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Sample records for ampk um antigo

  1. Targeting AMPK for cancer prevention and treatment

    PubMed Central

    Young, Matthew R.; Chen, Guohong; Hua, Baojin

    2015-01-01

    AMP-activated protein kinase (AMPK) is an important mediator in maintaining cellular energy homeostasis. AMPK is activated in response to a shortage of energy. Once activated, AMPK can promote ATP production and regulate metabolic energy. AMPK is a known target for treating metabolic syndrome and type-2 diabetes; however, recently AMPK is emerging as a possible metabolic tumor suppressor and target for cancer prevention and treatment. Recent epidemiological studies indicate that treatment with metformin, an AMPK activator reduces the incidence of cancer. In this article we review the role of AMPK in regulating inflammation, metabolism, and other regulatory processes with an emphasis on cancer, as well as, discuss the potential for targeting AMPK to treat various types of cancer. Activation of AMPK has been found to oppose tumor progression in several cancer types and offers a promising cancer therapy. This review evaluates the evidence linking AMPK with tumor suppressor function and analyzes the molecular mechanisms involved. AMPK activity opposes tumor development and progression in part by regulating inflammation and metabolism. PMID:25812084

  2. AMPK inhibition in health and disease

    PubMed Central

    Viollet, Benoit; Horman, Sandrine; Leclerc, Jocelyne; Lantier, Louise; Foretz, Marc; Billaud, Marc; Giri, Shailendra; Andreelli, Fabrizio

    2010-01-01

    All living organisms depend on dynamic mechanisms that repeatedly reassess the status of amassed energy, in order to adapt energy supply to demand. The AMP-activated protein kinase (AMPK) αβγ heterotrimer has emerged as an important integrator of signals managing energy balance. Control of AMPK activity involves allosteric AMP and ATP regulation, auto-inhibitory features and phosphorylation of its catalytic (α) and regulatory (β and γ) subunits. AMPK has a prominent role not only as a peripheral sensor but also in the central nervous system as a multifunctional metabolic regulator. AMPK represents an ideal second messenger for reporting cellular energy state. For this reason, activated AMPK acts as a protective response to energy stress in numerous systems. However, AMPK inhibition also actively participates in the control of whole body energy homeostasis. In this review, we discuss recent findings that support the role and function of AMPK inhibition under physiological and pathological states. PMID:20522000

  3. 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.

  4. AMPK in cardiovascular health and disease

    PubMed Central

    Shirwany, Najeeb A; Zou, Ming-Hui

    2010-01-01

    Adenosine Monophosphate-activated Protein Kinase (AMPK), a serine/threonine kinase and a member of the Snf1/AMPK protein kinase family, consists of three protein subunits that together make a functional enzyme. AMPK, which is expressed in a number of tissues, including the liver, brain, and skeletal muscle, is allosterically activated by a rise in the AMP: ATP ratio (ie in a low ATP or energy depleted state). The net effect of AMPK activation is to halt energy consuming (anabolic) pathways but to promote energy conserving (catabolic) cellular pathways. AMPK has therefore often been dubbed the “metabolic master switch”. AMPK also plays a critical physiological role in the cardiovascular system. Increasing evidence suggest that AMPK might also function as a sensor by responding to oxidative stress. Mostly importantly, AMPK modulates endogenous antioxidant gene expression and/or suppress the production of oxidants. AMPK promotes cardiovascular homeostasis by ensuring an optimum redox balance on the heart and vascular tissues. Dysfunctional AMPK is thought to underlie several cardiovascular pathologies. Here we review this kinase from its structure and discovery to current knowledge of its adaptive and maladaptive role in the cardiovascular system. PMID:20711221

  5. AMPK Function in Aging Process.

    PubMed

    Ruiz, Rocío; Pérez-Villegas, Eva María; Manuel Carrión, Ángel

    2016-01-01

    Aging involves the progressive deterioration of physiological functions, diminishing the individual's capacity for survival. Indeed, aging is the main risk factor for cancer, diabetes, cardiovascular disorders and neurodegenerative diseases. The discovery that the rate of aging is controlled by conserved genetic and biochemical pathways represented an unprecedented advance in aging research. The AMPK protein is a metabolic sensor that acts as a qualified cellular housekeeper, as well as controlling energy homeostasis and resistance to stress. Thus, the correct regulation of this factor enhances health and survival. In this manuscript we will review the molecular pathways regulated by AMPK that are related to the aging process, paying special attention to mitochondrial dysfunction, metabolic deregulation, cell senescence and autophagy.

  6. Salicylate improves macrophage cholesterol homeostasis via activation of Ampk[S

    PubMed Central

    Fullerton, Morgan D.; Ford, Rebecca J.; McGregor, Chelsea P.; LeBlond, Nicholas D.; Snider, Shayne A.; Stypa, Stephanie A.; Day, Emily A.; Lhoták, Šárka; Schertzer, Jonathan D.; Austin, Richard C.; Kemp, Bruce E.; Steinberg, Gregory R.

    2015-01-01

    Atherosclerosis stems from imbalances in lipid metabolism and leads to maladaptive inflammatory responses. The AMP-activated protein kinase (Ampk) is a highly conserved serine/threonine kinase that regulates many aspects of lipid and energy metabolism, although its specific role in controlling macrophage cholesterol homeostasis remains unclear. We sought to address this question by testing the effects of direct Ampk activators in primary bone marrow-derived macrophages from Ampk β1-deficient (β1−/−) mice. Macrophages from Ampk β1−/− mice had enhanced lipogenic capacity and diminished cholesterol efflux, although cholesterol uptake was unaffected. Direct activation of Ampk β1 via salicylate (the unacetylated form of aspirin) or A-769662 (a small molecule activator), decreased the synthesis of FAs and sterols in WT but not Ampk β1−/− macrophages. In lipid-laden macrophages, Ampk activation decreased cholesterol content (foam cell formation) and increased cholesterol efflux to HDL and apoA-I, effects that occurred in an Ampk β1-dependent manner. Increased cholesterol efflux was also associated with increased gene expression of the ATP binding cassette transporters, Abcg1 and Abca1. Moreover, in vivo reverse cholesterol transport was suppressed in mice that received Ampk β1−/− macrophages compared with the WT control. Our data highlight the therapeutic potential of targeting macrophage Ampk with new or existing drugs for the possible reduction in foam cell formation during the early stages of atherosclerosis. PMID:25773887

  7. AMPK activators: mechanisms of action and physiological activities.

    PubMed

    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

  8. 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

  9. AMPK: Lessons from transgenic and knockout animals

    PubMed Central

    Viollet, Benoit; Athea, Yoni; Mounier, Remi; Guigas, Bruno; Zarrinpashneh, Elham; Horman, Sandrine; Lantier, Louise; Hebrard, Sophie; Devin-Leclerc, Jocelyne; Beauloye, Christophe; Foretz, Marc; Andreelli, Fabrizio; Ventura-Clapier, Renee; Bertrand, Luc

    2009-01-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, has been proposed to function as a ‘fuel gauge’ to monitor cellular energy status in response to nutritional environmental variations. 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. Numerous observations obtained with pharmacological activators and agents that deplete intracellular ATP have been supportive of AMPK playing a role in the control of energy metabolism but none of these studies have provided conclusive evidence. Relatively recent developments in our understanding of precisely how AMPK complexes might operate to control energy metabolism is due in part to the development of transgenic and knockout mouse models. Although there are inevitable caveats with genetic models, some important findings have emerged. In the present review, we discuss recent findings obtained from animal models with inhibition or activation of AMPK signaling pathway. PMID:19273052

  10. Deubiquitination and Activation of AMPK by USP10.

    PubMed

    Deng, Min; Yang, Xu; Qin, Bo; Liu, Tongzheng; Zhang, Haoxing; Guo, Wei; Lee, Seung Baek; Kim, Jung Jin; Yuan, Jian; Pei, Huadong; Wang, Liewei; Lou, Zhenkun

    2016-02-18

    The AMP-activated protein kinase (AMPK) is the master regulator of metabolic homeostasis by sensing cellular energy status. When intracellular ATP levels decrease during energy stress, AMPK is initially activated through AMP or ADP binding and phosphorylation of a threonine residue (Thr-172) within the activation loop of its kinase domain. Here we report a key molecular mechanism by which AMPK activation is amplified under energy stress. We found that ubiquitination on AMPKα blocks AMPKα phosphorylation by LKB1. The deubiquitinase USP10 specifically removes ubiquitination on AMPKα to facilitate AMPKα phosphorylation by LKB1. Under energy stress, USP10 activity in turn is enhanced through AMPK-mediated phosphorylation of Ser76 of USP10. Thus, USP10 and AMPK form a key feedforward loop ensuring amplification of AMPK activation in response to fluctuation of cellular energy status. Disruption of this feedforward loop leads to improper AMPK activation and multiple metabolic defects.

  11. Hypothalamic AMPK as a Regulator of Energy Homeostasis

    PubMed Central

    Huynh, My Khanh Q.; Kinyua, Ann W.; Yang, Dong Joo

    2016-01-01

    Activated in energy depletion conditions, AMP-activated protein kinase (AMPK) acts as a cellular energy sensor and regulator in both central nervous system and peripheral organs. Hypothalamic AMPK restores energy balance by promoting feeding behavior to increase energy intake, increasing glucose production, and reducing thermogenesis to decrease energy output. Besides energy state, many hormones have been shown to act in concert with AMPK to mediate their anorexigenic and orexigenic central effects as well as thermogenic influences. Here we explore the factors that affect hypothalamic AMPK activity and give the underlying mechanisms for the role of central AMPK in energy homeostasis together with the physiological effects of hypothalamic AMPK on energy balance restoration. PMID:27547453

  12. Host AMPK Is a Modulator of Plasmodium Liver Infection.

    PubMed

    Ruivo, Margarida T Grilo; Vera, Iset Medina; Sales-Dias, Joana; Meireles, Patrícia; Gural, Nil; Bhatia, Sangeeta N; Mota, Maria M; Mancio-Silva, Liliana

    2016-09-01

    Manipulation of the master regulator of energy homeostasis AMP-activated protein kinase (AMPK) activity is a strategy used by many intracellular pathogens for successful replication. Infection by most pathogens leads to an activation of host AMPK activity due to the energetic demands placed on the infected cell. Here, we demonstrate that the opposite is observed in cells infected with rodent malaria parasites. Indeed, AMPK activity upon the infection of hepatic cells is suppressed and dispensable for successful infection. By contrast, an overactive AMPK is deleterious to intracellular growth and replication of different Plasmodium spp., including the human malaria parasite, P. falciparum. The negative impact of host AMPK activity on infection was further confirmed in mice under conditions that activate its function. Overall, this work establishes the role of host AMPK signaling as a suppressive pathway of Plasmodium hepatic infection and as a potential target for host-based antimalarial interventions. PMID:27568570

  13. Regulation and function of AMPK in physiology and diseases

    PubMed Central

    Jeon, Sang-Min

    2016-01-01

    5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that was originally identified as the key player in maintaining cellular energy homeostasis. Intensive research over the last decade has identified diverse molecular mechanisms and physiological conditions that regulate the AMPK activity. AMPK regulates diverse metabolic and physiological processes and is dysregulated in major chronic diseases, such as obesity, inflammation, diabetes and cancer. On the basis of its critical roles in physiology and pathology, AMPK is emerging as one of the most promising targets for both the prevention and treatment of these diseases. In this review, we discuss the current understanding of the molecular and physiological regulation of AMPK and its metabolic and physiological functions. In addition, we discuss the mechanisms underlying the versatile roles of AMPK in diabetes and cancer. PMID:27416781

  14. Hypothalamic AMPK as a Regulator of Energy Homeostasis.

    PubMed

    Huynh, My Khanh Q; Kinyua, Ann W; Yang, Dong Joo; Kim, Ki Woo

    2016-01-01

    Activated in energy depletion conditions, AMP-activated protein kinase (AMPK) acts as a cellular energy sensor and regulator in both central nervous system and peripheral organs. Hypothalamic AMPK restores energy balance by promoting feeding behavior to increase energy intake, increasing glucose production, and reducing thermogenesis to decrease energy output. Besides energy state, many hormones have been shown to act in concert with AMPK to mediate their anorexigenic and orexigenic central effects as well as thermogenic influences. Here we explore the factors that affect hypothalamic AMPK activity and give the underlying mechanisms for the role of central AMPK in energy homeostasis together with the physiological effects of hypothalamic AMPK on energy balance restoration. PMID:27547453

  15. Pharmacological activation of AMPK ameliorates perivascular adipose/endothelial dysfunction in a manner interdependent on AMPK and SIRT1.

