhang, Tianshun; Nakagawa, Kaku; Ashida, Hitoshi

    2014-08-05

    The present study demonstrates that glabridin, a prenylated isoflavone in licorice, stimulates glucose uptake through the adenosine monophosphate-activated protein kinase (AMPK) pathway in L6 myotubes. Treatment with glabridin for 4h induced glucose uptake in a dose-dependent manner accompanied by the translocation of glucose transporter type 4 (GLUT4) to the plasma membrane. Glabridin needed at least 4h to increase glucose uptake, while it significantly decreased glycogen and increased lactic acid within 15 min. Pharmacological inhibition of AMPK by Compound C suppressed the glabridin-induced glucose uptake, whereas phosphoinositide 3-kinase and Akt inhibition by LY294002 and Akt1/2 inhibitor, respectively, did not. Furthermore, glabridin induced AMPK phosphorylation, and siRNA for AMPK completely abolished glabridin-induced glucose uptake. We confirmed that glabridin-rich licorice extract prevent glucose intolerance accompanied by the AMPK-dependent GLUT4 translocation in the plasma membrane of mice skeletal muscle. These results indicate that glabridin may possess a therapeutic effect on metabolic disorders, such as diabetes and hyperglycemia, by modulating glucose metabolism through AMPK in skeletal muscle cells.

  19. The role of AMP-activated protein kinase in quercetin-induced apoptosis of HL-60 cells.

    PubMed

    Xiao, Jie; Niu, Guomin; Yin, Songmei; Xie, Shuangfeng; Li, Yiqing; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan

    2014-05-01

    Our previous studies have shown that quercetin inhibits Cox-2 and Bcl-2 expressions, and induces human leukemia HL-60 cell apoptosis. In order to investigate the role of AMP-activated protein kinase (AMPK) on quercetin-induced apoptosis of HL-60 cells, we used flow cytometry to detect cell apoptosis. The expressions of LKB1, phosphorylated AMPK (p-AMPK), and Cox-2 protein were detected in HL-60 cells and normal peripheral blood mononuclear cells (PBMCs) by western blot. The expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin. The expressions of p-AMPK were detected in HL-60 cells after culture with AMPK inhibitor Compound C. Then, the expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin alone or quercetin + Compound C. It was found that there was no significant difference in LKB1 between PBMCs and HL-60. p-AMPK in PBMCs was higher than that in HL-60, while Cox-2 was lower. After culture of HL-60 with quercetin, p-AMPK was increased, Cox-2 was decreased, but LKB1 remained unchanged. After culture of HL-60 with Compound C, p-AMPK was decreased. There was no significant difference in LKB1 between the quercetin-alone and the quercetin + Compound C groups. p-AMPK decreased more significantly, while Cox-2 increased more significantly in the quercetin + Compound C groups than those in the quercetin-alone groups. Taken together, these findings suggested that quercetin activates AMPK expression in HL-60 cells independent of LKB1 activation, inhibits Cox-2 expression by activating AMPK, and further regulates the Bcl-2-dependent pathways of apoptosis to exert its anti-leukemia effect.

  20. Metabolic switch and hypertrophy of cardiomyocytes following treatment with angiotensin II are prevented by AMP-activated protein kinase.

    PubMed

    Stuck, Bettina Johanna; Lenski, Matthias; Böhm, Michael; Laufs, Ulrich

    2008-11-21

    Angiotensin II induces cardiomyocyte hypertrophy, but its consequences on cardiomyocyte metabolism and energy supply are not completely understood. Here we investigate the effect of angiotensin II on glucose and fatty acid utilization and the modifying role of AMP-activated protein kinase (AMPK), a key regulator of metabolism and proliferation. Treatment of H9C2 cardiomyocytes with angiotensin II (Ang II, 1 microm, 4 h) increased [(3)H]leucine incorporation, up-regulated the mRNA expression of the hypertrophy marker genes MLC, ANF, BNP, and beta-MHC, and decreased the phosphorylation of the negative mTOR-regulator tuberin (TSC-2). Rat neonatal cardiomyocytes showed similar results. Western blot analysis revealed a time- and concentration-dependent down-regulation of AMPK-phosphorylation in the presence of angiotensin II, whereas the protein expression of the catalytic alpha-subunit remained unchanged. This was paralleled by membrane translocation of glucose-transporter type 4 (GLUT4), increased uptake of [(3)H]glucose and transient down-regulation of phosphorylation of acetyl-CoA carboxylase (ACC), whereas fatty acid uptake remained unchanged. Similarly, short-term transaortic constriction in mice resulted in down-regulation of P-AMPK and P-ACC but up-regulation of GLUT4 membrane translocation in the heart. Preincubation of cardiomyocytes with the AMPK stimulator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mM, 4 h) completely prevented the angiotensin II-induced cardiomyocytes hypertrophy. In addition, AICAR reversed the metabolic effects of angiotensin II: GLUT4 translocation was reduced, but ACC phosphorylation and TSC phosphorylation were elevated. In summary, angiotensin II-induced hypertrophy of cardiomyocytes is accompanied by decreased activation of AMPK, increased glucose uptake, and decreased mTOR inhibition. Stimulation with the AMPK activator AICAR reverses these metabolic changes, increases fatty acid utilization, and inhibits

  1. AMP-Activated Protein Kinase Is Essential for Survival in Chronic Hypoxia

    PubMed Central

    Borger, Darrell R.; Gavrilescu, L. Cristina; Bucur, Maria C.; Ivan, Mircea; DeCaprio, James A.

    2008-01-01

    This study was undertaken to interrogate cancer cell survival during long-term hypoxic stress. Two systems with relevance to carcinogenesis were employed: fully transformed BJ cells, and a renal carcinoma cell line (786-0). The dynamic of AMPK activity was consistent with a prosurvival role during chronic hypoxia. This was further supported by the effects of AMPK agonists and antagonists (AICAR and Compound C). Expression of a dominant-negative AMPK alpha resulted in decreased ATP level, and significantly compromised survival in hypoxia. Dose dependent pro-survival effects of rapamycin were consistent with mTOR inhibition being critical downstream of AMPK in persistent low oxygen. PMID:18359290

  2. AMP-activated protein kinase-dependent autophagy mediated the protective effect of sonic hedgehog pathway on oxygen glucose deprivation-induced injury of cardiomyocytes.

    PubMed

    Xiao, Qing; Yang, Ya; Qin, Yuan; He, Yan-Hua; Chen, Kui-Xiang; Zhu, Jian-Wei; Zhang, Gui-Ping; Luo, Jian-Dong

    2015-02-13

    Sonic hedgehog (Shh) pathway has been reported to protect cardiomyocytes in myocardial infarction (MI), but the underlying mechanism is not clear. Here, we provide evidence that Shh pathway induces cardiomyocytes survival through AMP-activated protein kinase-dependent autophagy. Shh pathway agonist SAG increased the expression of LC3-II, and induced the formation of autophagosomes in cultured H9c2 cardiomyocytes under oxygen glucose deprivation (OGD) 1 h and 4 h. Moreover, SAG induced a profound AMP-activated protein kinase (AMPK) activation, and then directly phosphorylated and activated the downstream autophagy initiator Ulk1, independent of the autophagy suppressor mammalian target of rapamycin (mTOR) complex 1. Taken together, our results have shown that Shh activates AMPK-dependent autophagy in cardiomyocytes under OGD, suggesting a role of autophagy in Shh-induced cellular protection.

  3. Coenzyme Q10 Attenuates High Glucose-Induced Endothelial Progenitor Cell Dysfunction through AMP-Activated Protein Kinase Pathways

    PubMed Central

    Tsai, Hsiao-Ya; Lin, Chih-Pei; Huang, Po-Hsun; Li, Szu-Yuan; Chen, Jia-Shiong; Lin, Feng-Yen; Chen, Jaw-Wen; Lin, Shing-Jong

    2016-01-01

    Coenzyme Q10 (CoQ10), an antiapoptosis enzyme, is stored in the mitochondria of cells. We investigated whether CoQ10 can attenuate high glucose-induced endothelial progenitor cell (EPC) apoptosis and clarified its mechanism. EPCs were incubated with normal glucose (5 mM) or high glucose (25 mM) enviroment for 3 days, followed by treatment with CoQ10 (10 μM) for 24 hr. Cell proliferation, nitric oxide (NO) production, and JC-1 assay were examined. The specific signal pathways of AMP-activated protein kinase (AMPK), eNOS/Akt, and heme oxygenase-1 (HO-1) were also assessed. High glucose reduced EPC functional activities, including proliferation and migration. Additionally, Akt/eNOS activity and NO production were downregulated in high glucose-stimulated EPCs. Administration of CoQ10 ameliorated high glucose-induced EPC apoptosis, including downregulation of caspase 3, upregulation of Bcl-2, and increase in mitochondrial membrane potential. Furthermore, treatment with CoQ10 reduced reactive oxygen species, enhanced eNOS/Akt activity, and increased HO-1 expression in high glucose-treated EPCs. These effects were negated by administration of AMPK inhibitor. Transplantation of CoQ10-treated EPCs under high glucose conditions into ischemic hindlimbs improved blood flow recovery. CoQ10 reduced high glucose-induced EPC apoptosis and dysfunction through upregulation of eNOS, HO-1 through the AMPK pathway. Our findings provide a potential treatment strategy targeting dysfunctional EPC in diabetic patients. PMID:26682233

  4. 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

  5. Down-regulation of Na+-coupled glutamate transporter EAAT3 and EAAT4 by AMP-activated protein kinase.

    PubMed

    Sopjani, Mentor; Alesutan, Ioana; Dërmaku-Sopjani, Miribane; Fraser, Scott; Kemp, Bruce E; Föller, Michael; Lang, Florian

    2010-06-01

    The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase (AMPK), a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4-expressing oocytes glutamate generated a current (I(g)), which was half maximal (K(M)) at 74 microM (EAAT3) or at 4 microM (EAAT4) glutamate. Co-expression of constitutively active (gammaR70Q)AMPK or of wild type AMPK did not affect K(M) but significantly decreased the maximal I(g) in both EAAT3- (by 34%) and EAAT4- (by 49%) expressing oocytes. Co-expression of the inactive mutant (alphaK45R)AMPK [alpha1(K45R)beta1gamma1] did not appreciably affect I(g). According to confocal microscopy and chemiluminescence co-expression of (gammaR70Q)AMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK down-regulates Na(+)-coupled glutamate transport.

  6. 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

  7. Metformin induces up-regulation of blood-brain barrier functions by activating AMP-activated protein kinase in rat brain microvascular endothelial cells.

    PubMed

    Takata, Fuyuko; Dohgu, Shinya; Matsumoto, Junichi; Machida, Takashi; Kaneshima, Shuji; Matsuo, Mai; Sakaguchi, Shinya; Takeshige, Yuki; Yamauchi, Atsushi; Kataoka, Yasufumi

    2013-04-19

    Blood-brain barrier (BBB) disruption occurs frequently in CNS diseases and injuries. Few drugs have been developed as therapeutic candidates for facilitating BBB functions. Here, we examined whether metformin up-regulates BBB functions using rat brain microvascular endothelial cells (RBECs). Metformin, concentration- and time-dependently increased transendothelial electrical resistance of RBEC monolayers, and decreased RBEC permeability to sodium fluorescein and Evans blue albumin. These effects of metformin were blocked by compound C, an inhibitor of AMP-activated protein kinase (AMPK). AMPK stimulation with an AMPK activator, AICAR, enhanced BBB functions. These findings indicate that metformin induces up-regulation of BBB functions via AMPK activation.

  8. AMP-activated protein kinase activation leads to lysome-mediated NA(+)/I(-)-symporter protein degradation in rat thyroid cells.

    PubMed

    Cazarin, J M; Andrade, B M; Carvalho, D P

    2014-05-01

    Iodide uptake by thyroid cells is mediated by a transmembrane glycoprotein known as the Na+/I--symporter (NIS). NIS-mediated iodide uptake plays important physiological role in thyroid gland function, as well as in diagnostic and treatment of Graves' disease and thyroid cancer. Although different studies investigated the transcriptional mechanisms of NIS expression, there is no report on the NIS post-translational regulation related to NIS protein degradation in thyroid cells. Recently, our group showed that AMP-activated protein kinase (AMPK) plays a pivotal role in the rat thyroid gland, downregulating iodide uptake, NIS protein, and mRNA content. Since several studies demonstrated that AMPK regulates post-transcriptional mechanisms, such as autophagy-mediated processes in different tissues, we hypothesized that AMPK activation could also regulate NIS protein degradation through the lysosome pathway in thyroid cells. Rat follicular thyroid PCCL3 cells cultivated in Ham's F12 supplemented with 5% calf serum and hormones were exposed to the AMPK pharmacological activator 5-aminoimidazole-4 carboxamide ribonucleoside (AICAR), in the presence or absence of Bafilomycin A1 or MG132 for 24 h. Treatment of PCCL3 cells with Bafilomycin A1 fully prevented the decrease of iodide uptake and NIS protein content mediated by AMPK activation. In contrast, the treatment with MG132 was unable to prevent the effects of AMPK activation on NIS. Our results show that AMPK activation significantly induces NIS protein degradation through a lysosome-mediated mechanism.

  9. Controls of Nuclear Factor-Kappa B Signaling Activity by 5’-AMP-Activated Protein Kinase Activation With Examples in Human Bladder Cancer Cells

    PubMed Central

    Kim, Jin

    2016-01-01

    Generally, both lipopolysaccharide (LPS)- and hypoxia-induced nuclear factor kappa B (NF-κB) effects are alleviated through differential posttranslational modification of NF-κB phosphorylation after pretreatment with 5´-AMP-activated protein kinase (AMPK) activators such as 5´-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or the hypoglycemic agent metformin. We found that AICAR or metformin acts as a regulator of LPS/NF-κB-or hypoxia/NF-κB-mediated cyclooxygenase induction by an AMPK-dependent mechanism with interactions between p65-NF-κB phosphorylation and acetylation, including in a human bladder cancer cell line (T24). In summary, we highlighted the regulatory interactions of AMPK activity on NF-κB induction, particularly in posttranslational phosphorylation and acetylation of NF-κB under inflammatory conditions or hypoxia environment. PMID:27706018

  10. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

    PubMed

    Tong, X; Kono, T; Evans-Molina, C

    2015-06-18

    The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

  11. C1q Tumor Necrosis Factor α-related Protein Isoform 5 Is Increased in Mitochondrial DNA-depleted Myocytes and Activates AMP-activated Protein Kinase*

    PubMed Central

    Park, Seung-Yoon; Choi, Jung Hyun; Ryu, Hyun Su; Pak, Youngmi Kim; Park, Kyong Soo; Lee, Hong Kyu; Lee, Wan

    2009-01-01

    Depletion of mtDNA in myocytes causes insulin resistance and alters nuclear gene expression that may be involved in rescuing processes against cellular stress. Here we show that the expression of C1q tumor necrosis factor α-related protein isoform 5 (C1QTNF5) is drastically increased following depletion of mtDNA in myocytes. C1QTNF5 is homologous to adiponectin in respect to domain structure, and its expression and secretion from myocytes correlated negatively with the cellular mtDNA content. Similar to adiponectin, C1QTNF5 induced the phosphorylation of AMP-activated protein kinase (AMPK), leading to increased cell surface recruitment of GLUT4 and increased glucose uptake. Treatment of cells with purified recombinant C1QTNF5 increased the phosphorylation of acetyl-CoA carboxylase and stimulated fatty acid oxidation. C1QTNF5-mediated phosphorylation of AMPK or acetyl-CoA carboxylase was unaffected by depletion of adiponectin receptors such as AdipoR1 or AdipoR2, which indicated that adiponectin receptors do not participate in C1QTNF5-induced activation of AMPK. Serum C1QTNF5 levels were significantly higher in obese/diabetic animals (OLETF rats, ob/ob mice, and db/db mice). These results highlight C1QTNF5 as a putative biomarker for mitochondrial dysfunction and a potent activator of AMPK. PMID:19651784

  12. C1q tumor necrosis factor alpha-related protein isoform 5 is increased in mitochondrial DNA-depleted myocytes and activates AMP-activated protein kinase.

    PubMed

    Park, Seung-Yoon; Choi, Jung Hyun; Ryu, Hyun Su; Pak, Youngmi Kim; Park, Kyong Soo; Lee, Hong Kyu; Lee, Wan

    2009-10-09

    Depletion of mtDNA in myocytes causes insulin resistance and alters nuclear gene expression that may be involved in rescuing processes against cellular stress. Here we show that the expression of C1q tumor necrosis factor alpha-related protein isoform 5 (C1QTNF5) is drastically increased following depletion of mtDNA in myocytes. C1QTNF5 is homologous to adiponectin in respect to domain structure, and its expression and secretion from myocytes correlated negatively with the cellular mtDNA content. Similar to adiponectin, C1QTNF5 induced the phosphorylation of AMP-activated protein kinase (AMPK), leading to increased cell surface recruitment of GLUT4 and increased glucose uptake. Treatment of cells with purified recombinant C1QTNF5 increased the phosphorylation of acetyl-CoA carboxylase and stimulated fatty acid oxidation. C1QTNF5-mediated phosphorylation of AMPK or acetyl-CoA carboxylase was unaffected by depletion of adiponectin receptors such as AdipoR1 or AdipoR2, which indicated that adiponectin receptors do not participate in C1QTNF5-induced activation of AMPK. Serum C1QTNF5 levels were significantly higher in obese/diabetic animals (OLETF rats, ob/ob mice, and db/db mice). These results highlight C1QTNF5 as a putative biomarker for mitochondrial dysfunction and a potent activator of AMPK.

  13. PD98059 and U0126 activate AMP-activated protein kinase by increasing the cellular AMP:ATP ratio and not via inhibition of the MAP kinase pathway.

    PubMed

    Dokladda, Kanchana; Green, Kevin A; Pan, David A; Hardie, D Grahame

    2005-01-03

    The MAP kinase pathway inhibitor U0126 caused phosphorylation and activation of AMP-activated protein kinase (AMPK) and increased phosphorylation of its downstream target acetyl-CoA carboxylase, in HEK293 cells. This effect only occurred in cells expressing the upstream kinase, LKB1. Of two other widely used MAP kinase pathway inhibitors not closely related in structure to U0126, PD98059 also activated AMPK but PD184352 did not. U0126 and PD98059, but not PD184352, also increased the cellular ADP:ATP and AMP:ATP ratios, accounting for their ability to activate AMPK. These results suggest the need for caution in interpreting experiments conducted using U0126 and PD98059.

  14. Activation of SIRT1 Attenuates Klotho Deficiency-Induced Arterial Stiffness and Hypertension by Enhancing AMP-Activated Protein Kinase Activity.

    PubMed

    Gao, Diansa; Zuo, Zhong; Tian, Jing; Ali, Quaisar; Lin, Yi; Lei, Han; Sun, Zhongjie

    2016-11-01

    Arterial stiffness is an independent risk factor for stroke and myocardial infarction. This study was designed to investigate the role of SIRT1, an important deacetylase, and its relationship with Klotho, a kidney-derived aging-suppressor protein, in the pathogenesis of arterial stiffness and hypertension. We found that the serum level of Klotho was decreased by ≈45% in patients with arterial stiffness and hypertension. Interestingly, Klotho haplodeficiency caused arterial stiffening and hypertension, as evidenced by significant increases in pulse wave velocity and blood pressure in Klotho-haplodeficient (KL(+/-)) mice. Notably, the expression and activity of SIRT1 were decreased significantly in aortic endothelial and smooth muscle cells in KL(+/-) mice, suggesting that Klotho deficiency downregulates SIRT1. Treatment with SRT1720 (15 mg/kg/d, IP), a specific SIRT1 activator, abolished Klotho deficiency-induced arterial stiffness and hypertension in KL(+/-) mice. Klotho deficiency was associated with significant decreases in activities of AMP-activated protein kinase α (AMPKα) and endothelial NO synthase (eNOS) in aortas, which were abolished by SRT1720. Furthermore, Klotho deficiency upregulated NADPH oxidase activity and superoxide production, increased collagen expression, and enhanced elastin fragmentation in the media of aortas. These Klotho deficiency-associated changes were blocked by SRT1720. In conclusion, this study provides the first evidence that Klotho deficiency downregulates SIRT1 activity in arterial endothelial and smooth muscle cells. Pharmacological activation of SIRT1 may be an effective therapeutic strategy for arterial stiffness and hypertension.

  15. Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells.

    PubMed

    Lee, Hak Joo; Mariappan, Meenalakshmi M; Feliers, Denis; Cavaglieri, Rita C; Sataranatarajan, Kavithalakshmi; Abboud, Hanna E; Choudhury, Goutam Ghosh; Kasinath, Balakuntalam S

    2012-02-10

    Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

  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.

  17. C1q/TNF-related protein-9 inhibits cytokine-induced vascular inflammation and leukocyte adhesiveness via AMP-activated protein kinase activation in endothelial cells.

    PubMed

    Jung, Chang Hee; Lee, Min Jung; Kang, Yu Mi; Lee, Yoo La; Seol, So Mi; Yoon, Hae Kyeong; Kang, Sang-Wook; Lee, Woo Je; Park, Joong-Yeol

    2016-01-05

    Although recent studies have reported cardioprotective effects of C1q/TNF-related protein 9 (CTRP9), the closet adiponectin paralog, its role on cytokine-induced endothelial inflammation is unknown. We investigated whether CTRP9 prevented inflammatory cytokine-induced nuclear factor-kappa B (NF-κB) activation and inhibited the expression of adhesion molecules and a chemokine in the vascular endothelial cell. We used human aortic endothelial cells (HAECs) to examine the effects of CTRP9 on NF-κB activation and the expression of NF-κB-mediated genes, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1). Tumor necrosis factor alpha (TNFα) was used as a representative proinflammatory cytokine. In an adhesion assay using THP-1 cells, CTRP9 reduced TNFα-induced adhesion of monocytes to HAECs. Treatment with CTRP9 significantly decreased TNFα-induced activation of NF-κB, as well as the expression of ICAM-1, VCAM-1, and MCP-1. In addition, treatment with CTRP9 significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), the downstream target of AMPK. The inhibitory effect of CTRP9 on the expression of ICAM-1, VCAM-1, and MCP-1 and monocyte adhesion to HAECs was abolished after transfection with an AMPKα1-specific siRNA. Our study is the first to demonstrate that CTRP9 attenuates cytokine-induced vascular inflammation in endothelial cells mediated by AMPK activation.

  18. Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases

    PubMed Central

    Capano, Michela; Crompton, Martin

    2005-01-01

    The cytosolic protein Bax plays a key role in apoptosis by migrating to mitochondria and releasing proapoptotic proteins from the mitochondrial intermembrane space. The present study investigates the movement of Bax in isolated rat neonatal cardiomyocytes subjected to simulated ischaemia (minus glucose, plus cyanide), using green fluorescent protein-tagged Bax as a means of imaging Bax movements. Simulated ischaemia induced Bax translocation from the cytosol to mitochondria, commencing within 20 min of simulated ischaemia and progressing for several hours. Under the same conditions, there was an increase in the active, phosphorylated forms of p38 MAPK (mitogen-activated protein kinase) and AMPK (AMP-activated protein kinase). The AMPK activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin also stimulated Bax translocation. Inhibition of p38 MAPK with SB203580 attenuated the phosphorylation of the downstream substrates, MAPK-activated protein kinases 2 and 3, but not that of the upstream MAPK kinase 3, nor of AMPK. Under all conditions (ischaemia, AICAR and metformin), SB203580 blocked Bax translocation completely. It is concluded that Bax translocation to mitochondria is an early step in ischaemia and that it occurs in response to activation of p38 MAPK downstream of AMPK. PMID:16321138

  19. 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.

  20. Krill Oil Supplementation Improves Dyslipidemia and Lowers Body Weight in Mice Fed a High-Fat Diet Through Activation of AMP-Activated Protein Kinase.

    PubMed

    Yang, Goowon; Lee, Jihyun; Lee, Sangsu; Kwak, Dongyun; Choe, Wonchae; Kang, Insug; Kim, Sung Soo; Ha, Joohun

    2016-12-01

    Krill oil is a novel, commercially available marine oil rich in long-chain polyunsaturated omega-3 fatty acids, particularly eicosapentaenoic acid and docosahexaenoic acid. Compared with fish oil, the effects of krill oil supplementation on human health and its underlying action mechanisms are currently poorly understood. In the present study, we examined the effect of krill oil supplementation on metabolic parameters of mice fed a high-fat diet (HFD). Krill oil supplementation in mice fed a HFD for 10 weeks resulted in an ∼15% lower body weight gain and a dramatic suppression of hepatic steatosis. These effects were associated with significantly lower serum triglyceride and low-density lipoprotein-cholesterol levels. We further uncovered a novel underlying mechanism, showing that AMP-activated protein kinase, a master regulator of glucose and lipid metabolism, mediates the beneficial effects of krill oil.

  1. meso-Dihydroguaiaretic acid inhibits hepatic lipid accumulation by activating AMP-activated protein kinase in human HepG2 cells.

    PubMed

    Lee, Myoung-Su; Kim, Kyung Jin; Kim, Daeyoung; Lee, Kyung-Eun; Hwang, Jae-Kwan

    2011-01-01

    Hepatic lipid accumulation is a major risk factor for dyslipidemia, nonalcoholic fatty liver disease, and insulin resistance. The present study was conducted to evaluate hypolipidemic effects of meso-dihydroguaiaretic acid (MDA), anti-oxidative and anti-inflammatory compound isolated from the Myristica fragrans HOUTT., by oil red O staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot. MDA significantly inhibited insulin-induced hepatic lipid accumulation in a dose-dependent manner. The lipid-lowering effect of MDA was accompanied by increased expression of proteins involved in fatty acid oxidation and decreased expression of lipid synthetic proteins. In addition, MDA activated AMP-activated protein kinase (AMPK) as determined by phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK. The effects of MDA on lipogenic protein expression were suppressed by pretreatment with compound C, an AMPK inhibitor. Taken together, these findings show that MDA inhibits insulin-induced lipid accumulation in human HepG2 cells by suppressing expression of lipogenic proteins through AMPK signaling, suggesting a potent lipid-lowering agent.

  2. 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.

  3. AMP-activated protein kinase is required for exercise-induced peroxisome proliferator-activated receptor co-activator 1 translocation to subsarcolemmal mitochondria in skeletal muscle.

    PubMed

    Smith, Brennan K; Mukai, Kazutaka; Lally, James S; Maher, Amy C; Gurd, Brendon J; Heigenhauser, George J F; Spriet, Lawrence L; Holloway, Graham P

    2013-03-15

    In skeletal muscle, mitochondria exist as two subcellular populations known as subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS mitochondria preferentially respond to exercise training, suggesting divergent transcriptional control of the mitochondrial genomes. The transcriptional co-activator peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and mitochondrial transcription factor A (Tfam) have been implicated in the direct regulation of the mitochondrial genome in mice, although SS and IMF differences may exist, and the potential signalling events regulating the mitochondrial content of these proteins have not been elucidated. Therefore, we examined the potential for PGC-1α and Tfam to translocate to SS and IMF mitochondria in human subjects, and performed experiments in rodents to identify signalling mechanisms regulating these translocation events. Acute exercise in humans and rats increased PGC-1α content in SS but not IMF mitochondria. Acute exposure to 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside in rats recapitulated the exercise effect of increased PGC-1α protein within SS mitochondria only, suggesting that AMP-activated protein kinase (AMPK) signalling is involved. In addition, rendering AMPK inactive (AMPK kinase dead mice) prevented exercise-induced PGC-1α translocation to SS mitochondria, further suggesting that AMPK plays an integral role in these translocation events. In contrast to the conserved PGC-1α translocation to SS mitochondria across species (humans, rats and mice), acute exercise only increased mitochondrial Tfam in rats. Nevertheless, in rat resting muscle PGC-1α and Tfam co-immunoprecipate with α-tubulin, suggesting a common cytosolic localization. These data suggest that exercise causes translocation of PGC-1α preferentially to SS mitochondria in an AMPK-dependent manner.

  4. Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers.

    PubMed

    Peng, Luying; Li, Zhong-Rong; Green, Robert S; Holzman, Ian R; Lin, Jing

    2009-09-01

    Butyrate, one of the SCFA, promotes the development of the intestinal barrier. However, the molecular mechanisms underlying the butyrate regulation of the intestinal barrier are unknown. To test the hypothesis that the effect of butyrate on the intestinal barrier is mediated by the regulation of the assembly of tight junctions involving the activation of the AMP-activated protein kinase (AMPK), we determined the effect of butyrate on the intestinal barrier by measuring the transepithelial electrical resistance (TER) and inulin permeability in a Caco-2 cell monolayer model. We further used a calcium switch assay to study the assembly of epithelial tight junctions and determined the effect of butyrate on the assembly of epithelial tight junctions and AMPK activity. We demonstrated that the butyrate treatment increased AMPK activity and accelerated the assembly of tight junctions as shown by the reorganization of tight junction proteins, as well as the development of TER. AMPK activity was also upregulated by butyrate during calcium switch-induced tight junction assembly. Compound C, a specific AMPK inhibitor, inhibited the butyrate-induced activation of AMPK. The facilitating effect of butyrate on the increases in TER in standard culture media, as well as after calcium switch, was abolished by compound C. We conclude that butyrate enhances the intestinal barrier by regulating the assembly of tight junctions. This dynamic process is mediated by the activation of AMPK. These results suggest an intriguing link between SCFA and the intracellular energy sensor for the development of the intestinal barrier.

  5. Berberine inhibits mouse insulin gene promoter through activation of AMP activated protein kinase and may exert beneficial effect on pancreatic β-cell.

    PubMed

    Shen, Ning; Huan, Yi; Shen, Zhu-fang

    2012-11-05

    Berberine is one of the main alkaloids of Rhizoma coptidis, proven to have anti-diabetic potentials through activation of AMP activated protein kinase (AMPK) in liver and muscle. However, the role of berberine on the insulin gene is unknown. Therefore, the effect of berberine on insulin gene transcription was investigated in the present study. Reporter gene assays were used in the mouse β-cell line NIT-1 to test the effect of berberine on the promoter of mouse insulin gene Ins2. The mRNA and protein levels of insulin were also detected. Diet induced glucose intolerant mice were used to explore the effect of berberine on blood glucose homeostasis and insulin resistance in vivo. The insulin content in islet was semi-quantified by an image analysis software in the immunohistochemistry sections. The results revealed that berberine caused a reversible concentration-dependent inhibition of insulin gene transcription in NIT-1 cells which showed a significant difference from the long term used AMPK activator metformin. Such inhibition on insulin promoter resulted in the reduction of mRNA and protein of insulin. Furthermore, the inhibition of insulin promoter was totally abolished by AMPK inhibitor Compound C. Berberine significantly improved insulin resistance and glucose intolerance of mice. Likewise, insulin content in islets of berberine treated mice was also decreased. Thus, the insulin gene represents a novel target of AMPK that may contribute to the action of berberine in type 2 diabetes mellitus.

  6. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis

    PubMed Central

    Polekhina, Galina; Feil, Susanne C.; Gupta, Abhilasha; O’Donnell, Paul; Stapleton, David; Parker, Michael W.

    2005-01-01

    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. PMID:16508085

  7. 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

  8. 24-hydroxyursolic acid from the leaves of the Diospyros kaki (Persimmon) induces apoptosis by activation of AMP-activated protein kinase.

    PubMed

    Khanal, Prem; Oh, Won-Keun; Thuong, Phuong Thien; Cho, Sung Dae; Choi, Hong Seok

    2010-05-01

    There are multiple lines of evidence that persimmon extract and its constituents have potent antitumor activity against human cancer cells. However, the molecular mechanisms of 24-hydroxyursolic acid, a triterpenoid found in persimmon, on antitumor activities are not yet understood. Here, we demonstrate that 24-hydroxyursolic acid inhibited cell proliferation, strongly activated AMP-activated protein kinase (AMPK) and mediated critical anticancer effects by inhibition of cyclooxygenase (COX-2) expression in HT-29 cells. In addition, 24-hydroxyursolic acid induced cellular apoptosis by activation of poly(ADP-ribose) polymerase (PARP), caspase-3, and phosphorylation of p53 at Ser15. It also strongly induced DNA fragmentation in HT-29 cells and thereby significantly inhibited colony formation of HT-29 cells in soft agar. In addition, 24-hydroxyursolic acid blocked the EGF-induced ERKs phosphorylation and led to the inhibition of AP-1 activity and cell transformation in JB6 CL41 cells. Collectively, these findings are the first to reveal a molecular basis for the anticarcinogenic action of 24-hydroxyursolic acid and might account for the reported chemopreventive and chemotherapic effects of persimmon extracts.

  9. Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase.

    PubMed

    Kim, Dong Eun; Kim, Yunha; Cho, Dong-Hyung; Jeong, Seong-Yun; Kim, Sung-Bae; Suh, Nayoung; Lee, Jung Shin; Choi, Eun Kyung; Koh, Jae-Young; Hwang, Jung Jin; Kim, Choung-Soo

    2015-01-01

    Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

  10. 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.

  11. Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells

    PubMed Central

    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

  12. A High-Concentrate Diet Induced Milk Fat Decline via Glucagon-Mediated Activation of AMP-Activated Protein Kinase in Dairy Cows

    PubMed Central

    Li, Lin; Cao, Yang; Xie, Zhenglu; Zhang, Yuanshu

    2017-01-01

    Dairy cows are often fed a high-concentrate (HC) diet to meet lactation demands; however, long-term concentrate feeding is unhealthy and decreases milk fat. Therefore, we investigated the effects of liver lipid metabolism on milk fat synthesis. Ten lactating Holstein cows were assigned randomly into HC and LC (low-concentrate) diet groups. After 20 weeks of feeding, milk fat declined, and lipopolysaccharide levels in the jugular, portal, and hepatic veins increased in the HC group. Liver consumption and release of nonesterified fatty acid (NEFA) into the bloodstream also decreased. AMP-activated protein kinase alpha (AMPKα) was up-regulated significantly in the livers of the HC-fed cows. The HC diet also up-regulated the expression of the transcription factor peroxisome proliferator-activated receptor α (PPARα) and its downstream targets involved in fatty acid oxidation, including carnitine palmitoyltransferase-1,2 (CPT-1, CPT-2), liver-fatty acid-binding protein (L-FABP), and acyl-CoA oxidase (ACO). The HC diet increased blood glucagon (GC) levels, and liver glucagon receptor (GCGR) expression was elevated. Cumulatively, a long-term HC diet decreased plasma concentrations of NEFA via the GC/GCGR-AMPK-PPARα signalling pathway and reduced their synthesis in the liver. The decreased NEFA concentration in the blood during HC feeding may explain the decline in the milk fat of lactating cows. PMID:28287130

  13. The AMP-activated protein kinase AAK-2 links energy levels and insulin-like signals to lifespan in C. elegans

    PubMed Central

    Apfeld, Javier; O'Connor, Greg; McDonagh, Tom; DiStefano, Peter S.; Curtis, Rory

    2004-01-01

    Although limiting energy availability extends lifespan in many organisms, it is not understood how lifespan is coupled to energy levels. We find that the AMP:ATP ratio, a measure of energy levels, increases with age in Caenorhabditis elegans and can be used to predict life expectancy. The C. elegans AMP-activated protein kinase α subunit AAK-2 is activated by AMP and functions to extend lifespan. In addition, either an environmental stressor that increases the AMP:ATP ratio or mutations that lower insulin-like signaling extend lifespan in an aak-2-dependent manner. Thus, AAK-2 is a sensor that couples lifespan to information about energy levels and insulin-like signals. PMID:15574588

  14. Metformin regulates hepatic lipid metabolism through activating AMP-activated protein kinase and inducing ATGL in laying hens.

    PubMed

    Chen, Wei-Lu; Wei, Hen-Wei; Chiu, Wen-Zan; Kang, Ching-Hui; Lin, Ting-Han; Hung, Chien-Ching; Chen, Ming-Chun; Shieh, Ming-Song; Lee, Chin-Cheng; Lee, Horng-Mo

    2011-12-05

    Although many clinical trials have showed that metformin improves non-alcoholic fatty liver disease, which is a common liver disease associated with hepatic enzyme abnormalities, an animal model is required to investigate the effects of altered gene expression and post-translational processing (proteins) in mediating the observed responses. Laying hens appear to develop fatty livers, as in the case in human beings, when ingesting energy in excess of maintenance, and they can be used as an animal model for observing hepatic steatosis. The aim of this study was to investigate whether metformin could improve the non-alcoholic fatty liver of laying hens and to examine the possible mechanisms of lipid-lowering effects. Forty-eight Leghorn laying hens of Hy-Line variety W-36 - 44 weeks with 64.8% hen-day egg production - were randomly assigned into 4 treatments, each receiving 0, 10, 30, or 100mg of metformin with saline per kg body weight by daily wing vein injection. Results showed that, compared with the control, significant decreases existed in the laying rates; plasma triglyceride, cholesterol, and insulin levels; body weights; abdominal fat weights; hepatic lipid contents; and hepatic fatty acid synthase expression of layers receiving 30 or 100mg per kg body weight, whereas significant increases in their hepatic 5'adenosine monophosphate-activated protein kinase, acyl-CoA carboxylase phosphorylation, adipose triglyceride lipase, and carnitine palmitoyl transferase-1 expression were observed. These data suggest that metformin could reduce lipid deposits in the liver and that the laying hen is a valuable animal model for studying hepatic steatosis.

  15. 5'AMP-activated protein kinase activity is increased in adipose tissue of northern elephant seal pups during prolonged fasting-induced insulin resistance.

    PubMed

    Viscarra, Jose A; Champagne, Cory D; Crocker, Daniel E; Ortiz, Rudy M

    2011-06-01

    Northern elephant seals endure a 2- to 3-month fast characterized by sustained hyperglycemia, hypoinsulinemia, and increased plasma cortisol and free fatty acids, conditions often seen in insulin-resistant humans. We had previously shown that adipose Glut4 expression and 5'AMP-activated protein kinase (AMPK) activity increase and plasma glucose decreases in fasting seals suggesting that AMPK activity contributes to glucose regulation during insulin-resistant conditions. To address the hypothesis that AMPK activity increases during fasting-induced insulin resistance, we performed glucose tolerance tests (GTT) on early (n=5) and late (n=8)-fasted seal pups and compared adipose tissue expression of insulin signaling proteins, peroxisome proliferator-activated receptor γ (PPARγ), and AMPK, in addition to plasma adiponectin, leptin, cortisol, insulin, and non-esterified fatty acid (NEFA) levels. Fasting was associated with decreased glucose clearance, plasma insulin and adiponectin, and intracellular insulin signaling, as well as increased plasma cortisol and NEFAs, supporting the suggestion that seals develop insulin resistance late in the fast. The expression of Glut4 and VAMP2 increased (52 and 63% respectively) with fasting but did not change significantly during the GTT. PPARγ and phosphorylated AMPK did not change in the early fasted seals, but increased significantly (73 and 50% respectively) in the late-fasted seals during the GTT. Increased AMPK activity along with the reduction in the activity of insulin-signaling proteins supports our hypothesis that AMPK activity is increased following the onset of insulin resistance. The association between increased AMPK activity and Glut4 expression suggests that AMPK plays a greater role in regulating glucose metabolism in mammals adapted to prolonged fasting than in non-fasting mammals.

  16. Angelica gigas Ameliorates Hyperglycemia and Hepatic Steatosis in C57BL/KsJ-db/db Mice via Activation of AMP-Activated Protein Kinase Signaling Pathway.

    PubMed

    Bae, Ui-Jin; Choi, Eun-Kyung; Oh, Mi-Ra; Jung, Su-Jin; Park, Joon; Jung, Tae-Sung; Park, Tae-Sun; Chae, Soo-Wan; Park, Byung-Hyun

    2016-01-01

    The prevention and management of type 2 diabetes mellitus has become a major global public health challenge. Decursin, an active compound of Angelica gigas Nakai roots, was recently reported to have a glucose-lowering activity. However, the antidiabetic effect of Angelica gigas Nakai extract (AGNE) has not yet been investigated. We evaluated the effects of AGNE on glucose homeostasis in type 2 diabetic mice and investigated the underlying mechanism by which AGNE acts. Male C57BL/KsJ-db/db mice were treated with either AGNE (10 mg/kg, 20 mg/kg, and 40 mg/kg) or metformin (100 mg/kg) for 8 weeks. AGNE supplementation (20 and 40 mg/kg) significantly decreased fasting glucose and insulin levels, decreased the areas under the curve of glucose in oral glucose tolerance and insulin tolerance tests, and improved homeostatic model assessment-insulin resistant (HOMA-IR) scores. AGNE also ameliorated hepatic steatosis, hyperlipidemia, and hypercholesterolemia. Mechanistic studies suggested that the glucose-lowering effect of AGNE was mediated by the activation of AMP activated protein kinase, Akt, and glycogen synthase kinase-3[Formula: see text]. AGNE can potentially improve hyperglycemia and hepatic steatosis in patients with type 2 diabetes.

  17. Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways.

    PubMed

    Huang, Bo; Yuan, Hai Dan; Kim, Do Yeon; Quan, Hai Yan; Chung, Sung Hyun

    2011-04-27

    Cinnamaldehyde (CA), one of the active components of cinnamon, has been known to exert several pharmacological effects such as anti-inflammatory, antioxidant, antitumor, and antidiabetic activities. However, its antiobesity effect has not been reported yet. This study investigated the antidifferentiation effect of CA on 3T3-L1 preadipocytes, and the antiobesity activity of CA was further explored using high-fat-diet-induced obese ICR mice. During 3T3-L1 preadipocytes were differentiated into adipocytes, 10-40 μM CA was treated and lipid contents were quantified by Oil Red O staining, along with changes in the expression of genes and proteins associated with adipocyte differentiation and adipogenesis. It was found that CA significantly reduced lipid accumulation and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding proteins α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) in concentration-dependent manners. Moreover, CA markedly up-regulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), and these effects were blunted in the presence of AMPK inhibitor, compound C. In the animal study, weight gains, insulin resistance index, plasma triglyceride (TG), nonesterified fatty acid (NEFA), and cholesterol levels in the 40 mg/kg of CA-administered group were significantly decreased by 67.3, 55, 39, 31, and 23%, respectively, when compared to the high-fat diet control group. In summary, these results suggest that CA exerts antiadipogenic effects through modulation of the PPAR-γ and AMPK signaling pathways.

  18. Berberine, a natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states.

    PubMed

    Lee, Yun S; Kim, Woo S; Kim, Kang H; Yoon, Myung J; Cho, Hye J; Shen, Yun; Ye, Ji-Ming; Lee, Chul H; Oh, Won K; Kim, Chul T; Hohnen-Behrens, Cordula; Gosby, Alison; Kraegen, Edward W; James, David E; Kim, Jae B

    2006-08-01

    Berberine has been shown to have antidiabetic properties, although its mode of action is not known. Here, we have investigated the metabolic effects of berberine in two animal models of insulin resistance and in insulin-responsive cell lines. Berberine reduced body weight and caused a significant improvement in glucose tolerance without altering food intake in db/db mice. Similarly, berberine reduced body weight and plasma triglycerides and improved insulin action in high-fat-fed Wistar rats. Berberine downregulated the expression of genes involved in lipogenesis and upregulated those involved in energy expenditure in adipose tissue and muscle. Berberine treatment resulted in increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 adipocytes and L6 myotubes, increased GLUT4 translocation in L6 cells in a phosphatidylinositol 3' kinase-independent manner, and reduced lipid accumulation in 3T3-L1 adipocytes. These findings suggest that berberine displays beneficial effects in the treatment of diabetes and obesity at least in part via stimulation of AMPK activity.

  19. 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

  20. AMP-Activated Protein Kinase α2 in Neutrophils Regulates Vascular Repair via Hypoxia-Inducible Factor-1α and a Network of Proteins Affecting Metabolism and Apoptosis

    PubMed Central

    Abdel Malik, Randa; Zippel, Nina; Frömel, Timo; Heidler, Juliana; Zukunft, Sven; Walzog, Barbara; Ansari, Nariman; Pampaloni, Francesco; Wingert, Susanne; Rieger, Michael A.; Wittig, Ilka; Fisslthaler, Beate

    2017-01-01

    Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair. Objective: To determine the role of the AMPKα2 subunit in vascular repair. Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2−/− versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice. Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia. PMID:27777247

  1. Interactome analysis of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells by affinity purification-mass spectrometry.

    PubMed

    Moon, Sungyoon; Han, Dohyun; Kim, Yikwon; Jin, Jonghwa; Ho, Won-Kyung; Kim, Youngsoo

    2014-03-14

    The heterotrimeric enzyme AMP-activated protein kinase (AMPK) is a major metabolic factor that regulates the homeostasis of cellular energy. In particular, AMPK mediates the insulin resistance that is associated with type 2 diabetes. Generally, cellular processes require tight regulation of protein kinases, which is effected through their formation of complex with other proteins and substrates. Despite their critical function in regulation and pathogenesis, there are limited data on the interaction of protein kinases. To identify proteins that interact with AMPK, we performed large-scale affinity purification (AP)-mass spectrometry (MS) of the AMPK-α1 and -β1 subunits. Through a comprehensive analysis, using a combination of immunoprecipitaion and ion trap mass spectrometry, we identified 381 unique proteins in the AMPKα/β interactomes: 325 partners of AMPK-α1 and 243 for AMPK-β1. Further, we identified 196 novel protein-protein interactions with AMPK-α1 and AMPK-β1. Notably, in our bioinformatics analysis, the novel interaction partners mediated functions that are related to the regulation of actin organization. Specifically, several such proteins were linked to pancreatic beta-cell functions, including glucose-stimulated insulin secretion, beta-cell development, beta-cell differentiation, and cell-cell communication.

  2. 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.

  3. The New 4-O-Methylhonokiol Analog GS12021 Inhibits Inflammation and Macrophage Chemotaxis: Role of AMP-Activated Protein Kinase α Activation

    PubMed Central

    Kim, Sora; Ka, Sun-O; Lee, Youngyi; Park, Byung-Hyun; Fei, Xiang; Jung, Jae-Kyung; Seo, Seung-Yong; Bae, Eun Ju

    2015-01-01

    Preventing pathologic tissue inflammation is key to treating obesity-induced insulin resistance and type 2 diabetes. Previously, we synthesized a series of methylhonokiol analogs and reported that compounds with a carbamate structure had inhibitory function against cyclooxygenase-2 in a cell-free enzyme assay. However, whether these compounds could inhibit the expression of inflammatory genes in macrophages has not been investigated. Here, we found that a new 4-O-methylhonokiol analog, 3′,5-diallyl-4′-methoxy-[1,1′-biphenyl]-2-yl morpholine-4-carboxylate (GS12021) inhibited LPS- or TNFα-stimulated inflammation in macrophages and adipocytes, respectively. LPS-induced phosphorylation of nuclear factor-kappa B (NF-κB)/p65 was significantly decreased, whereas NF-κB luciferase activities were slightly inhibited, by GS12021 treatment in RAW 264.7 cells. Either mitogen-activated protein kinase phosphorylation or AP-1 luciferase activity was not altered by GS12021. GS12021 increased the phosphorylation of AMP-activated protein kinase (AMPK) α and the expression of sirtuin (SIRT) 1. Inhibition of mRNA expression of inflammatory genes by GS12021 was abolished in AMPKα1-knockdown cells, but not in SIRT1 knockout cells, demonstrating that GS12021 exerts anti-inflammatory effects through AMPKα activation. The transwell migration assay results showed that GS12021 treatment of macrophages prevented the cell migration promoted by incubation with conditioned medium obtained from adipocytes. GS12021 suppression of p65 phosphorylation and macrophage chemotaxis were preserved in AMPKα1-knockdown cells, indicating AMPK is not required for these functions of GS12021. Identification of this novel methylhonokiol analog could enable studies of the structure-activity relationship of this class of compounds and further evaluation of its in vivo potential for the treatment of insulin-resistant states and other chronic inflammatory diseases. PMID:25706552

  4. AMP-activated protein kinase stimulates Warburg-like glycolysis and activation of satellite cells during muscle regeneration.

    PubMed

    Fu, Xing; Zhu, Mei-Jun; Dodson, Mike V; Du, Min

    2015-10-30

    Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration.

  5. 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.

  6. Naringin ameliorates metabolic syndrome by activating AMP-activated protein kinase in mice fed a high-fat diet.

    PubMed

    Pu, Peng; Gao, Dong-Mei; Mohamed, Salim; Chen, Jing; Zhang, Jing; Zhou, Xiao-Ya; Zhou, Nai-Jing; Xie, Jing; Jiang, Hong

    2012-02-01

    Metabolic syndrome is a low-grade inflammatory state in which oxidative stress is involved. Naringin, isolated from the Citrussinensis, is a phenolic compound with anti-oxidative and anti-inflammatory activities. The aim of this study was to explore the effects of naringin on metabolic syndrome in mice. The animal models, induced by high-fat diet in C57BL/6 mice, developed obesity, dyslipidemia, fatty liver, liver dysfunction and insulin resistance. These changes were attenuated by naringin. Further investigations revealed that the inhibitory effect on inflammation and insulin resistance was mediated by blocking activation of the MAPKs pathways and by activating IRS1; the lipid-lowering effect was attributed to inhibiting the synthesis way and increasing fatty acid oxidation; the hypoglycemic effect was due to the regulation of PEPCK and G6pase. The anti-oxidative stress of naringin also participated in the improvement of insulin resistance and lipogenesis. All of these depended on the AMPK activation. To confirm the results of the animal experiment, we tested primary hepatocytes exposed to high glucose system. Naringin was protective by phosphorylating AMPKα and IRS1. Taken together, these results suggested that naringin protected mice exposed to a high-fat diet from metabolic syndrome through an AMPK-dependent mechanism involving multiple types of intracellular signaling and reduction of oxidative damage.

  7. AICAR induces astroglial differentiation of neural stem cells via activating the JAK/STAT3 pathway independently of AMP-activated protein kinase.

    PubMed

    Zang, Yi; Yu, Li-Fang; Pang, Tao; Fang, Lei-Ping; Feng, Xu; Wen, Tie-Qiao; Nan, Fa-Jun; Feng, Lin-Yin; Li, Jia

    2008-03-07

    Neural stem cell differentiation and the determination of lineage decision between neuronal and glial fates have important implications in the study of developmental, pathological, and regenerative processes. Although small molecule chemicals with the ability to control neural stem cell fate are considered extremely useful tools in this field, few were reported. AICAR is an adenosine analog and extensively used to activate AMP-activated protein kinase (AMPK), a metabolic "fuel gauge" of the biological system. In the present study, we found an unrecognized astrogliogenic activity of AICAR on not only immortalized neural stem cell line C17.2 (C17.2-NSC), but also primary neural stem cells (NSCs) derived from post-natal (P0) rat hippocampus (P0-NSC) and embryonic day 14 (E14) rat embryonic cortex (E14-NSC). However, another AMPK activator, Metformin, did not alter either the C17.2-NSC or E14-NSC undifferentiated state although both Metformin and AICAR can activate the AMPK pathway in NSC. Furthermore, overexpression of dominant-negative mutants of AMPK in C17.2-NSC was unable to block the gliogenic effects of AICAR. We also found AICAR could activate the Janus kinase (JAK) STAT3 pathway in both C17.2-NSC and E14-NSC but Metformin fails. JAK inhibitor I abolished the gliogenic effects of AICAR. Taken together, these results suggest that the astroglial differentiation effect of AICAR on neural stem cells was acting independently of AMPK and that the JAK-STAT3 pathway is essential for the gliogenic effect of AICAR.

  8. 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.

  9. Short-chain fatty acids activate AMP-activated protein kinase and ameliorate ethanol-induced intestinal barrier dysfunction in Caco-2 cell monolayers.

    PubMed

    Elamin, Elhaseen E; Masclee, Ad A; Dekker, Jan; Pieters, Harm-Jan; Jonkers, Daisy M

    2013-12-01

    Short-chain fatty acids (SCFAs) have been shown to promote intestinal barrier function, but their protective effects against ethanol-induced intestinal injury and underlying mechanisms remain essentially unknown. The aim of the study was to analyze the influence of SCFAs on ethanol-induced barrier dysfunction and to examine the role of AMP-activated protein kinase (AMPK) as a possible mechanism using Caco-2 monolayers. The monolayers were treated apically with butyrate (2, 10, or 20 mmol/L), propionate (4, 20, or 40 mmol/L), or acetate (8, 40, or 80 mmol/L) for 1 h before ethanol (40 mmol/L) for 3 h. Barrier function was analyzed by measurement of transepithelial resistance and permeation of fluorescein isothiocyanate-labeled dextran. Distribution of the tight junction (TJ) proteins zona occludens-1, occludin, and filamentous-actin (F-actin) was examined by immunofluorescence. Metabolic stress was determined by measuring oxidative stress, mitochondrial function, and ATP using dichlorofluorescein diacetate, dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, and bioluminescence assay, respectively. AMPK was knocked down by small interfering RNA (siRNA), and its activity was assessed by a cell-based ELISA. Exposure to ethanol significantly impaired barrier function compared with controls (P < 0.0001), disrupted TJ and F-actin cytoskeleton integrity, and induced metabolic stress. However, pretreatment with 2 mmol/L butyrate, 4 mmol/L propionate, and 8 mmol/L acetate significantly alleviated the ethanol-induced barrier dysfunction, TJ and F-actin disruption, and metabolic stress compared with ethanol-exposed monolayers (P < 0.0001). The promoting effects on barrier function were abolished by inhibiting AMPK using either compound C or siRNA. These observations indicate that SCFAs exhibit protective effects against ethanol-induced barrier disruption via AMPK activation, suggesting a potential for SCFAs as prophylactic and/or therapeutic factors against ethanol

  10. 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.

  11. 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.

  12. Cordycepin inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production via activating amp-activated protein kinase (AMPK) signaling.

    PubMed

    Zhang, Jian-Li; Xu, Ying; Shen, Jie

    2014-07-08

    Tumor necrosis factor (TNF)-α is elevated during the acute phase of Kawasaki disease (KD), which damages vascular endothelial cells to cause systemic vasculitis. In the current study, we investigated the potential role of cordycepin on TNFα expression in both lipopolysaccharide (LPS)-stimulated macrophages and ex vivo cultured peripheral blood mononuclear cells (PBMCs) of KD patients. We found that cordycepin significantly suppressed LPS-induced TNFα expression and production in mouse macrophages (RAW 264.7 cells and bone marrow-derived macrophages (BMDMs)). Meanwhile, cordycepin alleviated TNFα production in KD patients' PBMCs. PBMCs from healthy controls had a much lower level of basal TNF-α content than that of KD patients. LPS-induced TNF-α production in healthy controls' PBMCs was also inhibited by cordycepin. For the mechanism study, we discovered that cordycepin activated AMP-activated protein kinase (AMPK) signaling in both KD patients' PBMCs and LPS-stimulated macrophages, which mediated cordycepin-induced inhibition against TNFα production. AMPK inhibition by its inhibitor (compound C) or by siRNA depletion alleviated cordycepin's effect on TNFα production. Further, we found that cordycepin inhibited reactive oxygen species (ROS) production and nuclear factor kappa B (NF-κB) activation in LPS-stimulate RAW 264.7 cells or healthy controls' PBMCs. PBMCs of KD patients showed higher basal level of ROS and NF-κB activation, which was also inhibited by cordycepin co-treatment. In conclusion, our data showed that cordycepin inhibited TNFα production, which was associated with AMPK activation as well as ROS and NF-κB inhibition. The results of this study should have significant translational relevance in managing this devastating disease.

  13. Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface*

    PubMed Central

    Jinadasa, Tushare; Szabó, Elöd Z.; Numata, Masayuki; Orlowski, John

    2014-01-01

    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

  14. Glucose de-repression by yeast AMP-activated protein kinase SNF1 is controlled via at least two independent steps.

    PubMed

    García-Salcedo, Raúl; Lubitz, Timo; Beltran, Gemma; Elbing, Karin; Tian, Ye; Frey, Simone; Wolkenhauer, Olaf; Krantz, Marcus; Klipp, Edda; Hohmann, Stefan

    2014-04-01

    The AMP-activated protein kinase, AMPK, controls energy homeostasis in eukaryotic cells but little is known about the mechanisms governing the dynamics of its activation/deactivation. The yeast AMPK, SNF1, is activated in response to glucose depletion and mediates glucose de-repression by inactivating the transcriptional repressor Mig1. Here we show that overexpression of the Snf1-activating kinase Sak1 results, in the presence of glucose, in constitutive Snf1 activation without alleviating glucose repression. Co-overexpression of the regulatory subunit Reg1 of the Glc-Reg1 phosphatase complex partly restores glucose regulation of Snf1. We generated a set of 24 kinetic mathematical models based on dynamic data of Snf1 pathway activation and deactivation. The models that reproduced our experimental observations best featured (a) glucose regulation of both Snf1 phosphorylation and dephosphorylation, (b) determination of the Mig1 phosphorylation status in the absence of glucose by Snf1 activity only and (c) a regulatory step directing active Snf1 to Mig1 under glucose limitation. Hence it appears that glucose de-repression via Snf1-Mig1 is regulated by glucose via at least two independent steps: the control of activation of the Snf1 kinase and directing active Snf1 to inactivating its target Mig1.

  15. Activation of AMP-activated protein kinase induce expression of FoxO1, FoxO3a, and myostatin after exercise-induced muscle damage.

    PubMed

    Lee, Kihyuk; Ochi, Eisuke; Song, Hongsun; Nakazato, Koichi

    2015-10-23

    AMP-activated protein kinase (AMPK) has been shown to regulate protein metabolism in skeletal muscle. We previously found that levels of Forkhead box proteins, FoxO1 and FoxO3a, and myostatin in rat gastrocnemius increased after exercise-induced muscle damage (EIMD). Eccentric muscle contractions (ECs), defined as elongation of muscle under tension, were used for inducing EIMD. The objective of this study was to clarify whether AMPK participates in activation and expression of FoxO proteins and myostatin in rat gastrocnemius muscle after EIMD. Wistar rats were randomly assigned into the following three groups; CON (n = 6), 180ECs group (ankle angular velocity, 180°/s; n = 6), and 30ECs group (ankle angular velocity, 30°/s; n = 6). 20 ECs were conducted with percutaneous electrical stimulation of gastrocnemius and simultaneous forced dorsiflexion of ankle joint (from 0° to 45°). To evaluate activation of AMPK, we measured the phosphorylated states of AMPK and acetyl CoA carboxylase. For evaluation of the direct relationships of AMPK and other proteins, we also examined contents of FoxOs and myostatin with stimulation of L6 myotube with AMPK agonist, 5 -aminoimidazole -4 -carboxamide -1-β-d-ribofuranoside (AICAR) (0.1, 0.5, 1, 1.5, and 2 mM). Western blotting was employed for protein analysis. Significant torque deficit was only observed in the 180ECs, suggesting EIMD. We also observed that phosphorylated AMPKα was induced in response to 180ECs (p < 0.01 vs. CON). Additionally, the level of phosphorylated acetyl CoA carboxylase was significantly higher in response to 180ECs and 30ECs. The phosphorylated states of FoxO1, FoxO3a, and myostatin expression were increased significantly in response to 180ECs. Furthermore, treatment of L6 myotubes with AICAR showed similar tendencies to that observed in in vivo gastrocnemius muscle treated with 180ECs. Therefore, we conclude that activation of AMPK plays a key role in increasing the level of FoxO1, FoxO3a

  16. AMP-activated protein kinase inhibits TGF-β-, angiotensin II-, aldosterone-, high glucose-, and albumin-induced epithelial-mesenchymal transition.

    PubMed

    Lee, Jang Han; Kim, Ji Hyun; Kim, Ja Seon; Chang, Jai Won; Kim, Soon Bae; Park, Jung Sik; Lee, Sang Koo

    2013-03-15

    The epithelial-mesenchymal transition (EMT) is a novel mechanism that promotes renal fibrosis. Transforming growth factor-β (TGF-β), angiotensin II, aldosterone, high glucose, and urinary albumin are well-known causes of EMT and renal fibrosis. We examined whether and how activation of AMP-activated protein kinase (AMPK) suppressed EMT induced by the above agents in tubular epithelial cells. All experiments were performed using HK-2 cells. Protein expression was measured by Western blot analysis. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. Exposure of tubular cells to TGF-β (10 ng/ml), angiotensin II (1 μM), aldosterone (100 nM), high glucose (30 mM), and albumin (5 mg/ml) for 5 days induced EMT, as shown by upregulation of α-smooth muscle actin and downregulation of E-cadherin. ROS and NADPH oxidase 4 (Nox4) expression were increased, and antioxidants such as tiron and N-acetylcysteine inhibited EMT induction. Metformin (the best known clinical activator of AMPK) suppressed EMT induction through inhibition of ROS via induction of heme oxygenase-1 and endogenous antioxidant thioredoxin. An AMPK inhibitor (compound C) and AMPK small interfering RNA blocked the effect of metformin, and another AMPK activator [5-aminoimidazole-4-carboxamide-1β riboside (AICAR)] exerted the same effects as metformin. In conclusion, AMPK activation might be beneficial in attenuating the tubulointerstitial fibrosis induced by TGF-β, angiotensin II, aldosterone, high glucose, and urinary albumin.

  17. α-Terpineol induces fatty liver in mice mediated by the AMP-activated kinase and sterol response element binding protein pathway.

    PubMed

    Choi, You-Jin; Sim, Woo-Cheol; Choi, Hyun Kyu; Lee, Seung-Ho; Lee, Byung-Hoon

    2013-05-01

    The use of herbal medicines in disease prevention and treatment is growing rapidly worldwide, without careful consideration of safety issues. α-Terpineol is a monoterpene alcoholic component of Melaleuca alternifolia, Salvia officinalis and Carthamus tinctorius that is used widely as a flavor and essential oil in food. The present study showed that α-terpineol induces fatty liver via the AMP-activated protein kinase (AMPK)-mTOR-sterol regulatory element-binding protein-1 (SREBP-1) pathway. α-Terpineol-treated hepatocytes had significantly increased neutral lipid accumulation. α-Terpineol suppressed AMPK phosphorylation, and increased p70S6 kinase (p70S6K) phosphorylation and SREBP-1 activation. It also increased luciferase activity in cells transfected with LXRE-tk-Luc and SRE-tk-Luc. Inhibition of mTOR signaling by co-treatment with rapamycin or co-transfection with dominant negative p70S6K blocked completely the effects of α-terpineol. α-Terpineol oral administration to mice for 2weeks led to decreased AMPK phosphorylation and increased SREBP-1 activation in the liver, followed by hepatic lipid accumulation. Conversely, rapamycin co-treatment reversed α-terpineol-induced SREBP-1 activation and fatty liver in mice. These data provide evidence that α-terpineol causes fatty liver, an effect mediated by the AMPK/mTOR/SREBP-1 pathway.

  18. Piperidine alkaloids from Piperretrofractum Vahl. protect against high-fat diet-induced obesity by regulating lipid metabolism and activating AMP-activated protein kinase

    SciTech Connect

    Kim, Kyung Jin; Lee, Myoung-Su; Jo, Keunae; Hwang, Jae-Kwan

    2011-07-22

    Highlights: {yields} Piperidine alkaloids from Piperretrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, are isolated as the anti-obesity constituents. {yields} PRPA administration significantly reduces body weight gain without altering food intake and fat pad mass. {yields} PRPA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} PRPAs attenuate HFD-induced obesity by activating AMPK and PPAR{delta}, and regulate lipid metabolism, suggesting their potential anti-obesity effects. -- Abstract: The fruits of Piperretrofractum 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 {delta} (PPAR{delta}) transactivation assay. The molecular mechanism was investigated in 3T3-L1 adipocytes and L6 myocytes. PRPA treatment activated AMP-activated protein kinase (AMPK) signaling and PPAR{delta} protein and also regulated the expression of lipid metabolism-related proteins. In the animal model, oral PRPA administration (50, 100, or 300 mg/kg/day for 8 weeks) 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

  19. C6 ceramide dramatically increases vincristine sensitivity both in vivo and in vitro, involving AMP-activated protein kinase-p53 signaling.

    PubMed

    Chen, Min-Bin; Jiang, Qin; Liu, Yuan-yuan; Zhang, Yan; He, Bang-shun; Wei, Mu-Xin; Lu, Jian-Wei; Ji, Yong; Lu, Pei-Hua

    2015-09-01

    Use of the conventional cancer chemotherapy (i.e. vincristine) is limited in tumor cells exhibiting pre-existing or acquired resistance. Here, we found that C6 ceramide (C6) dramatically sensitized vincristine's activity. In vitro, C6 and vincristine coadministration induced substantial necrosis and apoptosis in multiple human cancer cell lines, which were accompanied by a profound AMP-activated protein kinase (AMPK) activation, subsequent p53 activation, mTORC1 inactivation and Bcl-2/HIF-1α downregulation. Such synergistic effects were attenuated by AMPK inactivation through genetic mutation or short hairpin RNA silencing. Coadministration-activated p53 translocated to mitochondria, and formed a complex with cyclophilin-D, leading to mitochondrial permeability transition pore opening and cell necrosis. Disrupting p53-Cyp-D complexation through pharmacological or genetic means reduced costimulation-induced cytotoxicity. In vivo, a liposomal C6 was synthesized, which dramatically enhanced the antiproliferative activity of vincristine on HCT-116 or A2780 xenografts. Together, C6 sensitizes vincristine-induced anticancer activity in vivo and in vitro, involving activating AMPK-p53 signaling.

  20. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes

    PubMed Central

    Tamás, Peter; Hawley, Simon A.; Clarke, Rosemary G.; Mustard, Kirsty J.; Green, Kevin; Hardie, D. Grahame; Cantrell, Doreen A.

    2006-01-01

    The adenosine monophosphate (AMP)–activated protein kinase (AMPK) has a crucial role in maintaining cellular energy homeostasis. This study shows that human and mouse T lymphocytes express AMPKα1 and that this is rapidly activated in response to triggering of the T cell antigen receptor (TCR). TCR stimulation of AMPK was dependent on the adaptors LAT and SLP76 and could be mimicked by the elevation of intracellular Ca2+ with Ca2+ ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca2+ stimulation of AMPK required the activity of Ca2+–calmodulin-dependent protein kinase kinases (CaMKKs), whereas AMPK activation induced by increased AMP/ATP ratios did not. These experiments reveal two distinct pathways for the regulation of AMPK in T lymphocytes. The role of AMPK is to promote ATP conservation and production. The rapid activation of AMPK in response to Ca2+ signaling in T lymphocytes thus reveals that TCR triggering is linked to an evolutionally conserved serine kinase that regulates energy metabolism. Moreover, AMPK does not just react to cellular energy depletion but also anticipates it. PMID:16818670

  1. AMP-activated protein kinase at the nexus of therapeutic skeletal muscle plasticity in Duchenne muscular dystrophy.

    PubMed

    Ljubicic, Vladimir; Jasmin, Bernard J

    2013-10-01

    Recent studies have highlighted the potential of adenosine monophosphate-activated protein kinase (AMPK) to act as a central therapeutic target in Duchenne muscular dystrophy (DMD). Here, we review the role of AMPK as an important integrator of cell signaling pathways that mediate phenotypic plasticity within the context of dystrophic skeletal muscle. Pharmacological AMPK activation remodels skeletal muscle towards a slower, more oxidative phenotype, which is more pathologically resistant to the lack of dystrophin. Moreover, recent studies suggest that AMPK-activated autophagy may be beneficial for myofiber structure and function in mice with muscular dystrophy. Thus, AMPK may represent an ideal target for intervention because clinically approved pharmacological agonists exist, and because benefits can be derived via two independent yet, complementary biological pathways. The availability of several AMPK activators could therefore lead to the rapid development and implementation of novel and highly effective therapeutics aimed at altering the relentless progression of DMD.

  2. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions

    PubMed Central

    Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

    2016-01-01

    Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells

  3. Quercetin activates AMP-activated protein kinase by reducing PP2C expression protecting old mouse brain against high cholesterol-induced neurotoxicity.

    PubMed

    Lu, Jun; Wu, Dong-Mei; Zheng, Yuan-Lin; Hu, Bin; Zhang, Zi-Feng; Shan, Qun; Zheng, Zi-Hui; Liu, Chan-Min; Wang, Yong-Jian

    2010-10-01

    It is known that a high-cholesterol diet induces oxidative stress, inflammatory response, and beta-amyloid (Abeta) accumulation in mouse brain, resulting in neurodegenerative changes. Quercetin, a naturally occurring flavonoid, has been reported to possess numerous biological activities beneficial to health. Our previous studies have demonstrated that quercetin protects mouse brain against D-galactose-induced oxidative damage. Against this background, we evaluated the effect of quercetin on high-cholesterol-induced neurotoxicity in old mice and explored its potential mechanism. Our results showed that oral administration of quercetin significantly improved the behavioural performance of high-cholesterol-fed old mice in both a step-through test and the Morris water maze task. This is at least in part caused by decreasing ROS and protein carbonyl levels and restoring Cu--Zn superoxide dismutase (Cu, Zn-SOD) activity. Furthermore, quercetin also significantly activated the AMP-activated protein kinase (AMPK) via down-regulation of protein phosphatase 2C (PP2C), which reduced the integral optical density (IOD) of activated microglia cells and CD11b expression, down-regulated iNOS and cyclooxygenase-2 (COX-2) expression, and decreased IL-1beta, IL-6, and TNF-alpha expression in the brains of high-cholesterol-fed old mice through the suppression of NF-kappaB p65 nuclear translocation. Moreover, AMPK activation significantly increased 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase (ACC) phosphorylation and reduced fatty acid synthase (FAS) expression in the brains of high-cholesterol-fed old mice, which reduced cholesterol levels, down-regulated cholesterol 24-hydroxylase (CYP46A1) and beta-amyloid converting enzyme 1 (BACE1) expression, decreased eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation, and lowered Abeta deposits. However, the neuroprotective effect of quercetin was weakened by intraperitoneal

  4. Green tea extract intake during lactation modified cardiac macrophage infiltration and AMP-activated protein kinase phosphorylation in weanling rats from undernourished mother during gestation and lactation.

    PubMed

    Matsumoto, E; Kataoka, S; Mukai, Y; Sato, M; Sato, S

    2017-04-01

    Maternal dietary restriction is often associated with cardiovascular disease in offspring. The aim of this study was to investigate the effect of green tea extract (GTE) intake during lactation on macrophage infiltration, and activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and serine-threonine kinase Akt (Akt) in the hearts of weanlings exposed to maternal dietary protein restriction. Pregnant Wistar rats were fed control (C) or low-protein diets (LP) throughout gestation. Following delivery, the dams received a control or a GTE-containing control diet during lactation: control diet during gestation and lactation (CC), low-protein diet during gestation and lactation (LPC), low-protein diet during gestation and 0.12% GTE-containing low-protein diet during lactation (LPL), and low-protein diet during gestation and 0.24% GTE-containing low-protein diet during lactation (LPH). The female offspring were sacrificed at day 22. Biochemical parameters in the plasma, macrophage infiltration, degree of fibrosis and expression levels of AMPK and Akt were examined. The plasma insulin level increased in LPH compared with LPC. Percentage of the fibrotic areas and the number of macrophages in LPC were higher than those in CC. Conversely, the fibrotic areas and the macrophage number in LPH were smaller (21 and 56%, respectively) than those in LPC. The levels of phosphorylated AMPK in LPL and LPH, and Akt in LPH were greater than those in LPC. In conclusion, maternal protein restriction may induce macrophage infiltration and the decrease of insulin levels. However, GTE intake during lactation may suppress macrophage infiltration and restore insulin secretion function via upregulation of AMPK and insulin signaling in weanlings.

  5. Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca2+ concentration in rat carotid body glomus cells

    PubMed Central

    Kim, Donghee; Kang1,2, Dawon; Martin, Elizabeth A.; Kim, Insook; Carroll, John L.

    2014-01-01

    Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca2+ concentration ([Ca2+]i). Recent studies suggest that AMP-activated protein kinase (AMPK) mediates these effects of hypoxia by inhibiting the background K+ channels such as TASK. Here we studied the effects of modulators of AMPK on TASK activity in cell-attached patches. Activators of AMPK (1 mM AICAR and 0.1–0.5 mM A769662) did not inhibit TASK activity or cause depolarization during acute (10 min) or prolonged (2–3 hr) exposure. Hypoxia inhibited TASK activity by ~70% in cells pretreated with AICAR or A769662. Both AICAR and A769662 (15–40 min) failed to increase [Ca2+]i in glomus cells. Compound C (40 µM), an inhibitor of AMPK, showed no effect on hypoxia-induced inhibition of TASK. AICAR and A769662 phosphorylated AMPKα in PC12 cells, and Compound C blocked the phosphorylation. Our results suggest that AMPK does not affect TASK activity and is not involved in hypoxia-induced elevation of intracellular [Ca2+] in isolated rat carotid body glomus cells. PMID:24530802

  6. Skeletal muscle AMP-activated protein kinase γ1(H151R) overexpression enhances whole body energy homeostasis and insulin sensitivity.

    PubMed

    Schönke, Milena; Myers, Martin G; Zierath, Juleen R; Björnholm, Marie

    2015-10-01

    AMP-activated protein kinase (AMPK) is a major sensor of energy homeostasis and stimulates ATP-generating processes such as lipid oxidation and glycolysis in peripheral tissues. The heterotrimeric enzyme consists of a catalytic α-subunit, a β-subunit that is important for enzyme activity, and a noncatalytic γ-subunit that binds AMP and activates the AMPK complex. We generated a skeletal muscle Cre-inducible transgenic mouse model expressing a mutant γ1-subunit (AMPKγ1(H151R)), resulting in chronic AMPK activation. The expression of the predominant AMPKγ3 isoform in skeletal muscle was reduced in extensor digitorum longus (EDL) muscle (81-83%) of AMPKγ1(H151R) transgenic mice, whereas the abundance and phosphorylation of the AMPK target acetyl-CoA carboxylase was increased in tibialis anterior muscle. Glycogen content was increased 10-fold in gastrocnemius muscle. Whole body carbohydrate oxidation was increased by 11%, and whereas glucose tolerance was unaffected, insulin sensitivity was increased in AMPKγ1(H151R) transgenic mice. Furthermore, perigonadal white adipose tissue mass and serum leptin were reduced in female AMPKγ1(H151R) transgenic mice by 38 and 51% respectively. Conversely, in male AMPKγ1(H151R) transgenic mice, food intake was increased (14%), but body weight and body composition were unaltered, presumably because of increased energy expenditure. In conclusion, transgenic activation of skeletal muscle AMPKγ1 in this model plays an important sex-specific role in skeletal muscle metabolism and whole body energy homeostasis.

  7. In Vitro Anti-Echinococcal and Metabolic Effects of Metformin Involve Activation of AMP-Activated Protein Kinase in Larval Stages of Echinococcus granulosus

    PubMed Central

    Loos, Julia A.; Cumino, Andrea C.

    2015-01-01

    Metformin (Met) is a biguanide anti-hyperglycemic agent, which also exerts antiproliferative effects on cancer cells. This drug inhibits the complex I of the mitochondrial electron transport chain inducing a fall in the cell energy charge and leading 5'-AMP-activated protein kinase (AMPK) activation. AMPK is a highly conserved heterotrimeric complex that coordinates metabolic and growth pathways in order to maintain energy homeostasis and cell survival, mainly under nutritional stress conditions, in a Liver Kinase B1 (LKB1)-dependent manner. This work describes for the first time, the in vitro anti-echinococcal effect of Met on Echinococcus granulosus larval stages, as well as the molecular characterization of AMPK (Eg-AMPK) in this parasite of clinical importance. The drug exerted a dose-dependent effect on the viability of both larval stages. Based on this, we proceeded with the identification of the genes encoding for the different subunits of Eg-AMPK. We cloned one gene coding for the catalytic subunit (Eg-ampkɑ) and two genes coding for the regulatory subunits (Eg-ampkβ and Eg-ampkγ), all of them constitutively transcribed in E. granulosus protoscoleces and metacestodes. Their deduced amino acid sequences show all the conserved functional domains, including key amino acids involved in catalytic activity and protein-protein interactions. In protoscoleces, the drug induced the activation of AMPK (Eg-AMPKɑ-P176), possibly as a consequence of cellular energy charge depletion evidenced by assays with the fluorescent indicator JC-1. Met also led to carbohydrate starvation, it increased glucogenolysis and homolactic fermentation, and decreased transcription of intermediary metabolism genes. By in toto immunolocalization assays, we detected Eg-AMPKɑ-P176 expression, both in the nucleus and the cytoplasm of cells as in the larval tegument, the posterior bladder and the calcareous corpuscles of control and Met-treated protoscoleces. Interestingly, expression of Eg

  8. Role of AMP-activated protein kinase in cross-talk between apoptosis and autophagy in human colon cancer

    PubMed Central

    Song, X; Kim, S-Y; Zhang, L; Tang, D; Bartlett, D L; Kwon, Y T; Lee, Y J

    2014-01-01

    Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116 cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast (MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy, rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation, resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations identify a novel mechanism for AMPK-induced apoptosis through interplay

  9. Role of AMP-activated protein kinase in cross-talk between apoptosis and autophagy in human colon cancer.

    PubMed

    Song, X; Kim, S-Y; Zhang, L; Tang, D; Bartlett, D L; Kwon, Y T; Lee, Y J

    2014-10-30

    Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116 cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast (MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy, rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation, resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations identify a novel mechanism for AMPK-induced apoptosis through interplay

  10. 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

    2015-12-05

    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.