    PubMed

    Sun, Yan; Li, Jia; Xiao, Na; Wang, Meng; Kou, Junping; Qi, Lianwen; Huang, Fang; Liu, Baolin; Liu, Kang

    2014-11-01

    Adipose and endothelial dysfunction is tightly associated with cardiovascular diseases in obesity and insulin resistance. Because perivascular adipose tissue (PVAT) surrounds vessels directly and influences vessel functions through paracrine effect, and AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) show similarities in modulation of metabolic pathway, we hypothesized that activation of AMPK and SIRT1 in PVAT might regulate the endothelial function in pathological settings. Thus, in this study, we focused on the regulation of AMPK and SIRT1 activities implicated in adipocytokine expression and endothelial homeostasis under inflammatory conditions by using salicylate, metformin, AICA riboside (AICAR) and resveratrol as AMPK activating agents. We prepared conditioned medium (CM) by stimulating PVAT with palmitic acid (PA) and observed the effects of AMPK activating agents on adipocytokine expression and vessel vasodilation in rats. Moreover, we explored the effects of resveratrol and metformin in fructose-fed rats. We observed that PA stimulation induced inflammation and dysregulation of adipocytokine expression accompanied with reduced AMPK activity and SIRT1 abundance in PVAT. AMPK activating agents inhibited NF-κB p65 phosphorylation and suppressed gene expression of pro-inflammatory adipocytokines, and upregulated adiponectin and PPARγ expression in PVAT in an AMPK/SIRT1-interdependent manner. Meanwhile, CM stimulation impaired endothelium-dependent vasodilation in response to acetylcholine (ACh). Pretreatment of CM with AMPK-activating agents enhanced eNOS phosphorylation in the aorta and restored the loss of endothelium-dependent vasodilation, whereas this action was abolished by co-treatment with AMPK inhibitor compound C or SIRT1 inhibitor nicotinamide. Long-term fructose-feeding in rats induced dysregulation of adipocytokine expression in PVAT and the loss of endothelium-dependent vasodilation, whereas these alterations were reversed by oral

  16. AMPK as a Potential Anticancer Target – Friend or Foe?

    PubMed Central

    Chuang, Hsiao-Ching; Chou, Chih-Chien; Kulp, Samuel K.; Chen, Ching-Shih

    2014-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a key player in maintaining energy homeostasis in response to metabolic stress. Beyond diabetes and metabolic syndrome, there is a growing interest in the therapeutic exploitation of the AMPK pathway in cancer treatment in light of its unique ability to regulate cancer cell proliferation through the reprogramming of cell metabolism. Although many studies support the tumor-suppressive role of AMPK, emerging evidence suggests that the metabolic checkpoint function of AMPK might be overridden by stress or oncogenic signals so that tumor cells use AMPK activation as a survival strategy to gain growth advantage. These findings underscore the complexity in the cellular function of AMPK in maintaining energy homeostasis under physiological versus pathological conditions. Thus, this review aims to provide an overview of recent findings on the functional interplay of AMPK with different cell metabolic and signaling effectors, particularly histone deacetylases, in mediating downstream tumor suppressive or promoting mechanisms in different cell systems. Although AMPK activation inhibits tumor growth by targeting multiple signaling pathways relevant to tumorigenesis, under certain cellular contexts or certain stages of tumor development, AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation, low oxygen, and low pH, or as a downstream effectors of oncogenic proteins, including androgen receptor, hypoxia-inducible factor-1α, c-Src, and MYC. Thus, investigations to define at which stage(s) of tumorigenesis and cancer progression or for which genetic aberrations AMPK inhibition might represent a more relevant strategy than AMPK activation for cancer treatment are clearly warranted. PMID:23859619

  17. AMPK as a potential anticancer target - friend or foe?

    PubMed

    Chuang, Hsiao-Ching; Chou, Chih-Chien; Kulp, Samuel K; Chen, Ching-Shih

    2014-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a key player in maintaining energy homeostasis in response to metabolic stress. Beyond diabetes and metabolic syndrome, there is a growing interest in the therapeutic exploitation of the AMPK pathway in cancer treatment in light of its unique ability to regulate cancer cell proliferation through the reprogramming of cell metabolism. Although many studies support the tumor-suppressive role of AMPK, emerging evidence suggests that the metabolic checkpoint function of AMPK might be overridden by stress or oncogenic signals so that tumor cells use AMPK activation as a survival strategy to gain growth advantage. These findings underscore the complexity in the cellular function of AMPK in maintaining energy homeostasis under physiological versus pathological conditions. Thus, this review aims to provide an overview of recent findings on the functional interplay of AMPK with different cell metabolic and signaling effectors, particularly histone deacetylases, in mediating downstream tumor suppressive or promoting mechanisms in different cell systems. Although AMPK activation inhibits tumor growth by targeting multiple signaling pathways relevant to tumorigenesis, under certain cellular contexts or certain stages of tumor development, AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation, low oxygen, and low pH, or as downstream effectors of oncogenic proteins, including androgen receptor, hypoxia-inducible factor-1α, c-Src, and MYC. Thus, investigations to define at which stage(s) of tumorigenesis and cancer progression or for which genetic aberrations AMPK inhibition might represent a more relevant strategy than AMPK activation for cancer treatment are clearly warranted.

  18. A redox-dependent mechanism for regulation of AMPK activation by Thioredoxin1 during energy starvation.

    PubMed

    Shao, Dan; Oka, Shin-Ichi; Liu, Tong; Zhai, Peiyong; Ago, Tetsuro; Sciarretta, Sebastiano; Li, Hong; Sadoshima, Junichi

    2014-02-01

    5'-AMP-activated protein kinase (AMPK) is a key regulator of metabolism and survival during energy stress. Dysregulation of AMPK is strongly associated with oxidative-stress-related disease. However, whether and how AMPK is regulated by intracellular redox status remains unknown. Here we show that the activity of AMPK is negatively regulated by oxidation of Cys130 and Cys174 in its α subunit, which interferes with the interaction between AMPK and AMPK kinases (AMPKK). Reduction of Cys130/Cys174 is essential for activation of AMPK during energy starvation. Thioredoxin1 (Trx1), an important reducing enzyme that cleaves disulfides in proteins, prevents AMPK oxidation, serving as an essential cofactor for AMPK activation. High-fat diet consumption downregulates Trx1 and induces AMPK oxidation, which enhances cardiomyocyte death during myocardial ischemia. Thus, Trx1 modulates activation of the cardioprotective AMPK pathway during ischemia, functionally linking oxidative stress and metabolism in the heart.

  19. AMPK regulation of the growth of cultured human keratinocytes

    SciTech Connect

    Saha, Asish K. . E-mail: aksaha@bu.edu; Persons, Kelly; Safer, Joshua D.; Luo Zhijun; Holick, Michael F.; Ruderman, Neil B.

    2006-10-20

    AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR). At concentrations of 10{sup -4} and 10{sup -3} M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10{sup -6} M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D{sub 3} (10{sup -7} and 10{sup -6} M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D{sub 3} is AMPK-independent.

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

    PubMed Central

    Sinnett, Sarah E.; Brenman, Jay E.

    2014-01-01

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

  1. Structural basis of AMPK regulation by small molecule activators

    NASA Astrophysics Data System (ADS)

    Xiao, Bing; Sanders, Matthew J.; Carmena, David; Bright, Nicola J.; Haire, Lesley F.; Underwood, Elizabeth; Patel, Bhakti R.; Heath, Richard B.; Walker, Philip A.; Hallen, Stefan; Giordanetto, Fabrizio; Martin, Stephen R.; Carling, David; Gamblin, Steven J.

    2013-12-01

    AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.

  2. AMPK: a master energy regulator for gonadal function.

    PubMed

    Bertoldo, Michael J; Faure, Melanie; Dupont, Joëlle; Froment, Pascal

    2015-01-01

    From C. elegans to mammals (including humans), nutrition and energy metabolism significantly influence reproduction. At the cellular level, some detectors of energy status indicate whether energy reserves are abundant (obesity), or poor (diet restriction). One of these detectors is AMPK (5' AMP-activated protein kinase), a protein kinase activated by ATP deficiency but also by several natural substances such as polyphenols or synthetic molecules like metformin, used in the treatment of insulin resistance. AMPK is expressed in muscle and liver, but also in the ovary and testis. This review focuses on the main effects of AMPK identified in gonadal cells. We describe the role of AMPK in gonadal steroidogenesis, in proliferation and survival of somatic gonadal cells and in the maturation of oocytes or spermatozoa. We discuss also the role of AMPK in germ and somatic cell interactions within the cumulus-oocyte complex and in the blood testis barrier. Finally, the interface in the gonad between AMPK and modification of metabolism is reported and discussion about the role of AMPK on fertility, in regards to the treatment of infertility associated with insulin resistance (male obesity, polycystic ovary syndrome). PMID:26236179

  3. AMPK: a master energy regulator for gonadal function

    PubMed Central

    Bertoldo, Michael J.; Faure, Melanie; Dupont, Joëlle; Froment, Pascal

    2015-01-01

    From C. elegans to mammals (including humans), nutrition and energy metabolism significantly influence reproduction. At the cellular level, some detectors of energy status indicate whether energy reserves are abundant (obesity), or poor (diet restriction). One of these detectors is AMPK (5′ AMP-activated protein kinase), a protein kinase activated by ATP deficiency but also by several natural substances such as polyphenols or synthetic molecules like metformin, used in the treatment of insulin resistance. AMPK is expressed in muscle and liver, but also in the ovary and testis. This review focuses on the main effects of AMPK identified in gonadal cells. We describe the role of AMPK in gonadal steroidogenesis, in proliferation and survival of somatic gonadal cells and in the maturation of oocytes or spermatozoa. We discuss also the role of AMPK in germ and somatic cell interactions within the cumulus-oocyte complex and in the blood testis barrier. Finally, the interface in the gonad between AMPK and modification of metabolism is reported and discussion about the role of AMPK on fertility, in regards to the treatment of infertility associated with insulin resistance (male obesity, polycystic ovary syndrome). PMID:26236179

  4. Nilotinib induces autophagy in hepatocellular carcinoma through AMPK activation.

    PubMed

    Yu, Hui-Chuan; Lin, Chen-Si; Tai, Wei-Tien; Liu, Chun-Yu; Shiau, Chung-Wai; Chen, Kuen-Feng

    2013-06-21

    Hepatocellular carcinoma (HCC) is the most common liver cancer and the third-leading cause of cancer death worldwide. Nilotinib is an orally available receptor tyrosine kinase inhibitor approved for chronic myelogenous leukemia. This study investigated the effect of nilotinib on HCC. Nilotinib did not induce cellular apoptosis. Instead, staining with acridine orange and microtubule-associated protein 1 light chain 3 revealed that nilotinib induced autophagy in a dose- and time-dependent manner in HCC cell lines, including PLC5, Huh-7, and Hep3B. Moreover, nilotinib up-regulated the phosphryaltion of AMP-activated kinase (AMPK) and protein phosphatase PP2A inactivation were detected after nilotinib treatment. Up-regulating PP2A activity suppressed nilotinib-induced AMPK phosphorylation and autophagy, suggesting that PP2A mediates the effect of nilotinib on AMPK phosphorylation and autophagy. Our data indicate that nilotinib-induced AMPK activation is mediated by PP2A, and AMPK activation and subsequent autophagy might be a major mechanism of action of nilotinib. Growth of PLC5 tumor xenografts in BALB/c nude mice was inhibited by daily oral treatment with nilotinib. Western blot analysis showed both increased phospho-AMPK expression and decreased PP2A activity in vivo. Together, our results reveal that nilotinib induces autophagy, but not apoptosis in HCC, and that the autophagy-inducing activity is associated with PP2A-regulated AMPK phosphorylation.