  11. 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.

  12. Fatal infantile cardiac glycogenosis with phosphorylase kinase deficiency and a mutation in the gamma2-subunit of AMP-activated protein kinase.

    PubMed

    Akman, Hasan O; Sampayo, James N; Ross, Fiona A; Scott, John W; Wilson, Gregory; Benson, Lee; Bruno, Claudio; Shanske, Sara; Hardie, D Grahame; Dimauro, Salvatore

    2007-10-01

    A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the gamma2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and "pseudo PHK deficiency" prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patient's father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.

  13. p-Coumaric acid modulates glucose and lipid metabolism via AMP-activated protein kinase in L6 skeletal muscle cells.

    PubMed

    Yoon, Seon-A; Kang, Seong-Il; Shin, Hye-Sun; Kang, Seung-Woo; Kim, Jeong-Hwan; Ko, Hee-Chul; Kim, Se-Jae

    2013-03-22

    p-Coumaric acid (3-[4-hydroxyphenyl]-2-propenoic acid) is a ubiquitous plant metabolite with antioxidant, anti-inflammatory, and anticancer properties. In this study, we examined whether p-coumaric acid modulates glucose and lipid metabolism via AMP-activated protein kinase (AMPK) in L6 skeletal muscle cells. p-Coumaric acid increased the phosphorylation of AMPK in a dose-dependent manner in differentiated L6 skeletal muscle cells. It also increased the phosphorylation of acetyl-CoA carboxylase (ACC) and the expression of CPT-1 mRNA and PPARα, suggesting that it promotes the β-oxidation of fatty acids. Also, it suppressed oleic acid-induced triglyceride accumulation, and enhanced 2-NBDG uptake in differentiated L6 muscle cells. Pretreatment with compound C inhibited AMPK activation, reduced ACC phosphorylation and 2-NBDG uptake, and increased triglyceride accumulation. However, p-coumaric acid counterbalanced the inhibitory effects of compound C. Taken together, these results suggest that p-coumaric acid modulates glucose and lipid metabolism via AMPK activation in L6 skeletal muscle cells and that it has potentially beneficial effects in improving or treating metabolic disorders.

  14. 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.

  15. 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

  16. 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.

  17. AMP-activated Protein Kinase Deficiency Blocks the Hypoxic Ventilatory Response and Thus Precipitates Hypoventilation and Apnea

    PubMed Central

    Mahmoud, Amira D.; Lewis, Sophronia; Juričić, Lara; Udoh, Utibe-Abasi; Hartmann, Sandy; Jansen, Maurits A.; Ogunbayo, Oluseye A.; Puggioni, Paolo; Holmes, Andrew P.; Kumar, Prem; Navarro-Dorado, Jorge; Foretz, Marc; Viollet, Benoit; Dutia, Mayank B.; Marshall, Ian

    2016-01-01

    Rationale: Modulation of breathing by hypoxia accommodates variations in oxygen demand and supply during, for example, sleep and ascent to altitude, but the precise molecular mechanisms of this phenomenon remain controversial. Among the genes influenced by natural selection in high-altitude populations is one for the adenosine monophosphate–activated protein kinase (AMPK) α1-catalytic subunit, which governs cell-autonomous adaptations during metabolic stress. Objectives: We investigated whether AMPK-α1 and/or AMPK-α2 are required for the hypoxic ventilatory response and the mechanism of ventilatory dysfunctions arising from AMPK deficiency. Methods: We used plethysmography, electrophysiology, functional magnetic resonance imaging, and immediate early gene (c-fos) expression to assess the hypoxic ventilatory response of mice with conditional deletion of the AMPK-α1 and/or AMPK-α2 genes in catecholaminergic cells, which compose the hypoxia-responsive respiratory network from carotid body to brainstem. Measurements and Main Results: AMPK-α1 and AMPK-α2 deletion virtually abolished the hypoxic ventilatory response, and ventilatory depression during hypoxia was exacerbated under anesthesia. Rather than hyperventilating, mice lacking AMPK-α1 and AMPK-α2 exhibited hypoventilation and apnea during hypoxia, with the primary precipitant being loss of AMPK-α1 expression. However, the carotid bodies of AMPK-knockout mice remained exquisitely sensitive to hypoxia, contrary to the view that the hypoxic ventilatory response is determined solely by increased carotid body afferent input to the brainstem. Regardless, functional magnetic resonance imaging and c-fos expression revealed reduced activation by hypoxia of well-defined dorsal and ventral brainstem nuclei. Conclusions: AMPK is required to coordinate the activation by hypoxia of brainstem respiratory networks, and deficiencies in AMPK expression precipitate hypoventilation and apnea, even when carotid body

  18. Phosphorylation of hepatic AMP-activated protein kinase and liver kinase B1 is increased after a single oral dose of green tea extract to mice.

    PubMed

    Banerjee, Subhashis; Ghoshal, Sarbani; Porter, Todd D

    2012-12-01

    We have previously shown that green and black tea extracts increase the phosphorylation of AMP-activated protein kinase (AMPK) and HMG-CoA reductase in rat hepatoma cells in culture, concomitant with a decrease in cholesterol synthesis. In the present study, we evaluated the ability of a single oral dose of green or black tea extract to promote the phosphorylation of AMPK, liver kinase B1 (LKB1, an AMPK-kinase), and HMG-CoA reductase in mouse liver. Green tea extract administered by gavage at 50 and 100 mg/kg caused a 2- to 3-fold increase in hepatic AMPK phosphorylation at 3 and 6 hours after dosing and a 1.5- to 2-fold increase in LKB1 phosphorylation at these same time points. The phosphorylation of HMG-CoA reductase at these and later time points was not significantly increased. Black tea administered by gavage at up to 250 mg/kg was ineffective in increasing hepatic AMPK phosphorylation. Both green and black tea extracts increased LKB1 phosphorylation in hepatoma cells in culture at 15 μg/mL, and black tea also increased the phosphorylation of protein kinase A in hepatoma cells. These results suggest that compounds in both tea extracts activate AMPK by activating its upstream kinase, LKB1, and that black tea may do so by first activating protein kinase A, a known kinase for LKB1. Only green tea, at 50 and 100 mg/kg, was able to activate AMPK and LKB1 in mouse liver after oral dosing, suggesting that the polymerized catechins present in black tea do not reach the liver in sufficient concentration to affect AMPK activity.

  19. AMP-activated protein kinase is dispensable for maintaining ATP levels and for survival following inhibition of glycolysis, but promotes tumour engraftment of Ras-transformed fibroblasts

    PubMed Central

    Pelletier, Joffrey; Roux, Danièle; Viollet, Benoit

    2015-01-01

    Lactic acid generated by highly glycolytic tumours is exported by the MonoCarboxylate Transporters, MCT1 and MCT4, to maintain pHi and energy homeostasis. We report that MCT1 inhibition combined with Mct4 gene disruption severely reduced glycolysis and tumour growth without affecting ATP levels. Because of the key role of the 5′-AMP-activated protein kinase (AMPK) in energy homeostasis, we hypothesized that targeting glycolysis (MCT-blockade) in AMPK-null (Ampk−/−) cells should kill tumour cells from ‘ATP crisis’. We show that Ampk−/−-Ras-transformed mouse embryonic fibroblasts (MEFs) maintained ATP levels and viability when glycolysis was inhibited. In MCT-inhibited MEFs treated with OXPHOS inhibitors the ATP level and viability collapsed in both Ampk+/+ and Ampk−/− cells. We therefore propose that the intracellular acidification resulting from lactic acid sequestration mimicks AMPK by blocking mTORC1, a major component of an ATP consuming pathway, thereby preventing ‘ATP crisis’. Finally we showed that genetic disruption of Mct4 and/or Ampk dramatically reduced tumourigenicity in a xenograft mouse model suggesting a crucialrolefor these two actors in establishment of tumours in a nutrient-deprived environment. These findings demonstrated that blockade of lactate transport is an efficient anti-cancer strategy that highlights the potential in targeting Mct4 in a context of impaired AMPK activity. PMID:26059436

  20. Regulatory effect of AMP-activated protein kinase on pulmonary hypertension induced by chronic hypoxia in rats: in vivo and in vitro studies.

    PubMed

    Huang, Xiaoying; Fan, Rong; Lu, Yuanyuan; Yu, Chang; Xu, Xiaomei; Zhang, Xie; Liu, Panpan; Yan, Shuangquan; Chen, Chun; Wang, Liangxing

    2014-06-01

    Activation of AMP-activated protein kinase (AMPK) plays an important role in cardiovascular protection. It can inhibit arterial smooth muscle cell proliferation and cardiac fibroblast collagen synthesis induced by anoxia. However, the role of AMPK-dependent signalling cascades in the pulmonary vascular system is currently unknown. This study aims to determine the effects of AMPK on pulmonary hypertension and pulmonary vessel remodelling induced by hypoxia in rats using in vivo and in vitro studies. In vivo study: pulmonary hypertension, right ventricular hypertrophy and pulmonary vascular remodelling were found in hypoxic rats. Meanwhile, AMPKα1 and phosphorylated AMPKα1 were increased markedly in pulmonary arterioles and lung tissues. Mean pulmonary arterial pressure, index of right ventricular hypertrophy and parameters of pulmonary vascular remodelling, including vessel wall area/total area, density of nuclei in medial smooth muscle cells, and thickness of the medial smooth muscle cell layer were markedly suppressed by AICAR, an AMPK agonist. In vitro study: the expression of AMPKα1 and phosphorylated AMPKα1 was increased in pulmonary artery smooth muscle cells (PASMCs) under hypoxic conditions. The effects of PASMC proliferation stimulated by hypoxia were reinforced by treatment with Compound C, an AMPK inhibitor. AICAR inhibited the proliferation of PASMCs stimulated by hypoxia. These findings suggest that AMPK is involved in the formation of hypoxia-induced pulmonary hypertension and pulmonary vessel remodelling. Up-regulating AMPK can contribute to decreasing pulmonary vessel remodelling and pulmonary hypertension induced by hypoxia.

  1. HIV and Cocaine Impact Glial Metabolism: Energy Sensor AMP-activated protein kinase Role in Mitochondrial Biogenesis and Epigenetic Remodeling

    PubMed Central

    Samikkannu, Thangavel; Atluri, Venkata S. R.; Nair, Madhavan P. N.

    2016-01-01

    HIV infection and cocaine use have been identified as risk factors for triggering neuronal dysfunction. In the central nervous system (CNS), energy resource and metabolic function are regulated by astroglia. Glia is the major reservoir of HIV infection and disease progression in CNS. However, the role of cocaine in accelerating HIV associated energy deficit and its impact on neuronal dysfunction has not been elucidated yet. The aim of this study is to elucidate the molecular mechanism of HIV associated neuropathogenesis in cocaine abuse and how it accelerates the energy sensor AMPKs and its subsequent effect on mitochondrial oxidative phosphorylation (OXPHOS), BRSKs, CDC25B/C, MAP/Tau, Wee1 and epigenetics remodeling complex SWI/SNF. Results showed that cocaine exposure during HIV infection significantly increased the level of p24, reactive oxygen species (ROS), ATP-utilization and upregulated energy sensor AMPKs, CDC25B/C, MAP/Tau and Wee1 protein expression. Increased ROS production subsequently inhibits OCR/ECAR ratio and OXPHOS, and eventually upregulate epigenetics remodeling complex SWI/SNF in CHME-5 cells. These results suggest that HIV infection induced energy deficit and metabolic dysfunction is accelerated by cocaine inducing energy sensor AMPKs, mitochondrial biogenesis and chromatin remodeling complex SWI/SNF activation, which may lead to neuroAIDS disease progression. PMID:27535703

  2. Renoprotective Effects of Metformin are Independent of Organic Cation Transporters 1 &2 and AMP-activated Protein Kinase in the Kidney.

    PubMed

    Christensen, Michael; Jensen, Jonas B; Jakobsen, Steen; Jessen, Niels; Frøkiær, Jørgen; Kemp, Bruce E; Marciszyn, Allison L; Li, Hui; Pastor-Soler, Núria M; Hallows, Kenneth R; Nørregaard, Rikke

    2016-10-26

    The type-2 diabetes drug metformin has proven to have protective effects in several renal disease models. Here, we investigated the protective effects in a 3-day unilateral ureteral obstruction (3dUUO) mouse model. Compared with controls, ureteral obstructed animals displayed increased tubular damage and inflammation. Metformin treatment attenuated inflammation, increased the anti-oxidative response and decreased tubular damage. Hepatic metformin uptake depends on the expression of organic cation transporters (OCTs). To test whether the effects of metformin in the kidney are dependent on these transporters, we tested metformin treatment in OCT1/2(-/-) mice. Even though exposure of metformin in the kidney was severely decreased in OCT1/2(-/-) mice when evaluated with [(11)C]-Metformin and PET/MRI, we found that the protective effects of metformin were OCT1/2 independent when tested in this model. AMP-activated protein kinase (AMPK) has been suggested as a key mediator of the effects of metformin. When using an AMPK-β1 KO mouse model, the protective effects of metformin still occurred in the 3dUUO model. In conclusion, these results show that metformin has a beneficial effect in early stages of renal disease induced by 3dUUO. Furthermore, these effects appear to be independent of the expression of OCT1/2 and AMPK-β1, the most abundant AMPK-β isoform in the kidney.

  3. Renoprotective Effects of Metformin are Independent of Organic Cation Transporters 1 & 2 and AMP-activated Protein Kinase in the Kidney

    PubMed Central

    Christensen, Michael; Jensen, Jonas B.; Jakobsen, Steen; Jessen, Niels; Frøkiær, Jørgen; Kemp, Bruce E.; Marciszyn, Allison L.; Li, Hui; Pastor-Soler, Núria M.; Hallows, Kenneth R.; Nørregaard, Rikke

    2016-01-01

    The type-2 diabetes drug metformin has proven to have protective effects in several renal disease models. Here, we investigated the protective effects in a 3-day unilateral ureteral obstruction (3dUUO) mouse model. Compared with controls, ureteral obstructed animals displayed increased tubular damage and inflammation. Metformin treatment attenuated inflammation, increased the anti-oxidative response and decreased tubular damage. Hepatic metformin uptake depends on the expression of organic cation transporters (OCTs). To test whether the effects of metformin in the kidney are dependent on these transporters, we tested metformin treatment in OCT1/2−/− mice. Even though exposure of metformin in the kidney was severely decreased in OCT1/2−/− mice when evaluated with [11C]-Metformin and PET/MRI, we found that the protective effects of metformin were OCT1/2 independent when tested in this model. AMP-activated protein kinase (AMPK) has been suggested as a key mediator of the effects of metformin. When using an AMPK-β1 KO mouse model, the protective effects of metformin still occurred in the 3dUUO model. In conclusion, these results show that metformin has a beneficial effect in early stages of renal disease induced by 3dUUO. Furthermore, these effects appear to be independent of the expression of OCT1/2 and AMPK-β1, the most abundant AMPK-β isoform in the kidney. PMID:27782167

  4. Theaflavins, dimeric catechins, inhibit peptide transport across Caco-2 cell monolayers via down-regulation of AMP-activated protein kinase-mediated peptide transporter PEPT1.

    PubMed

    Takeda, Junko; Park, Ha-Young; Kunitake, Yuri; Yoshiura, Keiko; Matsui, Toshiro

    2013-06-15

    In the small intestine, peptide transporter 1 (PEPT1) plays a role in the transport of di- and tripeptides. In this study, we investigated whether theaflavins (TFs) affect the absorption of small peptides in human intestinal Caco-2 cells, since TFs do not penetrate through the cells and might be involved in intestinal transport systems. In transport experiments, the transport of glycyl-sarcosine (Gly-Sar, a model molecule for PEPT1 transport) and other dipeptides (Val-Tyr and Ile-Phe) were significantly reduced (P<0.05) in TFs-pretreated cells. In TF 3'-O-gallate-pretreated cells, Western blot analysis revealed attenuated expression of PEPT1 transporter and Gly-Sar transport was completely ameliorated by 10 μM Compound C, an AMP-activated protein kinase (AMPK) inhibitor. In conclusion, the present study demonstrated that TFs inhibit peptide transport across Caco-2 cell monolayers, probably through suppression of AMPK-mediated PEPT1 expression, which should be considered a new bioactivity of TFs in black tea.

  5. Role of the alpha2-isoform of AMP-activated protein kinase in the metabolic response of the heart to no-flow ischemia.

    PubMed

    Zarrinpashneh, Elham; Carjaval, Karla; Beauloye, Christophe; Ginion, Audrey; Mateo, Philippe; Pouleur, Anne-Catherine; Horman, Sandrine; Vaulont, Sophie; Hoerter, Jacqueline; Viollet, Benoit; Hue, Louis; Vanoverschelde, Jean-Louis; Bertrand, Luc

    2006-12-01

    AMP-activated protein kinase (AMPK) is a major sensor and regulator of the energetic state of the cell. Little is known about the specific role of AMPKalpha(2), the major AMPK isoform in the heart, in response to global ischemia. We used AMPKalpha(2)-knockout (AMPKalpha(2)(-/-)) mice to evaluate the consequences of AMPKalpha(2) deletion during normoxia and ischemia, with glucose as the sole substrate. Hemodynamic measurements from echocardiography of hearts from AMPKalpha(2)(-/-) mice during normoxia showed no significant modification compared with wild-type animals. In contrast, the response of hearts from AMPKalpha(2)(-/-) mice to no-flow ischemia was characterized by a more rapid onset of ischemia-induced contracture. This ischemic contracture was associated with a decrease in ATP content, lactate production, glycogen content, and AMPKbeta(2) content. Hearts from AMPKalpha(2)(-/-) mice were also characterized by a decreased phosphorylation state of acetyl-CoA carboxylase during normoxia and ischemia. Despite an apparent worse metabolic adaptation during ischemia, the absence of AMPKalpha(2) does not exacerbate impairment of the recovery of postischemic contractile function. In conclusion, AMPKalpha(2) is required for the metabolic response of the heart to no-flow ischemia. The remaining AMPKalpha(1) cannot compensate for the absence of AMPKalpha(2).

  6. 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

  7. 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

    Lin, Cheng-Hsiu; Kuo, Yueh-Hsiung; Shih, Chun-Ching

    2014-11-04

    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.

  8. Stanniocalcin-1 inhibits renal ischemia/reperfusion injury via an AMP-activated protein kinase-dependent pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AKI is associated with increased morbidity, mortality, and cost of care, and therapeutic options remain limited. Reactive oxygen species are critical for the genesis of ischemic AKI. Stanniocalcin-1 (STC1) suppresses superoxide generation through induction of uncoupling proteins (UCPs), and transgen...

  9. 5'-AMP activated protein kinase α2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase 4.

    PubMed

    Fritzen, Andreas Maechel; Lundsgaard, Anne-Marie; Jeppesen, Jacob; Christiansen, Mette Landau Brabaek; Biensø, Rasmus; Dyck, Jason R B; Pilegaard, Henriette; Kiens, Bente

    2015-11-01

    It is well known that exercise has a major impact on substrate metabolism for many hours after exercise. However, the regulatory mechanisms increasing lipid oxidation and facilitating glycogen resynthesis in the post-exercise period are unknown. To address this, substrate oxidation was measured after prolonged exercise and during the following 6 h post-exercise in 5´-AMP activated protein kinase (AMPK) α2 and α1 knock-out (KO) and wild-type (WT) mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post-exercise recovery, a lower lipid oxidation (P < 0.05) and higher glucose oxidation were observed in AMPKα2 KO (respiratory exchange ratio (RER) = 0.84 ± 0.02) than in WT and AMPKα1 KO (average RER = 0.80 ± 0.01) without genotype differences in muscle malonyl-CoA or free-carnitine concentrations. A similar increase in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO was observed following exercise, which is consistent with AMPKα2 deficiency not affecting the exercise-induced activation of the PDK4 transcriptional regulators HDAC4 and SIRT1. Interestingly, PDK4 protein content increased (63%, P < 0.001) in WT but remained unchanged in AMPKα2 KO. In accordance with the lack of increase in PDK4 protein content, lower (P < 0.01) inhibitory pyruvate dehydrogenase (PDH)-E1α Ser(293) phosphorylation was observed in AMPKα2 KO muscle compared to WT. These findings indicate that AMPKα2 regulates muscle metabolism post-exercise through inhibition of the PDH complex and hence glucose oxidation, subsequently creating conditions for increased fatty acid oxidation.

  10. Suppression of 5'-nucleotidase enzymes promotes AMP-activated protein kinase (AMPK) phosphorylation and metabolism in human and mouse skeletal muscle.

    PubMed

    Kulkarni, Sameer S; Karlsson, Håkan K R; Szekeres, Ferenc; Chibalin, Alexander V; Krook, Anna; Zierath, Juleen R

    2011-10-07

    The 5'-nucleotidase (NT5) family of enzyme dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. We hypothesized that gene silencing of NT5 enzymes to increase the intracellular availability of AMP would increase AMP-activated protein kinase (AMPK) activity and metabolism. We determined the role of cytosolic NT5 in metabolic responses linked to the development of insulin resistance in obesity and type 2 diabetes. Using siRNA to silence NT5C2 expression in cultured human myotubes, we observed a 2-fold increase in the AMP/ATP ratio, a 2.4-fold increase in AMPK phosphorylation (Thr(172)), and a 2.8-fold increase in acetyl-CoA carboxylase phosphorylation (Ser(79)) (p < 0.05). siRNA silencing of NT5C2 expression increased palmitate oxidation by 2-fold in the absence and by 8-fold in the presence of 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside. This was paralleled by an increase in glucose transport and a decrease in glucose oxidation, incorporation into glycogen, and lactate release from NT5C2-depleted myotubes. Gene silencing of NT5C1A by shRNA injection and electroporation in mouse tibialis anterior muscle reduced protein content (60%; p < 0.05) and increased phosphorylation of AMPK (60%; p < 0.05) and acetyl-CoA carboxylase (50%; p < 0.05) and glucose uptake (20%; p < 0.05). Endogenous expression of NT5C enzymes inhibited basal lipid oxidation and glucose transport in skeletal muscle. Reduction of 5'-nucleotidase expression or activity may promote metabolic flexibility in type 2 diabetes.

  11. 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

  12. Skeletal muscle AMP-activated protein kinase γ1H151R overexpression enhances whole body energy homeostasis and insulin sensitivity

    PubMed Central

    Schönke, Milena; Myers, Martin G.; Zierath, Juleen R.

    2015-01-01

    AMP-activated protein kinase (AMPK) is a major sensor of energy homeostasis and stimulates ATP-generating processes such as lipid oxidation and glycolysis in peripheral tissues. The heterotrimeric enzyme consists of a catalytic α-subunit, a β-subunit that is important for enzyme activity, and a noncatalytic γ-subunit that binds AMP and activates the AMPK complex. We generated a skeletal muscle Cre-inducible transgenic mouse model expressing a mutant γ1-subunit (AMPKγ1H151R), resulting in chronic AMPK activation. The expression of the predominant AMPKγ3 isoform in skeletal muscle was reduced in extensor digitorum longus (EDL) muscle (81–83%) of AMPKγ1H151R transgenic mice, whereas the abundance and phosphorylation of the AMPK target acetyl-CoA carboxylase was increased in tibialis anterior muscle. Glycogen content was increased 10-fold in gastrocnemius muscle. Whole body carbohydrate oxidation was increased by 11%, and whereas glucose tolerance was unaffected, insulin sensitivity was increased in AMPKγ1H151R transgenic mice. Furthermore, perigonadal white adipose tissue mass and serum leptin were reduced in female AMPKγ1H151R transgenic mice by 38 and 51% respectively. Conversely, in male AMPKγ1H151R transgenic mice, food intake was increased (14%), but body weight and body composition were unaltered, presumably because of increased energy expenditure. In conclusion, transgenic activation of skeletal muscle AMPKγ1 in this model plays an important sex-specific role in skeletal muscle metabolism and whole body energy homeostasis. PMID:26306597

  13. Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway.

    PubMed

    Song, Young Mi; Lee, Yong-ho; Kim, Ji-Won; Ham, Dong-Sik; Kang, Eun-Seok; Cha, Bong Soo; Lee, Hyun Chul; Lee, Byung-Wan

    2015-01-01

    Metformin activates both PRKA and SIRT1. Furthermore, autophagy is induced by either the PRKA-MTOR-ULK1 or SIRT1-FOXO signaling pathways. We aimed to elucidate the mechanism by which metformin alleviates hepatosteatosis by examining the molecular interplay between SIRT1, PRKA, and autophagy. ob/ob mice were divided into 3 groups: one with ad libitum feeding of a standard chow diet, one with 300 mg/kg intraperitoneal metformin injections, and one with 3 g/d caloric restriction (CR) for a period of 4 wk. Primary hepatocytes or HepG2 cells were treated with oleic acid (OA) plus high glucose in the absence or presence of metformin. Both CR and metformin significantly improved body weight and glucose homeostasis, along with hepatic steatosis, in ob/ob mice. Furthermore, CR and metformin both upregulated SIRT1 expression and also stimulated autophagy induction and flux in vivo. Metformin also prevented OA with high glucose-induced suppression of both SIRT1 expression and SIRT1-dependent activation of autophagy machinery, thereby alleviating intracellular lipid accumulation in vitro. Interestingly, metformin treatment upregulated SIRT1 expression and activated PRKA even after siRNA-mediated knockdown of PRKAA1/2 and SIRT1, respectively. Taken together, these results suggest that metformin alleviates hepatic steatosis through PRKA-independent, SIRT1-mediated effects on the autophagy machinery.

  14. Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis.

    PubMed

    Song, Zhenyuan; Deaciuc, Ion; Zhou, Zhanxiang; Song, Ming; Chen, Theresa; Hill, Daniell; McClain, Craig J

    2007-10-01

    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.

  15. 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

  16. AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin

    PubMed Central

    Aznar, Nicolas; Patel, Arjun; Rohena, Cristina C; Dunkel, Ying; Joosen, Linda P; Taupin, Vanessa; Kufareva, Irina; Farquhar, Marilyn G; Ghosh, Pradipta

    2016-01-01

    Loss of epithelial polarity impacts organ development and function; it is also oncogenic. AMPK, a key sensor of metabolic stress stabilizes cell-cell junctions and maintains epithelial polarity; its activation by Metformin protects the epithelial barrier against stress and suppresses tumorigenesis. How AMPK protects the epithelium remains unknown. Here, we identify GIV/Girdin as a novel effector of AMPK, whose phosphorylation at a single site is both necessary and sufficient for strengthening mammalian epithelial tight junctions and preserving cell polarity and barrier function in the face of energetic stress. Expression of an oncogenic mutant of GIV (cataloged in TCGA) that cannot be phosphorylated by AMPK increased anchorage-independent growth of tumor cells and helped these cells to evade the tumor-suppressive action of Metformin. This work defines a fundamental homeostatic mechanism by which the AMPK-GIV axis reinforces cell junctions against stress-induced collapse and also provides mechanistic insight into the tumor-suppressive action of Metformin. DOI: http://dx.doi.org/10.7554/eLife.20795.001 PMID:27813479

  17. Effects of fucoidan on proliferation, AMP-activated protein kinase, and downstream metabolism- and cell cycle-associated molecules in poorly differentiated human hepatoma HLF cells.

    PubMed

    Kawaguchi, Takumi; Hayakawa, Masako; Koga, Hironori; Torimura, Takuji

    2015-05-01

    Survival rates are low in patients with poorly differentiated hepatocellular carcinoma (HCC). Fucoidan, a sulfated polysaccharide derived from brown seaweed, has anticancer activity; however, the effects of fucoidan on poorly differentiated HCC remain unclear. In this study, we investigated the effects of fucoidan on AMP-activated protein kinase (AMPK), a proliferation regulator, and its downstream metabolism- and cell cycle-related molecules in a poorly differentiated human hepatoma HLF cell line. HLF cells were treated with fucoidan (10, 50, or 100 µg/ml; n=4) or phosphate buffered saline (control; n=4) for 96 h. Proliferation was evaluated by counting cells every 24 h. AMPK, TSC2, mTOR, GSK3β, acetyl-CoA carboxylase (ACC), ATP-citrate lyase, p53, cyclin D1, cyclin-dependent kinase (CDK) 4, and CDK6 expression and/or phosphorylation were examined by immunoblotting 24 h after treatment with 100 µg/ml fucoidan. Cell cycle progression was analyzed by fluorescence-activated cell sorter 48 h after treatment. Treatment with 50 or 100 µg/ml fucoidan significantly and dose- and time-dependently suppressed HLF cell proliferation (P<0.0001). Fucoidan induced AMPK phosphorylation on Ser172 24 h after treatment. Although no differences were seen in expression and phosphorylation levels of TSC2, mTOR, GSK3β, ATP-citrate lyase, and p53 between the control and fucoidan-treated HLF cells, fucoidan induced ACC phosphorylation on Ser79. Moreover, fucoidan decreased cyclin D1, CDK4 and CDK6 expression 24 h after treatment. Furthermore, HLF cells were arrested in the G1/S phase 48 h after fucoidan treatment. We demonstrated that fucoidan suppressed HLF cell proliferation with AMPK phosphorylation. We showed that fucoidan phosphorylated ACC and downregulated cyclin D1, CDK4 and CDK6 expression. Our findings suggest that fucoidan inhibits proliferation through AMPK-associated suppression of fatty acid synthesis and G1/S transition in HLF cells.

  18. The beneficial effects of betaine on dysfunctional adipose tissue and N6-methyladenosine mRNA methylation requires the AMP-activated protein kinase α1 subunit.

    PubMed

    Zhou, Xihong; Chen, Jingqing; Chen, Jin; Wu, Weiche; Wang, Xinxia; Wang, Yizhen

    2015-12-01

    The current study was conducted to determine whether betaine could improve fatty acid oxidation, mitochondrial function and N6-methyladenosine (m(6)A) mRNA methylation in adipose tissue in high-fat-induced mice and how AMP-activated protein kinase α1 subunit (AMPKα1) was involved. AMPKα1 knockout mice and wild-type mice were fed either a low-fat diet, high-fat diet or high-fat diet supplemented with betaine in the drinking water for 8weeks. Our results showed that mitochondrial genes (PGC1α) and β-oxidation-related genes (CPT1a) at protein level were increased in wild-type mice supplemented with betaine when compared with those in mice with high-fat diet. Betaine also decreased FTO expression and improved m(6)A methylation in adipose tissue of wild-type mice with high-fat diet. However, betaine failed to exert the abovementioned effects in AMPKα1 knockout mice. In adipocytes isolated from mice with high-fat diet, betaine treatment increased lipolysis and lipid oxidation. Moreover, betaine decreased FTO expression and increased m(6)A methylation. However, while AMPKα1 was knockdown, no remarkable changes in adipocytes were observed under betaine treatment. Our results indicated that betaine supplementation rectified mRNA hypomethylation and high FTO expression induced by high-fat diet, which may contribute to its beneficial effects on impaired adipose tissue function. Our results suggested that the AMPKα1 subunit is required for the beneficial effects of betaine on dysfunctional adipose tissue and m(6)A methylation. These results may provide the foundation for a mechanism that links m(6)A methylation status in RNA, AMPKα1 phosphorylation and dysfunctional adipose tissue induced by high-fat diet.

  19. AMP-Activated Protein Kinase Attenuates High Salt-Induced Activation of Epithelial Sodium Channels (ENaC) in Human Umbilical Vein Endothelial Cells.

    PubMed

    Zheng, Wei-Wan; Li, Xin-Yuan; Liu, Hui-Bin; Wang, Zi-Rui; Hu, Qing-Qing; Li, Yu-Xia; Song, Bin-Lin; Lou, Jie; Wang, Qiu-Shi; Ma, He-Ping; Zhang, Zhi-Ren

    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 (P O ). 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 P O . 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.

  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. PMID:27635187

  1. AMP-activated protein kinase (AMPK)α2 plays a role in determining the cellular fate of glucose in insulin-resistant mouse skeletal muscle

    PubMed Central

    Lee-Young, R.S.; Bonner, J.S.; Mayes, W.H.; Iwueke, I.; Barrick, B.A.; Hasenour, C.M.; Kang, L.; Wasserman, D.H.

    2014-01-01

    Aims/hypothesis We determined whether: (1) an acute lipid infusion impairs skeletal muscle AMP-activated protein kinase (AMPK)α2 activity, increases inducible nitric oxide synthase (iNOS) and causes peripheral insulin resistance in conscious, unstressed, lean mice; and (2) restoration of AMPKα2 activity during the lipid infusion attenuates the increase in iNOS and reverses the defect in insulin sensitivity in vivo. Methods Chow-fed, 18-week-old C57BL/6J male mice were surgically catheterised. After 5 days they received: (1) a 5 h infusion of 5 ml kg−1 h−1 Intralipid + 6U/h heparin (Lipid treatment) or saline (Control); (2) Lipid treatment or Control, followed by a 2 h hyperinsulinaemic–euglycaemic clamp (insulin clamp; 4 mU kg−1 min−1); and (3) infusion of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) (1 mg kg−1 min−1), or saline during Lipid treatment, followed by a 2 h insulin clamp. In a separate protocol, mice producing a muscle-specific kinase-dead AMPKα2 subunit (α2-KD) underwent an insulin clamp to determine the role of AMPKα2 in insulin-mediated muscle glucose metabolism. Results Lipid treatment decreased AMPKα2 activity, increased iNOS abundance/activation and reduced whole-body insulin sensitivity in vivo. AICAR increased AMPKα2 activity twofold; this did not suppress iNOS or improve whole-body or tissue-specific rates of glucose uptake during Lipid treatment. AICAR caused a marked increase in insulin-mediated glycogen synthesis in skeletal muscle. Consistent with this latter result, lean α2-KD mice exhibited impaired insulinstimulated glycogen synthesis even though muscle glucose uptake was not affected. Conclusions/interpretation Acute induction of insulin resistance via lipid infusion in healthy mice impairs AMPKα2, increases iNOS and causes insulin resistance in vivo. However, these changes do not appear to be interrelated. Rather, a functionally active AMPKα2 subunit is required for insulin

  2. Antidiabetic and Antihyperlipidemic Effects of Clitocybe nuda on Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Chen, Mei-Hsing; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2014-01-01

    The objective of this study was to evaluate the antihyperlipidemic and antihyperglycemic effects and mechanism of the extract of Clitocybe nuda (CNE), in high-fat- (HF-) fed mice. C57BL/6J was randomly divided into two groups: the control (CON) group was fed with a low-fat diet, whereas the experimental group was fed with a HF diet for 8 weeks. Then, the HF group was subdivided into five groups and was given orally CNE (including C1: 0.2, C2: 0.5, and C3: 1.0 g/kg/day extracts) or rosiglitazone (Rosi) or vehicle for 4 weeks. CNE effectively prevented HF-diet-induced increases in the levels of blood glucose, triglyceride, insulin (P < 0.001, P < 0.01, P < 0.05, resp.) and attenuated insulin resistance. By treatment with CNE, body weight gain, weights of white adipose tissue (WAT) and hepatic triacylglycerol content were reduced; moreover, adipocytes in the visceral depots showed a reduction in size. By treatment with CNE, the protein contents of glucose transporter 4 (GLUT4) were significantly increased in C3-treated group in the skeletal muscle. Furthermore, CNE reduces the hepatic expression of glucose-6-phosphatase (G6Pase) and glucose production. CNE significantly increases protein contents of phospho-AMP-activated protein kinase (AMPK) in the skeletal muscle and adipose and liver tissues. Therefore, it is possible that the activation of AMPK by CNE leads to diminished gluconeogenesis in the liver and enhanced glucose uptake in skeletal muscle. It is shown that CNE exhibits hypolipidemic effect in HF-fed mice by increasing ATGL expression, which is known to help triglyceride to hydrolyze. Moreover, antidiabetic properties of CNE occurred as a result of decreased hepatic glucose production via G6Pase downregulation and improved insulin sensitization. Thus, amelioration of diabetic and dyslipidemic states by CNE in HF-fed mice occurred by regulation of GLUT4, G6Pase, ATGL, and AMPK phosphorylation. PMID:24550994

  3. Targeting AMP-activated protein kinase in adipocytes to modulate obesity-related adipokine production associated with insulin resistance and breast cancer cell proliferation

    PubMed Central

    2011-01-01

    Background Adipokines, e.g. TNFα, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another adipokine is protective against breast cancer cell proliferation and insulin resistance. AMP-activated protein kinase (AMPK) activity has been found decreased in visceral adipose tissue of insulin-resistant patients. Lipopolysaccharides (LPS) link systemic inflammation to high fat diet-induced insulin resistance. Modulation of LPS-induced adipokine production by metformin and AMPK activation might represent an alternative way to treat both, insulin resistance and breast cancer. Methods Human preadipocytes obtained from surgical biopsies were expanded and differentiated in vitro into adipocytes, and incubated with siRNA targeting AMPKalpha1 (72 h), LPS (24 h, 100 μg/ml) and/or metformin (24 h, 1 mM) followed by mRNA extraction and analyses. Additionally, the supernatant of preadipocytes or derived-adipocytes in culture for 24 h was used as conditioned media to evaluate MCF-7 breast cancer cell proliferation. Results Conditioned media from preadipocyte-derived adipocytes, but not from undifferentiated preadipocytes, increased MCF-7 cell proliferation (p < 0.01). Induction of IL-6 mRNA by LPS was reduced by metformin (p < 0.01), while the LPS-induced mRNA expression of the naturally occurring anti-inflammatory cytokine interleukin 1 receptor antagonist was increased (p < 0.01). Silencing of AMPKalpha1 enhanced LPS-induced IL-6 and IL-8 mRNA expression (p < 0.05). Conclusions Adipocyte-secreted factors enhance breast cancer cell proliferation, while AMPK and metformin improve the LPS-induced adipokine imbalance. Possibly, AMPK activation may provide a new way not only to improve the obesity-related adipokine profile and insulin

  4. Inflammatory Role of ROS-Sensitive AMP-Activated Protein Kinase in the Hypersensitivity of Lung Vagal C Fibers Induced by Intermittent Hypoxia in Rats

    PubMed Central

    Yang, Chang-Huan; Shen, Yan-Jhih; Lai, Ching Jung; Kou, Yu Ru

    2016-01-01

    Obstructive sleep apnea (OSA), manifested by airway exposure to intermittent hypoxia (IH), is associated with excess reactive oxygen species (ROS) production in airways, airway inflammation, and hyperreactive airway diseases. The cause-effect relationship for these events remains unclear. We investigated the inflammatory role of ROS-sensitive AMP-activated protein kinase (AMPK) in IH-induced airway hypersensitivity mediated by lung vagal C fibers (LVCFs) in rats. Conscious rats were exposed to room air (RA) or IH with or without treatment with N-acetyl-L-cysteine (NAC, an antioxidant), Compound C (an AMPK inhibitor), ibuprofen (a cyclooxygenase inhibitor), or their vehicles. Immediately after exposure (24 h), we found that intravenous capsaicin, phenylbiguanide, or α,β-methylene-ATP evoked augmented LVCF-mediated apneic responses and LVCF afferent responses in rats subjected to IH exposure in comparison with those in RA rats. The potentiating effect of IH on LVCF responses decreased at 6 h after and vanished at 12 h after the termination of IH exposure. The potentiating effect of IH on LVCF-mediated apneic and LVCF afferent responses was significantly attenuated by treatment with NAC, compound C, or ibuprofen, but not by their vehicles. Further biochemical analysis revealed that rats exposed to IH displayed increased lung levels of lipid peroxidation (an index of oxidative stress), AMPK phosphorylation (an index of AMPK activation), and prostaglandin E2 (a cyclooxygenase metabolite), compared with those exposed to RA. IH-induced increase in lipid peroxidation was considerably suppressed by treatment with NAC but not by compound C or ibuprofen. IH-induced increase in AMPK phosphorylation was totally abolished by NAC or compound C but not by ibuprofen. IH-induced increase in prostaglandin E2 was considerably prevented by any of these three inhibitor treatments. The vehicles of these inhibitors exerted no significant effect on the three IH-induced responses. These

  5. Effects of heat stress on serum insulin, adipokines, AMP-activated protein kinase, and heat shock signal molecules in dairy cows.

    PubMed

    Min, Li; Cheng, Jian-bo; Shi, Bao-lu; Yang, Hong-jian; Zheng, Nan; Wang, Jia-qi

    2015-06-01

    Heat stress affects feed intake, milk production, and endocrine status in dairy cows. The temperature-humidity index (THI) is employed as an index to evaluate the degree of heat stress in dairy cows. However, it is difficult to ascertain whether THI is the most appropriate measurement of heat stress in dairy cows. This experiment was conducted to investigate the effects of heat stress on serum insulin, adipokines (leptin and adiponectin), AMP-activated protein kinase (AMPK), and heat shock signal molecules (heat shock transcription factor (HSF) and heat shock proteins (HSP)) in dairy cows and to research biomarkers to be used for better understanding the meaning of THI as a bioclimatic index. To achieve these objectives, two experiments were performed. The first experiment: eighteen lactating Holstein dairy cows were used. The treatments were: heat stress (HS, THI average=81.7, n=9) and cooling (CL, THI average=53.4, n=9). Samples of HS were obtained on August 16, 2013, and samples of CL were collected on April 7, 2014 in natural conditions. The second experiment: HS treatment cows (n=9) from the first experiment were fed for 8 weeks from August 16, 2013 to October 12, 2013. Samples for moderate heat stress, mild heat stress, and no heat stress were obtained, respectively, according to the physical alterations of the THI. Results showed that heat stress significantly increased the serum adiponectin, AMPK, HSF, HSP27, HSP70, and HSP90 (P<0.05). Adiponectin is strongly associated with AMPK. The increases of adiponectin and AMPK may be one of the mechanisms to maintain homeostasis in heat-stressed dairy cows. When heat stress treatment lasted 8 weeks, a higher expression of HSF and HSP70 was observed under moderate heat stress. Serum HSF and HSP70 are sensitive and accurate in heat stress and they could be potential indicators of animal response to heat stress. We recommend serum HSF and HSP70 as meaningful biomarkers to supplement the THI and evaluate moderate heat

  6. Effects of heat stress on serum insulin, adipokines, AMP-activated protein kinase, and heat shock signal molecules in dairy cows*

    PubMed Central

    Min, Li; Cheng, Jian-bo; Shi, Bao-lu; Yang, Hong-jian; Zheng, Nan; Wang, Jia-qi

    2015-01-01

    Heat stress affects feed intake, milk production, and endocrine status in dairy cows. The temperature-humidity index (THI) is employed as an index to evaluate the degree of heat stress in dairy cows. However, it is difficult to ascertain whether THI is the most appropriate measurement of heat stress in dairy cows. This experiment was conducted to investigate the effects of heat stress on serum insulin, adipokines (leptin and adiponectin), AMP-activated protein kinase (AMPK), and heat shock signal molecules (heat shock transcription factor (HSF) and heat shock proteins (HSP)) in dairy cows and to research biomarkers to be used for better understanding the meaning of THI as a bioclimatic index. To achieve these objectives, two experiments were performed. The first experiment: eighteen lactating Holstein dairy cows were used. The treatments were: heat stress (HS, THI average=81.7, n=9) and cooling (CL, THI average=53.4, n=9). Samples of HS were obtained on August 16, 2013, and samples of CL were collected on April 7, 2014 in natural conditions. The second experiment: HS treatment cows (n=9) from the first experiment were fed for 8 weeks from August 16, 2013 to October 12, 2013. Samples for moderate heat stress, mild heat stress, and no heat stress were obtained, respectively, according to the physical alterations of the THI. Results showed that heat stress significantly increased the serum adiponectin, AMPK, HSF, HSP27, HSP70, and HSP90 (P<0.05). Adiponectin is strongly associated with AMPK. The increases of adiponectin and AMPK may be one of the mechanisms to maintain homeostasis in heat-stressed dairy cows. When heat stress treatment lasted 8 weeks, a higher expression of HSF and HSP70 was observed under moderate heat stress. Serum HSF and HSP70 are sensitive and accurate in heat stress and they could be potential indicators of animal response to heat stress. We recommend serum HSF and HSP70 as meaningful biomarkers to supplement the THI and evaluate moderate heat

  7. A Novel Cardioprotective Agent in Cardiac Transplantation: Metformin Activation of AMP-Activated Protein Kinase Decreases Acute Ischemia-Reperfusion Injury and Chronic Rejection

    PubMed Central

    Chin, Jocelyn T.; Troke, Joshua J.; Kimura, Naoyuki; Itoh, Satoshi; Wang, Xi; Palmer, Owen P.; Robbins, Robert C.; Fischbein, Michael P.

    2011-01-01

    The main cause of mortality after the first year from cardiac transplantation is cardiac allograft vasculopathy (CAV), which leads to chronic rejection of the heart. To improve long-term outcomes in cardiac transplantation, treatments to prevent or diminish CAV are actively being researched. Ischemia-reperfusion (I-R) injury has been shown to be the strongest alloantigen-independent factor in the development of CAV. Here, we investigate the use of metformin in murine cardiac transplantation models as a novel cardioprotective agent to limit acute I-R injury and subsequent chronic rejection. We show that metformin treatment activates AMP-activated kinase (AMPK) in vitro and in vivo. In the acute transplantation model, metformin activation of AMPK resulted in significantly decreased apoptosis in cardiac allografts on postoperative day (POD) 1 and 8. In the chronic transplantation model, metformin pretreatment of allografts led to significantly improved graft function and significantly decreased CAV, as measured on POD 52. Taken together, our results in the acute and chronic rejection studies suggest a potential cardioprotective mechanism for metformin; we demonstrate a correlation between metformin-induced decrease in acute I-R injury and metformin-related decrease in chronic rejection. Thus, one of the ways by which metformin and AMPK activation may protect the transplanted heart from chronic rejection is by decreasing initial I-R injury inherent in donor organ preservation and implantation. Our findings suggest novel therapeutic strategies for minimizing chronic cardiac rejection via the use of metformin- and AMPK-mediated pathways to suppress acute I-R injury. PMID:22180679

  8. A novel cardioprotective agent in cardiac transplantation: metformin activation of AMP-activated protein kinase decreases acute ischemia-reperfusion injury and chronic rejection.

    PubMed

    Chin, Jocelyn T; Troke, Joshua J; Kimura, Naoyuki; Itoh, Satoshi; Wang, Xi; Palmer, Owen P; Robbins, Robert C; Fischbein, Michael P

    2011-12-01

    The main cause of mortality after the first year from cardiac transplantation is cardiac allograft vasculopathy (CAV), which leads to chronic rejection of the heart. To improve long-term outcomes in cardiac transplantation, treatments to prevent or diminish CAV are actively being researched. Ischemia-reperfusion (I-R) injury has been shown to be the strongest alloantigen-independent factor in the development of CAV. Here, we investigate the use of metformin in murine cardiac transplantation models as a novel cardioprotective agent to limit acute I-R injury and subsequent chronic rejection. We show that metformin treatment activates AMP-activated kinase (AMPK) in vitro and in vivo. In the acute transplantation model, metformin activation of AMPK resulted in significantly decreased apoptosis in cardiac allografts on postoperative day (POD) 1 and 8. In the chronic transplantation model, metformin pretreatment of allografts led to significantly improved graft function and significantly decreased CAV, as measured on POD 52. Taken together, our results in the acute and chronic rejection studies suggest a potential cardioprotective mechanism for metformin; we demonstrate a correlation between metformin-induced decrease in acute I-R injury and metformin-related decrease in chronic rejection. Thus, one of the ways by which metformin and AMPK activation may protect the transplanted heart from chronic rejection is by decreasing initial I-R injury inherent in donor organ preservation and implantation. Our findings suggest novel therapeutic strategies for minimizing chronic cardiac rejection via the use of metformin- and AMPK-mediated pathways to suppress acute I-R injury.

  9. AMP-activated protein kinase mediates T cell activation-induced expression of FasL and COX-2 via protein kinase C theta-dependent pathway in human Jurkat T leukemia cells.

    PubMed

    Lee, Jung Yeon; Choi, A-Young; Oh, Young Taek; Choe, Wonchae; Yeo, Eui-Ju; Ha, Joohun; Kang, Insug

    2012-06-01

    AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis, is known to be activated during T cell activation. T cell activation by T cell receptor (TCR) engagement or its pharmacological mimics, PMA plus ionomycin (PMA/Io), induces immunomodulatory FasL and cyclooxygenase-2 (COX-2) expression. In this study, we examined the role and mechanisms of AMPK in PMA/Io-induced expression of FasL and COX-2 in Jurkat T human leukemic cells. Inhibition of AMPK by a pharmacological agent, compound C, or AMPKα1 siRNA suppressed expression of FasL and COX-2 mRNAs and proteins in PMA/Io-activated Jurkat cells. It also reduced secretion of FasL protein and prostaglandin E2, a main product of COX-2, in Jurkat cells and peripheral blood lymphocytes activated with PMA/Io or monoclonal anti-CD3 plus anti-CD28. Consistently, inhibition of AMPK blocked promoter activities of FasL and COX-2 in activated Jurkat cells. As protein kinase C theta (PKCθ) is a central molecule for TCR signaling, we examined any possible cross-talk between AMPK and PKCθ in activated T cells. Of particular importance, we found that inhibition of AMPK blocked phosphorylation and activation of PKCθ, suggesting that AMPK is an upstream kinase of PKCθ. Moreover, we showed that AMPK was directly associated with PKCθ and phosphorylated Thr538 of PKCθ in PMA/Io-stimulated Jurkat cells. We also showed that inhibition of PKCθ by rottlerin or dominant negative PKCθ reduced AMPK-mediated transcriptional activation of NF-AT and AP-1 in activated Jurkat cells. Taken together, these results suggest that AMPK regulates expression of FasL and COX-2 via the PKCθ and NF-AT and AP-1 pathways in activated Jurkat cells.

  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.

  11. 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.

  12. Heat stress acutely activates insulin-independent glucose transport and 5′-AMP-activated protein kinase prior to an increase in HSP72 protein in rat skeletal muscle

    PubMed Central

    Goto, Ayumi; Egawa, Tatsuro; Sakon, Ichika; Oshima, Rieko; Ito, Kanata; Serizawa, Yasuhiro; Sekine, Keiichi; Tsuda, Satoshi; Goto, Katsumasa; Hayashi, Tatsuya

    2015-01-01

    Heat stress (HS) stimulates heat shock protein (HSP) 72 mRNA expression, and the period after an increase in HSP72 protein is characterized by enhanced glucose metabolism in skeletal muscle. We have hypothesized that, prior to an increase in the level of HSP72 protein, HS activates glucose metabolism by acutely stimulating 5′-AMP-activated protein kinase (AMPK). Rat epitrochlearis muscle was isolated and incubated either with or without HS (42°C) for 10 and 30 min. HS for 30 min led to an increase in the level of Hspa1a and Hspa1b mRNA but did not change the amount of HSP72 protein. However, HS for both 10 and 30 min led to a significant increase in the rate of 3-O-methyl-d-glucose (3MG) transport, and the stimulatory effect of 3MG transport was completely blocked by cytochalasin B. HS-stimulated 3MG transport was also inhibited by dorsomorphin but not by wortmannin. HS led to a decrease in the concentration of ATP, phosphocreatine, and glycogen, to an increase in the level of phosphorylation of AMPKα Thr172, and to an increase in the activity of both AMPKα1 and AMPKα2. HS did not affect the phosphorylation status of insulin receptor signaling or Ca2+/calmodulin-dependent protein kinase II. These results suggest that HS acts as a rapid stimulator of insulin-independent glucose transport, at least in part by stimulating AMPK via decreased energy status. Although further research is warranted, heat treatment of skeletal muscle might be a promising method to promote glucose metabolism acutely. PMID:26542263

  13. Mogrol Derived from Siraitia grosvenorii Mogrosides Suppresses 3T3-L1 Adipocyte Differentiation by Reducing cAMP-Response Element-Binding Protein Phosphorylation and Increasing AMP-Activated Protein Kinase Phosphorylation

    PubMed Central

    Harada, Naoki; Ishihara, Mikako; Horiuchi, Hiroko; Ito, Yuta; Tabata, Hiromitsu; Suzuki, Yasushi A.; Nakano, Yoshihisa; Yamaji, Ryoichi; Inui, Hiroshi

    2016-01-01

    This study investigated the effects of mogrol, an aglycone of mogrosides from Siraitia grosvenorii, on adipogenesis in 3T3-L1 preadipocytes. Mogrol, but not mogrosides, suppressed triglyceride accumulation by affecting early (days 0–2) and late (days 4–8), but not middle (days 2–4), differentiation stages. At the late stage, mogrol increased AMP-activated protein kinase (AMPK) phosphorylation and reduced glycerol-3-phosphate dehydrogenase activity. At the early stage, mogrol promoted AMPK phosphorylation, inhibited the induction of CCAAT/enhancer-binding protein β (C/EBPβ; a master regulator of adipogenesis), and reduced 3T3-L1 cell contents (e.g., clonal expansion). In addition, mogrol, but not the AMPK activator AICAR, suppressed the phosphorylation and activity of the cAMP response element-binding protein (CREB), which regulates C/EBPβ expression. These results indicated that mogrol suppressed adipogenesis by reducing CREB activation in the initial stage of cell differentiation and by activating AMPK signaling in both the early and late stages of this process. PMID:27583359

  14. Metformin inhibits advanced glycation end products (AGEs)-induced growth and VEGF expression in MCF-7 breast cancer cells by suppressing AGEs receptor expression via AMP-activated protein kinase.

    PubMed

    Ishibashi, Y; Matsui, T; Takeuchi, M; Yamagishi, S

    2013-05-01

    Metformin use has been reported to decrease breast cancer incidence and mortality in diabetic patients. We have previously shown that advanced glycation end products (AGEs) and their receptor (RAGE) interaction stimulate growth and/or migration of pancreatic cancer and melanoma cells. However, effects of metformin on AGEs-RAGE axis in breast cancers remain unknown. We examined here whether and how metformin could block the AGEs-induced growth and vascular endothelial growth factor (VEGF) expression in MCF-7 breast cancer cells. Cell proliferation was measured with an electron coupling reagent WST-1 based colorimetric assay. Gene expression level was evaluated by real-time reverse-transcription polymerase chain reactions. AGEs significantly increased cell proliferation of MCF-7 cells, which was completely prevented by the treatment with 0.01 or 0.1 mM metformin or anti-RAGE antibodies. Furthermore, metformin at 0.01 mM completely suppressed the AGEs-induced upregulation of RAGE and VEGF mRNA levels in MCF-7 cells. An inhibitor of AMP-activated protein kinase, compound C significantly blocked the growth-inhibitory and RAGE and VEGF suppressing effects of metformin in AGEs-exposed MCF-7 cells. Our present study suggests that metformin could inhibit the AGEs-induced growth and VEGF expression in MCF-7 breast cancer cells by suppressing RAGE gene expression via AMP-activated protein kinase pathway. Metformin may protect against breast cancer expansion in diabetic patients by blocking the AGEs-RAGE axis.

  15. Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators

    PubMed Central

    Rajamohan, Francis; Reyes, Allan R.; Frisbie, Richard K.; Hoth, Lise R.; Sahasrabudhe, Parag; Magyar, Rachelle; Landro, James A.; Withka, Jane M.; Caspers, Nicole L.; Calabrese, Matthew F.; Ward, Jessica; Kurumbail, Ravi G.

    2015-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1β1γ1, α1β2γ1, α1β2γ3, α2β1γ1, α2β2γ1 and α2β2γ3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were ∼25-fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr172/174). However, α2-complexes were more readily activated by AMP than α1-complexes. Compared with β1-containing heterotrimers, β2-containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases. PMID:26635351

  16. Activation of AMP-activated protein kinase signaling pathway by adiponectin and insulin in mouse adipocytes: requirement of acyl-CoA synthetases FATP1 and Acsl1 and association with an elevation in AMP/ATP ratio.

    PubMed

    Liu, Qingqing; Gauthier, Marie-Soleil; Sun, Lei; Ruderman, Neil; Lodish, Harvey

    2010-11-01

    Adiponectin activates AMP-activated protein kinase (AMPK) in adipocytes, but the underlying mechanism remains unclear. Here we tested the hypothesis that AMP, generated in activating fatty acids to their CoA derivatives, catalyzed by acyl-CoA synthetases, is involved in AMPK activation by adiponectin. Moreover, in adipocytes, insulin affects the subcellular localization of acyl-CoA synthetase FATP1. Thus, we also tested whether insulin activates AMPK in these cells and, if so, whether it activates through a similar mechanism. We examined these hypotheses by measuring the AMP/ATP ratio and AMPK activation on adiponectin and insulin stimulation and after knocking down acyl-CoA synthetases in adipocytes. We show that adiponectin activation of AMPK is accompanied by an ∼2-fold increase in the cellular AMP/ATP ratio. Moreover, FATP1 and Acsl1, the 2 major acyl-CoA synthetase isoforms in adipocytes, are essential for AMPK activation by adiponectin. We also show that after 40 min. insulin activated AMPK in adipocytes, which was coupled with a 5-fold increase in the cellular AMP/ATP ratio. Knockdown studies show that FATP1 and Acsl1 are required for these processes, as well as for stimulation of long-chain fatty acid uptake by adiponection and insulin. These studies demonstrate that a change in cellular energy state is associated with AMPK activation by both adiponectin and insulin, which requires the activity of FATP1 and Acsl1.

  17. Activation of AMP-activated protein kinase by kainic acid mediates brain-derived neurotrophic factor expression through a NF-kappaB dependent mechanism in C6 glioma cells

    SciTech Connect

    Yoon, Hana; Oh, Young Taek; Lee, Jung Yeon; Choi, Ji Hyun; Lee, Ju Hie; Baik, Hyung Hwan; Kim, Sung Soo; Choe, Wonchae; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug

    2008-07-04

    AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca{sup 2+}/calmodulin-dependent protein kinase kinase (CaMKK) {beta}. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPK{alpha}1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-{kappa}B) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-{kappa}B activation.

  18. Fraxetin Induces Heme Oxygenase-1 Expression by Activation of Akt/Nrf2 or AMP-activated Protein Kinase α/Nrf2 Pathway in HaCaT Cells

    PubMed Central

    Kundu, Juthika; Chae, In Gyeong; Chun, Kyung-Soo

    2016-01-01

    Background Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative, has been reported to possess antioxidative, anti-inflammatory and neuroprotective effects. A number of recent observations suggest that the induction of heme oxygenase-1 (HO-1) inhibits inflammation and tumorigenesis. In the present study, we determined the effect of fraxetin on HO-1 expression in HaCaT human keratinocytes and investigated its underlying molecular mechanisms. Methods Reverse transcriptase-PCR and Western blot analysis were performed to detect HO-1 mRNA and protein expression, respectively. Cell viability was measured by the MTS test. The induction of intracellular reactive oxygen species (ROS) by fraxetin was evaluated by 2′,7′-dichlorofluorescin diacetate staining. Results Fraxetin upregulated mRNA and protein expression of HO-1. Incubation with fraxetin induced the localization of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus and increased the antioxidant response element-reporter gene activity. Fraxetin also induced the phosphorylation of Akt and AMP-activated protein kinase (AMPK)α and diminished the expression of phosphatase and tensin homolog, a negative regulator of Akt. Pharmacological inhibition of Akt and AMPKα abrogated fraxetin-induced expression of HO-1 and nuclear localization of Nrf2. Furthermore, fraxetin generated ROS in a concentration-dependent manner. Conclusions Fraxetin induces HO-1 expression through activation of Akt/Nrf2 or AMPKα/Nrf2 pathway in HaCaT cells. PMID:27722139

  19. Characterization of the effects of metformin on porcine oocyte meiosis and on AMP-activated protein kinase activation in oocytes and cumulus cells.

    PubMed

    Bilodeau-Goeseels, Sylvie; Magyara, Nora; Collignon, Coralie

    2014-05-01

    The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.

  20. S-allyl cysteine attenuates free fatty acid-induced lipogenesis in human HepG2 cells through activation of the AMP-activated protein kinase-dependent pathway.

    PubMed

    Hwang, Yong Pil; Kim, Hyung Gyun; Choi, Jae Ho; Do, Minh Truong; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2013-08-01

    S-Allyl cysteine (SAC), a nontoxic garlic compound, has a variety of pharmacological properties, including antioxidant and hepatoprotective properties. In this report, we provide evidence that SAC prevented free fatty acid (FFA)-induced lipid accumulation and lipotoxicity in hepatocytes. SAC significantly reduced FFA-induced generation of reactive oxygen species, caspase activation and subsequent cell death. Also, SAC mitigated total cellular lipid and triglyceride accumulation in steatotic HepG2 cells. SAC significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in HepG2 cells. Additionally, SAC down-regulated the levels of sterol regulatory element binding protein-1 (SREBP-1) and its target genes, including ACC and fatty acid synthase. Use of a specific inhibitor showed that SAC activated AMPK via calcium/calmodulin-dependent kinase kinase (CaMKK) and silent information regulator T1. Our results demonstrate that SAC activates AMPK through CaMKK and inhibits SREBP-1-mediated hepatic lipogenesis. Therefore, SAC has therapeutic potential for preventing nonalcoholic fatty liver disease.

  1. Melatonin reverses flow shear stress-induced injury in bone marrow mesenchymal stem cells via activation of AMP-activated protein kinase signaling.

    PubMed

    Yang, Yang; Fan, Chongxi; Deng, Chao; Zhao, Lin; Hu, Wei; Di, Shouyin; Ma, Zhiqiang; Zhang, Yu; Qin, Zhigang; Jin, Zhenxiao; Yan, Xiaolong; Jiang, Shuai; Sun, Yang; Yi, Wei

    2016-03-01

    Tissue-engineered heart valves (TEHVs) are a promising treatment for valvular heart disease, although their application is limited by high flow shear stress (FSS). Melatonin has a wide range of physiological functions and is currently under clinical investigation for expanded applications; moreover, extensive protective effects on the cardiovascular system have been reported. In this study, we investigated the protection conferred by melatonin supplementation against FSS-induced injury in bone marrow mesenchymal stem cells (BMSCs) and elucidated the potential mechanism in this process. Melatonin markedly reduced BMSC apoptotic death in a concentration-dependent manner while increasing the levels of transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and B-cell lymphoma 2 (Bcl2), and decreasing those of Bcl-2-associated X protein (Bax), p53 upregulated modulator of apoptosis (PUMA), and caspase 3. Notably, melatonin exerted its protective effects by upregulating the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), which promotes acetyl-CoA carboxylase (ACC) phosphorylation. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked the anti-FSS injury (anti-FSSI) effects of melatonin. Inhibition of AMPK by Compound C also counteracted the protective effects of melatonin, suggesting that melatonin reverses FSSI in BMSCs through the AMPK-dependent pathway. Overall, our findings indicate that melatonin contributes to the amelioration of FSS-induced BMSC injury by activating melatonin receptors and AMPK/ACC signaling. Our findings may provide a basis for the design of more effective strategies that promote the use of TEHCs in patients.

  2. 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.

  3. A new role for AMP-activated protein kinase in the circadian regulation of L-type voltage-gated calcium channels in late-stage embryonic retinal photoreceptors.

    PubMed

    Huang, Cathy C Y; Shi, Liheng; Lin, Chia-Hung; Kim, Andy Jeesu; Ko, Michael L; Ko, Gladys Y-P

    2015-11-01

    AMP-activated protein kinase (AMPK) is a cellular energy sensor, which is activated when the intracellular ATP production decreases. The activities of AMPK display circadian rhythms in various organs and tissues, indicating that AMPK is involved in the circadian regulation of cellular metabolism. In vertebrate retina, the circadian clocks regulate many aspects of retinal function and physiology, including light/dark adaption, but whether and how AMPK was involved in the retinal circadian rhythm was not known. We hypothesized that the activation of AMPK (measured as phosphorylated AMPK) in the retina was under circadian control, and AMPK might interact with other intracellular signaling molecules to regulate photoreceptor physiology. We combined ATP assays, western blots, immunostaining, patch-clamp recordings, and pharmacological treatments to decipher the role of AMPK in the circadian regulation of photoreceptor physiology. We found that the overall retinal ATP content displayed a diurnal rhythm that peaked at early night, which was nearly anti-phase to the diurnal and circadian rhythms of AMPK phosphorylation. AMPK was also involved in the circadian phase-dependent regulation of photoreceptor L-type voltage-gated calcium channels (L-VGCCs), the ion channel essential for sustained neurotransmitter release. The activation of AMPK dampened the L-VGCC currents at night with a corresponding decrease in protein expression of the L-VGCCα1 pore-forming subunit, while inhibition of AMPK increased the L-VGCC current during the day. AMPK appeared to be upstream of extracellular-signal-regulated kinase and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) but downstream of adenylyl cyclase in regulating the circadian rhythm of L-VGCCs. Hence, as a cellular energy sensor, AMPK integrates into the cell signaling network to regulate the circadian rhythm of photoreceptor physiology. We found that in chicken embryonic retina, the activation of AMP-activated protein

  4. Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells.

    PubMed

    Cho, Sung-Yun; Lee, Hyo-Jeong; Lee, Hyo-Jung; Jung, Deok-Beom; Kim, Hyunseok; Sohn, Eun Jung; Kim, Bonglee; Jung, Ji Hoon; Kwon, Byoung-Mog; Kim, Sung-Hoon

    2013-01-01

    Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPK α blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPK α in hypoxic SW620 cells, implying cross-talk between ERK and AMPK α . Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1 α and Akt/mTOR and the activation of AMPK α and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPK α in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPK α and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.

  5. Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells

    PubMed Central

    Cho, Sung-Yun; Lee, Hyo-Jeong; Lee, Hyo-Jung; Jung, Deok-Beom; Kim, Hyunseok; Sohn, Eun Jung; Kim, Bonglee; Jung, Ji Hoon; Kwon, Byoung-Mog; Kim, Sung-Hoon

    2013-01-01

    Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPKα blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPKα in hypoxic SW620 cells, implying cross-talk between ERK and AMPKα. Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1α and Akt/mTOR and the activation of AMPKα and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPKα in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPKα and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells. PMID:23589723

  6. Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes*

    PubMed Central

    Im, Ilkyun; Jang, Mi-jin; Park, Seung Ju; Lee, Sang-Hee; Choi, Jin-Ho; Yoo, Han-Wook; Kim, Seyun; Han, Yong-Mahn

    2015-01-01

    A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD+/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver. PMID:26491018

  7. Stimulation of AMP-activated protein kinase and enhancement of basal glucose uptake in muscle cells by quercetin and quercetin glycosides, active principles of the antidiabetic medicinal plant Vaccinium vitis-idaea.

    PubMed

    Eid, Hoda M; Martineau, Louis C; Saleem, Ammar; Muhammad, Asim; Vallerand, Diane; Benhaddou-Andaloussi, Ali; Nistor, Lidia; Afshar, Arvind; Arnason, John T; Haddad, Pierre S

    2010-07-01

    Several medicinal plants that stimulate glucose uptake in skeletal muscle cells were identified from among species used by the Cree of Eeyou Istchee of northern Quebec to treat symptoms of diabetes. This study aimed to elucidate the mechanism of action of one of these products, the berries of Vaccinium vitis idaea, as well as to isolate and identify its active constituents using a classical bioassay-guided fractionation approach. Western immunoblot analysis in C2C12 muscle cells revealed that the ethanol extract of the berries stimulated the insulin-independent AMP-activated protein kinase (AMPK) pathway. The extract mildly inhibited ADP-stimulated oxygen consumption in isolated mitochondria, an effect consistent with metabolic stress and the ensuing stimulation of AMPK. This mechanism is highly analogous to that of Metformin. Fractionation guided by glucose uptake activity resulted in the isolation of ten compounds. The two most active, quercetin-3-O-glycosides, enhanced glucose uptake by 38-59% (50 muM; 18 h treatment) in the absence of insulin. Quercetin aglycone, a minor constituent, stimulated uptake by 37%. The quercetin glycosides and the aglycone stimulated the AMPK pathway at concentrations of 25-100 muM, but only the aglycone inhibited ATP synthase in isolated mitochondria (by 34 and 79% at 25 and 100 muM, respectively). This discrepancy suggests that the activity of the glycosides may require hydrolysis to the aglycone form. These findings indicate that quercetin and quercetin 3-O-glycosides are responsible for the antidiabetic activity of V. vitis crude berry extract mediated by AMPK. These common plant products may thus have potential applications for the prevention and treatment of insulin resistance and other metabolic diseases.

  8. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    SciTech Connect

    Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  9. AMP-activated protein kinase attenuates oxLDL uptake in macrophages through PP2A/NF-κB/LOX-1 pathway.

    PubMed

    Chen, Bo; Li, Jin; Zhu, Haibo

    2016-10-01

    The differentiation of macrophages into lipid-laden foam cells is a hallmark in early-stage atherosclerosis. The developmental role of adenosine monophosphate-activated protein kinase (AMPK) in a transformation of foam cells, especially in macrophage cholesterol uptake that remains undetermined. Here we demonstrate that AMPK activation in response to IMM-H007 or AICAR resulted in a decrease in macrophage cholesterol uptake and thus inhibited foam cell formation in macrophages mediated by oxidized low-density lipoprotein (oxLDL). This functional change was caused by a downregulation of mRNA and protein expression of LOX-1 but not other scavenger receptors, including scavenger receptor-A (SR-A), CD36 and scavenger receptor-BI (SR-BI). The expression of LOX-1 was regulated by AMPK activation induced decreased phosphorylation of nuclear transcription factor NF-κB, since siRNA interference or dominant negative AMPK overexpression significantly promotes Ser536 dephosphorylation of NF-κB p65 and thus increases LOX-1 expression. Moreover, pharmacological AMPK activation was shown to promote protein phosphatase 2A (PP2A) activity and the specific PP2A inhibitor, okadaic acid, could prevent the effects of IMM-H007 or AICAR on NF-κB and LOX-1. In vivo, pharmacological AMPK activation reduced the lesion size of atherosclerosis and the expression of LOX-1 in aortas in apolipoprotein E-deficient mice. Our current findings suggest a novel mechanism of LOX-1 regulation by AMPK to attenuate macrophage oxLDL uptake and atherosclerosis.

  10. Autophagy activation: a novel mechanism of atorvastatin to protect mesenchymal stem cells from hypoxia and serum deprivation via AMP-activated protein kinase/mammalian target of rapamycin pathway.

    PubMed

    Zhang, Qian; Yang, Yue-Jin; Wang, Hong; Dong, Qiu-Ting; Wang, Tian-Jie; Qian, Hai-Yan; Xu, Hui

    2012-05-20

    Autophagy is a complex "self-eating" process and could be utilized for cell survival under stresses. Statins, which could reduce apoptosis in mesenchymal stem cells (MSCs) during both ischemia and hypoxia/serum deprivation (H/SD), have been proved to induce autophagy in some cell lines. We have previously shown that atorvastatin (ATV) could regulate AMP-activated protein kinase (AMPK), a positive modulator of autophagy, in MSCs. Thus, we hypothesized that autophagy activation through AMPK and its downstream molecule mammalian target of rapamycin (mTOR) may be a novel mechanism of ATV to protect MSCs from apoptosis during H/SD. Here, we demonstrated that H/SD induced autophagy in MSCs significantly as identified by increasing acidic vesicular organelle-positive cells, type II of light chain 3 (LC3-II) expression, and autophagosome formation. The levels of H/SD-induced apoptosis were increased by autophagy inhibitor 3-methyladenine (3-MA) while decreased by rapamycin, an autophagic inducer. ATV further enhanced the autophagic activity observed in MSCs exposed to H/SD. Treatment with 3-MA attenuated ATV-induced autophagy and abrogated the protective effects of ATV on MSC apoptosis, while rapamycin failed to cause additional effects on either autophagy or apoptosis compared with ATV alone. The phosphorylation of AMPK was upregulated whereas the phosphorylation of mTOR was downregulated in ATV-treated MSCs, which were both attenuated by AMPK inhibitor compound C. Further, treatment with compound C reduced the ATV-induced autophagy in MSCs under H/SD. These data suggest that autophagy plays a protective role in H/SD-induced apoptosis of MSCs, and ATV could effectively activate autophagy via AMPK/mTOR pathway to enhance MSC survival during H/SD.

  11. 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

  12. AMP-activated protein kinase is required for cell survival and growth in HeLa-S3 cells in vivo.

    PubMed

    Song, Xuhong; Huang, Dongyang; Liu, Yanmin; Pan, Xiaokang; Zhang, Jing; Liang, Bin

    2014-06-01

    Activation of the AMP-dependent protein kinase (AMPK) is linked to cancer cell survival in a variety of cancer cell lines, particularly under conditions of stress. As a potent activator of AMPK, metformin has become a hot topic of discussion for its effect on cancer cell. Here, we report that AMPK activated by metformin promotes HeLa-S3 cell survival and growth in vivo. Our results show that metformin inhibited cell proliferation in MCF-7 cells, but not in LKB1-deficient HeLa-S3 cells. Re-expression of LKB-1 in HeLa-S3 cells restored the growth inhibitory effect of metformin, indicating a requirement for LKB-1 in metformin-induced growth inhibition. Moreover, AMPK activation exerted a protective effect in HeLa-S3 cells by relieving ER stress, modulating ER Ca(2+) storage, and finally contributing to cellular adaptation and resistance to apoptosis. Our findings identify a link between AMPK activation and cell survival in HeLa-S3 cells, which demonstrates a beneficial effect of AMPK activated by metformin in cancer cell, and suggests a discrete re-evaluation on the role of metformin/AMPK activation on tumor cell growth, proliferation, and on clinical application in cancer therapy.

  13. The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis

    SciTech Connect

    Li, Wei; Fu, Jianfang; Zhang, Shun; Zhao, Jie; Xie, Nianlin; Cai, Guoqing

    2015-06-01

    Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from the elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli–germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli–germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation. - Highlights: • Intermittent treatment with BTZ caused fertility impairment in adult mice. • BTZ treatment elicited apoptosis during early phase of testicular recovery. • Up-regulation of oxidative stress by BTZ treatment

  14. Theaflavins enhance intestinal barrier of Caco-2 Cell monolayers through the expression of AMP-activated protein kinase-mediated Occludin, Claudin-1, and ZO-1.

    PubMed

    Park, Ha-Young; Kunitake, Yuri; Hirasaki, Naoto; Tanaka, Mitsuru; Matsui, Toshiro

    2015-01-01

    We investigated the effect of theaflavins (TFs) on membrane barrier of Caco-2 cells. For fluorescein-transport experiments, the apparent permeability (Papp) of fluorescein in Caco-2 cells pretreated with 20 μM TFs were significantly decreased compared with that in untreated cells. Although the respective monomeric catechins did not show any Papp reduction, purpurogallin pretreatment resulted in a significant Papp reduction similar to that of TF-3'-O-gallate (TF3'G) pretreatment. This indicates that the benzotropolone moiety may play a crucial role in the Papp reduction or tight junction (TJ)-closing effect induced by TFs. In TF-3'-O-gallate-pretreated Caco-2 cells, fluorescein transport was completely restored by compound C (AMPK inhibitor). In addition, TF3'G significantly increased both the mRNA and protein expression of TJ-related proteins (occludin, claudin-1, and ZO-1) as well as the phosphorylation of AMPK. It was, thus, concluded that TFs could enhance intestinal barrier function by increasing the expression of TJ-related proteins through the activation of AMPK in Caco-2 cells.