  5. AMPK at the Nexus of Energetics and Aging

    PubMed Central

    Burkewitz, Kristopher; Zhang, Yue; Mair, William B.

    2014-01-01

    When energy supply is low, organisms respond by slowing aging and increasing resistance to diverse age-related pathologies. Targeting the mechanisms underpinning this response may therefore treat multiple disorders through a single intervention. Here we discuss AMP-activated protein kinase (AMPK) as an integrator and mediator of several pathways and processes linking energetics to longevity. Activated by low energy, AMPK is both pro-longevity and druggable, but its role in some pathologies may not be beneficial. As such, activating AMPK may modulate multiple longevity pathways to promote healthy aging, but unlocking its full potential may require selective targeting towards substrates involved in longevity-assurance. PMID:24726383

  6. AMPK in the small intestine in normal and pathophysiological conditions.

    PubMed

    Harmel, Elodie; Grenier, Emilie; Bendjoudi Ouadda, Ali; El Chebly, Mounib; Ziv, Ehud; Beaulieu, Jean François; Sané, Alain; Spahis, Schohraya; Laville, Martine; Levy, Emile

    2014-03-01

    The role of AMPK in regulating energy storage and depletion remains unexplored in the intestine. This study will to define its status, composition, regulation and lipid function, as well as to examine the impact of insulin resistance and type 2 diabetes on intestinal AMPK activation, insulin sensitivity, and lipid metabolism. Caco-2/15 cells and Psammomys obesus (P. obesus) animal models were experimented. We showed the predominance of AMPKα1 and the prevalence of α1/β2/γ1 heterotrimer in Caco-2/15 cells. The activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside and metformin resulted in increased phospho(p)-ACC. However, the down-regulation of p-AMPK by compound C and high glucose lowered p-ACC without affecting 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Administration of metformin to P. obesus with insulin resistance and type 2 diabetes led to 1) an up-regulation of intestinal AMPK signaling pathway typified by ascending p-AMPKα(-Thr172); 2) a reduction in ACC activity; 3) an elevation of carnitine palmitoyltransferase 1; 4) a trend of increase in insulin sensitivity portrayed by augmentation of p-Akt and phospho-glycogen synthetase kinase 3β; 5) a reduced phosphorylation of p38-MAPK and ERK1/2; and 6) a decrease in diabetic dyslipidemia following lowering of intracellular events that govern lipoprotein assembly. These data suggest that AMPK fulfills key functions in metabolic processes in the small intestine.

  7. AMPK and autophagy in glucose/glycogen metabolism.

    PubMed

    Ha, Joohun; Guan, Kun-Liang; Kim, Joungmok

    2015-12-01

    Glucose/glycogen metabolism is a primary metabolic pathway acting on a variety of cellular needs, such as proliferation, growth, and survival against stresses. The multiple regulatory mechanisms underlying a specific metabolic fate have been documented and explained the molecular basis of various pathophysiological conditions, including metabolic disorders and cancers. AMP-activated protein kinase (AMPK) has been appreciated for many years as a central metabolic regulator to inhibit energy-consuming pathways as well as to activate the compensating energy-producing programs. In fact, glucose starvation is a potent physiological AMPK activating condition, in which AMPK triggers various subsequent metabolic events depending on cells or tissues. Of note, the recent studies show bidirectional interplay between AMPK and glycogen. A growing number of studies have proposed additional level of metabolic regulation by a lysosome-dependent catabolic program, autophagy. Autophagy is a critical degradative pathway not only for maintenance of cellular homeostasis to remove potentially dangerous constituents, such as protein aggregates and dysfunctional subcellular organelles, but also for adaptive responses to metabolic stress, such as nutrient starvation. Importantly, many lines of evidence indicate that autophagy is closely connected with nutrient signaling modules, including AMPK, to fine-tune the metabolic pathways in response to many different cellular cues. In this review, we introduce the studies demonstrating the role of AMPK and autophagy in glucose/glycogen metabolism. Also, we describe the recent advances on their contributions to the metabolic disorders.

  8. A Fluorescent Reporter of AMPK activity and Cellular Energy Stress

    PubMed Central

    Tsou, Peiling; Zheng, Bin; Hsu, Chia-Hsien; Sasaki, Atsuo T; Cantley, Lewis C.

    2011-01-01

    SUMMARY AMP-activated protein kinase (AMPK) is activated when the AMP/ATP ratio in cells is elevated due to energy stress. Here we describe a biosensor, AMPKAR, which exhibits enhanced fluorescence resonance energy transfer (FRET) in response to phosphorylation by AMPK, allowing spatio-temporal monitoring of AMPK activity in single cells. We show that this reporter responds to a variety of stimuli that are known to induce energy stress and that the response is dependent on AMPK α1 & α2 and on the upstream kinase, LKB1. Interestingly we found that AMPK activation is confined to the cytosol in response to energy stress but can be observed in both the cytosol and nucleus in response to calcium elevation. Finally, using this probe with U2OS cells in a microfluidics device, we observed a very high cell-to-cell variability in the amplitude and time course of AMPK activation and recovery in response to pulses of glucose deprivation. PMID:21459332

  9. Berberine Improves Kidney Function in Diabetic Mice via AMPK Activation

    PubMed Central

    Zhao, Long; Sun, Li-Na; Nie, Hui-Bin; Wang, Xue-Ling; Guan, Guang-Ju

    2014-01-01

    Diabetic nephropathy is a major cause of morbidity and mortality in diabetic patients. Effective therapies to prevent the development of this disease are required. Berberine (BBR) has several preventive effects on diabetes and its complications. However, the molecular mechanism of BBR on kidney function in diabetes is not well defined. Here, we reported that activation of AMP-activated protein kinase (AMPK) is required for BBR-induced improvement of kidney function in vivo. AMPK phosphorylation and activity, productions of reactive oxygen species (ROS), kidney function including serum blood urea nitrogen (BUN), creatinine clearance (Ccr), and urinary protein excretion, morphology of glomerulus were determined in vitro or in vivo. Exposure of cultured human glomerulus mesangial cells (HGMCs) to BBR time- or dose-dependently activates AMPK by increasing the thr172 phosphorylation and its activities. Inhibition of LKB1 by siRNA or mutant abolished BBR-induced AMPK activation. Incubation of cells with high glucose (HG, 30 mM) markedly induced the oxidative stress of HGMCs, which were abolished by 5-aminoimidazole-4-carboxamide ribonucleoside, AMPK gene overexpression or BBR. Importantly, the effects induced by BBR were bypassed by AMPK siRNA transfection in HG-treated HGMCs. In animal studies, streptozotocin-induced hyperglycemia dramatically promoted glomerulosclerosis and impaired kidney function by increasing serum BUN, urinary protein excretion, and decreasing Ccr, as well as increased oxidative stress. Administration of BBR remarkably improved kidney function in wildtype mice but not in AMPKα2-deficient mice. We conclude that AMPK activation is required for BBR to improve kidney function in diabetic mice. PMID:25409232

  10. Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate.

    PubMed

    Ducommun, Serge; Deak, Maria; Sumpton, David; Ford, Rebecca J; Núñez Galindo, Antonio; Kussmann, Martin; Viollet, Benoit; Steinberg, Gregory R; Foretz, Marc; Dayon, Loïc; Morrice, Nicholas A; Sakamoto, Kei

    2015-05-01

    AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets.

  11. AMPK: A cellular metabolic and redox sensor. A minireview

    PubMed Central

    Shirwany, Najeeb A; Zou, Ming-Hui

    2014-01-01

    AMPK is a serine/threonine kinase that is found in all eukaryotes and is ubiquitously expressed in all organ systems. Once activated, AMPK stimulates hepatic fatty acid oxidation and ketogenesis, inhibits cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibits adipocyte lipolysis and lipogenesis, stimulates skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulates insulin secretion by the pancreas. Thus its importance in many critical cellular processes is well established. For cells it is critical that energy supply and demand are closely matched. AMPK is recognized as a critical integrator of this balance. It is known to be allosterically activated by an increased AMP:ATP ratio. Activation of the kinase switches on catabolic pathways while switching off anabolic ones. It also acts as a redox sensor in endothelial cells where oxidative stress can disturb NO signaling. Abnormal NO signaling leads to disturbed vasodilatory responses. By inhibiting the formation of reactive oxygen species in the endothelium, AMPK can optimize the redox balance in the vasculature. Here, we review the role of AMPK in the cell. PMID:24389195

  12. AMP as a low-energy charge signal autonomously initiates assembly of AXIN-AMPK-LKB1 complex for AMPK activation.

    PubMed

    Zhang, Ya-Lin; Guo, Huiling; Zhang, Chen-Song; Lin, Shu-Yong; Yin, Zhenyu; Peng, Yongying; Luo, Hui; Shi, Yuzhe; Lian, Guili; Zhang, Cixiong; Li, Mengqi; Ye, Zhiyun; Ye, Jing; Han, Jiahuai; Li, Peng; Wu, Jia-Wei; Lin, Sheng-Cai

    2013-10-01

    The AMP-activated protein kinase (AMPK) is a master regulator of metabolic homeostasis by sensing cellular energy status. AMPK is mainly activated via phosphorylation by LKB1 when cellular AMP/ADP levels are increased. However, how AMP/ADP brings about AMPK phosphorylation remains unclear. Here, we show that it is AMP, but not ADP, that drives AXIN to directly tether LKB1 to phosphorylate AMPK. The complex formation of AXIN-AMPK-LKB1 is greatly enhanced in glucose-starved or AICAR-treated cells and in cell-free systems supplemented with exogenous AMP. Depletion of AXIN abrogated starvation-induced AMPK-LKB1 colocalization. Importantly, adenovirus-based knockdown of AXIN in the mouse liver impaired AMPK activation and caused exacerbated fatty liver after starvation, underscoring an essential role of AXIN in AMPK activation. These findings demonstrate an initiating role of AMP and demonstrate that AXIN directly transmits AMP binding of AMPK to its activation by LKB1, uncovering the mechanistic route for AMP to elicit AMPK activation by LKB1.

  13. AMPK-dependent and independent effects of AICAR and compound C on T-cell responses

    PubMed Central

    Rao, Enyu; Zhang, Yuwen; Li, Qiang; Hao, Jiaqing; Egilmez, Nejat K.; Suttles, Jill; Li, Bing

    2016-01-01

    As a master metabolic sensor, AMP-activated protein kinase (AMPK) is involved in different fundamental cellular processes. Regulation of AMPK activity either by agonists (e.g., AICAR) or by antagonists (e.g., Compound C) has been widely employed to study the physiological functions of AMPK. However, mounting evidence indicates AMPK-independent effects for these chemicals and how they regulate immune cell functions remains largely unknown. Herein, using T cells from AMPK conditional knockout mice and their wild type littermates, we demonstrate that AICAR and Compound C can, indeed, activate or inhibit AMPK activity in T cells, respectively. Specifically, AICAR inhibits, but Compound C promotes, Ca2+-induced T cell death in an AMPK-dependent manner. In contrast, our data also demonstrate that AICAR and Compound C inhibit T cell activation and cytokine production in an AMPK-independent manner. Moreover, we find that the AMPK-independent activity of AICAR and Compound Cis mediated via the mTOR signaling pathway in activated T cells. Our results not only reveal the critical role of AMPK in regulating T cell survival and function, but also demonstrate AMPK-dependent and independent rolesof AICAR/Compound C in regulating T cell responses, thus suggesting a context-dependent effect of these “AMPK regulators”. PMID:27177226

  14. Expanding roles for AMPK in skeletal muscle plasticity.

    PubMed

    Mounier, Rémi; Théret, Marine; Lantier, Louise; Foretz, Marc; Viollet, Benoit

    2015-06-01

    Skeletal muscle possesses a remarkable plasticity and responds to environmental and physiological challenges by changing its phenotype in terms of size, composition, and metabolic properties. Muscle fibers rapidly adapt to drastic changes in energy demands during exercise through fine-tuning of the balance between catabolic and anabolic processes. One major sensor of energy demand in exercising muscle is AMP-activated protein kinase (AMPK). Recent advances have shed new light on the relevance of AMPK both as a multitask gatekeeper and as an energy regulator in skeletal muscle. Here we summarize recent findings on the function of AMPK in skeletal muscle adaptation to contraction and highlight its role in the regulation of energy metabolism and the control of skeletal muscle regeneration post-injury. PMID:25818360

  15. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    SciTech Connect

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; Melcher, Karsten

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allosteric AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.

  16. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    DOE PAGES

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; et al

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allostericmore » AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.« less

  17. Thiamine deficiency induces anorexia by inhibiting hypothalamic AMPK.

    PubMed

    Liu, M; Alimov, A P; Wang, H; Frank, J A; Katz, W; Xu, M; Ke, Z-J; Luo, J

    2014-05-16

    Obesity and eating disorders are prevailing health concerns worldwide. It is important to understand the regulation of food intake and energy metabolism. Thiamine (vitamin B1) is an essential nutrient. Thiamine deficiency (TD) can cause a number of disorders in humans, such as Beriberi and Wernicke-Korsakoff syndrome. We demonstrated here that TD caused anorexia in C57BL/6 mice. After feeding a TD diet for 16days, the mice displayed a significant decrease in food intake and an increase in resting energy expenditure (REE), which resulted in a severe weight loss. At the 22nd day, the food intake was reduced by 69% and 74% for male and female mice, respectively in TD group. The REE increased by ninefolds in TD group. The loss of body weight (17-24%) was similar between male and female animals and mainly resulted from the reduction of fat mass (49% decrease). Re-supplementation of thiamine (benfotiamine) restored animal's appetite, leading to a total recovery of body weight. The hypothalamic adenosine monophosphate-activated protein kinase (AMPK) is a critical regulator of food intake. TD inhibited the phosphorylation of AMPK in the arcuate nucleus (ARN) and paraventricular nucleus (PVN) of the hypothalamus without affecting its expression. TD-induced inhibition of AMPK phosphorylation was reversed once thiamine was re-supplemented. In contrast, TD increased AMPK phosphorylation in the skeletal muscle and upregulated the uncoupling protein (UCP)-1 in brown adipose tissues which was consistent with increased basal energy expenditure. Re-administration of thiamine stabilized AMPK phosphorylation in the skeletal muscle as well as energy expenditure. Taken together, TD may induce anorexia by inhibiting hypothalamic AMPK activity. With a simultaneous increase in energy expenditure, TD caused an overall body weight loss. The results suggest that the status of thiamine levels in the body may affect food intake and body weight.

  18. AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain.

    PubMed

    Thaiparambil, Jose T; Eggers, Carrie M; Marcus, Adam I

    2012-08-01

    The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.

  19. Development of Potent Adenosine Monophosphate Activated Protein Kinase (AMPK) Activators.

    PubMed

    Dokla, Eman M E; Fang, Chun-Sheng; Lai, Po-Ting; Kulp, Samuel K; Serya, Rabah A T; Ismail, Nasser S M; Abouzid, Khaled A M; Chen, Ching-Shih

    2015-11-01

    Previously, we reported the identification of a thiazolidinedione-based adenosine monophosphate activated protein kinase (AMPK) activator, compound 1 (N-[4-({3-[(1-methylcyclohexyl)methyl]-2,4-dioxothiazolidin-5-ylidene}methyl)phenyl]-4-nitro-3-(trifluoromethyl)benzenesulfonamide), which provided a proof of concept to delineate the intricate role of AMPK in regulating oncogenic signaling pathways associated with cell proliferation and epithelial-mesenchymal transition (EMT) in cancer cells. In this study, we used 1 as a scaffold to conduct lead optimization, which generated a series of derivatives. Analysis of the antiproliferative and AMPK-activating activities of individual derivatives revealed a distinct structure-activity relationship and identified 59 (N-(3-nitrophenyl)-N'-{4-[(3-{[3,5-bis(trifluoromethyl)phenyl]methyl}-2,4-dioxothiazolidin-5-ylidene)methyl]phenyl}urea) as the optimal agent. Relative to 1, compound 59 exhibits multifold higher potency in upregulating AMPK phosphorylation in various cell lines irrespective of their liver kinase B1 (LKB1) functional status, accompanied by parallel changes in the phosphorylation/expression levels of p70S6K, Akt, Foxo3a, and EMT-associated markers. Consistent with its predicted activity against tumors with activated Akt status, orally administered 59 was efficacious in suppressing the growth of phosphatase and tensin homologue (PTEN)-null PC-3 xenograft tumors in nude mice. Together, these findings suggest that 59 has clinical value in therapeutic strategies for PTEN-negative cancer and warrants continued investigation in this regard.

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

    PubMed

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

    2011-02-01

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

  1. 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.

  2. Miltefosine Suppresses Hepatic Steatosis by Activating AMPK Signal Pathway

    PubMed Central

    Zhu, Yaqin; Tong, Xing; Li, Kexue; Bai, Hui; Li, Xiaoyu; Ben, Jingjing; Zhang, Hanwen; Yang, Qing; Chen, Qi

    2016-01-01

    Background and Purpose It has been accepted that AMPK (Adenosine monophosphate–activated protein kinase) activation exhibits many beneficial effects on glucolipid metabolism. Lysophosphatidylcholine (LPC) is an important lysophospholipid which can improve blood glucose levels in diabetic mice and attenuate inflammation by activating AMPK signal pathway in macrophages. Synthetic alkylphospholipids (ALPs), such as miltefosine, is used as an alternate of LPC for the clinical application. Here, we investigated whether miltefosine could have an impact on hepatic steatosis and related metabolic disorders. Experimental Approach Mice were fed with high fat diet (HFD) for 16 weeks to generate an obese model. Next, the obese mice were randomly divided into three groups: saline-treated and miltefosine-treated (2.5 or 5 mg/kg/d) groups. Miltefosine was intraperitoneally administrated into mice for additional 4 weeks plus HFD treatment. Key Results It was shown that miltefosine treatment could substantially improve glucose metabolism, prevented hepatic lipid accumulation, and inhibited liver inflammation in HFD-fed mice by activating AMPK signal pathway. In vitro, miltefosine stimulated AMPKα phosphorylation both in time and dose dependent manner and decreased lipid accumulation in liver cells. When a specific AMPK inhibitor compound C was used to treat mice, the antagonistic effects of miltefosine on HFD-induced mouse hyperlipidaemia and liver steatosis were abolished. Treatment with miltefosine also dramatically inhibited the HFD-induced liver inflammation in mice. Conclusions and Implications Here we demonstrated that miltefosine might be a new activator of AMPK signal pathway in vivo and in vitro and be useful for treatment of hepatic steatosis and related metabolic disorders. PMID:27681040

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

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

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

  4. A role for AMPK in the inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids

    SciTech Connect

    Kohan, Alison B.; Talukdar, Indrani; Walsh, Callee M.; Salati, Lisa M.

    2009-10-09

    Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as {beta}-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser{sup 307} phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.

  5. Dissecting the Dual Role of AMPK in Cancer: From Experimental to Human Studies.

    PubMed

    Zadra, Giorgia; Batista, Julie L; Loda, Massimo

    2015-07-01

    The precise role of 5'AMP-activated kinase (AMPK) in cancer and its potential as a therapeutic target is controversial. Although it is well established that activation of this energy sensor inhibits the main anabolic processes that sustain cancer cell proliferation and growth, AMPK activation can confer on cancer cells the plasticity to survive under metabolic stress such as hypoxia and glucose deprivation, which are commonly observed in fast growing tumors. Thus, AMPK is referred to as both a "conditional" tumor suppressor and "contextual" oncogene. To add a further layer of complexity, AMPK activation in human cancer tissues and its correlation with tumor aggressiveness and progression appears to vary in different contexts. The current review discusses the different faces of this metabolic regulator, the therapeutic implications of its modulation, and provides an overview of the most relevant data available on AMPK activation and AMPK-activating drugs in human studies.