  15. Grape seed proanthocyanidin extracts enhance endothelial nitric oxide synthase expression through 5'-AMP activated protein kinase/Surtuin 1-Krüpple like factor 2 pathway and modulate blood pressure in ouabain induced hypertensive rats.

    PubMed

    Cui, Xiaopei; Liu, Xiangju; Feng, Hua; Zhao, Shaohua; Gao, Haiqing

    2012-01-01

    Grape seed proanthocyanidin extracts (GSPE) belonging to polyphenols, possess various biological effects including anti-inflammation, anti-oxidant, anti-aging, anti-atherosclerosis, etc. GSPE is potential in regulating endothelial function. However, the underlying mechanism is not clear yet. In this study, by small interfering RNA (siRNA) knocking down, we proved that GSPE increase endothelial nitric oxide synthase (eNOS) expression in human umbilical vessel cells (HUVECs) in vitro, which was attributed to its transcription factor Krüpple like factor 2 (KLF2) induction. Furthermore, GSPE activate 5'-AMP activated protein kinase (AMPK) and increase surtuin 1 (SIRT1) protein level, critical for KLF2 induction. We also illuminated the role of GSPE in hypertension treatment. By chronic administration of GSPE in ouabain induced hypertensive rats model, we access the effect of GSPE on blood pressure regulation and the possible mechanisms involved. After 5 weeks feeding, GSPE significantly block the ouabain induced blood pressure increase. The aortic NO production impaired by ouabain was improved. In conclusion, GSPE increase eNOS expression and NO production in an AMPK/SIRT1 dependent manner through KLF2 induction, and attenuate ouabain induced hypertension.

  16. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    SciTech Connect

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 {mu}g/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  17. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase.

    PubMed

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

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

    PubMed

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

    2015-10-01

    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 Thr(172) phosphorylation, α-isoform-specific AMPK activity, and 3-O-methylglucose (3MG) transport. In contrast, caffeine inhibited basal and contraction-stimulated Akt Ser(473) 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.

  19. Hydrogen-rich water attenuates amyloid β-induced cytotoxicity through upregulation of Sirt1-FoxO3a by stimulation of AMP-activated protein kinase in SK-N-MC cells.

    PubMed

    Lin, Chih-Li; Huang, Wen-Nung; Li, Hsin-Hua; Huang, Chien-Ning; Hsieh, Sam; Lai, Copper; Lu, Fung-Jou

    2015-10-05

    Amyloid β (Aβ) peptides are identified in cause of neurodegenerative diseases such as Alzheimer's disease (AD). Previous evidence suggests Aβ-induced neurotoxicity is linked to the stimulation of reactive oxygen species (ROS) production. The accumulation of Aβ-induced ROS leads to increased mitochondrial dysfunction and triggers apoptotic cell death. This suggests antioxidant therapies may be beneficial for preventing ROS-related diseases such as AD. Recently, hydrogen-rich water (HRW) has been proven effective in treating oxidative stress-induced disorders because of its ROS-scavenging abilities. However, the precise molecular mechanisms whereby HRW prevents neuronal death are still unclear. In the present study, we evaluated the putative pathways by which HRW protects against Aβ-induced cytotoxicity. Our results indicated that HRW directly counteracts oxidative damage by neutralizing excessive ROS, leading to the alleviation of Aβ-induced cell death. In addition, HRW also stimulated AMP-activated protein kinase (AMPK) in a sirtuin 1 (Sirt1)-dependent pathway, which upregulates forkhead box protein O3a (FoxO3a) downstream antioxidant response and diminishes Aβ-induced mitochondrial potential loss and oxidative stress. Taken together, our findings suggest that HRW may have potential therapeutic value to inhibit Aβ-induced neurotoxicity.

  20. 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.

  1. Role of AMP-activated protein kinase activators in antiproliferative multi-drug pituitary tumour therapies: effects of combined treatments with compounds affecting the mTOR-p70S6 kinase axis in cultured pituitary tumour cells.

    PubMed

    Tulipano, G; Faggi, L; Cacciamali, A; Spinello, M; Cocchi, D; Giustina, A

    2015-01-01

    AMP-activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP levels and elevate AMP levels. We have recently shown that AMPK can represent a valid target for improving the medical treatment of growth hormone (GH)-secreting pituitary adenomas and the effects of its activation or inhibition in pituitary tumour cells are worthy of further characterisation. We aimed to determine whether AMPK may have a role in combined antiproliferative therapies based on multiple drugs targeting cell anabolic functions at different levels in pituitary tumour cells to overcome the risk of cell growth escape phenomena. Accordingly, we tried to determine whether a rationale exists in combining compounds activating AMPK with compounds targeting the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR/p70S6K signalling pathway. AMPK down-regulation by specific small-interfering RNAs confirmed that activated AMPK had a role in restraining growth of GH3 cells. Hence, we compared the effects of compounds directly targeting the mTOR-p70S6K axis, namely the mTOR inhibitor rapamycin and the p70S6K inhibitor PF-4708671, with the effects of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on cell signalling and cell growth, in rat pituitary GH3 cells. AICAR was able to reduce growth factor-induced p70S6K activity, as shown by the decrease of phospho-p70S6K levels. However, it was far less effective than rapamycin and PF-4708671. We observed significant differences between the growth inhibitory effects of the three compounds in GH3 and GH1 cells. Interestingly, PF-4708671 was devoid of any effect. AICAR was at least as effective as rapamycin and the co-treatment was more effective than single treatments. AICAR induced apoptosis of GH3 cells, whereas rapamycin caused preferentially a decrease of cell proliferation. Finally, AICAR and rapamycin differed in their actions on growth factor-induced extracellular signal regulated kinase 1/2 phosphorylation

  2. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-01-01

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated

  3. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice.

    PubMed

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-06-03

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated

  4. A revised model for AMP-activated protein kinase structure: The alpha-subunit binds to both the beta- and gamma-subunits although there is no direct binding between the beta- and gamma-subunits.

    PubMed

    Wong, Kelly A; Lodish, Harvey F

    2006-11-24

    The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.

  5. (−)-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

  6. Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5' AMP-activated protein kinase (AMPK).

    PubMed

    Córdova, Alex; Strobel, Pablo; Vallejo, Andrés; Valenzuela, Pamela; Ulloa, Omar; Burgos, Rafael A; Menarim, Bruno; Rodríguez-Gil, Joan Enric; Ratto, Marcelo; Ramírez-Reveco, Alfredo

    2014-12-01

    This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 μM compound C AMPK inhibitor or 2 mM AMP+40 μM compound C. Our results showed that the utilization of a hypometabolic TRIS extender supplemented or not with AMP or metformin significantly improves stallion sperm freezability when compared with a commercial extender. Additionally, high cooling rates do not affect stallion sperm quality after cooling and post-thawing. Finally, stallion spermatozoa present several putative AMPK sperm isoforms that do not seem to respond to classical activators, but do respond to the Compound C inhibitor.

  7. Transgenic Mouse Model of Ventricular Preexcitation and Atrioventricular Reentrant Tachycardia Induced by an AMP-Activated Protein Kinase Loss-of-Function Mutation Responsible for Wolff-Parkinson-White Syndrome

    PubMed Central

    Sidhu, Jasvinder S.; Rajawat, Yadavendra S.; Rami, Tapan G.; Gollob, Michael H.; Wang, Zhinong; Yuan, Ruiyong; Marian, A.J.; DeMayo, Francesco J.; Weilbacher, Donald; Taffet, George E.; Davies, Joanna K.; Carling, David; Khoury, Dirar S.; Roberts, Robert

    2010-01-01

    Background We identified a gene (PRKAG2) that encodes the γ-2 regulatory subunit of AMP-activated protein kinase (AMPK) with a mutation (Arg302Gln) responsible for familial Wolff-Parkinson-White (WPW) syndrome. The human phenotype consists of ventricular preexcitation, conduction abnormalities, and cardiac hypertrophy. Methods and Results To elucidate the molecular basis for the phenotype, transgenic mice were generated by cardiac-restricted expression of the wild-type (TGWT) and mutant(TGR302Q) PRKAG2 gene with the cardiac-specific promoter α-myosin heavy chain. ECG recordings and intracardiac electrophysiology studies demonstrated the TGR302Q mice to have ventricular preexcitation (PR interval 10±2 versus 33±5 ms in TGWT, P<0.05) and a prolonged QRS (20±5 versus 10±1 ms in TGWT, P<0.05). A distinct AV accessory pathway was confirmed by electrical and pharmacological stimulation and substantiated by induction of orthodromic AV reentrant tachycardia. Enzymatic activity of AMPK in the mutant heart was significantly reduced (0.009±0.003 versus 0.025±0.001 nmol · min−1 · g−1 in nontransgenic mice), presumably owing to the mutation disrupting the AMP binding site. Excessive cardiac glycogen was observed. Hypertrophy was confirmed by increases in heart weight (296 versus 140 mg in TGWT) and ventricular wall thickness. Conclusions We have developed a genetic animal model of WPW that expresses a mutation responsible for a familial form of WPW syndrome with a phenotype identical to that of the human, including induction of supraventricular arrhythmia. The defect is due to loss of function of AMPK. Elucidation of the molecular basis should provide insight into development of the cardiac conduction system and accessory pathways. PMID:15611370

  8. Regulation of ion channels and transporters by AMP-activated kinase (AMPK)

    PubMed Central

    Lang, Florian; Föller, Michael

    2014-01-01

    The energy-sensing AMP-activated kinase AMPK ensures survival of energy-depleted cells by stimulating ATP production and limiting ATP utilization. Both energy production and energy consumption are profoundly influenced by transport processes across the cell membane including channels, carriers and pumps. Accordingly, AMPK is a powerful regulator of transport across the cell membrane. AMPK regulates diverse K+ channels, Na+ channels, Ca2+ release activated Ca2+ channels, Cl- channels, gap junctional channels, glucose carriers, Na+/H+-exchanger, monocarboxylate-, phosphate-, creatine-, amino acid-, peptide- and osmolyte-transporters, Na+/Ca2+-exchanger, H+-ATPase and Na+/K+-ATPase. AMPK activates ubiquitin ligase Nedd4–2, which labels several plasma membrane proteins for degradation. AMPK further regulates transport proteins by inhibition of Rab GTPase activating protein (GAP) TBC1D1. It stimulates phosphatidylinositol 3-phosphate 5-kinase PIKfyve and inhibits phosphatase and tensin homolog (PTEN) via glycogen synthase kinase 3β (GSK3β). Moreover, it stabilizes F-actin as well as downregulates transcription factor NF-κB. All those cellular effects serve to regulate transport proteins. PMID:24366036

  9. DAF-16/FoxO Directly Regulates an Atypical AMP-Activated Protein Kinase Gamma Isoform to Mediate the Effects of Insulin/IGF-1 Signaling on Aging in Caenorhabditis elegans

    PubMed Central

    Tullet, Jennifer M. A.; Araiz, Caroline; Sanders, Matthew J.; Au, Catherine; Benedetto, Alexandre; Papatheodorou, Irene; Clark, Emily; Schmeisser, Kathrin; Jones, Daniel; Schuster, Eugene F.; Thornton, Janet M.; Gems, David

    2014-01-01

    The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, β and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK β subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved

  10. Ergostatrien-3β-ol from Antrodia camphorata inhibits diabetes and hyperlipidemia in high-fat-diet treated mice via regulation of hepatic related genes, glucose transporter 4, and AMP-activated protein kinase phosphorylation.

    PubMed

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2015-03-11

    This study was designed to explore the effects and mechanism of ergostatrien-3β-ol (EK100) from the submerged whole broth of Antrodia camphorata on diabetes and dyslipidemia in high fat diet (HFD)-fed mice for 12 weeks. The C57BL/6J mouse fed with a high fat diet (HFD) could induce insulin resistance and hyperlipidemia. After 8 week of induction, mice were receiving EK100 (at three dosages) or fenofibrate (Feno) or rosiglitazone (Rosi) or vehicle by oral gavage 4 weeks afterward. HFD-fed mice display increased blood glucose, glycated hemoglobin (HbA1c), total cholesterol (TC), triglyceride (TG), insulin, and leptin levels. These blood markers were significantly lower in EK100-treated mice, and finally ameliorated insulin resistance. EK100 treatment exhibited reduced hepatic ballooning degeneration and size of visceral adipocytes. Glucose transporter 4 (GLUT4) proteins and phosphorylation of Akt in skeletal muscle were significantly increased in EK100- and Rosi-treated mice. EK100, Feno, and Rosi treatment led to significant increases in phosphorylation of AMP-activated protein kinase (phospho-AMPK) protein in both skeletal muscle and liver. Moreover, EK100 caused a decrease in hepatic expressions of phosphenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6 Pase), and decreased glucose production. EK100 lowered blood TG level by inhibition of hepatic fatty acid synthesis by dampening sterol response element binding protein-1c (SREBP-1c) but increasing expression of peroxisome proliferator activated receptor α (PPARα). Moreover, EK100-treated mice reduced blood TC levels by decreased hepatic expressions of SREBP2, which plays a major role in the regulation of cholesterol synthesis. EK100 increased high-density lipoprotein cholesterol (HDL-C) concentrations by increasing expressions of apolipoprotein A-I (apo A-I) in liver tissue. Our findings manifest that EK100 may have therapeutic potential in treating type 2 diabetes associated with hyperlipidemia

  11. Ca2+/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy.

    PubMed

    Pfisterer, Simon G; Mauthe, Mario; Codogno, Patrice; Proikas-Cezanne, Tassula

    2011-12-01

    Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca(2+)-mediated autophagy modulation. We imposed cellular stress by starvation or administration of etoposide (0.5-50 μM), sorafenib (1-40 μM), staurosporine (20-500 nM), or thapsigargin (20-500 nM) (1, 2, or 3 h) and measured the formation of WIPI-1 positive autophagosomal membranes. Automated analysis of up to 5000 individual cells/treatment demonstrated that Ca(2+) chelation by BAPTA-AM (10 and 30 μM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca(2+)/calmodulin-dependent kinase kinase (CaMKK) α/β and calmodulin-dependent kinase (CaMK) I/II/IV inhibitors 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609; 10-30 μg/ml) and 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93; 1-10 μM), respectively, significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca(2+) mobilization upon autophagy induction involves CaMKI/IV. By small interefering RNA (siRNA)-mediated down-regulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMP-activated protein kinase (AMPK) α(1)/α(2)-deficient mouse embryonic fibroblasts upon nutrient starvation, whereas basal autophagy was prominently reduced. However, transient down

  12. Perspectives of the AMP-activated kinase (AMPK) signalling pathway in thyroid cancer.

    PubMed

    Andrade, Bruno Moulin; de Carvalho, Denise Pires

    2014-04-01

    Approximately 90% of non-medullary thyroid malignancies originate from the follicular cell and are classified as papillary or follicular (well-differentiated) thyroid carcinomas, showing an overall favourable prognosis. However, recurrence or persistence of the disease occurs in some cases associated with the presence of loco-regional or distant metastatic lesions that generally become resistant to radioiodine therapy, while glucose uptake and metabolism are increased. Recent advances in the field of tumor progression have shown that CTC (circulating tumour cells) are metabolic and genetically heterogeneous. There is now special interest in unravelling the mechanisms that allow the reminiscence of dormant tumour lesions that might be related to late disease progression and increased risk of recurrence. AMPK (AMP-activated protein kinase) is activated by the depletion in cellular energy levels and allows adaptive changes in cell metabolism that are fundamental for cell survival in a stressful environment; nevertheless, the activation of this kinase also decreases cell proliferation rate and induces tumour cell apoptosis. In the thyroid field, AMPK emerged as a novel important intracellular pathway, since it regulates both iodide and glucose uptakes in normal thyroid cells. Furthermore, it has recently been demonstrated that the AMPK pathway is highly activated in papillary thyroid carcinomas, although the clinical significance of these findings remains elusive. Herein we review the current knowledge about the role of AMPK activation in thyroid physiology and pathophysiology, with special focus on thyroid cancer.

  13. Perspectives of the AMP-activated kinase (AMPK) signalling pathway in thyroid cancer

    PubMed Central

    Andrade, Bruno Moulin; de Carvalho, Denise Pires

    2014-01-01

    Approximately 90% of non-medullary thyroid malignancies originate from the follicular cell and are classified as papillary or follicular (well-differentiated) thyroid carcinomas, showing an overall favourable prognosis. However, recurrence or persistence of the disease occurs in some cases associated with the presence of loco-regional or distant metastatic lesions that generally become resistant to radioiodine therapy, while glucose uptake and metabolism are increased. Recent advances in the field of tumor progression have shown that CTC (circulating tumour cells) are metabolic and genetically heterogeneous. There is now special interest in unravelling the mechanisms that allow the reminiscence of dormant tumour lesions that might be related to late disease progression and increased risk of recurrence. AMPK (AMP-activated protein kinase) is activated by the depletion in cellular energy levels and allows adaptive changes in cell metabolism that are fundamental for cell survival in a stressful environment; nevertheless, the activation of this kinase also decreases cell proliferation rate and induces tumour cell apoptosis. In the thyroid field, AMPK emerged as a novel important intracellular pathway, since it regulates both iodide and glucose uptakes in normal thyroid cells. Furthermore, it has recently been demonstrated that the AMPK pathway is highly activated in papillary thyroid carcinomas, although the clinical significance of these findings remains elusive. Herein we review the current knowledge about the role of AMPK activation in thyroid physiology and pathophysiology, with special focus on thyroid cancer. PMID:27919039

  14. cAMP-activated chloride currents in amphibian retinal pigment epithelial cells.

    PubMed Central

    Hughes, B A; Segawa, Y

    1993-01-01

    1. The effect of cAMP on whole-cell currents in isolated retinal pigment epithelial (RPE) cells of the bullfrog and marine toad was investigated by means of the perforated patch clamp technique. 2. Superfusing cells with either cAMP or forskolin led to the development of a time-independent current that had a linear current-voltage (I-V) relationship. The reversal potential of (Vrev) of the cAMP-activated current was unaffected by the removal of either Na+ or HCO3- from the external and internal solutions or by the addition of extracellular barium, but it was near the Cl- equilibrium potential (ECl) over a wide range of extracellular Cl- concentrations, suggesting the presence of a Cl(-)-selective channel. 3. The anion permeability sequence of the cAMP-activated conductance calculated from biionic reversal potentials was NO3- = I- > Br- > Cl- >> HCO3- > methanesulphonate. 4. The conductance was blocked by a variety of Cl- transport inhibitors, including 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), 4,4'-dinitro-2,2'- stilbene disulphonic acid (DNDS), frusemide, N-phenylanthranilic acid (DPC) and niflumic acid. 5. The present study demonstrates that cAMP activates a Cl(-)-selective channel that most probably resides in the basolateral membrane. PMID:8410715

  15. AMP-activated protein kinase couples 3-bromopyruvate-induced energy depletion to apoptosis via activation of FoxO3a and upregulation of proapoptotic Bcl-2 proteins.

    PubMed

    Bodur, Cagri; Karakas, Bahriye; Timucin, Ahmet Can; Tezil, Tugsan; Basaga, Huveyda

    2016-11-01

    Most tumors primarily rely on glycolysis rather than mitochondrial respiration for ATP production. This phenomenon, also known as Warburg effect, renders tumors more sensitive to glycolytic disturbances compared to normal cells. 3-bromopyruvate is a potent inhibitor of glycolysis that shows promise as an anticancer drug candidate. Although investigations revealed that 3-BP triggers apoptosis through ATP depletion and subsequent AMPK activation, the underlying molecular mechanisms coupling AMPK to apoptosis are poorly understood. We showed that 3-BP leads to a rapid ATP depletion which was followed by growth inhibition and Bax-dependent apoptosis in HCT116 cells. Apoptosis was accompanied with activation of caspase-9 and -3 while pretreatment with a general caspase inhibitor attenuated cell death. AMPK, p38, JNK, and Akt were phosphorylated immediately upon treatment. Pharmacological inhibition and silencing of AMPK largely inhibited 3-BP-induced apoptosis and reversed phosphorylation of JNK. Transcriptional activity of FoxO3a was dramatically increased subsequent to AMPK-mediated phosphorylation of FoxO3a at Ser413. Cell death analysis of cells transiently transfected with wt or AMPK-phosphorylation-deficient FoxO3 expression plasmids verified the contributory role of AMPK-FoxO3a axis in 3-BP-induced apoptosis. In addition, expression of proapoptotic Bcl-2 proteins Bim and Bax were upregulated in an AMPK-dependent manner. Bim was transcriptionally activated in association with FoxO3a activity, while Bax upregulation was abolished in p53-null cells. Together, these data suggest that AMPK couples 3-BP-induced metabolic disruption to intrinsic apoptosis via modulation of FoxO3a-Bim axis and Bax expression. © 2015 Wiley Periodicals, Inc.

  16. 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.

  17. Activation of AMP-activated protein kinase, inhibition of pyruvate dehydrogenase activity, and redistribution of substrate partitioning mediate the acute insulin-sensitizing effects of troglitazone in skeletal muscle cells.

    PubMed

    Fediuc, S; Pimenta, A S; Gaidhu, M P; Ceddia, R B

    2008-05-01

    The aim of this study was to investigate the acute effects of troglitazone on several pathways of glucose and fatty acid (FA) partitioning and the molecular mechanisms involved in these processes in skeletal muscle. Exposure of L6 myotubes to troglitazone for 1 h significantly increased phosphorylation of AMPK and ACC, which was followed by approximately 30% and approximately 60% increases in palmitate oxidation and carnitine palmitoyl transferase-1 (CPT-1) activity, respectively. Troglitazone inhibited basal ( approximately 25%) and insulin-stimulated ( approximately 35%) palmitate uptake but significantly increased basal and insulin-stimulated glucose uptake by approximately 2.2- and 2.7-fold, respectively. Pharmacological inhibition of AMPK completely prevented the effects of troglitazone on palmitate oxidation and glucose uptake. Interestingly, even though troglitazone exerted an insulin sensitizing effect, it reduced basal and insulin-stimulated rates of glycogen synthesis, incorporation of glucose into lipids, and glucose oxidation to values corresponding to approximately 30%, approximately 60%, and 30% of the controls, respectively. These effects were accompanied by an increase in basal and insulin-stimulated phosphorylation of Akt(Thr308), Akt(Ser473), and GSK3alpha/beta. Troglitazone also powerfully suppressed pyruvate decarboxylation, which was followed by a significant increase in basal ( approximately 3.5-fold) and insulin-stimulated ( approximately 5.5-fold) rates of lactate production by muscle cells. In summary, we provide novel evidence that troglitazone exerts acute insulin sensitizing effects by increasing FA oxidation, reducing FA uptake, suppressing pyruvate dehydrogenase activity, and shifting glucose metabolism toward lactate production in muscle cells. These effects seem to be at least partially dependent on AMPK activation and may account for potential acute PPAR-gamma-independent anti-diabetic effects of thiazolidinediones in skeletal

  18. 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.

  19. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility.

    PubMed

    Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

    2016-09-27

    AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

  20. Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)2] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38MAPK/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment.

    PubMed

    Filomeni, Giuseppe; Cardaci, Simone; Da Costa Ferreira, Ana Maria; Rotilio, Giuseppe; Ciriolo, Maria Rosa

    2011-08-01

    We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N']copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

  1. Loss of a neural AMP-activated kinase mimics the effects of elevated serotonin on fat, movement, and hormonal secretions.

    PubMed

    Cunningham, Katherine A; Bouagnon, Aude D; Barros, Alexandre G; Lin, Lin; Malard, Leandro; Romano-Silva, Marco Aurélio; Ashrafi, Kaveh

    2014-06-01

    AMP-activated protein kinase (AMPK) is an evolutionarily conserved master regulator of metabolism and a therapeutic target in type 2 diabetes. As an energy sensor, AMPK activity is responsive to both metabolic inputs, for instance the ratio of AMP to ATP, and numerous hormonal cues. As in mammals, each of two genes, aak-1 and aak-2, encode for the catalytic subunit of AMPK in C. elegans. Here we show that in C. elegans loss of aak-2 mimics the effects of elevated serotonin signaling on fat reduction, slowed movement, and promoting exit from dauer arrest. Reconstitution of aak-2 in only the nervous system restored wild type fat levels and movement rate to aak-2 mutants and reconstitution in only the ASI neurons was sufficient to significantly restore dauer maintenance to the mutant animals. As in elevated serotonin signaling, inactivation of AAK-2 in the ASI neurons caused enhanced secretion of dense core vesicles from these neurons. The ASI neurons are the site of production of the DAF-7 TGF-β ligand and the DAF-28 insulin, both of which are secreted by dense core vesicles and play critical roles in whether animals stay in dauer or undergo reproductive development. These findings show that elevated levels of serotonin promote enhanced secretions of systemic regulators of pro-growth and differentiation pathways through inactivation of AAK-2. As such, AMPK is not only a recipient of hormonal signals but can also be an upstream regulator. Our data suggest that some of the physiological phenotypes previously attributed to peripheral AAK-2 activity on metabolic targets may instead be due to the role of this kinase in neural serotonin signaling.

  2. Globular adiponectin inhibits ethanol-induced reactive oxygen species production through modulation of NADPH oxidase in macrophages: involvement of liver kinase B1/AMP-activated protein kinase pathway.

    PubMed

    Kim, Mi Jin; Nagy, Laura E; Park, Pil-Hoon

    2014-09-01

    Adiponectin, an adipokine predominantly secreted from adipocytes, has been shown to play protective roles against chronic alcohol consumption. Although excessive reactive oxygen species (ROS) production in macrophages is considered one of the critical events for ethanol-induced damage in various target tissues, the effect of adiponectin on ethanol-induced ROS production is not clearly understood. In the present study, we investigated the effect of globular adiponectin (gAcrp) on ethanol-induced ROS production and the potential mechanisms underlying these effects of gAcrp in macrophages. Here we demonstrated that gAcrp prevented ethanol-induced ROS production in both RAW 264.7 macrophages and primary murine peritoneal macrophages. Globular adiponectin also inhibited ethanol-induced activation of NADPH oxidase. In addition, gAcrp suppressed ethanol-induced increase in the expression of NADPH oxidase subunits, including Nox2 and p22(phox), via modulation of nuclear factor-κB pathway. Furthermore, pretreatment with compound C, a selective inhibitor of AMPK, or knockdown of AMPK by small interfering RNA restored suppression of ethanol-induced ROS production and Nox2 expression by gAcrp. Finally, we found that gAcrp treatment induced phosphorylation of liver kinase B1 (LKB1), an upstream signaling molecule mediating AMPK activation. Knockdown of LKB1 restored gAcrp-suppressed Nox2 expression, suggesting that LKB1/AMPK pathway plays a critical role in the suppression of ethanol-induced ROS production and activation of NADPH oxidase by gAcrp. Taken together, these results demonstrate that globular adiponectin prevents ethanol-induced ROS production, at least in part, via modulation of NADPH oxidase in macrophages. Further, LKB1/AMPK axis plays an important role in the suppression of ethanol-induced NADPH oxidase activation by gAcrp in macrophages.

  3. A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.

    PubMed

    Delgado, R; Hidalgo, P; Diaz, F; Latorre, R; Labarca, P

    1991-01-15

    Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening.

  4. A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.

    PubMed Central

    Delgado, R; Hidalgo, P; Diaz, F; Latorre, R; Labarca, P

    1991-01-01

    Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening. PMID:1846445

  5. Quercetin, luteolin, and epigallocatechin gallate promote glucose disposal in adipocytes with regulation of AMP-activated kinase and/or sirtuin 1 activity.

    PubMed

    Xiao, Na; Mei, Fan; Sun, Yan; Pan, Guojun; Liu, Baolin; Liu, Kang

    2014-08-01

    Quercetin, luteolin, and epigallocatechin gallate are flavonoids abundant in edible and medicinal plants with beneficial effects on glucose homeostasis. This study explored the action of these flavonoids on glucose disposal in adipocytes. Quercetin, luteolin, and epigallocatechin gallate enhanced glucose consumption with the positive regulation of AMP-activated kinase phosphorylation, and the AMP-activated kinase inhibitor compound C abolished their effects on glucose consumption. Luteolin and epigallocatechin gallate, but not quercetin, increased sirtuin 1 abundance, and their regulation of glucose consumption was also attenuated by co-treatment with sirtuin 1 inhibitor nicotinamide. Quercetin, luteolin, and epigallocatechin gallate suppressed nuclear factor-κB activation by inhibition of p65 phosphorylation with beneficial regulation of adipokine expression, whereas these actions were diminished by coincubation with compound C. The sirtuin 1 inhibitor nicotinamide attenuated the effects of luteolin and EGCG on p65 phosphorylation and adipokine expression without any influence on the activity of quercetin. Results of Western blot and fluorescence microscopy also showed that quercetin, luteolin, and epigallocatechin gallate increased Akt substrate of 160 kDa phosphorylation and promoted 2-deoxy-D-glucose uptake by adipocytes under basal and inflammatory conditions. These findings suggested that quercetin, luteolin, and epigallocatechin gallate inhibited inflammation and promoted glucose disposal in adipocytes with the regulation of AMP-activated kinase and/or sirtuin 1.

  6. Amyloid-β Oligomers Transiently Inhibit AMP-activated kinase and Cause Metabolic Defects in Hippocampal Neurons.

    PubMed

    Seixas da Silva, Gisele S; Melo, Helen M; Lourenco, Mychael V; Lyra E Silva, Natalia de M; de Carvalho, Marcelo B; Alves-Leon, Soniza; de Souza, Jorge M; Klein, William L; da-Silva, Wagner S; Ferreira, Sergio T; De Felice, Fernanda G

    2017-03-16

    AMP-activated kinase (AMPK) is a key player in energy sensing and metabolic reprogramming under cellular energy restriction. Several studies have linked impaired AMPK function to peripheral metabolic diseases such as diabetes. However, the impact of neurological disorders, such as Alzheimer disease (AD), on AMPK function and downstream effects of altered AMPK activity on neuronal metabolism have been investigated only recently. Here, we report the impact of A β oligomers (AβOs), synaptotoxins that accumulate in AD brains, on neuronal AMPK activity. Short-term exposure of cultured rat hippocampal neurons or ex vivo human cortical slices to AβOs transiently decreased intracellular ATP levels and AMPK activity, as evaluated by its phosphorylation at threonine residue 172 (AMPKpThr172). The AβO-dependent reduction in AMPKpThr172 levels was mediated by glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype, and resulted in removal of glucose transporters (GLUTs) from the surfaces of dendritic processes in hippocampal neurons. Importantly, insulin prevented the AβO-induced inhibition of AMPK. Our results establish a novel toxic impact of A βOs on neuronal metabolism and suggest that AβO-induced, NMDA receptor-mediated AMPK inhibition may play a key role in early brain metabolic defects in AD.

  7. The Role of Phosphatidylinositol-3-Kinase and AMP-Activated Kinase in the Rapid Estrogenic Attenuation of Cannabinoid-Induced Changes in Energy Homeostasis

    PubMed Central

    Jeffery, Garrett S.; Peng, Kelly C.; Wagner, Edward J.

    2011-01-01

    We sought to determine the involvement of phosphatidyl inositol 3-kinase (PI3K) and AMP-activated protein kinase (AMPK) in the estrogenic antagonism of the cannabinoid regulation of energy homeostasis. Food intake and body weight were evaluated in ovariectomized female guinea pigs treated s.c. with estradiol benzoate (EB) or its sesame oil vehicle, or the CB1 receptor antagonist AM251 or its cremephor/ethanol/0.9% saline vehicle. AMPK catalytic subunit, PI3K p85α regulatory subunit and proopiomelanocortin (POMC) gene expression was assessed via quantitative RT-PCR in microdissected hypothalamic tissue. Whole-cell patch clamp recordings were performed in hypothalamic slices. Both EB and AM251 decreased food intake and weight gain, and increased AMPKα1, AMPKα2 and PI3K p85α gene expression in the mediobasal hypothalamus. 17β-Estradiol rapidly and markedly attenuated the decreases in glutamatergic miniature excitatory postsynaptic current (mEPSC) frequency caused by the cannabinoid receptor agonist WIN 55,212-2 in POMC neurons. This rapid estrogenic diminution of cannabinoid-induced decreases in mEPSC frequency was blocked by the estrogen receptor (ER) antagonist ICI 182,780 and the PI3K inhibitor PI 828, the latter of which also prevented the AM251-induced increase in mEPSC frequency. In addition, the AMPK activator metformin reversed the EB-induced decreases in food intake and weight gain and restored the ability of WIN 55,212-2 to reduce mEPSC frequency. These data reveal that estrogens physiologically antagonize cannabinoid-induced changes in appetite and POMC neuronal activity by activating PI3K and inhibiting AMPK. As such, they provide insight into the neuroanatomical substrates and signal transduction mechanisms upon which these counter-regulatory factors converge in the control of energy homeostasis.

  8. Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels

    PubMed Central

    Maertens, Chantal; Wei, Lin; Tytgat, Jan; Droogmans, Guy; Nilius, Bernd

    2000-01-01

    It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15–21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, ICl,swell, was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16–21 significantly inhibited ICl,swell (n=4–5). Ca2+-activated Cl− currents, ICl,Ca, activated by loading T84 cells via the patch pipette with 1 μM free Ca2+, were not inhibited by any of the tested fractions (15–21), (n=2–5). Chlorotoxin (625 nM) did neither effect ICl,swell nor ICl,Ca (n=4–5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 μM chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca2+-activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin. PMID:10683204

  9. A rapid interference between glucocorticoids and cAMP-activated signalling in hypothalamic neurones prevents binding of phosphorylated cAMP response element binding protein and glucocorticoid receptor at the CRE-Like and composite GRE sites of thyrotrophin-releasing hormone gene promoter.

    PubMed

    Díaz-Gallardo, M Y; Cote-Vélez, A; Charli, J L; Joseph-Bravo, P

    2010-04-01

    Glucocorticoids or cAMP increase, within minutes, thyrotrophin-releasing hormone (TRH) transcription in hypothalamic primary cultures, although this effect is prevented if cells are simultaneously incubated with both drugs. Rat TRH promoter contains a CRE site at -101/-94 bp and a composite GRE element (cGRE) at -218/-197 bp. Nuclear extracts of hypothalamic cells incubated with 8Br-cAMP or dexamethasone, and not their combination, bind to oligonucleotides containing the CRE or cGRE sequences. Adjacent to CRE are Sp/Krüppel response elements, and flanking the GRE half site, two AP1 binding sites. The present study aimed to identify the hypothalamic transcription factors that bind to these sites. We verified that the effects of glucocorticoid were not mimicked by corticosterone-bovine serum albumin. Footprinting and chromatin immunoprecipitation (ChIP) assays were used to examine the interaction of cAMP- and glucocorticoid-mediated regulation of TRH transcription at the CRE and cGRE regions of the TRH promoter. Nuclear extracts from hypothalamic cells incubated for 1 h with cAMP or glucocorticoids protected CRE. The GRE half site was recognised by nuclear proteins from cells stimulated with glucocorticoids and, for the adjacent AP-1 sites, by nuclear proteins from cells stimulated with cAMP or phorbol esters. Protection of CRE or cGRE was lost if cells were coincubated with dexamethasone and 8Br-cAMP. ChIP assays revealed phospho-CREB, c-Jun, Sp1, c-Fos and GR antibodies bound the TRH promoter of cells treated with cAMP or glucocorticoids; anti:RNA-polymerase II immunoprecipitated TRH promoter in a similar proportion as anti:pCREB or anti:GR. Recruitment of pCREB, SP1 or GR was lost when cells were exposed simultaneously to 8Br-cAMP and glucocorticoids. The data show that while pCREB and Sp1 bind to CRE-2, or GR to cGRE of the TRH promoter, the mutual antagonism between cAMP and glucocorticoid signalling, which prevent their binding to TRH promoter, could serve as

  10. A novel pineal-specific product of the oligopeptide transporter PepT1 gene: circadian expression mediated by cAMP activation of an intronic promoter.

    PubMed

    Gaildrat, Pascaline; Møller, Morten; Mukda, Sujira; Humphries, Ann; Carter, David A; Ganapathy, Vadivel; Klein, David C

    2005-04-29

    The oligopeptide transporter 1, PepT1, is a member of the Slc15 family of 12 membrane-spanning domain transporters; PepT1 has proton/peptide cotransport activity and is selectively expressed in intestinal epithelial cells, where it is responsible for the nutritional absorption of di- and tri-peptides. Here, a novel PepT1 gene product has been identified in the rat pineal gland, termed pgPepT1. It encodes a 150-amino acid protein encompassing the C-terminal 3 membrane-spanning domains of intestinal PepT1 protein, with 3 additional N-terminal residues. Expression of pgPepT1 appears to be restricted to the pineal gland and follows a marked circadian pattern with >100-fold higher levels of mRNA occurring at night; this is accompanied by an accumulation of membrane-associated pgPepT1 protein ( approximately 16 kDa). The daily rhythm in pgPepT1 mRNA is regulated by the well described neural pathway that controls pineal melatonin production. This includes the retina, the circadian clock in the suprachiasmatic nucleus, central structures, and projections from the superior cervical ganglia; activation of this pathway results in the release of norepinephrine. Here it was found that pgPepT1 expression is mediated by a norepinephrine-->cyclic AMP mechanism that activates an alternative promoter located in intron 20 of the gene. pgPepT1 protein was found to have transporter-modulator activity; it could contribute to circadian changes in pineal function through this mechanism.

  11. ESTRADIOL AND THE ESTRADIOL METABOLITE, 2-HYDROXYESTRADIOL, ACTIVATE AMP-ACTIVATED PROTEIN KINASE IN C2C12 MYOTUBES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Systemic loss of estradiol (E2) during menopause is associated with increased adiposity which can be prevented with E2 replacement. Rodent studies suggest that E2, or lack of, is a key mediator in menopause-related metabolic changes. We have previously demonstrated that E2 treatment produces a rap...

  12. cAMP-activated chloride channels in a CFTR-transfected pancreatic adenocarcinoma-derived cell line, pANS6.

    PubMed

    Smith, A N; Wardle, C J; Winpenny, J P; Verdon, B; Gray, M A; Argent, B E; Harris, A

    1995-06-09

    Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR.