  6. Localisation of AMPK γ subunits in cardiac and skeletal muscles.

    PubMed

    Pinter, Katalin; Grignani, Robert T; Watkins, Hugh; Redwood, Charles

    2013-12-01

    The trimeric protein AMP-activated protein kinase (AMPK) is an important sensor of energetic status and cellular stress, and mutations in genes encoding two of the regulatory γ subunits cause inherited disorders of either cardiac or skeletal muscle. AMPKγ2 mutations cause hypertrophic cardiomyopathy with glycogen deposition and conduction abnormalities; mutations in AMPKγ3 result in increased skeletal muscle glycogen. In order to gain further insight into the roles of the different γ subunits in muscle and into possible disease mechanisms, we localised the γ2 and γ3 subunits, along with the more abundant γ1 subunit, by immunofluorescence in cardiomyocytes and skeletal muscle fibres. The predominant cardiac γ2 variant, γ2-3B, gave a striated pattern in cardiomyocytes, aligning with the Z-disk but with punctate staining similar to T-tubule (L-type Ca(2+) channel) and sarcoplasmic reticulum (SERCA2) markers. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca(2+) influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be co-ordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

  7. AMPK Regulates Metabolic Actions of Glucocorticoids by Phosphorylating the Glucocorticoid Receptor through p38 MAPK

    PubMed Central

    Nader, Nancy; Ng, Sinnie Sin Man; Lambrou, George I.; Pervanidou, Panagiota; Wang, Yonghong; Chrousos, George P.; Kino, Tomoshige

    2010-01-01

    Glucocorticoids play central roles in the regulation of energy metabolism by shifting it toward catabolism, whereas AMP-activated protein kinase (AMPK) is the master regulator of energy homeostasis, sensing energy depletion and stimulating pathways of increasing fuel uptake and saving on peripheral supplies. We showed here that AMPK regulates glucocorticoid actions on carbohydrate metabolism by targeting the glucocorticoid receptor (GR) and modifying transcription of glucocorticoid-responsive genes in a tissue- and promoter-specific fashion. Activation of AMPK in rats reversed glucocorticoid-induced hepatic steatosis and suppressed glucocorticoid-mediated stimulation of glucose metabolism. Transcriptomic analysis in the liver suggested marked overlaps between the AMPK and glucocorticoid signaling pathways directed mostly from AMPK to glucocorticoid actions. AMPK accomplishes this by phosphorylating serine 211 of the human GR indirectly through phosphorylation and consequent activation of p38 MAPK and by altering attraction of transcriptional coregulators to DNA-bound GR. In human peripheral mononuclear cells, AMPK mRNA expression positively correlated with that of glucocorticoid-responsive glucocorticoid-inducible leucine zipper protein, which correlated also positively with the body mass index of subjects. These results indicate that the AMPK-mediated energy control system modulates glucocorticoid action at target tissues. Because increased action of glucocorticoids is associated with the development of metabolic disorders, activation of AMPK could be a promising target for developing pharmacological interventions to these pathologies. PMID:20660302

  8. VASP Increases Hepatic Fatty Acid Oxidation by Activating AMPK in Mice

    PubMed Central

    Tateya, Sanshiro; Rizzo-De Leon, Norma; Handa, Priya; Cheng, Andrew M.; Morgan-Stevenson, Vicki; Ogimoto, Kayoko; Kanter, Jenny E.; Bornfeldt, Karin E.; Daum, Guenter; Clowes, Alexander W.; Chait, Alan; Kim, Francis

    2013-01-01

    Activation of AMP-activated protein kinase (AMPK) signaling reduces hepatic steatosis and hepatic insulin resistance; however, its regulatory mechanisms are not fully understood. In this study, we sought to determine whether vasodilator-stimulated phosphoprotein (VASP) signaling improves lipid metabolism in the liver and, if so, whether VASP’s effects are mediated by AMPK. We show that disruption of VASP results in significant hepatic steatosis as a result of significant impairment of fatty acid oxidation, VLDL-triglyceride (TG) secretion, and AMPK signaling. Overexpression of VASP in hepatocytes increased AMPK phosphorylation and fatty acid oxidation and reduced hepatocyte TG accumulation; however, these responses were suppressed in the presence of an AMPK inhibitor. Restoration of AMPK phosphorylation by administration of 5-aminoimidazole-4-carboxamide riboside in Vasp−/− mice reduced hepatic steatosis and normalized fatty acid oxidation and VLDL-TG secretion. Activation of VASP by the phosphodiesterase-5 inhibitor, sildenafil, in db/db mice reduced hepatic steatosis and increased phosphorylated (p-)AMPK and p-acetyl CoA carboxylase. In Vasp−/− mice, however, sildendafil treatment did not increase p-AMPK or reduce hepatic TG content. These studies identify a role of VASP to enhance hepatic fatty acid oxidation by activating AMPK and to promote VLDL-TG secretion from the liver. PMID:23349495

  9. Insulin Resistance Prevents AMPK-induced Tau Dephosphorylation through Akt-mediated Increase in AMPKSer-485 Phosphorylation.

    PubMed

    Kim, Bhumsoo; Figueroa-Romero, Claudia; Pacut, Crystal; Backus, Carey; Feldman, Eva L

    2015-07-31

    Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including obesity, diabetes, and dyslipidemia, and insulin resistance (IR) is the central feature of MetS. Recent studies suggest that MetS is a risk factor for Alzheimer disease (AD). AMP-activated kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme and a key player in regulating energy metabolism. In this report, we examined the role of IR on the regulation of AMPK phosphorylation and AMPK-mediated Tau phosphorylation. We found that AMPK(Ser-485), but not AMPK(Thr-172), phosphorylation is increased in the cortex of db/db and high fat diet-fed obese mice, two mouse models of IR. In vitro, treatment of human cortical stem cell line (HK-5320) and primary mouse embryonic cortical neurons with the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), induced AMPK phosphorylation at both Thr-172 and Ser-485. AMPK activation also triggered Tau dephosphorylation. When IR was mimicked in vitro by chronically treating the cells with insulin, AICAR specifically induced AMPK(Ser-485), but not AMPK(Thr-172), hyperphosphorylation whereas AICAR-induced Tau dephosphorylation was inhibited. IR also resulted in the overactivation of Akt by AICAR treatment; however, preventing Akt overactivation during IR prevented AMPK(Ser-485) hyperphosphorylation and restored AMPK-mediated Tau dephosphorylation. Transfection of AMPK(S485A) mutant caused similar results. Therefore, our results suggest the following mechanism for the adverse effect of IR on AD pathology: IR → chronic overactivation of Akt → AMPK(Ser-485) hyperphosphorylation → inhibition of AMPK-mediated Tau dephosphorylation. Together, our results show for the first time a possible contribution of IR-induced AMPK(Ser-485) phosphorylation to the increased risk of AD in obesity and diabetes.

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

    PubMed Central

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

    2016-01-01

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

  11. The AMPK activator R419 improves exercise capacity and skeletal muscle insulin sensitivity in obese mice

    PubMed Central

    Marcinko, Katarina; Bujak, Adam L.; Lally, James S.V.; Ford, Rebecca J.; Wong, Tammy H.; Smith, Brennan K.; Kemp, Bruce E.; Jenkins, Yonchu; Li, Wei; Kinsella, Todd M.; Hitoshi, Yasumichi; Steinberg, Gregory R.

    2015-01-01

    Objective Skeletal muscle AMP-activated protein kinase (AMPK) is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. R419 is a mitochondrial complex-I inhibitor that has recently been shown to acutely activate AMPK in myotubes. Our main objective was to examine whether R419 treatment improves insulin sensitivity and exercise capacity in obese insulin resistant mice and whether skeletal muscle AMPK was important for mediating potential effects. Methods Glucose homeostasis, insulin sensitivity, exercise capacity, and electron transport chain content/activity were examined in wildtype (WT) and AMPK β1β2 muscle-specific null (AMPK-MKO) mice fed a high-fat diet (HFD) with or without R419 supplementation. Results There was no change in weight gain, adiposity, glucose tolerance or insulin sensitivity between HFD-fed WT and AMPK-MKO mice. In both HFD-fed WT and AMPK-MKO mice, R419 enhanced insulin tolerance, insulin-stimulated glucose disposal, skeletal muscle 2-deoxyglucose uptake, Akt phosphorylation and glucose transporter 4 (GLUT4) content independently of alterations in body mass. In WT, but not AMPK-MKO mice, R419 improved treadmill running capacity. Treatment with R419 increased muscle electron transport chain content and activity in WT mice; effects which were blunted in AMPK-MKO mice. Conclusions Treatment of obese mice with R419 improved skeletal muscle insulin sensitivity through a mechanism that is independent of skeletal muscle AMPK. R419 also increases exercise capacity and improves mitochondrial function in obese WT mice; effects that are diminished in the absence of skeletal muscle AMPK. These findings suggest that R419 may be a promising therapy for improving whole-body glucose homeostasis and exercise capacity. PMID:26413470

  12. AMPK induces vascular smooth muscle cell senescence via LKB1 dependent pathway

    SciTech Connect

    Sung, Jin Young; Woo, Chang-Hoon; Kang, Young Jin; Lee, Kwang Youn; Choi, Hyoung Chul

    2011-09-16

    Highlights: {yields} An aging model was established by stimulating VSMC with adriamycin. {yields} Adriamycin increased p-LKB1, p-AMPK, p53 and p21 expressions. {yields} Inhibition of AMPK diminished SA-{beta}-gal staining and restored VSMC proliferation. {yields} p53 and p21 siRNA attenuated adriamycin-induced SA-{beta}-gal staining in VSMC. {yields} p53-p21 pathway is a mediator of LKB1/AMPK induced VSMC senescence. -- Abstract: Vascular cells have a limited lifespan with limited cell proliferation and undergo cellular senescence. The functional changes associated with cellular senescence are thought to contribute to age-related vascular disorders. AMP-activated protein kinase (AMPK) has been discussed in terms of beneficial or harmful effects for aging-related diseases. However, the detailed functional mechanisms of AMPK are largely unclear. An aging model was established by stimulating vascular smooth muscle cell (VSMC) with adriamycin. Adriamycin progressively increased the mRNA and protein expressions of AMPK. The phosphorylation levels of LKB1 and acetyl-CoA carboxylase (ACC), the upstream and downstream of AMPK, were dramatically increased by adriamycin stimulation. The expressions of p53 and p21, which contribute to vascular senescence, were also increased. Inhibition of AMPK diminished senescence-associated {beta}-galactosidase (SA-{beta}-gal) staining, and restored VSMC proliferation. Cytosolic translocation of LKB1 by adriamycin could be a mechanism for AMPK activation in senescence. Furthermore, p53 siRNA and p21 siRNA transfection attenuated adriamycin-induced SA-{beta}-gal staining. These results suggest that LKB1 dependent AMPK activation elicits VSMC senescence and p53-p21 pathway is a mediator of LKB1/AMPK-induced senescence.

  13. Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice

    SciTech Connect

    Li, Yuan-Yuan; Yu, Li-Fang; Zhang, Li-Na; Qiu, Bei-Ying; Su, Ming-Bo; Wu, Fang; Chen, Da-Kai; Pang, Tao; Gu, Min; Zhang, Wei; Ma, Wei-Ping; Jiang, Hao-Wen; Li, Jing-Ya Nan, Fa-Jun Li, Jia

    2013-12-01

    AMP-activated protein kinase (AMPK), which is a pivotal guardian of whole-body energy metabolism, has become an attractive therapeutic target for metabolic syndrome. Previously, using a homogeneous scintillation proximity assay, we identified the small-molecule AMPK activator C24 from an optimization based on the original allosteric activator PT1. In this paper, the AMPK activation mechanism of C24 and its potential beneficial effects on glucose and lipid metabolism on db/db mice were investigated. C24 allosterically stimulated inactive AMPK α subunit truncations and activated AMPK heterotrimers by antagonizing autoinhibition. In primary hepatocytes, C24 increased the phosphorylation of AMPK downstream target acetyl-CoA carboxylase dose-dependently without changing intracellular AMP/ATP ratio, indicating its allosteric activation in cells. Through activating AMPK, C24 decreased glucose output by down-regulating mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary hepatocytes. C24 also decreased the triglyceride and cholesterol contents in HepG2 cells. Due to its improved bioavailability, chronic oral treatment with multiple doses of C24 significantly reduced blood glucose and lipid levels in plasma, and improved the glucose tolerance of diabetic db/db mice. The hepatic transcriptional levels of PEPCK and G6Pase were reduced. These results demonstrate that this orally effective activator of AMPK represents a novel approach to the treatment of metabolic syndrome. - Highlights: • C24 activates AMPK through antagonizing autoinhibition within α subunit. • C24 activates AMPK in hepatocytes and decreases glucose output via AMPK. • C24 exerts beneficial effects on diabetic db/db mice. • C24 represents a novel therapeutic for treatment of metabolic syndrome.

  14. Inositol-Requiring Enzyme 1-Dependent Activation of AMPK Promotes Brucella abortus Intracellular Growth

    PubMed Central

    Liu, Ning; Li, Yingying; Dong, Chunyan; Xu, Xiaohan; Wei, Pan; Sun, Wanchun

    2016-01-01

    ABSTRACT AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is well conserved during evolution. AMPK activation inhibits production of reactive oxygen species (ROS) in cells via suppression of NADPH oxidase. However, the role of AMPK during the process of Brucella infection remains unknown. Our data demonstrate that B. abortus infection induces AMPK activation in HeLa cells in a time-dependent manner. The known AMPK kinases LKB1, CAMKKβ, and TAK1 are not required for the activation of AMPK by B. abortus infection. Instead, this activation is dependent on the RNase activity of inositol-requiring enzyme 1 (IRE1). Moreover, we also found that B. abortus infection-induced IRE1-dependent activation of AMPK promotes B. abortus intracellular growth with peritoneal macrophages via suppression of NADPH-derived ROS production. IMPORTANCE Previous studies showed that B. abortus infection does not promote any oxidative burst regulated by NADPH oxidase. However, the underlying mechanism remains elusive. We report for the first time that AMPK activation caused by B. abortus infection plays important role in NADPH oxidase-derived ROS production. PMID:26755628

  15. The AMPK-related kinase SNARK regulates muscle mass and myocyte survival

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-r...

  16. Metformin Prevents Nigrostriatal Dopamine Degeneration Independent of AMPK Activation in Dopamine Neurons

    PubMed Central

    Bayliss, Jacqueline A.; Lemus, Moyra B.; Santos, Vanessa V.; Deo, Minh; Davies, Jeffrey S.; Kemp, Bruce E.; Elsworth, John D.

    2016-01-01

    Metformin is a widely prescribed drug used to treat type-2 diabetes, although recent studies show it has wide ranging effects to treat other diseases. Animal and retrospective human studies indicate that Metformin treatment is neuroprotective in Parkinson’s Disease (PD), although the neuroprotective mechanism is unknown, numerous studies suggest the beneficial effects on glucose homeostasis may be through AMPK activation. In this study we tested whether or not AMPK activation in dopamine neurons was required for the neuroprotective effects of Metformin in PD. We generated transgenic mice in which AMPK activity in dopamine neurons was ablated by removing AMPK beta 1 and beta 2 subunits from dopamine transporter expressing neurons. These AMPK WT and KO mice were then chronically exposed to Metformin in the drinking water then exposed to MPTP, the mouse model of PD. Chronic Metformin treatment significantly attenuated the MPTP-induced loss of Tyrosine Hydroxylase (TH) neuronal number and volume and TH protein concentration in the nigrostriatal pathway. Additionally, Metformin treatment prevented the MPTP-induced elevation of the DOPAC:DA ratio regardless of genotype. Metformin also prevented MPTP induced gliosis in the Substantia Nigra. These neuroprotective actions were independent of genotype and occurred in both AMPK WT and AMPK KO mice. Overall, our studies suggest that Metformin’s neuroprotective effects are not due to AMPK activation in dopaminergic neurons and that more research is required to determine how metformin acts to restrict the development of PD. PMID:27467571

  17. The AMPK gamma1 R70Q mutant regulates multiple metabolic and growth pathways in neonatal cardiac myocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although mutations in the gamma-subunit of AMP-activated protein kinase (AMPK) can result in excessive glycogen accumulation and cardiac hypertrophy, the mechanisms by which this occurs have not been well defined. Because >65% of cardiac AMPK activity is associated with the gamma1-subunit of AMPK, w...

  18. Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells.

    PubMed

    Sozio, Margaret S; Lu, Changyue; Zeng, Yan; Liangpunsakul, Suthat; Crabb, David W

    2011-10-01

    AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPAR-α) are critical regulators of short-term and long-term fatty acid oxidation, respectively. We examined whether the activities of these molecules were coordinately regulated. H4IIEC3 cells were transfected with PPAR-α and PPAR-γ expression plasmids and a peroxisome-proliferator-response element (PPRE) luciferase reporter plasmid. The cells were treated with PPAR agonists (WY-14,643 and rosiglitazone), AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAR) and metformin, and the AMPK inhibitor compound C. Both AICAR and metformin decreased basal and WY-14,643-stimulated PPAR-α activity; compound C increased agonist-stimulated reporter activity and partially reversed the effect of the AMPK activators. Similar effects on PPAR-γ were seen, with both AICAR and metformin inhibiting PPRE reporter activity. Compound C increased basal PPAR-γ activity and rosiglitazone-stimulated activity. In contrast, retinoic acid receptor-α (RAR-α), another nuclear receptor that dimerizes with retinoid X receptor (RXR), was largely unaffected by the AMPK activators. Compound C modestly increased AM580 (an RAR agonist)-stimulated activity. The AMPK activators did not affect PPAR-α binding to DNA, and there was no consistent correlation between effects of the AMPK activators and inhibitor on PPAR and the nuclear localization of AMPK-α subunits. Expression of either a constitutively active or dominant negative AMPK-α inhibited basal and WY-14,643-stimulated PPAR-α activity and basal and rosiglitazone-stimulated PPAR-γ activity. We concluded that the AMPK activators AICAR and metformin inhibited transcriptional activities of PPAR-α and PPAR-γ, whereas inhibition of AMPK with compound C activated both PPARs. The effects of AMPK do not appear to be mediated through effects on RXR or on PPAR/RXR binding to DNA. These effects are independent of kinase activity and instead appear to

  19. Toona Sinensis ameliorates insulin resistance via AMPK and PPARγ pathways.

    PubMed

    Liu, Hung-Wen; Huang, Wen-Cheng; Yu, Wen-Jen; Chang, Sue-Joan

    2015-06-01

    Toona Sinensis leaf (TSL) extract with a beneficial effect for managing hyperglycemia has been reported, however the underlying mechanism by which TSL extract acts as an insulin sensitizer remains uncertain, especially in peripheral tissues. TSL 95% ethanol extract exhibited the highest transactivity of PPARγ and contained the highest amounts of natural PPARγ ligands including palmitic acid, linoleic acid, and α-linolenic acid among different TSL ethanol extracts (0, 10, 50, 70, and 95%). The efficacy and the mechanism of TSL ethanol extract (95%) mediated anti-diabetic effects were examined by both in vivo and in vitro models in this study. An improved whole-body insulin sensitivity was observed in high-fat diet-fed (HFD) mice after 14 weeks of TSL treatment, as evidenced by a faster rate of plasma glucose clearing. The improved insulin sensitivity was through direct stimulation of PPARγ and adiponectin expression in adipose tissues of HFD mice. In addition to the PPARγ pathway, TSL stimulated glucose uptake via directly inducing AMPKα but not AS160 activation in C2C12 myotubes under palmitate-induced insulin resistance. TSL successfully induced sirtuin 1 and restored PGC1α, but failed to restore mitochondrial electron transport complexes I, III, IV and V in mRNA levels. Loss of the mitochondrial membrane potential coupled with AMPK activation suggests that TSL acts as a mitochondrial inhibitor to stimulate AMPK-mediated glucose uptake. This study demonstrated that TSL stimulated glucose uptake via AMPK activation in skeletal muscles and promoted PPARγ and normalized adiponectin expression in adipose tissues, thereby ameliorating insulin resistance.

  20. 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

  1. Synthesis and biological evaluation of arctigenin ester and ether derivatives as activators of AMPK.

    PubMed

    Shen, Sida; Zhuang, Jingjing; Chen, Yijia; Lei, Min; Chen, Jing; Shen, Xu; Hu, Lihong

    2013-07-01

    A series of new arctigenin and 9-deoxy-arctigenin derivatives bearing different ester and ether side chains at the phenolic hydroxyl positions are designed, synthesized, and evaluated for activating AMPK potency in L6 myoblasts. Initial biological evaluation indicates that some alkyl ester and phenethyl ether arctigenin derivatives display potential activities in AMPK phosphorylation improvement. Further structure-activity relationship analysis shows that arctigenin ester derivatives 3a, 3h and 9-deoxy-arctigenin phenethyl ether derivatives 6a, 6c, 6d activate AMPK more potently than arctigenin. Moreover, the 2-(3,4-dimethoxyphenyl)ethyl ether moiety of 6c has been demonstrated as a potential functional group to improve the effect of AMPK phosphorylation. The structural optimization of arctigenin leads to the identification of 6c as a promising lead compound that exhibits excellent activity in AMPK activation.

  2. Synthesis and biological evaluation of arctigenin ester and ether derivatives as activators of AMPK.

    PubMed

    Shen, Sida; Zhuang, Jingjing; Chen, Yijia; Lei, Min; Chen, Jing; Shen, Xu; Hu, Lihong

    2013-07-01

    A series of new arctigenin and 9-deoxy-arctigenin derivatives bearing different ester and ether side chains at the phenolic hydroxyl positions are designed, synthesized, and evaluated for activating AMPK potency in L6 myoblasts. Initial biological evaluation indicates that some alkyl ester and phenethyl ether arctigenin derivatives display potential activities in AMPK phosphorylation improvement. Further structure-activity relationship analysis shows that arctigenin ester derivatives 3a, 3h and 9-deoxy-arctigenin phenethyl ether derivatives 6a, 6c, 6d activate AMPK more potently than arctigenin. Moreover, the 2-(3,4-dimethoxyphenyl)ethyl ether moiety of 6c has been demonstrated as a potential functional group to improve the effect of AMPK phosphorylation. The structural optimization of arctigenin leads to the identification of 6c as a promising lead compound that exhibits excellent activity in AMPK activation. PMID:23673223

  3. Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM.

    PubMed

    Chennell, George; Willows, Robin J W; Warren, Sean C; Carling, David; French, Paul M W; Dunsby, Chris; Sardini, Alessandro

    2016-01-01

    We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation. PMID:27548185

  4. Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

    PubMed Central

    Chennell, George; Willows, Robin J. W.; Warren, Sean C.; Carling, David; French, Paul M. W.; Dunsby, Chris; Sardini, Alessandro

    2016-01-01

    We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation. PMID:27548185

  5. Inhibitory crosstalk between ERK and AMPK in the growth and proliferation of cardiac fibroblasts

    SciTech Connect

    Du Jianhai; Guan Tongju; Zhang Hui; Xia Yi; Liu Fei; Zhang Youyi

    2008-04-04

    Extracellular signal-regulated kinase (ERK) is one of the key protein kinases that regulate the growth and proliferation in cardiac fibroblasts (CFs). As an energy sensor of cellular metabolism, AMP-activated protein kinase (AMPK) is found recently to be involved in myocardial remodeling. In this study, we investigated the crosstalk between ERK and AMPK in the growth and proliferation of CFs. In neonatal rat cardiac fibroblasts (NRCFs), we found that serum significantly inhibited basal AMPK phosphorylation between 10 min and 24 h and also partially inhibited AMPK phosphorylation by AMPK activator, 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR). Furthermore, ERK inhibitor could greatly reverse the inhibition of AMPK by serum. Conversely, activation of AMPK by AICAR also showed a significant inhibition of basal and serum-induced ERK phosphorylation but it showed a delayed and steadfast inhibition which appeared after 60 min and lasted until 12 h. Moreover, inhibition of ERK could repress the activation of p70S6K, an important kinase in cardiac proliferation, and AICAR could also inhibit p70S6K phosphorylation. In addition, under both serum and serum-free medium, AICAR significantly inhibited the DNA synthesis and cell numbers, and reduced cells at S phase. In conclusion, AMPK activation with AICAR inhibited growth and proliferation in cardiac fibroblasts, which involved inhibitory interactions between ERK and AMPK. This is the first report that AMPK could be a target of ERK in growth factors-induced proliferation, which may give a new mechanism that growth factors utilize in their promotion of proliferation in cardiac fibroblasts.