  13. Liver AMP-Activated Protein Kinase Is Unnecessary for Gluconeogenesis but Protects Energy State during Nutrient Deprivation

    PubMed Central

    Hasenour, Clinton M.; Ridley, D. Emerson; James, Freyja D.; Hughey, Curtis C.; Donahue, E. Patrick; Viollet, Benoit; Foretz, Marc; Young, Jamey D.; Wasserman, David H.

    2017-01-01

    AMPK is an energy sensor that protects cellular energy state by attenuating anabolic and promoting catabolic processes. AMPK signaling is purported to regulate hepatic gluconeogenesis and substrate oxidation; coordination of these processes is vital during nutrient deprivation or pathogenic during overnutrition. Here we directly test hepatic AMPK function in regulating metabolic fluxes that converge to produce glucose and energy in vivo. Flux analysis was applied in mice with a liver-specific deletion of AMPK (L-KO) or floxed control littermates to assess rates of hepatic glucose producing and citric acid cycle (CAC) fluxes. Fluxes were assessed in short and long term fasted mice; the latter condition is a nutrient stressor that increases liver AMP/ATP. The flux circuit connecting anaplerosis with gluconeogenesis from the CAC was unaffected by hepatic AMPK deletion in short and long term fasting. Nevertheless, depletion of hepatic ATP was exacerbated in L-KO mice, corresponding to a relative elevation in citrate synthase flux and accumulation of branched-chain amino acid-related metabolites. L-KO mice also had a physiological reduction in flux from glycogen to G6P. These results demonstrate AMPK is unnecessary for maintaining gluconeogenic flux from the CAC yet is critical for stabilizing liver energy state during nutrient deprivation. PMID:28107516

  14. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  15. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  16. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 1.2Mb region responsible for variation in AD...

  17. Timing of mitogen-activated protein kinase (MAPK) activation in the rat pineal gland.

    PubMed

    Ho, A K; Price, D M; Terriff, D; Chik, C L

    2006-06-27

    Activation of members of the mitogen-activated protein kinase (MAPK) family of signaling cascades is a tightly controlled event in rat pinealocytes. Cell culture studies indicate that whereas the NE-->cGMP activation of p42/44MAPK is rapid and transient, the NE-->cAMP activation of p38MAPK is slower and more sustained. The decline in the p42/44MAPK response is in part due to the induction of MAPK phosphatase-1 by NE. In comparison, p38MAPK activation is tightly coupled to the synthesis and degradation of an upstream element in its activation cascade. Whole animal studies confirm activation of p42/44MAPK occurring during the early part of night and precedes p38MAPK activation. Studies with selective MAPK inhibitors reveal a modulating effect of MAPKs on arylalkylamine-N-acetyltransferse (AA-NAT) activity, with involvement of p42/44MAPK in the induction of AA-NAT and p38MAPK participating in the amplitude and duration of the AA-NAT response. These effects of p42/44MAPK and p38MAPK on AA-NAT activity match their timing of activation. Taken together, our studies on the timing of MAPK activation and regulation of AA-NAT by MAPKs add to the importance of MAPKs in regulating the circadian biology of the pineal gland.

  18. Pravastatin activates activator protein 2 alpha to argument the angiotensin II-induced abdominal aortic aneurysms.

    PubMed

    Ma, Hui; Liang, Wen-Jing; Shan, Mei-Rong; Wang, Xue-Qing; Zhou, Sheng-Nan; Chen, Yuan; Guo, Tao; Li, Peng; Yu, Hai-Ya; Liu, Chao; Yin, Ya-Ling; Wang, Yu-Lin; Dong, Bo; Pang, Xin-Yan; Wang, Shuang-Xi

    2017-02-04

    We have previously reported that activation of AMP-activated kinase alpha 2 (AMPKα2) by nicotine or angiotensin II (AngII) instigates formation of abdominal aortic aneurysms (AAA) in Apoe-/- mice. Statins, used to treat hyperlipidemia widely, activate AMPK in vascular cells. We sought to examine the effects of pravastatin on AAA formation and uncover the molecular mechanism. The AAA model was induced by AngII and evaluated by incidence, elastin degradation, and maximal abdominal aortic diameter in Apoe-/- mice. The phosphorylated levels of AMPKα2 and activator protein 2 alpha (AP-2α) were examined in cultured vascular smooth muscle cells (VSMCs) or in mice. We observed that pravastatin (50 mg/kg/day, 8 weeks) remarkably increased the AngII-induced AAA incidence in mice. In VSMCs, pravastatin increased the levels of pAMPK, pAP-2α, and MMP2 in both basal and AngII-stressed conditions, which were abolished by tempol and compound C. Pravastatin-upregulated MMP2 was abrogated by AMPKα2 or AP-2α siRNA. Lentivirus-mediated gene silence of AMPKα2 or AP-2α abolished pravastatin-worsened AAA formations in AngII-infused Apoe-/- mice. Clinical investigations demonstrated that both AMPKα2 and AP-2α phosphorylations were increased in AAA patients or human subjects taking pravastatin. In conclusion, pravastatin promotes AAA formation through AMPKα2-dependent AP-2α activations.

  19. Protein Kinase A-independent Ras Protein Activation Cooperates with Rap1 Protein to Mediate Activation of the Extracellular Signal-regulated Kinases (ERK) by cAMP.

    PubMed

    Li, Yanping; Dillon, Tara J; Takahashi, Maho; Earley, Keith T; Stork, Philip J S

    2016-10-07

    Cyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1, have been proposed to mediate this activation, with either Ras or Rap1 acting in distinct cell types. Using Hek293 cells, we show that both Ras and Rap1 are required for cAMP signaling to ERKs. The roles of Ras and Rap1 were distinguished by their mechanism of activation, dependence on the cAMP-dependent protein kinase (PKA), and the magnitude and kinetics of their effects on ERKs. Ras was required for the early portion of ERK activation by cAMP and was activated independently of PKA. Ras activation required the Ras/Rap guanine nucleotide exchange factor (GEF) PDZ-GEF1. Importantly, this action of PDZ-GEF1 was disrupted by mutation within its putative cyclic nucleotide-binding domain within PDZ-GEF1. Compared with Ras, Rap1 activation of ERKs was of longer duration. Rap1 activation was dependent on PKA and required Src family kinases and the Rap1 exchanger C3G. This is the first report of a mechanism for the cooperative actions of Ras and Rap1 in cAMP activation of ERKs. One physiological role for the sustained activation of ERKs is the transcription and stabilization of a range of transcription factors, including c-FOS. We show that the induction of c-FOS by cAMP required both the early and sustained phases of ERK activation, requiring Ras and Rap1, as well as for each of the Raf isoforms, B-Raf and C-Raf.

  20. Corosolic acid protects hepatocytes against ethanol-induced damage by modulating mitogen-activated protein kinases and activating autophagy.

    PubMed

    Guo, Xiaolan; Cui, Ruibing; Zhao, Jianjian; Mo, Rui; Peng, Lei; Yan, Ming

    2016-11-15

    The reactive oxygen species(ROS)/mitogen-activated protein kinase (MAPK) destroyed autophagy and the reactive oxygen species/mitogen-activated protein kinase (MAPK) pathway are considered closely related to ethanol-induced hepatocellular injury. Previous work indicated that corosolic acid, the natural extracts of leaves of the banaba tree, Lagerstroemia speciosa L., could protect the liver against ethanol-induced damage, but the underlying mechanism is unclear. In the study we found that corosolic acid significantly inhibited ethanol-induced apoptosis, increased level of tumor necrosis factor-α(TNF-α) and reactive oxygen species accumulation in vitro. Corosolic acid inhibited ethanol-activated p38 and c-Jun N-terminal kinase MAPK signaling in BRL-3A and HepG2 cells as well as in experimental rats. Corosolic acid restored the ethanol-suppressed expression of autophagy-related genes, including beclin-1 and the ratio of microtubule-associated protein light chain 3II/I (LC3II/I) via AMP-activated protein kinase (AMPK) activation both in vitro and in vivo. In experimental rats, corosolic acid ameliorated the detrimental histopathological findings. Corosolic acid may protect the liver against ethanol-induced injury by modulation of MAPK signaling and autophagy activation. These findings suggested that corosolic acid might be a promising agent in treatment of alcoholic liver diseases.

  1. Sucralose activates an ERK1/2-ribosomal protein S6 signaling axis.

    PubMed

    Guerra, Marcy L; Kalwat, Michael A; McGlynn, Kathleen; Cobb, Melanie H

    2017-02-01

    The sweetener sucralose can signal through its GPCR receptor to induce insulin secretion from pancreatic β cells, but the downstream signaling pathways involved are not well-understood. Here we measure responses to sucralose, glucagon-like peptide 1, and amino acids in MIN6 β cells. Our data suggest a signaling axis, whereby sucralose induces calcium and cAMP, activation of ERK1/2, and site-specific phosphorylation of ribosomal protein S6. Interestingly, sucralose acted independently of mTORC1 or ribosomal S6 kinase (RSK). These results suggest that sweeteners like sucralose can influence β-cell responses to secretagogues like glucose through metabolic as well as GPCR-mediated pathways. Future investigation of novel sweet taste receptor signaling pathways in β cells will have implications for diabetes and other emergent fields involving these receptors.

  2. Adenosine monophosphate-activated protein kinase activation and suppression of inflammatory response by cell stretching in rabbit synovial fibroblasts.

    PubMed

    Kunanusornchai, Wanlop; Muanprasat, Chatchai; Chatsudthipong, Varanuj

    2016-12-01

    Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca(2+) influx via a mechanosensitive L-type Ca(2+) channel, which subsequently raises intracellular Ca(2+) and activates AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca(2+)-channel-CaMKKβ-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.

  3. Prostate Cancer Cell Growth: Stimulatory Role of Neurotensin And Mechanism of Inhibition by Flavonoids as Related to Protein Kinase C

    DTIC Science & Technology

    2007-01-01

    Denderen B, Jennings IG, Iseli T, Michell BJ, Witters LA. AMP-activated protein kinase, super metabolic regulator. Biochem Soc Trans 2003;31(Pt 1):162–8...Forgez P. Neurotensin counteracts apoptosis in breast cancer cells. Biochem Biophys Res Commun 2002;295:482–8. [78] Souaze F, Viardot- Foucault V, Roullet N

  4. Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1▿

    PubMed Central

    Liu, Yang; Xu, Xinjing; Carlson, Marian

    2011-01-01

    The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change. PMID:21216941

  5. Separating proteins with activated carbon.

    PubMed

    Stone, Matthew T; Kozlov, Mikhail

    2014-07-15

    Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon.

  6. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    SciTech Connect

    Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung; Chung, Sung Woon; Hong, Ki Whan; Kim, Chi Dae; Bae, Sun Sik

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  7. Depletion of the cereblon gene activates the unfolded protein response and protects cells from ER stress-induced cell death.

    PubMed

    Lee, Kwang Min; Yang, Seung-Joo; Park, Sojung; Choi, Yoo Duk; Shin, Hwa Kyoung; Pak, Jhang Ho; Park, Chul-Seung; Kim, Inki

    2015-02-27

    Previous studies showed that cereblon (CRBN) binds to various cellular target proteins, implying that CRBN regulates a wide range of cell responses. In this study, we found that deletion of the Crbn gene desensitized mouse embryonic fibroblast cells to various cell death-promoting stimuli, including endoplasmic reticulum stress inducers. Mechanistically, deletion of Crbn activates pathways involved in the unfolded protein response prior to ER stress induction. Loss of Crbn activated PKR-like ER kinase (PERK) with enhanced phosphorylation of eIF2α. Following ER stress induction, loss of Crbn delayed dephosphorylation of eIF2α, while reconstitution of Crbn reversed enhanced phosphorylation of PERK and eIF2α. Lastly, we found that activation of the PERK/eIF2α pathway following Crbn deletion is caused by activation of AMP-activated protein kinase (AMPK). We propose that CRBN plays a role in cellular stress signaling, including the unfolded protein response, by controlling the activity of AMPK.

  8. Acadesine Inhibits Tissue Factor Induction and Thrombus Formation by Activating the Phosphoinositide 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Zhang, Weiyu; Wang, Jianguo; Wang, Huan; Tang, Rong; Belcher, John D.; Viollet, Benoit; Geng, Jian-Guo; Zhang, Chunxiang; Wu, Chaodong; Slungaard, Arne; Zhu, Chuhong; Huo, Yuqing

    2013-01-01

    Objective Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation. Methods and Results Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A2A receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A2A receptor or α1–AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-κB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation. Conclusion Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis. PMID:20185792

  9. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex

    SciTech Connect

    Nakagawa, Koji; Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie; Asaka, Masahiro; Takeda, Hiroshi; Kobayashi, Masanobu

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer The nuclear protein Artemis physically interacts with AMPK{alpha}2. Black-Right-Pointing-Pointer Artemis co-localizes with AMPK{alpha}2 in the nucleus. Black-Right-Pointing-Pointer Artemis promotes phosphorylation and activation of AMPK. Black-Right-Pointing-Pointer The interaction between AMPK{alpha}2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic {alpha} subunit and regulatory {beta} and {gamma} subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the {alpha}-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPK{alpha}2-binding protein. Artemis was found to co-immunoprecipitate with AMPK{alpha}2, and the co-localization of Artemis with AMPK{alpha}2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK{alpha}2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPK{alpha}2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.

  10. Protein profiling reveals antioxidant and signaling activities of NAP (Davunetide) in rodent hippocampus exposed to hypobaric hypoxia.

    PubMed

    Sethy, Niroj Kumar; Sharma, Narendra Kumar; Das, Mainak; Bhargava, Kalpana

    2014-11-01

    NAP (davunetide) is a clinical octapeptide and reportedly possesses neuroprotective, neurotrophic and cognitive protective properties. The information for NAP-mediated neuroproteome changes and associated signaling pathways during hypoxia will help in drug development programmes across the world. In the present study, we have evaluated the antioxidant activities of NAP in rat hippocampus exposed to hypobaric hypoxia (25,000 ft, 282 mm Hg) for 3, 6 and 12 h respectively. Using 2D-gel electrophoresis (2D-GE) with matrix-assisted laser desorption ionization time of flight (MALDI-TOF/TOF) mass spectrometry, we have identified altered expression of 80 proteins in NAP-supplemented hippocampus after hypoxia. Pathway analysis revealed that NAP supplementation significantly regulated oxidative stress response, oxidoreductase activity and cellular response to stress pathways during hypoxia. Additionally, NAP supplementation also regulated energy production pathways along with AMP-activated protein kinase (AMPK) signaling and signaling by Rho family GTPases pathways. We observed higher expression of antioxidant Sod1, Eno1, Prdx2 and Prdx5 proteins that were subsequently validated by Western blotting. A higher level of Prdx2 was also observed by immunohistochemistry in NAP-supplemented hippocampus during hypoxia. In corroboration, we are able to detect significant lower level of protein carbonyls in NAP-supplemented hypoxic hippocampus suggesting amelioration of oxidant molecules by NAP supplementation. These results emphasize the antioxidant and signaling properties of NAP in rodent hippocampus during hypobaric hypoxia.

  11. GROWTH AND DEVELOPMENT SYMPOSIUM: Adenosine monophosphate-activated protein kinase and mitochondria in Rendement Napole pig growth.

    PubMed

    Scheffler, T L; Gerrard, D E

    2016-09-01

    The Rendement Napole mutation (RN-), which is well known to influence pork quality, also has a profound impact on metabolic characteristics of muscle. Pigs with RN- possess a SNP in the γ3 subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK); AMPK, a key energy sensor in skeletal muscle, modulates energy producing and energy consuming pathways to maintain cellular homeostasis. Importantly, AMPK regulates not only acute response to energy stress but also facilitates long-term adaptation via changes in gene and protein expression. The RN- allele increases AMPK activity, which alters the metabolic phenotype of skeletal muscle by increasing mitochondrial content and oxidative capacity. Fibers with greater oxidative capacity typically exhibit increased protein turnover and smaller fiber size, which indicates that RN- pigs may exhibit decreased efficiency and growth potential. However, whole body and muscle growth of RN- pigs appear similar to that of wild-type pigs and despite increased oxidative capacity, fibers maintain the capacity for hypertrophic growth. This indicates that compensatory mechanisms may allow RN- pigs to achieve rates of muscle growth similar to those of wild-type pigs. Intriguingly, lipid oxidation and mitochondria function are enhanced in RN- pig muscle. Thus far, characteristics of RN- muscle are largely based on animals near market weight. To better understand interaction between energy signaling and protein accretion in muscle, further work is needed to define age-dependent relationships between AMPK signaling, metabolism, and muscle growth.

  12. Activated AMPK boosts the Nrf2/HO-1 signaling axis—A role for the unfolded protein response

    PubMed Central

    Zimmermann, Kristin; Baldinger, Johannes; Mayerhofer, Barbara; Atanasov, Atanas G.; Dirsch, Verena M.; Heiss, Elke H.

    2015-01-01

    In light of the emerging interplay between redox and metabolic signaling pathways we investigated the potential cross talk between nuclear factor E2-related factor 2 (Nrf2) and AMP-activated kinase (AMPK), central regulators of the cellular redox and energy balance, respectively. Making use of xanthohumol (XN) as an activator of both the AMPK and the Nrf2 signaling pathway we show that AMPK exerts a positive influence on Nrf2/heme oxygenase (HO)-1 signaling in mouse embryonic fibroblasts. Genetic ablation and pharmacological inhibition of AMPK blunts Nrf2-dependent HO-1 expression by XN already at the mRNA level. XN leads to AMPK activation via interference with mitochondrial function and activation of liver kinase B1 as upstream AMPK kinase. The subsequent AMPK-mediated enhancement of the Nrf2/HO-1 response does not depend on inhibition of the mammalian target of rapamycin, inhibition of glycogen synthase kinase 3β, or altered abundance of Nrf2 (total and nuclear). However, reduced endoplasmic reticulum stress was identified and elaborated as a step in the AMPK-augmented Nrf2/HO-1 response. Overall, we shed more light on the hitherto incompletely understood cross talk between the LKB1/AMPK and the Nrf2/HO-1 axis revealing for the first time involvement of the unfolded protein response as an additional player and suggesting tight cooperation between signaling pathways controlling cellular redox, energy, or protein homeostasis. PMID:25843659

  13. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  14. 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.

  15. Comparative transcriptomic analysis of mice liver treated with different AMPK activators in a mice model of atherosclerosis.

    PubMed

    Ma, Ang; Wang, Dongmei; An, Yuanyuan; Fang, Wei; Zhu, Haibo

    2017-02-02

    Atherosclerosis is known to be the primary underlying factor responsible for the development of cardiovascular diseases. Suppression of AMP-activated protein kinase stimulates arterial deposition of excess lipids, resulting in the development of atherosclerotic lesions. In this study we successfully developed the disease model of mice and mimicked the therapeutic effect, for that we chose three different AMP-activated protein kinase activators (IMM-H007, A-769662 and Metformin) to identify which one has a superior effect in the atherosclerosis model. We combined the transcriptomes of four groups of mice liver including high-fat diet group and the experimental groups treated with different AMP-activated protein kinase activators. We analyzed the increased genes to candidate metabolic and disease pathways. Compared to the high-fat diet group, a total of 799 differentially expressed genes were identified in treatment groups. There were 291, 473, and 323 differentially expressed genes in H007, Metformin, and A-769662 group respectively. And seven statistically significant pathways were observed in both H007 and Metformin groups. We expect that gene expression profiling in the mice model would extend our understanding of atherosclerosis in the molecular level. This study provides a fundamental framework for future clinical research on human atherosclerosis and new clues for developing novel drugs for the treatment of atherosclerosis.

  16. [Protein nutrition and physical activity].

    PubMed

    Navarro, M P

    1992-09-01

    The relationship between physical exercise and diet in order to optimize performance is getting growing interest. This review examines protein needs and protein intakes as well as the role of protein in the body and the metabolic changes occurring at the synthesis and catabolic levels during exercise. Protein synthesis in muscle or liver, amino acids oxidation, glucose production via gluconeogenesis from amino acids, etc., are modified, and consequently plasma and urinary nitrogen metabolites are affected. A brief comment on the advantages, disadvantages and forms of different protein supplements for sportsmen is given.

  17. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  18. Biologically active proteins from natural product extracts.

    PubMed

    O'Keefe, B R

    2001-10-01

    The term "biologically active proteins" is almost redundant. All proteins produced by living creatures are, by their very nature, biologically active to some extent in their homologous species. In this review, a subset of these proteins will be discussed that are biologically active in heterologous systems. The isolation and characterization of novel proteins from natural product extracts including those derived from microorganisms, plants, insects, terrestrial vertebrates, and marine organisms will be reviewed and grouped into several distinct classes based on their biological activity and their structure.

  19. Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

    PubMed

    Shimoda, Yoshiaki; Matsuo, Kiyonari; Kitamura, Youhei; Ono, Kazunori; Ueyama, Tomomi; Matoba, Satoaki; Yamada, Hiroyuki; Wu, Tongbin; Chen, Ju; Emoto, Noriaki; Ikeda, Koji

    2015-01-01

    Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.

  20. Rational Design of Protein C Activators

    PubMed Central

    Barranco-Medina, Sergio; Murphy, Mary; Pelc, Leslie; Chen, Zhiwei; Di Cera, Enrico; Pozzi, Nicola

    2017-01-01

    In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance. PMID:28294177

  1. Cbl proteins in platelet activation.

    PubMed

    Buitrago, Lorena; Tsygankov, Alexander; Sanjay, Archana; Kunapuli, Satya P

    2013-01-01

    Platelets play a fundamental role in hemostasis. Their functional responses have to be tightly controlled as any disturbance may lead to bleeding disorders or thrombosis. It is thus important to clearly identify and understand the signaling mechanisms involved in platelet function. An important role of c-Cbl and Cbl-b ubiquitin ligases in platelet functional responses and in hematological malignancies has been recently described. Cbl proteins perform negative and positive regulation of several signaling pathways in platelets. In this review, we explore the role of Cbl proteins in platelet functional responses.

  2. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  3. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  4. Remotely activated protein-producing nanoparticles.

    PubMed

    Schroeder, Avi; Goldberg, Michael S; Kastrup, Christian; Wang, Yingxia; Jiang, Shan; Joseph, Brian J; Levins, Christopher G; Kannan, Sneha T; Langer, Robert; Anderson, Daniel G

    2012-06-13

    The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.

  5. Dietary protein considerations to support active aging.

    PubMed

    Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

    2014-11-01

    Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging.

  6. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  7. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  8. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  9. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

    PubMed

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes.

  10. Activated protein C: biased for translation

    PubMed Central

    Zlokovic, Berislav V.; Mosnier, Laurent O.

    2015-01-01

    The homeostatic blood protease, activated protein C (APC), can function as (1) an antithrombotic on the basis of inactivation of clotting factors Va and VIIIa; (2) a cytoprotective on the basis of endothelial barrier stabilization and anti-inflammatory and antiapoptotic actions; and (3) a regenerative on the basis of stimulation of neurogenesis, angiogenesis, and wound healing. Pharmacologic therapies using recombinant human and murine APCs indicate that APC provides effective acute or chronic therapies for a strikingly diverse range of preclinical injury models. APC reduces the damage caused by the following: ischemia/reperfusion in brain, heart, and kidney; pulmonary, kidney, and gastrointestinal inflammation; sepsis; Ebola virus; diabetes; and total lethal body radiation. For these beneficial effects, APC alters cell signaling networks and gene expression profiles by activating protease-activated receptors 1 and 3. APC’s activation of these G protein–coupled receptors differs completely from thrombin’s activation mechanism due to biased signaling via either G proteins or β-arrestin-2. To reduce APC-associated bleeding risk, APC variants were engineered to lack >90% anticoagulant activity but retain normal cell signaling. Such a neuroprotective variant, 3K3A-APC (Lys191-193Ala), has advanced to clinical trials for ischemic stroke. A rich data set of preclinical knowledge provides a solid foundation for potential translation of APC variants to future novel therapies. PMID:25824691

  11. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    PubMed

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

  12. Synaptic Vesicle Proteins and Active Zone Plasticity

    PubMed Central

    Kittel, Robert J.; Heckmann, Manfred

    2016-01-01

    Neurotransmitter is released from synaptic vesicles at the highly specialized presynaptic active zone (AZ). The complex molecular architecture of AZs mediates the speed, precision and plasticity of synaptic transmission. Importantly, structural and functional properties of AZs vary significantly, even for a given connection. Thus, there appear to be distinct AZ states, which fundamentally influence neuronal communication by controlling the positioning and release of synaptic vesicles. Vice versa, recent evidence has revealed that synaptic vesicle components also modulate organizational states of the AZ. The protein-rich cytomatrix at the active zone (CAZ) provides a structural platform for molecular interactions guiding vesicle exocytosis. Studies in Drosophila have now demonstrated that the vesicle proteins Synaptotagmin-1 (Syt1) and Rab3 also regulate glutamate release by shaping differentiation of the CAZ ultrastructure. We review these unexpected findings and discuss mechanistic interpretations of the reciprocal relationship between synaptic vesicles and AZ states, which has heretofore received little attention. PMID:27148040

  13. Lipid Dependent Mechanisms of Protein Pump Activity

    DTIC Science & Technology

    1989-05-23

    properties which result form the colligative interactions of many lipid molecules. Important materials properties include . . . i I I II II I i I 1 the...d identify by olock number) *This project is aime at investigating if a lipid elastic property , known as the spontaneous radius of curvature Ro’, is...a regulated membrane property and if its value modulates membrane protein activity. Specific aims reported on here include: 1) Correlation of ion pump

  14. AMPK activators: mechanisms of action and physiological activities

    PubMed Central

    Kim, Joungmok; Yang, Goowon; Kim, Yeji; Kim, Jin; Ha, Joohun

    2016-01-01

    AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, which coordinates metabolic pathways and thus balances nutrient supply with energy demand. Because of the favorable physiological outcomes of AMPK activation on metabolism, AMPK has been considered to be an important therapeutic target for controlling human diseases including metabolic syndrome and cancer. Thus, activators of AMPK may have potential as novel therapeutics for these diseases. In this review, we provide a comprehensive summary of both indirect and direct AMPK activators and their modes of action in relation to the structure of AMPK. We discuss the functional differences among isoform-specific AMPK complexes and their significance regarding the development of novel AMPK activators and the potential for combining different AMPK activators in the treatment of human disease. PMID:27034026

  15. Adenosine monophosphate activated protein kinase (AMPK), a mediator of estradiol-induced apoptosis in long-term estrogen deprived breast cancer cells.

    PubMed

    Chen, Haiyan; Wang, Ji-Ping; Santen, Richard J; Yue, Wei

    2015-06-01

    Estrogens stimulate growth of hormone-dependent breast cancer but paradoxically induce tumor regress under certain circumstances. We have shown that long-term estrogen deprivation (LTED) enhances the sensitivity of hormone dependent breast cancer cells to estradiol (E2) so that physiological concentrations of estradiol induce apoptosis in these cells. E2-induced apoptosis involve both intrinsic and extrinsic pathways but precise mechanisms remain unclear. We found that exposure of LTED MCF-7 cells to E2 activated AMP activated protein kinase (AMPK). In contrast, E2 inhibited AMPK activation in wild type MCF-7 cells where E2 prevents apoptosis. As a result of AMPK activation, the transcriptional activity of FoxO3, a downstream factor of AMPK, was up-regulated in E2 treatment of LTED. Increased activity of FoxO3 was demonstrated by up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45α. Among them, Bim and FasL mediate intrinsic and extrinsic apoptosis respectively and Gadd45α causes cell cycle arrest at the G2/M phase. To further confirm the role of AMPK in apoptosis, we used AMPK activator AICAR in wild type MCF-7 cells and examined apoptosis, proliferation and expression of Bim, FasL, and Gadd45α. The effects of AICAR on these parameters recapitulated those observed in E2-treated LTED cells. Activation of AMPK by AICAR also increased expression of Bax in MCF-7 cells and its localization to mitochondria, which is a required process for apoptosis. These results reveal that AMPK is an important factor mediating E2-induced apoptosis in LTED cells, which is implicative of therapeutic potential for relapsing breast cancer after hormone therapy.

  16. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    NASA Technical Reports Server (NTRS)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  17. Indazole-type alkaloids from Nigella sativa seeds exhibit antihyperglycemic effects via AMPK activation in vitro.

    PubMed

    Yuan, Tao; Nahar, Pragati; Sharma, Meenakshi; Liu, Ke; Slitt, Angela; Aisa, H A; Seeram, Navindra P

    2014-10-24

    Six rare naturally occurring indazole-type alkaloids including two new compounds, 17-O-(β-d-glucopyranosyl)-4-O-methylnigellidine (1) and nigelanoid (2), and four known compounds (3-6) were isolated from a defatted extract of Nigella sativa (black cumin) seeds. 17-O-(β-d-Glucopyranosyl)-4-O-methylnigellidine (1) increased glucose consumption by liver hepatocytes (HepG2 cells) through activation of AMP-activated protein kinase (AMPK). Also, this is the first report of compounds 4 and 6 from a natural source.

  18. Comparison of Metalloproteinase Protein and Activity Profiling

    PubMed Central

    Giricz, Orsi; Lauer, Janelle L.; Fields, Gregg B.

    2010-01-01

    Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The present study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer and one normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media, both in amount and in variety. TIMP-1 was produced in all cell lines. MMP activity assays with three different FRET substrates were then utilized to determine if protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was only confirmed in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays. PMID:20920458

  19. EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1.

    PubMed

    Lobo, Miguel J; Amaral, Margarida D; Zaccolo, Manuela; Farinha, Carlos M

    2016-07-01

    Cyclic AMP (cAMP) activates protein kinase A (PKA) but also the guanine nucleotide exchange factor 'exchange protein directly activated by cAMP' (EPAC1; also known as RAPGEF3). Although phosphorylation by PKA is known to regulate CFTR channel gating - the protein defective in cystic fibrosis - the contribution of EPAC1 to CFTR regulation remains largely undefined. Here, we demonstrate that in human airway epithelial cells, cAMP signaling through EPAC1 promotes CFTR stabilization at the plasma membrane by attenuating its endocytosis, independently of PKA activation. EPAC1 and CFTR colocalize and interact through protein adaptor NHERF1 (also known as SLC9A3R1). This interaction is promoted by EPAC1 activation, triggering its translocation to the plasma membrane and binding to NHERF1. Our findings identify a new CFTR-interacting protein and demonstrate that cAMP activates CFTR through two different but complementary pathways - the well-known PKA-dependent channel gating pathway and a new mechanism regulating endocytosis that involves EPAC1. The latter might constitute a novel therapeutic target for treatment of cystic fibrosis.

  20. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  1. Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein.

    PubMed

    Kong, Yuelin; Tong, Yue; Gao, Mingming; Chen, Chen; Gao, Xiangdong; Yao, Wenbing

    2016-01-01

    Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.

  2. Activation and activities of the p53 tumour suppressor protein

    PubMed Central

    Bálint, É; Vousden, K H

    2001-01-01

    The p53 tumour suppressor protein inhibits malignant progression by mediating cell cycle arrest, apoptosis or repair following cellular stress. One of the major regulators of p53 function is the MDM2 protein, and multiple forms of cellular stress activate p53 by inhibiting the MDM2-mediated degradation of p53. Mutations in p53, or disruption of the pathways that allow activation of p53, seem to be a general feature of all cancers. Here we review recent advances in our understanding of the pathways that regulate p53 and the pathways that are induced by p53, as well as their implications for cancer therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11747320

  3. Interaction of Protein Inhibitor of Activated STAT (PIAS) Proteins with the TATA-binding Protein, TBP*

    PubMed Central

    Prigge, Justin R.; Schmidt, Edward E.

    2007-01-01

    Transcription activators often recruit promoter-targeted assembly of a pre-initiation complex; many repressors antagonize recruitment. These activities can involve direct interactions with proteins in the pre-initiation complex. We used an optimized yeast two-hybrid system to screen mouse pregnancy-associated libraries for proteins that interact with TATA-binding protein (TBP). Screens revealed an interaction between TBP and a single member of the zinc finger family of transcription factors, ZFP523. Two members of the protein inhibitor of activated STAT (PIAS) family, PIAS1 and PIAS3, also interacted with TBP in screens. Endogenous PIAS1 and TBP co-immunoprecipitated from nuclear extracts, suggesting the interaction occurred in vivo. In vitro-translated PIAS1 and TBP coimmunopreciptated, which indicated that other nuclear proteins were not required for the interaction. Deletion analysis mapped the PIAS-interacting domain of TBP to the conserved TBPCORE and the TBP-interacting domain on PIAS1 to a 39-amino acid C-terminal region. Mammals issue seven known PIAS proteins from four pias genes, pias1, pias3, piasx, and piasy, each with different cell type-specific expression patterns; the TBP-interacting domain reported here is the only part of the PIAS C-terminal region shared by all seven PIAS proteins. Direct analyses indicated that PIASx and PIASy also interacted with TBP. Our results suggest that all PIAS proteins might mediate situation-specific regulatory signaling at the TBP interface and that previously unknown levels of complexity could exist in the gene regulatory interplay between TBP, PIAS proteins, ZFP523, and other transcription factors. PMID:16522640

  4. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  5. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  6. Activated Protein C action in inflammation

    PubMed Central

    Sarangi, Pranita P.; Lee, Hyun-wook; Kim, Minsoo

    2010-01-01

    Summary Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factor Va and VIIIa. In addition to its anticoagulation functions, APC also has cytoprotective effects such as anti-inflammatory, anti-apoptotic, and endothelial barrier protection. Recently, a recombinant form of human APC (rhAPC or drotrecogin alfa activated; known commercially as “Xigris”) was approved by the US Federal Drug Administration for treatment of severe sepsis associated with a high risk of mortality. Sepsis, also known as systemic inflammatory response syndrome (SIRS) resulting from infection, is a serious medical condition in critical care patients. In sepsis, hyperactive and dysregulated inflammatory responses lead to secretion of pro- and anti-inflammatory cytokines, activation and migration of leucocytes, activation of coagulation, inhibition of fibrinolysis, and increased apoptosis. Although initial hypotheses focused on antithrombotic and profibrinolytic functions of APC in sepsis, other agents with more potent anticoagulation functions were not effective in treating severe sepsis. Furthermore, APC therapy is also associated with the risk of severe bleeding in treated patients. Therefore, the cytoprotective effects, rather than the anticoagulant effect of APC are postulated to be responsible for the therapeutic benefit of APC in the treatment of severe sepsis. PMID:19995397

  7. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  8. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  9. Protein-Protein Interactions Suggest Novel Activities of Human Cytomegalovirus Tegument Protein pUL103

    PubMed Central

    Ortiz, Daniel A.; Glassbrook, James E.

    2016-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, coimmunoprecipitation (co-IP) and proximity biotinylation (BioID), to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high-value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, supports the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expands our understanding of the multifunctional repertoire of pUL103: we detected HCMV pUL103 in nuclei of infected cells and identified an ALIX-binding domain within the pUL103 sequence. IMPORTANCE Human cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein pUL103 is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and

  10. Human carotid atherosclerotic plaque protein(s) change HDL protein(s) composition and impair HDL anti-oxidant activity.

    PubMed

    Cohen, Elad; Aviram, Michael; Khatib, Soliman; Volkova, Nina; Vaya, Jacob

    2016-01-01

    High density lipoprotein (HDL) anti-atherogenic functions are closely associated with cardiovascular disease risk factor, and are dictated by its composition, which is often affected by environmental factors. The present study investigates the effects of the human carotid plaque constituents on HDL composition and biological functions. To this end, human carotid plaques were homogenized and incubated with HDL. Results showed that after incubation, most of the apolipoprotein A1 (Apo A1) protein was released from the HDL, and HDL diameter increased by an average of approximately 2 nm. In parallel, HDL antioxidant activity was impaired. In response to homogenate treatment HDL could not prevent the accelerated oxidation of LDL caused by the homogenate. Boiling of the homogenate prior to its incubation with HDL abolished its effects on HDL composition changes. Moreover, tryptophan fluorescence quenching assay revealed an interaction between plaque component(s) and HDL, an interaction that was reduced by 50% upon using pre-boiled homogenate. These results led to hypothesize that plaque protein(s) interacted with HDL-associated Apo A1 and altered the HDL composition. Immuno-precipitation of Apo A1 that was released from the HDL after its incubation with the homogenate revealed a co-precipitation of three isomers of actin. However, beta-actin alone did not significantly affect the HDL composition, and yet the active protein within the plaque was elusive. In conclusion then, protein(s) in the homogenate interact with HDL protein(s), leading to release of Apo A1 from the HDL particle, a process that was associated with an increase in HDL diameter and with impaired HDL anti-oxidant activity.

  11. Transcription activation by the adenovirus E1a protein

    NASA Astrophysics Data System (ADS)

    Lillie, James W.; Green, Michael R.

    1989-03-01

    The adenovirus Ela protein stimulates transcription of a wide variety of viral and cellular genes. It is shown here that Ela has the two functions characteristic of a typical cellular activator: one direct Ela to the promoter, perhaps by interacting with a DMA-bound protein, and the other, an activating region, enables the bound activator to stimulate transcription.

  12. DNA helicase activity in purified human RECQL4 protein.

    PubMed

    Suzuki, Takahiro; Kohno, Toshiyuki; Ishimi, Yukio

    2009-09-01

    Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'-5' direction. These results suggest that the hRECQL4 protein exhibits DNA helicase activity.

  13. Nandinine, a Derivative of Berberine, Inhibits Inflammation and Reduces Insulin Resistance in Adipocytes via Regulation of AMP-Kinase Activity.

    PubMed

    Zhao, Wenwen; Ge, Haixia; Liu, Kang; Chen, Xiuping; Zhang, Jian; Liu, Baolin

    2017-02-01

    Nandinine is a derivative of berberine that has high efficacy for treating cardiovascular diseases. This study investigated the effects of berberine and nandinine on the regulation of insulin sensitivity in adipocytes. Through treatment with macrophage-derived conditioned medium in 3T3-L1 adipocytes, dysregulation of adipokine production and activation of the IκB kinase β/nuclear factor-kappa B pathway was induced. However, these phenomena were effectively reversed by berberine, nandinine, and salicylate pretreatments. Furthermore, both berberine and nandinine inhibited serine phosphorylation of insulin receptor substrate-1 induced by IκB kinase β and increased tyrosine phosphorylation of insulin receptor substrate-1 to activate the PI3K/Akt pathway, which finally led to insulin-mediated glucose uptake. In addition, berberine and nandinine significantly increased AMP-activated protein kinase activity, thereby contributing to their anti-inflammatory effect by inhibiting IκB kinase β activation. Finally, in vivo studies demonstrated that both berberine (100 or 200 mg/kg) and nandinine (100 or 200 mg/kg) effectively ameliorated glucose intolerance and induced the insulin sensitivity index in mice. In conclusion, berberine and nandinine attenuated insulin resistance in adipocytes by inhibiting inflammation in an AMP-activated protein kinase-dependent manner. Berberine and nandinine may be used as dietary supplements and nandinine is a new candidate for obesity treatment.

  14. Trithorax group proteins: switching genes on and keeping them active.

    PubMed

    Schuettengruber, Bernd; Martinez, Anne-Marie; Iovino, Nicola; Cavalli, Giacomo

    2011-11-23

    Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology.

  15. [Increased fibrinolytic activity during cardiopulmonary bypass is caused by activated protein C system].

    PubMed

    Gando, S; Tedo, I; Masio, H; Goda, Y; Kawahigashi, H

    1994-06-01

    To examine the hypothesis that activated protein C system during cardiopulmonary bypass surgery may increase fibrinolytic activity during cardiopulmonary bypass, protein C activity, protein C antigen and thrombomodulin of sixteen patients undergoing elective cardiopulmonary bypass surgery were investigated after induction of anesthesia, before and after cardiopulmonary bypass, and at the end of operation. Protein C activity decreased and thrombomodulin increased significantly after the cardiopulmonary bypass. There were no significant correlations of thrombomodulin with protein C activity and protein C antigen. In conclusion, we have demonstrated that protein C system is activated and circulating thrombomodulin appears in the systemic circulation during cardiopulmonary bypass surgery and this enhanced activation of protein C system is possibly related to the reported increase of fibrinolytic activity during cardiopulmonary bypass.

  16. Identification of highly active flocculant proteins in bovine blood.

    PubMed

    Piazza, George J; Nuñez, Alberto; Garcia, Rafael A

    2012-03-01

    Synthetic polymeric flocculants are used extensively for wastewater remediation, soil stabilization, and reduction in water leakage from unlined canals. Sources of highly active, inexpensive, renewable flocculants are needed to replace synthetic flocculants. High kaolin flocculant activity was documented for bovine blood (BB) and blood plasma with several anticoagulant treatments. BB serum also had high flocculant activity. To address the hypothesis that some blood proteins have strong flocculating activity, the BB proteins were separated by SEC. Then, the major proteins of the flocculant-active fractions were separated by SDS-PAGE. Identity of the major protein components was determined by tryptic digestion and peptide analysis by MALDI TOF MS. The sequence of selected peptides was confirmed using TOF/TOF-MS/MS fragmentation. Hemoglobin dimer (subunits α and β) was identified as the major protein component of the active fraction in BB; its high flocculation activity was confirmed by testing a commercial sample of hemoglobin. In the same manner, three proteins from blood plasma (fibrinogen, γ-globulin, α-2-macroglobulin) were found to be highly active flocculants, but bovine serum albumin, α-globulin, and β-globulin were not flocculants. On a mass basis, hemoglobin, γ-globulin, α-2-macroglobulin were as effective as anionic polyacrylamide (PAM), a widely used synthetic flocculant. The blood proteins acted faster than PAM, and unlike PAM, the blood proteins flocculants did not require calcium salts for their activity.

  17. Global Analysis of Protein Activities Using Proteome Chips

    NASA Astrophysics Data System (ADS)

    Zhu, Heng; Bilgin, Metin; Bangham, Rhonda; Hall, David; Casamayor, Antonio; Bertone, Paul; Lan, Ning; Jansen, Ronald; Bidlingmaier, Scott; Houfek, Thomas; Mitchell, Tom; Miller, Perry; Dean, Ralph A.; Gerstein, Mark; Snyder, Michael

    2001-09-01

    To facilitate studies of the yeast proteome, we cloned 5800 open reading frames and overexpressed and purified their corresponding proteins. The proteins were printed onto slides at high spatial density to form a yeast proteome microarray and screened for their ability to interact with proteins and phospholipids. We identified many new calmodulin- and phospholipid-interacting proteins; a common potential binding motif was identified for many of the calmodulin-binding proteins. Thus, microarrays of an entire eukaryotic proteome can be prepared and screened for diverse biochemical activities. The microarrays can also be used to screen protein-drug interactions and to detect posttranslational modifications.

  18. Functional and cardioprotective effects of simultaneous and individual activation of protein kinase A and Epac

    PubMed Central

    Bond, Mark; James, Andrew F; Dyar, Zara; Amini, Raheleh; Johnson, Jason L; Suleiman, M‐Saadeh

    2017-01-01

    Background and Purpose Myocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cAMP activates two independent signalling pathways, PKA and Epac. This study investigated the cardiac effects of activating PKA and/or Epac and their involvement in cardioprotection against I/R. Experimental Approach Hearts from male rats were used either for determination of PKA and PKC activation or perfused in the Langendorff mode for either cardiomyocyte isolation or used to monitor functional activity at basal levels and after 30 min global ischaemia and 2 h reperfusion. Functional recovery and myocardial injury during reperfusion (LDH release and infarct size) were evaluated. Activation of PKA and/or Epac in perfused hearts was induced using cell permeable cAMP analogues in the presence or absence of inhibitors of PKA, Epac and PKC. H9C2 cells and cardiomyocytes were used to assess activation of Epac and effect on Ca2+ transients. Key Results Selective activation of either PKA or Epac was found to trigger a positive inotropic effect, which was considerably enhanced when both pathways were simultaneously activated. Only combined activation of PKA and Epac induced marked cardioprotection against I/R injury. This was accompanied by PKCε activation and repressed by inhibitors of PKA, Epac or PKC. Conclusion and Implications Simultaneous activation of both PKA and Epac induces an additive inotropic effect and confers optimal and marked cardioprotection against I/R injury. The latter effect is mediated by PKCε activation. This work has introduced a new therapeutic approach and targets to protect the heart against cardiac insults. PMID:28071786

  19. Mass spectrometry data from a quantitative analysis of protein expression in gills of immuno-challenged blue mussels (Mytilus edulis).

    PubMed

    Hörnaeus, K; Guillemant, J; Mi, J; Hernroth, B; Bergquist, J; Lind, S Bergström

    2016-09-01

    Here, we provide the dataset associated with our research article on the potential effects of ocean acidification on antimicrobial peptide (AMP) activity in the gills of Mytilus edulis, "Impact of ocean acidification on antimicrobial activity in gills of the blue mussel (Mytilus edulis)" [1]. Blue mussels were stimulated with lipopolysaccharides and samples were collected at different time points post injection. Protein extracts were prepared from the gills, digested using trypsin and a full in-depth proteome investigation was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Protein identification and quantification was performed using the MaxQuant 1.5.1.2 software, "MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification" [2].

  20. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    PubMed

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

  1. AKAP-Lbc nucleates a protein kinase D activation scaffold.

    PubMed

    Carnegie, Graeme K; Smith, F Donelson; McConnachie, George; Langeberg, Lorene K; Scott, John D

    2004-09-24

    The transmission of cellular signals often proceeds through multiprotein complexes where enzymes are positioned in proximity to their upstream activators and downstream substrates. In this report we demonstrate that the A-kinase anchoring protein AKAP-Lbc assembles an activation complex for the lipid-dependent enzyme protein kinase D (PKD). Using a combination of biochemical, enzymatic, and immunofluorescence techniques, we show that the anchoring protein contributes to PKD activation in two ways: it recruits an upstream kinase PKCeta and coordinates PKA phosphorylation events that release activated protein kinase D. Thus, AKAP-Lbc synchronizes PKA and PKC activities in a manner that leads to the activation of a third kinase. This configuration illustrates the utility of kinase anchoring as a mechanism to constrain the action of broad-spectrum enzymes.

  2. Regulation of the activity of protein kinases by endogenous heat stable protein inhibitors.

    PubMed

    Szmigielski, A

    1985-01-01

    Protein kinase activities are regulated by endogenous thermostable protein inhibitors. Type I inhibitor is a protein of MW 22,000-24,000 which inhibits specifically cyclic AMP-(cAMP) dependent protein kinase (APK) as a competitive inhibitor of catalytic subunits of the enzyme. Type I inhibitor activity changes inversely according to the activation of adenylate cyclase and the changes in cAMP content in tissues. It seems that type I inhibitor serves as a factor preventing spontaneous cAMP-dependent phosphorylation in unstimulated cell. The other thermostable protein which inhibits APK activity has been found in Sertoli cell-enriched testis (testis inhibitor). Physiological role of the testis inhibitor is unknown. Type II inhibitor is a protein of MW 15,000 which blocks phosphorylation mediated by cAMP and cyclic GMP (cGMP) dependent (APK and GPK) and cyclic nucleotide independent protein kinases as a competitive inhibitor of substrate proteins. Activity of this inhibitor specifically changes in reciprocal manner to the changes in cGMP content. It seems that type II inhibitor serves as a factor preventing the phosphorylation catalyzed by GPK when cGMP content is low. Stimulation of guanylate cyclase and activation of GPK is followed by a decrease of type II inhibitor activity. This change in relationship between activities of GPK and type II inhibitor allows for effective phosphorylation catalyzed by this enzyme when cGMP content is increased.

  3. The specific activation of TRPC4 by Gi protein subtype.

    PubMed

    Jeon, Jae-Pyo; Lee, Kyu Pil; Park, Eun Jung; Sung, Tae Sik; Kim, Byung Joo; Jeon, Ju-Hong; So, Insuk

    2008-12-12

    The classical type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Especially, TRPC4 has the similar properties to Ca(2+)-permeable nonselective cation channels (NSCCs) activated by muscarinic stimulation in visceral smooth muscles. In visceral smooth muscles, NSCCs activated by muscarinic stimulation were blocked by anti-Galphai/o antibodies. However, there is still no report which Galpha proteins are involved in the activation process of TRPC4. Among Galpha proteins, only Galphai protein can activate TRPC4 channel. The activation effect of Galphai was specific for TRPC4 because Galphai has no activation effect on TRPC5, TRPC6 and TRPV6. Coexpression with muscarinic receptor M2 induced TRPC4 current activation by muscarinic stimulation with carbachol, which was inhibited by pertussis toxin. These results suggest that Galphai is involved specifically in the activation of TRPC4.

  4. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation

    PubMed Central

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3β (GSK-3β) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  5. Protein Crystal Growth Activities on STS-42

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The Protein Crystal Growth (PCG) middeck payload is currently manifested to fly on STS-42 in January 1992. This payload is a joint effort between NASA s Office of Commercial Programs (OCP) and Office of Space Science and Applications (OSSA). The PCG experiments are managed by the Center for Macromolecular Crystallography (CMC), a NASA Center for the Commercial Development of Space (CCDS) based at the University of Alabama at Birmingham (UAB). This is the eighth flight of a payload in the PCG program that is jointly sponsored by the OCP and the OSSA. The flight hardware for STS-42 includes six Vapor Diffusion Apparatus (VDA) trays stored in two Refrigerator/Incubator Modules (R/TM s). The VDA trays will simultaneously conduct 120 experiments involving 15 different protein compounds, four of which are sponsored by the OCP, the UAB CCDS, and four co-investigators.

  6. Breadboard activities for advanced protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Banish, Michael

    1993-01-01

    The proposed work entails the design, assembly, testing, and delivery of a turn-key system for the semi-automated determination of protein solubilities as a function of temperature. The system will utilize optical scintillation as a means of detecting and monitoring nucleation and crystallite growth during temperature lowering (or raising, with retrograde solubility systems). The deliverables of this contract are: (1) turn-key scintillation system for the semi-automatic determination of protein solubilities as a function of temperature, (2) instructions and software package for the operation of the scintillation system, and (3) one semi-annual and one final report including the test results obtained for ovostatin with the above scintillation system.

  7. New constitutive latex osmotin-like proteins lacking antifungal activity.

    PubMed

    Freitas, Cleverson D T; Silva, Maria Z R; Bruno-Moreno, Frederico; Monteiro-Moreira, Ana C O; Moreira, Renato A; Ramos, Márcio V

    2015-11-01

    Proteins that share similar primary sequences to the protein originally described in salt-stressed tobacco cells have been named osmotins. So far, only two osmotin-like proteins were purified and characterized of latex fluids. Osmotin from Carica papaya latex is an inducible protein lacking antifungal activity, whereas the Calotropis procera latex osmotin is a constitutive antifungal protein. To get additional insights into this subject, we investigated osmotins in latex fluids of five species. Two potential osmotin-like proteins in Cryptostegia grandiflora and Plumeria rubra latex were detected by immunological cross-reactivity with polyclonal antibodies produced against the C. procera latex osmotin (CpOsm) by ELISA, Dot Blot and Western Blot assays. Osmotin-like proteins were not detected in the latex of Thevetia peruviana, Himatanthus drasticus and healthy Carica papaya fruits. Later, the two new osmotin-like proteins were purified through immunoaffinity chromatography with anti-CpOsm immobilized antibodies. Worth noting the chromatographic efficiency allowed for the purification of the osmotin-like protein belonging to H. drasticus latex, which was not detectable by immunoassays. The identification of the purified proteins was confirmed after MS/MS analyses of their tryptic digests. It is concluded that the constitutive osmotin-like proteins reported here share structural similarities to CpOsm. However, unlike CpOsm, they did not exhibit antifungal activity against Fusarium solani and Colletotrichum gloeosporioides. These results suggest that osmotins of different latex sources may be involved in distinct physiological or defensive events.

  8. On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

    PubMed Central

    Smirnov, M D; Triplett, D T; Comp, P C; Esmon, N L; Esmon, C T

    1995-01-01

    Phosphatidylethanolamine (PE) is an important membrane component for supporting activated protein C anticoagulant activity but has little influence on prothrombin activation. This difference constitutes a potential mechanism for selective inhibition of the protein C anticoagulant pathway by lupus anticoagulants and/or antiphospholipid antibodies. In this study, we demonstrate that the presence of PE augments lupus anticoagulant activity. In the plasma of some patients with lupus anticoagulants, activated protein C anticoagulant activity is more potently inhibited than prothrombin activation. As a result, in the presence of activated protein C and PE, these patient plasmas clot faster than normal plasma. Patients with minimal lupus anticoagulant activity are identified whose plasma potently inhibits activated protein C anticoagulant activity. This process is also PE dependent. In three patient plasmas, these phenomena are shown to be due to immunoglobulins. The PE requirement in the expression of activated protein C anticoagulant activity and the PE dependence of some antiphospholipid antibodies provide a mechanistic basis for the selective inhibition of the protein C pathway. Inhibition of activated protein C function may be a common mechanism contributing to increased thrombotic risk in certain patients with antiphospholipid antibodies. PMID:7814631

  9. Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor.

    PubMed

    Mikulík, Karel; Bobek, Jan; Bezousková, Silvia; Benada, Oldrich; Kofronová, Olga

    2002-11-29

    Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.

  10. Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity.

    PubMed

    Ger, Marija; Zitkus, Zigmantas; Valius, Mindaugas

    2011-10-01

    Adaptor protein Nck1 binds a number of intracellular proteins and influences various signaling pathways. Here we show that Nck1 directly binds and activates the GTPase-activating protein of Ras (RasGAP), which is responsible for the down-regulation of Ras. The first and the third SH3 domains of Nck1 and the NH(2)-terminal proline-rich sequence of RasGAP contribute most to the complex formation causing direct molecular interaction between the two proteins. Cell adhesion to the substrate is obligatory for the Nck1 and RasGAP association, as cell detachment makes RasGAP incapable of associating with Nck1. This leads to the complex dissipation, decrease of RasGAP activity and the increase of H-Ras-GTP level in the detached cells. Our findings reveal unexpected feature of adaptor protein Nck1 as the regulator of RasGAP activity.

  11. In situ protein folding and activation in bacterial inclusion bodies.

    PubMed

    Gonzalez-Montalban, Nuria; Natalello, Antonino; García-Fruitós, Elena; Villaverde, Antonio; Doglia, Silvia Maria

    2008-07-01

    Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.

  12. Protein stability and enzyme activity at extreme biological temperatures.

    PubMed

    Feller, Georges

    2010-08-18

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  13. ATPase activity of the cystic fibrosis transmembrane conductance regulator.

    PubMed

    Li, C; Ramjeesingh, M; Wang, W; Garami, E; Hewryk, M; Lee, D; Rommens, J M; Galley, K; Bear, C E

    1996-11-08

    The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel thought to be critical for salt and water transport by epithelial cells. Plausible models exist to describe a role for ATP hydrolysis in CFTR channel activity; however, biochemical evidence that CFTR possesses intrinsic ATPase activity is lacking. In this study, we report the first measurements of the rate of ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D resides within a motif conserved in many nucleotidases and is known to cause severe human disease. Following reconstitution the mutant protein exhibited both defective ATP hydrolysis and channel gating, providing direct evidence that CFTR utilizes ATP to gate its channel activity.

  14. Controlled Activation of Protein Rotational Dynamics Using Smart Hydrogel Tethering

    SciTech Connect

    Beech, Brenda M.; Xiong, Yijia; Boschek, Curt B.; Baird, Cheryl L.; Bigelow, Diana J.; Mcateer, Kathleen; Squier, Thomas C.

    2014-09-05

    Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications to take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a polyethylene glycol (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with [13C] and [15N], permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy. Upon initial formation of hydrogels protein dynamics are suppressed, with concomitant increases in protein stability. Relaxation of the hydrogel matrix following transient heating results in the activation of protein dynamics and restoration of substrate-induced large-amplitude domain motions necessary for substrate binding.

  15. Cloning of three novel neuronal Cdk5 activator binding proteins.

    PubMed

    Ching, Y P; Qi, Z; Wang, J H

    2000-01-25

    Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana.

  16. The lipid peroxidation product 4-hydroxy-trans-2-nonenal causes protein synthesis in cardiac myocytes via activated mTORC1-p70S6K-RPS6 signaling.

    PubMed

    Calamaras, Timothy D; Lee, Charlie; Lan, Fan; Ido, Yasuo; Siwik, Deborah A; Colucci, Wilson S

    2015-05-01

    Reactive oxygen species (ROS) are elevated in the heart in response to hemodynamic and metabolic stress and promote hypertrophic signaling. ROS also mediate the formation of lipid peroxidation-derived aldehydes that may promote myocardial hypertrophy. One lipid peroxidation by-product, 4-hydroxy-trans-2-nonenal (HNE), is a reactive aldehyde that covalently modifies proteins thereby altering their function. HNE adducts directly inhibit the activity of LKB1, a serine/threonine kinase involved in regulating cellular growth in part through its interaction with the AMP-activated protein kinase (AMPK), but whether this drives myocardial growth is unclear. We tested the hypothesis that HNE promotes myocardial protein synthesis and if this effect is associated with impaired LKB1-AMPK signaling. In adult rat ventricular cardiomyocytes, exposure to HNE (10 μM for 1h) caused HNE-LKB1 adduct formation and inhibited LKB1 activity. HNE inhibited the downstream kinase AMPK, increased hypertrophic mTOR-p70S6K-RPS6 signaling, and stimulated protein synthesis by 27.1 ± 3.5%. HNE also stimulated Erk1/2 signaling, which contributed to RPS6 activation but was not required for HNE-stimulated protein synthesis. HNE-stimulated RPS6 phosphorylation was completely blocked using the mTOR inhibitor rapamycin. To evaluate if LKB1 inhibition by itself could promote the hypertrophic signaling changes observed with HNE, LKB1 was depleted in adult rat ventricular myocytes using siRNA. LKB1 knockdown did not replicate the effect of HNE on hypertrophic signaling or affect HNE-stimulated RPS6 phosphorylation. Thus, in adult cardiac myocytes HNE stimulates protein synthesis by activation of mTORC1-p70S6K-RPS6 signaling most likely mediated by direct inhibition of AMPK. Because HNE in the myocardium is commonly increased by stimuli that cause pathologic hypertrophy, these findings suggest that therapies that prevent activation of mTORC1-p70S6K-RPS6 signaling may be of therapeutic value.

  17. Metaproteomics: Evaluation of protein extraction from activated sludge.

    PubMed

    Hansen, Susan Hove; Stensballe, Allan; Nielsen, Per Halkjaer; Herbst, Florian-Alexander

    2014-11-01

    Metaproteomic studies of full-scale activated sludge systems require reproducible protein extraction methods. A systematic evaluation of three different extractions protocols, each in combination with three different methods of cell lysis, and a commercial kit were evaluated. Criteria used for comparison of each method included the extracted protein concentration and the number of identified proteins and peptides as well as their phylogenetic, cell localization and functional distribution and quantitative reproducibility. Furthermore, the advantage of using specific metagenomes and a 2-step database approach was illustrated. The results recommend a protocol for protein extraction from activated sludge based on the protein extraction reagent B-Per and bead beating. The data have been deposited to the ProteomeXchange with identifier PXD000862 (http://proteomecentral.proteomexchange.org/dataset/PXD000862).

  18. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms.

    PubMed

    Massip, L; Garand, C; Labbé, A; Perreault, E; Turaga, R V N; Bohr, V A; Lebel, M

    2010-03-11

    Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.

  19. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

    PubMed Central

    Massip, L; Garand, C; Labbé, A; Perreault, È; Turaga, RVN; Bohr, VA; Lebel, M

    2015-01-01

    Werner’s syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCδ and PKCβII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases. PMID:19966859

  20. SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity.

    PubMed

    Wei, Wei-Yen; Li, Hui-Chun; Chen, Chiung-Yao; Yang, Chee-Hing; Lee, Shen-Kao; Wang, Chia-Wen; Ma, Hsin-Chieh; Juang, Yue-Li; Lo, Shih-Yen

    2012-04-01

    The pathogenesis of SARS-CoV remains largely unknown. To study the function of the SARS-CoV nucleocapsid protein, we have conducted a yeast two-hybrid screening experiment to identify cellular proteins that may interact with the SARS-CoV nucleocapsid protein. Pyruvate kinase (liver) was found to interact with SARS-CoV nucleocapsid protein in this experiment. The binding domains of these two proteins were also determined using the yeast two-hybrid system. The physical interaction between the SARS-CoV nucleocapsid and cellular pyruvate kinase (liver) proteins was further confirmed by GST pull-down assay, co-immunoprecipitation assay and confocal microscopy. Cellular pyruvate kinase activity in hepatoma cells was repressed by SARS-CoV nucleocapsid protein in either transiently transfected or stably transfected cells. PK deficiency in red blood cells is known to result in human hereditary non-spherocytic hemolytic anemia. It is reasonable to assume that an inhibition of PKL activity due to interaction with SARS-CoV N protein is likely to cause the death of the hepatocytes, which results in the elevation of serum alanine aminotransferase and liver dysfunction noted in most SARS patients. Thus, our results suggest that SARS-CoV could reduce pyruvate kinase activity via its nucleocapsid protein, and this may in turn cause disease.

  1. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    SciTech Connect

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  2. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    DOE PAGES

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; ...

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

  3. Specific modulation of protein activity by using a bioorthogonal reaction.

    PubMed

    Warner, John B; Muthusamy, Anand K; Petersson, E James

    2014-11-24

    Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a "click" reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z-containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active-site occlusion and allosteric mechanisms. We have termed this a "clickable magic bullet" strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.

  4. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  5. Hypoglycemia-activated GLUT2 neurons of the nucleus tractus solitarius stimulate vagal activity and glucagon secretion.

    PubMed

    Lamy, Christophe M; Sanno, Hitomi; Labouèbe, Gwenaël; Picard, Alexandre; Magnan, Christophe; Chatton, Jean-Yves; Thorens, Bernard

    2014-03-04

    Glucose-sensing neurons in the brainstem participate in the regulation of energy homeostasis but have been poorly characterized because of the lack of specific markers to identify them. Here we show that GLUT2-expressing neurons of the nucleus of the tractus solitarius form a distinct population of hypoglycemia-activated neurons. Their response to low glucose is mediated by reduced intracellular glucose metabolism, increased AMP-activated protein kinase activity, and closure of leak K(+) channels. These are GABAergic neurons that send projections to the vagal motor nucleus. Light-induced stimulation of channelrhodospin-expressing GLUT2 neurons in vivo led to increased parasympathetic nerve firing and glucagon secretion. Thus GLUT2 neurons of the nucleus tractus solitarius link hypoglycemia detection to counterregulatory response. These results may help identify the cause of hypoglycemia-associated autonomic failure, a major threat in the insulin treatment of diabetes.

  6. Auto-phosphorylation Represses Protein Kinase R Activity

    PubMed Central

    Wang, Die; de Weerd, Nicole A.; Willard, Belinda; Polekhina, Galina; Williams, Bryan R. G.; Sadler, Anthony J.

    2017-01-01

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity. PMID:28281686

  7. HMG Proteins and DNA Flexibility in Transcription Activation

    PubMed Central

    Ross, Eric D.; Hardwidge, Philip R.; Maher, L. James

    2001-01-01

    The relative stiffness of naked DNA is evident from measured values of longitudinal persistence length (∼150 bp) and torsional persistence length (∼180 bp). These parameters predict that certain arrangements of eukaryotic transcription activator proteins in gene promoters should be much more effective than others in fostering protein-protein interactions with the basal RNA polymerase II transcription apparatus. Thus, if such interactions require some kind of DNA looping, DNA loop energies should depend sensitively on helical phasing of protein binding sites, loop size, and intrinsic DNA curvature within the loop. Using families of artificial transcription templates where these parameters were varied, we were surprised to find that the degree of transcription activation by arrays of Gal4-VP1 transcription activators in HeLa cell nuclear extract was sensitive only to the linear distance separating a basal promoter from an array of bound activators on DNA templates. We now examine the hypothesis that this unexpected result is due to factors in the extract that act to enhance apparent DNA flexibility. We demonstrate that HeLa cell nuclear extract is rich in a heat-resistant activity that dramatically enhances apparent DNA longitudinal and torsional flexibility. Recombinant mammalian high-mobility group 2 (HMG-2) protein can substitute for this activity. We propose that the abundance of HMG proteins in eukaryotic nuclei provides an environment in which DNA is made sufficiently flexible to remove many constraints on protein binding site arrangements that would otherwise limit efficient transcription activation to certain promoter geometries. PMID:11533247

  8. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

    PubMed

    Soares, Alexandra Martins dos Santos; de Araújo, Sandra Alves; Lopes, Suzana Gomes; Costa Junior, Livio Martins

    2015-01-01

    The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts.

  9. Steady-state compartmentalization of lipid membranes by active proteins.

    PubMed Central

    Sabra, M C; Mouritsen, O G

    1998-01-01

    Using a simple microscopic model of lipid-protein interactions, based on the hydrophobic matching principle, we study some generic aspects of lipid-membrane compartmentalization controlled by a dispersion of active integral membrane proteins. The activity of the proteins is simulated by conformational excitations governed by an external drive, and the deexcitation is controlled by interaction of the protein with its lipid surroundings. In response to the flux of energy into the proteins from the environment and the subsequent dissipation of energy into the lipid bilayer, the lipid-protein assembly reorganizes into a steady-state structure with a typical length scale determined by the strength of the external drive. In the specific case of a mixed dimyristoylphosphatidylcholine-distearoylphosphatidylcholine bilayer in the gel-fluid coexistence region, it is shown explicitly by computer simulation that the activity of an integral membrane protein can lead to a compartmentalization of the lipid-bilayer membrane. The compartmentalization is related to the dynamical process of phase separation and lipid domain formation. PMID:9533687

  10. Modeling the SHG activities of diverse protein crystals

    PubMed Central

    Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J.

    2012-01-01

    A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-­magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices. PMID:23090400

  11. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins.

  12. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  13. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  14. Utilizing avidity to improve antifreeze protein activity: a type III antifreeze protein trimer exhibits increased thermal hysteresis activity.

    PubMed

    Can, Özge; Holland, Nolan B

    2013-12-03

    Antifreeze proteins (AFPs) are ice growth inhibitors that allow the survival of several species living at temperatures colder than the freezing point of their bodily fluids. AFP activity is commonly defined in terms of thermal hysteresis, which is the difference observed for the solution freezing and melting temperatures. Increasing the thermal hysteresis activity of these proteins, particularly at low concentrations, is of great interest because of their wide range of potential applications. In this study, we have designed and expressed one-, two-, and three-domain antifreeze proteins to improve thermal hysteresis activity through increased binding avidity. The three-domain type III AFP yielded significantly greater activity than the one- and two-domain proteins, reaching a thermal hysteresis of >1.6 °C at a concentration of <1 mM. To elucidate the basis of this increase, the data were fit to a multidomain protein adsorption model based on the classical Langmuir isotherm. Fits of the data to the modified isotherms yield values for the equilibrium binding constants for the adsorption of AFP to ice and indicate that protein surface coverage is proportional to thermal hysteresis activity.

  15. Heated Proteins are Still Active in a Functionalized Nanoporous Support

    SciTech Connect

    Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Lei, Chenghong; Liu, Jun

    2013-07-08

    We report that even under the heated condition, the conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction, although the hoarded protein was not exhibiting the full protein activity, the protein released subsequently still maintained its native conformation and activity. Glucose oxidase (GOX) was spontaneously and largely entrapped in aminopropyl-functionalized mesoporous silica (NH2-FMS) at 20 oC via a dominant electrostatic interaction. Although FMS-GOX displayed 45% activity of the free enzyme in solution, the GOX released from FMS exhibited its 100% activity prior to the entrapment. Surprisingly, the released GOX from FMS still maintained 89% of its initial activity prior to the entrapment after FMS-GOX was incubated at 60 oC for 1 h prior to release, while the free GOX in solution lost nearly all activity under the same incubation. Intrinsic fluorescence emission of GOX and native electrophoresis demonstrated that the heating resulted in significant conformational changes and oligomeric structures of the free GOX, but FMS efficiently maintained the thermal stability of GOX therein and resisted the thermal denaturation and oligomeric aggregation.

  16. Counteracting Protein Kinase Activity in the Heart: The Multiple Roles of Protein Phosphatases

    PubMed Central

    Weber, Silvio; Meyer-Roxlau, Stefanie; Wagner, Michael; Dobrev, Dobromir; El-Armouche, Ali

    2015-01-01

    Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PKs) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional, and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control toward this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of PPs which in turn can be used to fine-tune misleaded PK activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth. PMID:26617522

  17. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  18. Enzymatic Activity of the Scaffold Protein Rapsyn for Synapse Formation.

    PubMed

    Li, Lei; Cao, Yu; Wu, Haitao; Ye, Xinchun; Zhu, Zhihui; Xing, Guanglin; Shen, Chengyong; Barik, Arnab; Zhang, Bin; Xie, Xiaoling; Zhi, Wenbo; Gan, Lin; Su, Huabo; Xiong, Wen-Cheng; Mei, Lin

    2016-12-07

    Neurotransmission is ensured by a high concentration of neurotransmitter receptors at the postsynaptic membrane. This is mediated by scaffold proteins that bridge the receptors with cytoskeleton. One such protein is rapsyn (receptor-associated protein at synapse), which is essential for acetylcholine receptor (AChR) clustering and NMJ (neuromuscular junction) formation. We show that the RING domain of rapsyn contains E3 ligase activity. Mutation of the RING domain that abolishes the enzyme activity inhibits rapsyn- as well as agrin-induced AChR clustering in heterologous and muscle cells. Further biological and genetic studies support a working model where rapsyn, a classic scaffold protein, serves as an E3 ligase to induce AChR clustering and NMJ formation, possibly by regulation of AChR neddylation. This study identifies a previously unappreciated enzymatic function of rapsyn and a role of neddylation in synapse formation, and reveals a potential target of therapeutic intervention for relevant neurological disorders.

  19. The role of adapter proteins in T cell activation.

    PubMed

    Koretzky, G A; Boerth, N J

    1999-12-01

    Engagement of antigen receptors on lymphocytes leads to a myriad of complex signal transduction cascades. Recently, work from several laboratories has led to the identification and characterization of novel adapter molecules, proteins with no intrinsic enzymatic activity but which integrate signal transduction pathways by mediating protein-protein interactions. Interestingly, it appears that many of these adapter proteins play as critical a role as the effector enzymes themselves in both lymphocyte development and activation. This review describes some of the biochemical and molecular features of several of these newly identified hematopoietic cell-specific adapter molecules highlighting their importance in regulating (both positively and negatively) signal transduction mediated by the T cell antigen receptor.

  20. Protein profiling of mouse livers with peroxisome proliferator-activated receptor alpha activation.

    PubMed

    Chu, Ruiyin; Lim, Hanjo; Brumfield, Laura; Liu, Hong; Herring, Chris; Ulintz, Peter; Reddy, Janardan K; Davison, Matthew

    2004-07-01

    Peroxisome proliferator-activated receptor alpha (PPARalpha) is important in the induction of cell-specific pleiotropic responses, including the development of liver tumors, when it is chronically activated by structurally diverse synthetic ligands such as Wy-14,643 or by unmetabolized endogenous ligands resulting from the disruption of the gene encoding acyl coenzyme A (CoA) oxidase (AOX). Alterations in gene expression patterns in livers with PPARalpha activation were delineated by using a proteomic approach to analyze liver proteins of Wy-14,643-treated and AOX(-/-) mice. We identified 46 differentially expressed proteins in mouse livers with PPARalpha activation. Up-regulated proteins, including acetyl-CoA acetyltransferase, farnesyl pyrophosphate synthase, and carnitine O-octanoyltransferase, are involved in fatty acid metabolism, whereas down-regulated proteins, including ketohexokinase, formiminotransferase-cyclodeaminase, fructose-bisphosphatase aldolase B, sarcosine dehydrogenase, and cysteine sulfinic acid decarboxylase, are involved in carbohydrate and amino acid metabolism. Among stress response and xenobiotic metabolism proteins, selenium-binding protein 2 and catalase showed a dramatic approximately 18-fold decrease in expression and a modest approximately 6-fold increase in expression, respectively. In addition, glycine N-methyltransferase, pyrophosphate phosphohydrolase, and protein phosphatase 1D were down-regulated with PPARalpha activation. These observations establish proteomic profiles reflecting a common and predictable pattern of differential protein expression in livers with PPARalpha activation. We conclude that livers with PPARalpha activation are transcriptionally geared towards fatty acid combustion.

  1. Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

    PubMed Central

    Ulloa, R M; Mesri, E; Esteva, M; Torres, H N; Téllez-Iñón, M T

    1988-01-01

    A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band. Images Fig. 7. Fig. 8. PMID:2848508

  2. [Antimodification activity of the ArdA and Ocr proteins].

    PubMed

    Zavil'gel'skiĭ, G V; Kotova, V Iu; Rastorguev, S M

    2011-02-01

    The ArdA and Ocr antirestriction proteins, whose genes are in transmissible plasmids (ardA) and bacteriophage genomes (0.3 (ocr)), specifically inhibit type I restriction-modification enzymes. The Ocr protein (T7 bacteriophage) was shown to inhibit both restriction (endonuclease) and modification (methylase) activities of the EcoKI enzyme in a broad range of intracellular concentrations (starting from 10-20 molecules per cell). In contrast to Ocr, the ArdA protein (ColIb-P9 transmissible plasmid) inhibited both of the EcoKI activities only at high intracellular concentrations (30000-40000 molecules per cell). When the ArdA concentration was several fold lower, only endonuclease activity of EcoKI was inhibited. It was assumed that a poorer ArdA ability to inhibit EcoKI modification activity is related to the substantial difference in life cycle between transmissible plasmids (symbiosis with the bacterial cell) and bacteriophages (infection and lysis of bacteria). The Ocr and ArdA mutants that inhibited exclusively endonuclease activity of EcoKI were obtained. Antirestriction proteins incapable of homodimerization were assumed to inhibit only endonuclease activity of type I restriction-modification enzymes.

  3. Hydrodynamic collective effects of active proteins in biological membranes

    NASA Astrophysics Data System (ADS)

    Koyano, Yuki; Kitahata, Hiroyuki; Mikhailov, Alexander S.

    2016-08-01

    Lipid bilayers forming biological membranes are known to behave as viscous two-dimensional fluids on submicrometer scales; usually they contain a large number of active protein inclusions. Recently, it was shown [A. S. Mikhailov and R. Kapral, Proc. Natl. Acad. Sci. USA 112, E3639 (2015), 10.1073/pnas.1506825112] that such active proteins should induce nonthermal fluctuating lipid flows leading to diffusion enhancement and chemotaxislike drift for passive inclusions in biomembranes. Here, a detailed analytical and numerical investigation of such effects is performed. The attention is focused on the situations when proteins are concentrated within lipid rafts. We demonstrate that passive particles tend to become attracted by active rafts and are accumulated inside them.

  4. [Regulation of G protein-coupled receptor kinase activity].

    PubMed

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  5. Ubp8 and SAGA Regulate Snf1 AMP Kinase Activity

    PubMed Central

    Wilson, Marenda A.; Koutelou, Evangelia; Hirsch, Calley; Akdemir, Kadir; Schibler, Andria; Barton, Michelle Craig; Dent, Sharon Y. R.

    2011-01-01

    Posttranslational modifications of histone proteins play important roles in the modulation of gene expression. The Saccharomyces cerevisiae (yeast) 2-MDa SAGA (Spt-Ada-Gcn5) complex, a well-studied multisubunit histone modifier, regulates gene expression through Gcn5-mediated histone acetylation and Ubp8-mediated histone deubiquitination. Using a proteomics approach, we determined that the SAGA complex also deubiquitinates nonhistone proteins, including Snf1, an AMP-activated kinase. Ubp8-mediated deubiquitination of Snf1 affects the stability and phosphorylation state of Snf1, thereby affecting Snf1 kinase activity. Others have reported that Gal83 is phosphorylated by Snf1, and we found that deletion of UBP8 causes decreased phosphorylation of Gal83, which is consistent with the effects of Ubp8 loss on Snf1 kinase functions. Overall, our data indicate that SAGA modulates the posttranslational modifications of Snf1 in order to fine-tune gene expression levels. PMID:21628526

  6. [Histidine triad protein superfamily--biological function and enzymatic activity].