  6. Metformin inhibits inflammatory response via AMPK-PTEN pathway in vascular smooth muscle cells

    SciTech Connect

    Kim, Sun Ae; Choi, Hyoung Chul

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer PTEN was induced by metformin and inhibited by compound C and AMPK siRNA. Black-Right-Pointing-Pointer Metformin suppressed TNF-{alpha}-induced COX-2 and iNOS mRNA expression. Black-Right-Pointing-Pointer Compound C and bpv (pic) increased iNOS and COX-2 protein expression. Black-Right-Pointing-Pointer NF-{kappa}B activation was restored by inhibiting AMPK and PTEN. Black-Right-Pointing-Pointer AMPK and PTEN regulated TNF-{alpha}-induced ROS production in VSMCs. -- Abstract: Atherosclerosis is a chronic inflammation of the coronary arteries. Vascular smooth muscle cells (VSMCs) stimulated by cytokines and chemokines accelerate the inflammatory response and migrate to the injured endothelium during the progression of atherosclerosis. Activation of AMP activated protein kinase (AMPK), a key sensor maintaining metabolic homeostasis, suppresses the inflammatory response. However, how AMPK regulates the inflammatory response is poorly understood. To identify the mechanism of this response, we focused on phosphatase and tensin homolog (PTEN), which is a negative regulator of inflammation. We investigated that activation of AMPK-induced PTEN expression and suppression of the inflammatory response through the AMPK-PTEN pathway in VSMCs. We treated with the well-known AMPK activator metformin to induce PTEN expression. PTEN was induced by metformin (2 mM) and inhibited by compound C (10 {mu}M) and AMPK siRNA. Tumor necrosis factor-alpha (TNF-{alpha}) was used to induce inflammation. The inflammatory response was confirmed by cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) expression, and activation of nuclear factor (NF)-{kappa}B. Metformin suppressed COX-2 and iNOS mRNA and protein expression dose dependently. Treatment with compound C and bpv (pic) in the presence of metformin, iNOS and COX-2 protein expression increased. NF-{kappa}B activation decreased in response to metformin and was restored by inhibiting AMPK

  7. OSU53 Rescues Human OB-6 Osteoblastic Cells from Dexamethasone through Activating AMPK Signaling.

    PubMed

    Xu, Dawei; Zhao, Wei; Zhu, Xinhui; Fan, Jianbo; Cui, Shengyu; Sun, Yuyu; Chen, Xiang; Liu, Wei; Cui, Zhi-Ming

    2016-01-01

    Excessive dexamethasone (Dex) application causes osteoblast cell death, which could lead to osteoporosis or osteonecrosis. AMP-activated protein kinase (AMPK) activation is shown to protect osteoblasts/osteoblastic cells from Dex. In this report, we tested the potential effect of OSU53, a novel AMPK activator, in Dex-treated osteoblastic cells. We show that OSU53 activated AMPK signaling in human OB-6 osteoblastic cells. Further, Dex-induced osteoblastic OB-6 cell death and apoptosis were largely attenuated with pre-treatment with OSU53. OSU53 was more efficient than other known AMPK activators (A-769662 and Compound 13) in protecting OB-6 cells against Dex. AMPK activation is required for OSU53-induced actions in OB-6 cells. AMPKα shRNA knockdown or dominant-negative mutation (dn-AMPKα T172A) almost completely blocked OSU53-induced AMPK activation and OB-6 cell protection against Dex. Further studies showed that OSU53 increased NADPH (nicotinamide adenine dinucleotide phosphate) activity and alleviated Dex-induced oxidative stress in OB-6 cells. Such effects by OSU53 were again almost abolished with AMPKα shRNA or dn-AMPKα in OB-6 cells. Together, these results demonstrate that OSU53 protects osteoblastic cells from Dex possibly via activating AMPK-dependent signaling. PMID:27632213

  8. Argininosuccinate synthetase regulates hepatic AMPK linking protein catabolism and ureagenesis to hepatic lipid metabolism

    PubMed Central

    Madiraju, Anila K.; Alves, Tiago; Zhao, Xiaojian; Cline, Gary W.; Zhang, Dongyan; Bhanot, Sanjay; Samuel, Varman T.; Kibbey, Richard G.; Shulman, Gerald I.

    2016-01-01

    A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma β-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism. PMID:27247419

  9. A role for AMPK in increased insulin action after serum starvation.

    PubMed

    Ching, James Kain; Rajguru, Pooja; Marupudi, Nandhini; Banerjee, Sankha; Fisher, Jonathan S

    2010-11-01

    Serum starvation is a common cell culture procedure for increasing cellular response to insulin, though the mechanism for the serum starvation effect is not understood. We hypothesized that factors known to potentiate insulin action [e.g., AMP-activated protein kinase (AMPK) and p38] or to be involved in insulin signaling leading to glucose transport [e.g., Akt, PKCζ, AS160, and ataxia telangiectasia mutated (ATM)] would be phosphorylated during serum starvation and would be responsible for increased insulin action after serum starvation. L6 myotubes were incubated in serum-containing or serum-free medium for 3 h. Levels of phosphorylated AMPK, Akt, and ATM were greater in serum-starved cells than in control cells. Serum starvation did not affect p38, PKCζ, or AS160 phosphorylation or insulin-stimulated Akt or AS160 phosphorylation. Insulin had no effect on glucose transport in control cells but caused an increase in glucose uptake for serum-starved cells that was preventable by compound C (an AMPK inhibitor), by expression of dominant negative AMPK (AMPK-DN), and by KU55933 (an ATM inhibitor). ATM protein levels increased during serum starvation, and this increase in ATM was prevented by compound C and AMPK-DN. Thus, it appears that AMPK is required for the serum starvation-related increase in insulin-stimulated glucose transport, with ATM as a possible downstream effector.

  10. Molecular Pathways: Is AMPK a Friend or a Foe in Cancer?

    PubMed Central

    Hardie, D. Grahame

    2015-01-01

    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status expressed in essentially all eukaryotic cells. Once activated by energetic stress via a mechanism that detects increases in AMP:ATP and ADP:ATP ratios, AMPK acts to restore energy homeostasis by switching on catabolic pathways that generate ATP, while switching off ATP-consuming processes, including anabolic pathways required for cell growth and proliferation. AMPK activation promotes the glucose-sparing, oxidative metabolism utilized by most quiescent cells, rather than the rapid glucose uptake and glycolysis used by most proliferating cells. Numerous pharmacological activators of AMPK are known, including drugs in long use such as salicylate and metformin, and there is evidence that regular use of either of the latter provides protection against development of cancer. Tumor cells appear to be under selection pressure to down-regulate AMPK, thus limiting its restraining influence on cell growth and proliferation, and several interesting mechanisms by which this occurs are discussed. Paradoxically, however, a complete loss of AMPK function, which appears to be rare in human cancers, may be deleterious to survival of tumor cells. AMPK can therefore either be a friend and a foe in cancer, depending on the context. PMID:26152739

  11. Argininosuccinate synthetase regulates hepatic AMPK linking protein catabolism and ureagenesis to hepatic lipid metabolism.

    PubMed

    Madiraju, Anila K; Alves, Tiago; Zhao, Xiaojian; Cline, Gary W; Zhang, Dongyan; Bhanot, Sanjay; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

    2016-06-14

    A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma β-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism. PMID:27247419

  12. OSU53 Rescues Human OB-6 Osteoblastic Cells from Dexamethasone through Activating AMPK Signaling

    PubMed Central

    Xu, Dawei; Zhao, Wei; Zhu, Xinhui; Fan, Jianbo; Cui, Shengyu; Sun, Yuyu; Chen, Xiang; Liu, Wei; Cui, Zhi-ming

    2016-01-01

    Excessive dexamethasone (Dex) application causes osteoblast cell death, which could lead to osteoporosis or osteonecrosis. AMP-activated protein kinase (AMPK) activation is shown to protect osteoblasts/osteoblastic cells from Dex. In this report, we tested the potential effect of OSU53, a novel AMPK activator, in Dex-treated osteoblastic cells. We show that OSU53 activated AMPK signaling in human OB-6 osteoblastic cells. Further, Dex-induced osteoblastic OB-6 cell death and apoptosis were largely attenuated with pre-treatment with OSU53. OSU53 was more efficient than other known AMPK activators (A-769662 and Compound 13) in protecting OB-6 cells against Dex. AMPK activation is required for OSU53-induced actions in OB-6 cells. AMPKα shRNA knockdown or dominant-negative mutation (dn-AMPKα T172A) almost completely blocked OSU53-induced AMPK activation and OB-6 cell protection against Dex. Further studies showed that OSU53 increased NADPH (nicotinamide adenine dinucleotide phosphate) activity and alleviated Dex-induced oxidative stress in OB-6 cells. Such effects by OSU53 were again almost abolished with AMPKα shRNA or dn-AMPKα in OB-6 cells. Together, these results demonstrate that OSU53 protects osteoblastic cells from Dex possibly via activating AMPK-dependent signaling. PMID:27632213

  13. AMPK-associated signaling to bridge the gap between fuel metabolism and hepatocyte viability

    PubMed Central

    Yang, Yoon Mee; Han, Chang Yeob; Kim, Yoon Jun; Kim, Sang Geon

    2010-01-01

    The adenosine monophosphate-activated protein kinase (AMPK) and p70 ribosomal S6 kinase-1 pathway may serve as a key signaling flow that regulates energy metabolism; thus, this pathway becomes an attractive target for the treatment of liver diseases that result from metabolic derangements. In addition, AMPK emerges as a kinase that controls the redox-state and mitochondrial function, whose activity may be modulated by antioxidants. A close link exists between fuel metabolism and mitochondrial biogenesis. The relationship between fuel metabolism and cell survival strongly implies the existence of a shared signaling network, by which hepatocytes respond to challenges of external stimuli. The AMPK pathway may belong to this network. A series of drugs and therapeutic candidates enable hepatocytes to protect mitochondria from radical stress and increase cell viability, which may be associated with the activation of AMPK, liver kinase B1, and other molecules or components. Consequently, the components downstream of AMPK may contribute to stabilizing mitochondrial membrane potential for hepatocyte survival. In this review, we discuss the role of the AMPK pathway in hepatic energy metabolism and hepatocyte viability. This information may help identify ways to prevent and/or treat hepatic diseases caused by the metabolic syndrome. Moreover, clinical drugs and experimental therapeutic candidates that directly or indirectly modulate the AMPK pathway in distinct manners are discussed here with particular emphasis on their effects on fuel metabolism and mitochondrial function. PMID:20698033

  14. A novel direct activator of AMPK inhibits prostate cancer growth by blocking lipogenesis

    PubMed Central

    Zadra, Giorgia; Photopoulos, Cornelia; Tyekucheva, Svitlana; Heidari, Pedram; Weng, Qing Ping; Fedele, Giuseppe; Liu, Hong; Scaglia, Natalia; Priolo, Carmen; Sicinska, Ewa; Mahmood, Umar; Signoretti, Sabina; Birnberg, Neal; Loda, Massimo