    PubMed

    Krakowiak, Agnieszka; Fryc, Izabela

    2012-01-01

    The HIT superfamily consists of proteins that share the histidine triad motif, His-X-His-X-His-X-X (where X is a hydrophobic amino acid), which constitutes enzymatic catalytic center. These enzymes act as nucleotidylyl hydrolase or transferase, and the mutation of the second histidine in the triad abolishes their activity. HIT proteins were found ubiquitous in all organisms and they were classified into 5 branches, which are represented by human proteins: HINT1, FHIT, Aprataxin, GALT and DCPS. Because HINT1 orthologs, which belong to the evolutionally oldest family branch, were found from prokaryotes to eukaryotes, it is clear that HIT motif was conserved during the evolution what means that the enzymatic activity is necessary for functions of these proteins. However, in few cases, e.g. HINT1 and FHIT, the connection between the biological function and the enzymatic activity is still obscure. In this review, the relations between biology and activity for 7 HIT proteins, which were found in human, are highlighted.

  7. Signal peptides are allosteric activators of the protein translocase

    PubMed Central

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G.; Economou, Anastassios

    2010-01-01

    Extra-cytoplasmic polypeptides are usually synthesized as “preproteins” carrying aminoterminal, cleavable signal peptides1 and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA2,3. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA2,3. Preprotein targeting to SecA is thought to involve signal peptides4 and chaperones like SecB5,6. Here we reveal that signal peptides have a novel role beyond targeting: they are essential allosteric activators of the translocase. Upon docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, “triggering” that drives the translocase to a lower activation energy state; then “trapping” that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus and, finally, “secretion” during which trapped mature domains undergo multiple turnovers of translocation in segments7. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  8. Bitter melon seed oil-attenuated body fat accumulation in diet-induced obese mice is associated with cAMP-dependent protein kinase activation and cell death in white adipose tissue.

    PubMed

    Chen, Pei-Hsuan; Chen, Gou-Chun; Yang, Mei-Fang; Hsieh, Cheng-Hsien; Chuang, Shu-Han; Yang, Hsin-Ling; Kuo, Yueh-Hsiung; Chyuan, Jong-Ho; Chao, Pei-Min

    2012-07-01

    The aim of this study was to investigate the antiadiposity effect of bitter melon seed oil (BMSO), which is rich in the cis-9, trans-11, trans-13 isomer of conjugated linolenic acid. In Expt. 1, C57BL/6J mice were fed a butter-based, high-fat diet [HB; 29% butter + 1% soybean oil (SBO)] for 10 wk to induce obesity. They then continued to receive that diet or were switched to an SBO-based, high-fat diet alone (HS; 30% SBO) or containing bitter melon seed oil (BMSO) (HBM; 15% SBO + 15% BMSO) for 5 wk. The body fat percentage was significantly lower in mice fed the HBM diet (21%), but not the HS diet, compared with mice fed the HB diet. In Expt. 2, mice were fed an SBO-based, high-fat diet containing 0 (HS), 5 (LBM), 10 (MBM), or 15% (HBM) BMSO for 10 wk. In the LBM, MBM, and HBM groups, the body fat percentage was significantly lower by 32, 35, and 65%, respectively, compared with the HS control. The reduction in the HBM group was significantly greater than that in the LBM or MBM group. BMSO administration increased phosphorylation of acetyl-CoA carboxylase, cAMP-activated protein kinase (PKA), and signal transducer and activator of transcription 3 in the white adipose tissue (WAT), suggesting that PKA and leptin signaling might be involved in the BMSO-mediated reduction in lipogenesis and increase in thermogenesis and lipolysis. However, compared with the HS control, the HBM group had a significantly higher TNFα concentration in the WAT accompanied by TUNEL-positive nuclei. We conclude that BMSO is effective in attenuating body fat accumulation through mechanisms associated with PKA activation and programmed cell death in the WAT, but safety concerns need to be carefully addressed.

  9. Optimizing intramuscular adaptations to aerobic exercise: effects of carbohydrate restriction and protein supplementation on mitochondrial biogenesis.

    PubMed

    Margolis, Lee M; Pasiakos, Stefan M

    2013-11-01

    Mitochondrial biogenesis is a critical metabolic adaptation to aerobic exercise training that results in enhanced mitochondrial size, content, number, and activity. Recent evidence has shown that dietary manipulation can further enhance mitochondrial adaptations to aerobic exercise training, which may delay skeletal muscle fatigue and enhance exercise performance. Specifically, studies have demonstrated that combining carbohydrate restriction (endogenous and exogenous) with a single bout of aerobic exercise potentiates the beneficial effects of exercise on markers of mitochondrial biogenesis. Additionally, studies have demonstrated that high-quality protein supplementation enhances anabolic skeletal muscle intracellular signaling and mitochondrial protein synthesis following a single bout of aerobic exercise. Mitochondrial biogenesis is stimulated by complex intracellular signaling pathways that appear to be primarily regulated by 5'AMP-activated protein kinase and p38 mitogen-activated protein kinase mediated through proliferator-activated γ receptor co-activator 1 α activation, resulting in increased mitochondrial DNA expression and enhanced skeletal muscle oxidative capacity. However, the mechanisms by which concomitant carbohydrate restriction and dietary protein supplementation modulates mitochondrial adaptations to aerobic exercise training remains unclear. This review summarizes intracellular regulation of mitochondrial biogenesis and the effects of carbohydrate restriction and protein supplementation on mitochondrial adaptations to aerobic exercise.

  10. AMPK activity regulates trafficking of mitochondria to the leading edge during cell migration and matrix invasion

    PubMed Central

    Cunniff, Brian; McKenzie, Andrew J.; Heintz, Nicholas H.; Howe, Alan K.

    2016-01-01

    Cell migration is a complex behavior involving many energy-expensive biochemical events that iteratively alter cell shape and location. Mitochondria, the principal producers of cellular ATP, are dynamic organelles that fuse, divide, and relocate to respond to cellular metabolic demands. Using ovarian cancer cells as a model, we show that mitochondria actively infiltrate leading edge lamellipodia, thereby increasing local mitochondrial mass and relative ATP concentration and supporting a localized reversal of the Warburg shift toward aerobic glycolysis. This correlates with increased pseudopodial activity of the AMP-activated protein kinase (AMPK), a critically important cellular energy sensor and metabolic regulator. Furthermore, localized pharmacological activation of AMPK increases leading edge mitochondrial flux, ATP content, and cytoskeletal dynamics, whereas optogenetic inhibition of AMPK halts mitochondrial trafficking during both migration and the invasion of three-dimensional extracellular matrix. These observations indicate that AMPK couples local energy demands to subcellular targeting of mitochondria during cell migration and invasion. PMID:27385336

  11. Germ cell mitogenic activity is associated with nerve growth factor-like protein(s).

    PubMed

    Onoda, M; Pflug, B; Djakiew, D

    1991-12-01

    The mitogenicity of germ cell proteins released from round spermatids (RS) and pachytene spermatocytes (PS) was investigated. Germ cells were isolated by centrifugal elutriation from 90-day-old rat testes and incubated in a supplement enriched culture media that lacked exogenous proteins. The conditioned culture media of RS and PS were dialysed/concentrated and lyophilized to prepare RS protein (RSP) and PS protein (PSP). Mitogenic activity of RSP and PSP was determined by 3H-thymidine incorporation into Swiss 3T3 fibroblasts. RSP and PSP stimulated 3H-thymidine incorporation by fibroblasts in a dose-dependent manner. At a higher concentration of RSP (300 micrograms/ml), fibroblast proliferation was stimulated from 6- to 20-fold of control cultures, whereas PSP (300 micrograms/ml) stimulated fibroblast proliferation 2.5-fold of control cultures. Since RSP exhibited substantially greater mitogenic activity than PSP we further investigated the RSP mitogenic substance(s) by immunoneutralization with antibodies against several growth factors. The mitogenic activity of RSP was significantly reduced by treatment with nerve growth factor (NGF) antibody, while neither the treatment of RSP with acidic fibroblast growth factor (aFGF) antibody, nor basic fibroblast growth factor (bFGF) antibody significantly modified the mitogenic activity of RSP. Interestingly, murine NGF-beta, recombinant human NGF-beta, and bovine serum albumin (BSA) did not exhibit mitogenic activity on 3T3 fibroblasts. Nevertheless, the presence of a NGF-like protein in RS and PS was confirmed by indirect immunofluorescence staining with a murine NGF antibody. Subsequently, a Western blot analysis with the NGF antibody identified two immunoreactive bands of 41 +/- 2 kDa and 51 +/- 1 kDa in both RSP and PSP under reduced conditions. These germ cell NGF-like proteins were apparently different from similarly prepared murine and human NGFs (13 kDa) in their molecular weight. Furthermore, neurite outgrowth

  12. Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein–Protein Interactions

    PubMed Central

    Tetlow, Ian J.; Wait, Robin; Lu, Zhenxiao; Akkasaeng, Rut; Bowsher, Caroline G.; Esposito, Sergio; Kosar-Hashemi, Behjat; Morell, Matthew K.; Emes, Michael J.

    2004-01-01

    Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with γ-32P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated 32P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule–associated phosphoproteins after incubation of intact amyloplasts with γ-32P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein–protein

  13. Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80.

    PubMed Central

    Salmeron, J M; Langdon, S D; Johnston, S A

    1989-01-01

    In Saccharomyces cerevisiae, transcriptional activation mediated by the GAL4 regulatory protein is repressed in the absence of galactose by the binding of the GAL80 protein, an interaction that requires the carboxy-terminal 28 amino acids of GAL4. The homolog of GAL4 from Kluyveromyces lactis, LAC9, activates transcription in S. cerevisiae and is highly similar to GAL4 in its carboxyl terminus but is not repressed by wild-type levels of GAL80 protein. Here we show that GAL80 does repress LAC9-activated transcription in S. cerevisiae if overproduced. We sought to determine the molecular basis for the difference in the responses of the LAC9 and GAL4 proteins to GAL80. Our results indicate that this difference is due primarily to the fact that under wild-type conditions, the level of LAC9 protein in S. cerevisiae is much higher than that of GAL4, which suggests that LAC9 escapes GAL80-mediated repression by titration of GAL80 protein in vivo. The difference in response to GAL80 is not due to amino acid sequence differences between the LAC9 and GAL4 carboxyl termini. We discuss the implications of these results for the mechanism of galactose metabolism regulation in S. cerevisiae and K. lactis. Images PMID:2550790

  14. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  15. Methods to distinguish various types of protein phosphatase activity

    SciTech Connect

    Brautigan, D.L.; Shriner, C.L.

    1988-01-01

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on /sup 32/P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of /sup 32/P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble (/sup 32/P)phosphate.

  16. Multiple switches in G protein-coupled receptor activation.

    PubMed

    Ahuja, Shivani; Smith, Steven O

    2009-09-01

    The activation mechanism of G protein-coupled receptors has presented a puzzle that finally may be close to solution. These receptors have a relatively simple architecture consisting of seven transmembrane helices that contain just a handful of highly conserved amino acids, yet they respond to light and a range of chemically diverse ligands. Recent NMR structural studies on the active metarhodopsin II intermediate of the visual receptor rhodopsin, along with the recent crystal structure of the apoprotein opsin, have revealed multiple structural elements or 'switches' that must be simultaneously triggered to achieve full activation. The confluence of several required structural changes is an example of "coincidence counting", which is often used by nature to regulate biological processes. In ligand-activated G protein-coupled receptors, the presence of multiple switches may provide an explanation for the differences between full, partial and inverse agonists.

  17. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    PubMed

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation.

  18. Detergent activation of the binding protein in the folate radioassay

    SciTech Connect

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with ..beta..-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to ..beta..-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants (lipids/detergents).

  19. [Modulators of the regulatory protein activity acting at microdoses].

    PubMed

    Iamskova, V P; Krasnov, M S; Skripnikova, V S; Moliavka, A A; Il'ina, A P; Margasiuk, D V; Borisenko, A V; Berezin, B B; Iamskov, I A

    2009-01-01

    New, previously not studied bioregulators active in the ultra low doses corresponding of 10(-8) - 10(-17) mg/ml have been isolated from vitreoretinal tissue of eye. It has been shown that these bioregulators comprise some regulatory peptides-modulators represented by proteins with molecular weights 15-70 KDa one of which is bovine serum albumin. Correlation between the nanosize of bioregulators and their ability to show activity in ultra low doses is established.

  20. STAT5-Interacting Proteins: A Synopsis of Proteins that Regulate STAT5 Activity

    PubMed Central

    Able, Ashley A.; Burrell, Jasmine A.; Stephens, Jacqueline M.

    2017-01-01

    Signal Transducers and Activators of Transcription (STATs) are key components of the JAK/STAT pathway. Of the seven STATs, STAT5A and STAT5B are of particular interest for their critical roles in cellular differentiation, adipogenesis, oncogenesis, and immune function. The interactions of STAT5A and STAT5B with cytokine/hormone receptors, nuclear receptors, transcriptional regulators, proto-oncogenes, kinases, and phosphatases all contribute to modulating STAT5 activity. Among these STAT5 interacting proteins, some serve as coactivators or corepressors to regulate STAT5 transcriptional activity and some proteins can interact with STAT5 to enhance or repress STAT5 signaling. In addition, a few STAT5 interacting proteins have been identified as positive regulators of STAT5 that alter serine and tyrosine phosphorylation of STAT5 while other proteins have been identified as negative regulators of STAT5 via dephosphorylation. This review article will discuss how STAT5 activity is modulated by proteins that physically interact with STAT5. PMID:28287479

  1. Protein kinase A regulates the osteogenic activity of Osterix.

    PubMed

    He, Siyuan; Choi, You Hee; Choi, Joong-Kook; Yeo, Chang-Yeol; Chun, ChangJu; Lee, Kwang Youl

    2014-10-01

    Osterix belongs to the SP gene family and is a core transcription factor responsible for osteoblast differentiation and bone formation. Activation of protein kinase A (PKA), a serine/threonine kinase, is essential for controlling bone formation and BMP-induced osteoblast differentiation. However, the relationship between Osterix and PKA is still unclear. In this report, we investigated the precise role of the PKA pathway in regulating Osterix during osteoblast differentiation. We found that PKA increased the protein level of Osterix; PKA phosphorylated Osterix, increased protein stability, and enhanced the transcriptional activity of Osterix. These results suggest that Osterix is a novel target of PKA, and PKA modulates osteoblast differentiation partially through the regulation of Osterix.

  2. Installing hydrolytic activity into a completely de novo protein framework

    NASA Astrophysics Data System (ADS)

    Burton, Antony J.; Thomson, Andrew R.; Dawson, William M.; Brady, R. Leo; Woolfson, Derek N.

    2016-09-01

    The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

  3. A designed supramolecular protein assembly with in vivo enzymatic activity.

    PubMed

    Song, Woon Ju; Tezcan, F Akif

    2014-12-19

    The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(k(cat)/K(m))/k(uncat)] for ampicillin hydrolysis of 2.3 × 10(6) and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.

  4. New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma.

    PubMed

    Alshaikh, N A; Rosing, J; Thomassen, M C L G D; Castoldi, E; Simioni, P; Hackeng, T M

    2017-02-17

    Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation.

  5. Protein S as an in vivo cofactor to activated protein C in prevention of microarterial thrombosis in rabbits.

    PubMed Central

    Arnljots, B; Dahlbäck, B

    1995-01-01

    The antithrombotic effects of bovine activated protein C (APC) and protein S were investigated in a rabbit model of microarterial thrombosis. Because of the species specificity of the APC-protein S interaction, bovine APC expresses potent anticoagulant activity in rabbit plasma only when bovine protein S is also present. This provided a way to assess the contribution of bovine protein S to the antithrombotic effect of bovine APC. Rabbits were infused with boluses of activated protein C (0.1, 0.2, 0.4, or 0.8 mg/kg), protein S (0.5 mg/kg), or activated protein C (0.1 or 0.01 mg/kg) plus protein S (0.5 mg/kg). APC alone produced a dose-dependent antithrombotic effect, but only the group receiving the highest dose differed significantly from controls. While a low dose of activated protein C (0.1 mg/kg) alone had no antithrombotic effect, together with protein S (0.5 mg/kg) it produced a potent response. The presented results demonstrate the in vivo significance of protein S as a cofactor to activated protein C. The data show that a potent antithrombotic effect, without hemorrhagic side effects or significant systemic anticoagulation, may be achieved by low doses of activated protein C when combined with protein S. Images PMID:7738165

  6. Monitoring Brain Activity with Protein Voltage and Calcium Sensors

    PubMed Central

    Storace, Douglas A.; Braubach, Oliver R.; Jin, Lei; Cohen, Lawrence B.; Sung, Uhna

    2015-01-01

    Understanding the roles of different cell types in the behaviors generated by neural circuits requires protein indicators that report neural activity with high spatio-temporal resolution. Genetically encoded fluorescent protein (FP) voltage sensors, which optically report the electrical activity in distinct cell populations, are, in principle, ideal candidates. Here we demonstrate that the FP voltage sensor ArcLight reports odor-evoked electrical activity in the in vivo mammalian olfactory bulb in single trials using both wide-field and 2-photon imaging. ArcLight resolved fast odorant-responses in individual glomeruli, and distributed odorant responses across a population of glomeruli. Comparisons between ArcLight and the protein calcium sensors GCaMP3 and GCaMP6f revealed that ArcLight had faster temporal kinetics that more clearly distinguished activity elicited by individual odorant inspirations. In contrast, the signals from both GCaMPs were a saturating integral of activity that returned relatively slowly to the baseline. ArcLight enables optical electrophysiology of mammalian neuronal population activity in vivo. PMID:25970202

  7. Visible-Light-Triggered Activation of a Protein Kinase Inhibitor.

    PubMed

    Wilson, Danielle; Li, Jason W; Branda, Neil R

    2017-02-20

    A photoresponsive small molecule undergoes a ring-opening reaction when exposed to visible light and becomes an active inhibitor of the enzyme protein kinase C. This "turning on" of enzyme inhibition with light puts control into the hands of the user, creating the opportunity to regulate when and where enzyme catalysis takes place.

  8. Cholesterol-Lowering Activity of Tartary Buckwheat Protein.

    PubMed

    Zhang, Chengnan; Zhang, Rui; Li, Yuk Man; Liang, Ning; Zhao, Yimin; Zhu, Hanyue; He, Zouyan; Liu, Jianhui; Hao, Wangjun; Jiao, Rui; Ma, Ka Ying; Chen, Zhen-Yu

    2017-03-08

    Previous research has shown that Tartary buckwheat flour is capable of reducing plasma cholesterol. The present study was to examine the effect of rutin and Tartary buckwheat protein on plasma total cholesterol (TC) in hypercholesterolemia hamsters. In the first animal experiment, 40 male hamsters were divided into four groups fed either the control diet or one of the three experimental diets containing 8.2 mmol rutin, 8.2 mmol quercetin, or 2.5 g kg(-1) cholestyramine, respectively. Results showed that only cholestyramine but not rutin and its aglycone quercetin decreased plasma TC, which suggested that rutin was not the active ingredient responsible for plasma TC-lowering activity of Tartary buckwheat flour. In the second animal experiment, 45 male hamsters were divided into five groups fed either the control diet or one of the four experimental diets containing 24% Tartary buckwheat protein, 24% rice protein, 24% wheat protein, or 5 g kg(-1) cholestyramine, respectively. Tartary buckwheat protein reduced plasma TC more effectively than cholestyramine (45% versus 37%), while rice and wheat proteins only reduced plasma TC by 10-13%. Tartary buckwheat protein caused 108% increase in the fecal excretion of total neutral sterols and 263% increase in the fecal excretion of total acidic sterols. real-time polymerase chain reaction and Western blotting analyses showed that Tartary buckwheat protein affected the gene expression of intestinal Niemann-Pick C1-like protein 1 (NPC1L1), acyl CoA:cholesterol acyltransferase 2 (ACAT2), and ATP binding cassette transporters 5 and 8 (ABCG5/8) in a down trend, whereas it increased the gene expression of hepatic cholesterol-7α -hydroxylase (CYP7A1). It was concluded that Tartary buckwheat protein was at least one of the active ingredients in Tartary buckwheat flour to lower plasma TC, mainly mediated by enhancing the excretion of bile acids via up-regulation of hepatic CYP7A1 and also by inhibiting the absorption of dietary

  9. Reassessing the Potential Activities of Plant CGI-58 Protein.

    PubMed

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.

  10. Reassessing the Potential Activities of Plant CGI-58 Protein

    PubMed Central

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed. PMID:26745266

  11. Pivotal Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in Inflammatory Pulmonary Diseases

    PubMed Central

    Qian, Feng; Deng, Jing; Wang, Gang; Ye, Richard D.; Christman, John W.

    2016-01-01

    Mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) is exclusively regulated by p38 MAPK in vivo. Upon activation of p38 MAPK, MK2 binds with p38 MAPK, leading to phosphorylation of TTP, Hsp27, Akt and Cdc25 that are involved in regulation of various essential cellular functions. In this review, we discuss current knowledge about molecular mechanisms of MK2 in regulation of TNF-α production, NADPH oxidase activation, neutrophil migration, and DNA-damage-induced cell cycle arrest which are involved in the molecular pathogenesis of acute lung injury, pulmonary fibrosis, and non-small-cell lung cancer. Collectively current and emerging new information indicate that developing MK2 inhibitors and blocking MK2-mediated signal pathways is a potential therapeutic strategy for treatment of inflammatory and fibrotic lung diseases and lung cancer. PMID:26119506

  12. Turnover of whole body proteins and myofibrillar proteins in middle-aged active men

    SciTech Connect

    Zackin, M.; Meredith, C.; Frontera, W.; Evans, W.

    1986-03-05

    Endurance-trained older men have a higher proportion of lean tissue and greater muscle cell oxidative capacity, reversing age-related trends and suggesting major changes in protein metabolism. In this study, protein turnover was determined in 6 middle-aged (52+/-1 yr) men who were well trained (VO/sub 2/ max 55.2+/-5.0 ml O/sub 2//kg.min) and lean (body fat 18.9+/-2.8%, muscle mass 36.6+/-0.6%). The maintained habitual exercise while consuming 0.6, 0.9 or 1.2 g protein/kg.day for 10-day periods. N flux was measured from /sup 15/N in urea after oral /sup 15/N-glycine administration. Myofibrillar protein breakdown was estimated from urinary 3-methyl-histidine. Dietary protein had no effect on turnover rates, even when N balance was negative. Whole body protein synthesis was 3.60+/-0.12 g/kg.day and breakdown was 3.40+/-0.14 g/kg.day for all N intakes. Whole body protein flux, synthesis and breakdown were similar to values reported for sedentary young (SY) or sedentary old (SO) men on comparable diets. 3-me-his (3.67+/-0.14 ..mu..mol/kg.day) was similar to values reported for SY but higher (p<0.01) than for SO. Myofibrillar protein breakdown per unit muscle mass (185+/-7 ..mu..mol 3-me-his/g creatinine) was higher (p<0.01) than for SY or SO. In active middle-aged men, myofibrillar proteins may account for a greater proportion of whole body protein turnover, despite an age-related reduction in muscle mass.

  13. A Novel Method for Assessing the Chaperone Activity of Proteins

    PubMed Central

    Hristozova, Nevena; Tompa, Peter; Kovacs, Denes

    2016-01-01

    Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families–molecules expressed during adverse conditions, infection, and diseases–chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function. PMID:27564234

  14. Design of a Split Intein with Exceptional Protein Splicing Activity

    PubMed Central

    2016-01-01

    Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques. PMID:26854538

  15. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  16. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

    PubMed

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  17. Notch1 receptor regulates AKT protein activation loop (Thr308) dephosphorylation through modulation of the PP2A phosphatase in phosphatase and tensin homolog (PTEN)-null T-cell acute lymphoblastic leukemia cells.

    PubMed

    Hales, Eric C; Orr, Steven M; Larson Gedman, Amanda; Taub, Jeffrey W; Matherly, Larry H

    2013-08-02

    Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr(308) phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr(308) phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr(308) phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr(308) by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr(172) and p70S6K-Thr(389), both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.

  18. Protein Corona of Magnetic Hydroxyapatite Scaffold Improves Cell Proliferation via Activation of Mitogen-Activated Protein Kinase Signaling Pathway.

    PubMed

    Zhu, Yue; Yang, Qi; Yang, Minggang; Zhan, Xiaohui; Lan, Fang; He, Jing; Gu, Zhongwei; Wu, Yao

    2017-03-21

    The beneficial effect of magnetic scaffolds on the improvement of cell proliferation has been well documented. Nevertheless, the underlying mechanisms about the magnetic scaffolds stimulating cell proliferation remain largely unknown. Once the scaffold enters into the biological fluids, a protein corona forms and directly influences the biological function of scaffold. This study aimed at investigating the formation of protein coronas on hydroxyapatite (HA) and magnetic hydroxyapatite (MHA) scaffolds in vitro and in vivo, and consequently its effect on regulating cell proliferation. The results demonstrated that magnetic nanoparticles (MNP)-infiltrated HA scaffolds altered the composition of protein coronas and ultimately contributed to increased concentration of proteins related to calcium ions, G-protein coupled receptors (GPCRs), and MAPK/ERK cascades as compared with pristine HA scaffolds. Noticeably, the enriched functional proteins on MHA samples could efficiently activate of the MAPK/ERK signaling pathway, resulting in promoting MC3T3-E1 cell proliferation, as evidenced by the higher expression levels of the key proteins in the MAPK/ERK signaling pathway, including mitogen-activated protein kinase kinases1/2 (MEK1/2) and extracellular signal regulated kinase 1/2 (ERK1/2). Artificial down-regulation of MEK expression can significantly down-regulate the MAPK/ERK signaling and consequently suppress the cell proliferation on MHA samples. These findings not only provide a critical insight into the molecular mechanism underlying cellular proliferation on magnetic scaffolds, but also have important implications in the design of magnetic scaffolds for bone tissue engineering.

  19. Plasmodium falciparum heat shock protein 70 lacks immune modulatory activity.

    PubMed

    Pooe, Ofentse Jacob; Köllisch, Gabriele; Heine, Holger; Shonhai, Addmore

    2017-02-14

    Heat shock protein 70 (Hsp70) family are conserved molecules that constitute a major part of the cell's protein folding machinery. The role of Hsp70s of parasitic origin in host cell immune modulation has remained contentious. This is largely due to the fact that several studies implicating Hsp70 in immune modulation rely on the use of recombinant protein derived from bacteria which is often fraught contamination. Thus, in the current study, we expressed recombinant Plasmodium falciparum Hsp70 (PfHsp70) using in three bacterial expression hosts: E. coli XL1 Blue, E. coli ClearColi BL21 and Brevibacillus choshinensis, respectively. We further investigated the immunostimulatory capability of the protein by assessing cytokine production by murine immune cells cultured in the presence of the protein. Recombinant PfHsp70 obtained from E. coli XL1 Blue expression host induced IL6 and IL8 cytokines. On the other hand, PfHsp70 produced in E. coli ClearColi and B. choshinensis expression systems was associated with no detectable traces of LPS and exhibited no immunomodulatory activity. Our findings suggest that PfHsp70 does not possess immunomodulatory function. Furthermore, our study suggests that E. coli ClearColi and B. choshinensis are versatile for the production of recombinant protein for use in immunomodulatory studies.

  20. Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein

    PubMed Central

    Ader, Nadine; Brindley, Melinda; Avila, Mislay; Örvell, Claes; Horvat, Branka; Hiltensperger, Georg; Schneider-Schaulies, Jürgen; Vandevelde, Marc; Zurbriggen, Andreas; Plemper, Richard K.

    2013-01-01

    The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering. PMID:23077316

  1. Pharmacological activities in thermal proteins: relationships in molecular evolution

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Hefti, F.; Hartikka, J.; Junard, E.; Przybylski, A. T.; Vaughan, G.

    1987-01-01

    The model of protobiological events that has been presented in these pages has increasing relevance to pharmacological research. The thermal proteins that function as key substances in the proteinoid theory have recently been found to prolong the survival of rat forebrain neurons in culture and to stimulate the growth of neurites. A search for such activity in thermal proteins added to cultures of modern neurons was suggested by the fact that some of the microspheres assembled from proteinoids rich in hydrophobic amino acids themselves generate fibrous outgrowths.

  2. Pharmacokinetics of activated protein C in guinea pigs

    SciTech Connect

    Berger, H. Jr.; Kirstein, C.G.; Orthner, C.L. )

    1991-05-15

    Protein C is a vitamin K-dependent zymogen of the serine protease, activated protein C (APC), an important regulatory enzyme in hemostasis. In view of the potential of human APC as an anticoagulant and profibrinolytic agent, the pharmacokinetics and tissue distribution of APC were studied in guinea pigs. The plasma elimination of a trace dose of {sup 125}I-APC was biphasic following an initial rapid elimination of approximately 15% of the injected dose within 1 to 2 minutes. This rapid removal of {sup 125}I-APC from the circulation was found to be a result of an association with the liver regardless of the route of injection. Essentially identical results were obtained with active site-blocked forms of APC generated with either diisopropylfluorophosphate or D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, which indicates that the active site was not essential for the liver association. Accumulation of all three forms of APC in the liver peaked at 30 minutes and then declined as increasing amounts of degraded radiolabeled material appeared in the gastrointestinal tract and urine. Removal of the gamma-carboxyglutamic acid (gla) domain of diisopropylphosphoryl-APC resulted in a 50% reduction in the association with liver and an accumulation in the kidneys. Protein C and protein S were cleared from the circulation at rates approximately one-half and one-fourth, respectively, that of APC. Both in vitro and in vivo, APC was found to form complexes with protease inhibitors present in guinea pig plasma. Complex formation resulted in a more rapid disappearance of the enzymatic activity of APC than elimination of the protein moiety. These findings indicate two distinct mechanisms for the elimination of APC. One mechanism involves reaction with plasma protease inhibitors and subsequent elimination by specific hepatic receptors. (Abstract Truncated)

  3. Novel condensation products having high activity to insolubilize proteins and protein-insolubilized products

    SciTech Connect

    Krasnobajew, V.; Boeniger, R.

    1980-01-01

    According to the invention a substantially more active product with respect to the fixing or insolubilization pf proteins, including enzymes, is obtained when 1,3 phenylenediamine is condensed with glutardialdehyde. One application of the process is the enzymatic hydrolysis of lactose in milk products by lactase.

  4. The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development.

    PubMed

    Dickerman, Benjamin K; White, Christine L; Kessler, Patricia M; Sadler, Anthony J; Williams, Bryan R G; Sen, Ganes C

    2015-12-01

    The murine double-stranded RNA-binding protein termed protein kinase R (PKR)-associated protein X (RAX) and the human homolog, protein activator of PKR (PACT), were originally characterized as activators of PKR. Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax(-/-) mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eukaryotic translation initiation factor 2 α subunit (eIF2α) (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax(-/-) mice. Generating rax(-/-) mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax(-/-) defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax(-/-) mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax(-/-) defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21(WAF1/CIP1). These results demonstrate that PKR kinase activity is required for onset of the rax(-/-) phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development.

  5. Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding

    PubMed Central

    Langendorf, Christopher G.; Ngoei, Kevin R. W.; Scott, John W.; Ling, Naomi X. Y.; Issa, Sam M. A.; Gorman, Michael A.; Parker, Michael W.; Sakamoto, Kei; Oakhill, Jonathan S.; Kemp, Bruce E.

    2016-01-01

    The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases. PMID:26952388

  6. Solubilized placental membrane protein inhibits insulin receptor tyrosine kinase activity

    SciTech Connect

    Strout, H.V. Jr.; Slater, E.E.

    1987-05-01

    Regulation of insulin receptor (IR) tyrosine kinase (TK) activity may be important in modulating insulin action. Utilizing an assay which measures IR phosphorylation of angiotensin II (AII), the authors investigated whether fractions of TX-100 solubilized human placental membranes inhibited IR dependent AII phosphorylation. Autophosphorylated IR was incubated with membrane fractions before the addition of AII, and kinase inhibition measured by the loss of TSP incorporated in AII. An inhibitory activity was detected which was dose, time, and temperature dependent. The inhibitor was purified 200-fold by sequential chromatography on wheat germ agglutinin, DEAE, and hydroxyapatite. This inhibitory activity was found to correlate with an 80 KD protein which was electroeluted from preparative slab gels and rabbit antiserum raised. Incubation of membrane fractions with antiserum before the IRTK assay immunoprecipitated the inhibitor. Protein immunoblots of crude or purified fractions revealed only the 80 KD protein. Since IR autophosphorylation is crucial to IRTK activity, the authors investigated the state of IR autophosphorylation after treatment with inhibitor; no change was detected by phosphoamino acid analysis.

  7. Methods of measuring Protein Disulfide Isomerase activity: a critical overview

    NASA Astrophysics Data System (ADS)

    Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

    2014-09-01

    Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

  8. Analysis of antifreeze protein activity using colorimetric gold nanosensors

    NASA Astrophysics Data System (ADS)

    Jing, Xu; Choi, Ho-seok; Park, Ji-In; Kim, Young-Pil

    2015-07-01

    High activity and long stability of antifreeze proteins (AFPs), also known as ice-binding proteins (IBPs), are necessary for exerting their physiological functions in biotechnology and cryomedicine. Here we report a simple analysis of antifreeze protein activity and stability based on self-assembly of gold nanoparticles (AuNPs) via freezing and thawing cycles. While the mercaptosuccinic acid-capped AuNP (MSA-AuNP) was easily self-assembled after a freezing/thawing cycle, due to the mechanical attack of ice crystal on the MSA-AuNP surface, the presence of AFP impeded the self-assembly of MSA-AuNP via the interaction of AFP with ice crystals via freezing and thawing cycles, which led to a strong color in the MSA-AuNP solution. As a result, the aggregation parameter (E520/E650) of MSA-AuNP showed the rapid detection of both activity and stability of AFPs. We suggest that our newly developed method is very suitable for measuring antifreeze activity and stability in a simple and rapid manner with reliable quantification.

  9. Synthetic phosphorylation of p38α recapitulates protein kinase activity.

    PubMed

    Chooi, K Phin; Galan, Sébastien R G; Raj, Ritu; McCullagh, James; Mohammed, Shabaz; Jones, Lyn H; Davis, Benjamin G

    2014-02-05

    Through a "tag-and-modify" protein chemical modification strategy, we site-selectively phosphorylated the activation loop of protein kinase p38α. Phosphorylation at natural (180) and unnatural (172) sites created two pure phospho-forms. p38α bearing only a single phosphocysteine (pCys) as a mimic of pThr at 180 was sufficient to switch the kinase to an active state, capable of processing natural protein substrate ATF2; 172 site phosphorylation did not. In this way, we chemically recapitulated triggering of a relevant segment of the MAPK-signaling pathway in vitro. This allowed detailed kinetic analysis of global and stoichiometric phosphorylation events catalyzed by p38α and revealed that site 180 is a sufficient activator alone and engenders dominant mono-phosphorylation activity. Moreover, a survey of kinase inhibition using inhibitors with different (Type I/II) modes (including therapeutically relevant) revealed unambiguously that Type II inhibitors inhibit phosphorylated p38α and allowed discovery of a predictive kinetic analysis based on cooperativity to distinguish Type I vs II.

  10. DEC1 negatively regulates AMPK activity via LKB1

    PubMed Central

    Sato, Fuyuki; Muragaki, Yasuteru; Zhang, Yanping

    2016-01-01

    Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) is one of the clock genes that show a circadian rhythm in various tissues. AMP-activated protein kinase (AMPK) activity plays important roles in the metabolic process and in cell death induced by glucose depletion. Recent reports have shown that AMPK activity exhibited a circadian rhythm. However, little is known regarding the regulatory mechanisms involved in the circadian rhythm of AMPK activity. The aim of this study is to investigate whether there is a direct correlation between DEC1 expression and AMPK activity. DEC1 protein and AMPK activity showed a circadian rhythm in the mouse liver with different peak levels. Knocking down DEC1 expression increased AMPK activity, whereas overexpression of DEC1 decreased it. Overexpressing the DEC1 basic mutants had little effect on the AMPK activity. DEC1 bound to the E-box of the LKB1 promoter, decreased LKB1 activity and total protein levels. There was an inverse relationship between DEC1 expression and AMPK activity. Our results suggest that DEC1 negatively regulates AMPK activity via LKB1. PMID:26498531

  11. Optimizing Intramuscular Adaptations to Aerobic Exercise: Effects of Carbohydrate Restriction and Protein Supplementation on Mitochondrial Biogenesis12

    PubMed Central

    Margolis, Lee M.; Pasiakos, Stefan M.

    2013-01-01

    Mitochondrial biogenesis is a critical metabolic adaptation to aerobic exercise training that results in enhanced mitochondrial size, content, number, and activity. Recent evidence has shown that dietary manipulation can further enhance mitochondrial adaptations to aerobic exercise training, which may delay skeletal muscle fatigue and enhance exercise performance. Specifically, studies have demonstrated that combining carbohydrate restriction (endogenous and exogenous) with a single bout of aerobic exercise potentiates the beneficial effects of exercise on markers of mitochondrial biogenesis. Additionally, studies have demonstrated that high-quality protein supplementation enhances anabolic skeletal muscle intracellular signaling and mitochondrial protein synthesis following a single bout of aerobic exercise. Mitochondrial biogenesis is stimulated by complex intracellular signaling pathways that appear to be primarily regulated by 5′AMP-activated protein kinase and p38 mitogen-activated protein kinase mediated through proliferator-activated γ receptor co-activator 1 α activation, resulting in increased mitochondrial DNA expression and enhanced skeletal muscle oxidative capacity. However, the mechanisms by which concomitant carbohydrate restriction and dietary protein supplementation modulates mitochondrial adaptations to aerobic exercise training remains unclear. This review summarizes intracellular regulation of mitochondrial biogenesis and the effects of carbohydrate restriction and protein supplementation on mitochondrial adaptations to aerobic exercise. PMID:24228194

  12. Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 in Angiotensin II–Induced Inflammation and Hypertension

    PubMed Central

    Ebrahimian, Talin; Li, Melissa Wei; Lemarié, Catherine A.; Simeone, Stefania M.C.; Pagano, Patrick J.; Gaestel, Matthias; Paradis, Pierre; Wassmann, Sven; Schiffrin, Ernesto L.

    2015-01-01

    Vascular oxidative stress and inflammation play an important role in angiotensin II–induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase–activated protein kinase 2 (MK2), a downstream target of p38 mitogen–activated protein kinase, is involved in angiotensin II–induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II–induced 40% increase of MK2 expression. Mk2 kn