    2014-01-01

    5′AMP-activated kinase (AMPK) constitutes a hub for cellular metabolic and growth control, thus representing an ideal therapeutic target for prostate cancers (PCas) characterized by increased lipogenesis and activation of mTORC1 pathway. However, whether AMPK activation itself is sufficient to block cancer cell growth remains to be determined. A small molecule screening was performed and identified MT 63–78, a specific and potent direct AMPK activator. Here, we show that direct activation of AMPK inhibits PCa cell growth in androgen sensitive and castration resistant PCa (CRPC) models, induces mitotic arrest, and apoptosis. In vivo, AMPK activation is sufficient to reduce PCa growth, whereas the allelic loss of its catalytic subunits fosters PCa development. Importantly, despite mTORC1 blockade, the suppression of de novo lipogenesis is the underpinning mechanism responsible for AMPK-mediated PCa growth inhibition, suggesting AMPK as a therapeutic target especially for lipogenesis-driven PCas. Finally, we demonstrate that MT 63–78 enhances the growth inhibitory effect of AR signaling inhibitors MDV3100 and abiraterone. This study thus provides a rationale for their combined use in CRPC treatment. PMID:24497570

  15. Survival advantage of AMPK activation to androgen-independent prostate cancer cells during energy stress.

    PubMed

    Chhipa, Rishi Raj; Wu, Yue; Mohler, James L; Ip, Clement

    2010-10-01

    Androgen-independent prostate cancer usually develops as a relapse following androgen ablation therapy. Removing androgen systemically causes vascular degeneration and nutrient depletion of the prostate tumor tissue. The fact that the malignancy later evolves to androgen-independence suggests that some cancer cells are able to survive the challenge of energy/nutrient deprivation. AMP-activated protein kinase (AMPK) is an important manager of energy stress. The present study was designed to investigate the role of AMPK in contributing to the survival of the androgen-independent phenotype. Most of the experiments were carried out in the androgen-dependent LNCaP cells and the androgen-independent C4-2 cells. These two cell lines have the same genetic background, since the C4-2 line is derived from the LNCaP line. Glucose deprivation (GD) was instituted to model energy stress encountered by these cells. The key findings are as follows. First, the activation of AMPK by GD was much stronger in C4-2 cells than in LNCaP cells, and the robustness of AMPK activation was correlated favorably with cell viability. Second, the response of AMPK was specific to energy deficiency rather than to amino acid deficiency. The activation of AMPK by GD was functional, as demonstrated by appropriate phosphorylation changes of mTOR and mTOR downstream substrates. Third, blocking AMPK activation by chemical inhibitor or dominant negative AMPK led to increased apoptotic cell death. The observation that similar results were found in other androgen-independent prostate cancer cell lines, including CW22Rv1 abd VCaP, provided further assurance that AMPK is a facilitator on the road to androgen-independence of prostate cancer cells.

  16. AMPK signaling in skeletal muscle during exercise: Role of reactive oxygen and nitrogen species.

    PubMed

    Morales-Alamo, David; Calbet, Jose A L

    2016-09-01

    Reactive oxygen and nitrogen species (RONS) are generated during exercise depending on intensity, duration and training status. A greater amount of RONS is released during repeated high-intensity sprint exercise and when the exercise is performed in hypoxia. By activating adenosine monophosphate-activated kinase (AMPK), RONS play a critical role in the regulation of muscle metabolism but also in the adaptive responses to exercise training. RONS may activate AMPK by direct an indirect mechanisms. Directly, RONS may activate or deactivate AMPK by modifying RONS-sensitive residues of the AMPK-α subunit. Indirectly, RONS may activate AMPK by reducing mitochondrial ATP synthesis, leading to an increased AMP:ATP ratio and subsequent Thr(172)-AMPK phosphorylation by the two main AMPK kinases: LKB1 and CaMKKβ. In presence of RONS the rate of Thr(172)-AMPK dephosphorylation is reduced. RONS may activate LKB1 through Sestrin2 and SIRT1 (NAD(+)/NADH.H(+)-dependent deacetylase). RONS may also activate CaMKKβ by direct modification of RONS sensitive motifs and, indirectly, by activating the ryanodine receptor (Ryr) to release Ca(2+). Both too high (hypoxia) and too low (ingestion of antioxidants) RONS levels may lead to Ser(485)-AMPKα1/Ser(491)-AMPKα2 phosphorylation causing inhibition of Thr(172)-AMPKα phosphorylation. Exercise training increases muscle antioxidant capacity. When the same high-intensity training is applied to arm and leg muscles, arm muscles show signs of increased oxidative stress and reduced mitochondrial biogenesis, which may be explained by differences in RONS-sensing mechanisms and basal antioxidant capacities between arm and leg muscles. Efficient adaptation to exercise training requires optimal exposure to pulses of RONS. Inappropriate training stimulus may lead to excessive RONS formation, oxidative inactivation of AMPK and reduced adaptation or even maladaptation. Theoretically, exercise programs should be designed taking into account the

  17. CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

    PubMed

    Scerif, Miski; Füzesi, Tamás; Thomas, Julia D; Kola, Blerina; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta

    2013-10-01

    AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess.

  18. AMPK Dilates Resistance Arteries via Activation of SERCA and BKCa Channels in Smooth Muscle.

    PubMed

    Schneider, Holger; Schubert, Kai Michael; Blodow, Stephanie; Kreutz, Claus-Peter; Erdogmus, Serap; Wiedenmann, Margarethe; Qiu, Jiehua; Fey, Theres; Ruth, Peter; Lubomirov, Lubomir T; Pfitzer, Gabriele; Mederos Y Schnitzler, Michael; Hardie, D Grahame; Gudermann, Thomas; Pohl, Ulrich

    2015-07-01

    The protective effects of 5'-AMP-activated protein kinase (AMPK) on the metabolic syndrome may include direct effects on resistance artery vasomotor function. However, the precise actions of AMPK on microvessels and their potential interaction are largely unknown. Thus, we set to determine the effects of AMPK activation on vascular smooth muscle tone and the underlying mechanisms. Resistance arteries isolated from hamster and mouse exhibited a pronounced endothelium-independent dilation on direct pharmacological AMPK activation by 2 structurally unrelated compounds (PT1 and A769662). The dilation was associated with a decrease of intracellular-free calcium [Ca(2+)]i in vascular smooth muscle cell. AMPK stimulation induced activation of BKCa channels as assessed by patch clamp studies in freshly isolated hamster vascular smooth muscle cell and confirmed by direct proof of membrane hyperpolarization in intact arteries. The BKCa channel blocker iberiotoxin abolished the hyperpolarization but only partially reduced the dilation and did not affect the decrease of [Ca(2+)]i. By contrast, the sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) inhibitor thapsigargin largely reduced these effects, whereas combined inhibition of SERCA and BKCa channels virtually abolished them. AMPK stimulation significantly increased the phosphorylation of the SERCA modulator phospholamban at the regulatory T17 site. Stimulation of smooth muscle AMPK represents a new, potent vasodilator mechanism in resistance vessels. AMPK directly relaxes vascular smooth muscle cell by a decrease of [Ca(2+)]i. This is achieved by calcium sequestration via SERCA activation, as well as activation of BKCa channels. There is in part a mutual compensation of both calcium-lowering mechanisms. However, SERCA activation which involves an AMPK-dependent phosphorylation of phospholamban is the predominant mechanism in resistance vessels.

  19. CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

    PubMed

    Scerif, Miski; Füzesi, Tamás; Thomas, Julia D; Kola, Blerina; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta

    2013-10-01

    AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess. PMID:23884964

  20. Blockade of MerTK Activation by AMPK Inhibits RPE Cell Phagocytosis.

    PubMed

    Qin, Suofu

    2016-01-01

    Timely removal of shed photoreceptor outer segments by retinal pigment epithelial cells (RPE) plays a key role in biological renewal of these highly peroxidizable structures and in maintenance of retina health. How environmental stress cause RPE cell dysfunction is undefined however. AMP-activated protein kinase (AMPK), a heterotrimer of a catalytic α subunit and regulatory β and γ subunits, maintains energy homeostasis by limiting energy utilization and/or promoting energy production when energy supply is compromised. Intriguingly, AMPK has been shown to be important in functions of RPE cells. In this mini-review, the role and mechanisms of AMPK in controlling RPE cell phagocytosis are discussed.

  1. Metabolic Roles of AMPK and Metformin in Cancer Cells

    PubMed Central

    Choi, Yeon Kyung; Park, Keun-Gyu

    2013-01-01

    Metformin is one of the most widely used anti-diabetic agents in the world, and a growing body of evidence suggests that it may also be effective as an anti-cancer drug. Observational studies have shown that metformin reduces cancer incidence and cancer-related mortality in multiple types of cancer. These results have drawn attention to the mechanisms underlying metformin’s anti-cancer effects, which may include triggering of the AMP-activated protein kinase (AMPK) pathway, resulting in vulnerability to an energy crisis (leading to cell death under conditions of nutrient deprivation) and a reduction in circulating insulin/IGF-1 levels. Clinical trials are currently underway to determine the benefits, appropriate dosage, and tolerability of metformin in the context of cancer therapy. This review highlights fundamental aspects of the molecular mechanisms underlying metformin’s anti-cancer effects, describes the epidemiological evidence and ongoing clinical challenges, and proposes directions for future translational research. PMID:23794020

  2. A Novel MEK-ERK-AMPK Signaling Axis Controls Chemokine Receptor CCR7-dependent Survival in Human Mature Dendritic Cells*

    PubMed Central

    López-Cotarelo, Pilar; Escribano-Díaz, Cristina; González-Bethencourt, Ivan Luis; Gómez-Moreira, Carolina; Deguiz, María Laura; Torres-Bacete, Jesús; Gómez-Cabañas, Laura; Fernández-Barrera, Jaime; Delgado-Martín, Cristina; Mellado, Mario; Regueiro, José Ramón; Miranda-Carús, María Eugenia; Rodríguez-Fernández, José Luis

    2015-01-01

    Chemokine receptor CCR7 directs mature dendritic cells (mDCs) to secondary lymph nodes where these cells regulate the activation of T cells. CCR7 also promotes survival in mDCs, which is believed to take place largely through Akt-dependent signaling mechanisms. We have analyzed the involvement of the AMP-dependent kinase (AMPK) in the control of CCR7-dependent survival. A pro-apoptotic role for AMPK is suggested by the finding that pharmacological activators induce apoptosis, whereas knocking down of AMPK with siRNA extends mDC survival. Pharmacological activation of AMPK also induces apoptosis of mDCs in the lymph nodes. Stimulation of CCR7 leads to inhibition of AMPK, through phosphorylation of Ser-485, which was mediated by Gi/Gβγ, but not by Akt or S6K, two kinases that control the phosphorylation of AMPK on Ser-485 in other settings. Using selective pharmacological inhibitors, we show that CCR7-induced phosphorylation of AMPK on Ser-485 is mediated by MEK and ERK. Coimmunoprecipitation analysis and proximity ligation assays indicate that AMPK associates with ERK, but not with MEK. These results suggest that in addition to Akt-dependent signaling mechanisms, CCR7 can also promote survival of mDCs through a novel MEK1/2-ERK1/2-AMPK signaling axis. The data also suggest that AMPK may be a potential target to modulate mDC lifespan and the immune response. PMID:25425646

  3. Cardioprotective actions of Notch1 against myocardial infarction via LKB1-dependent AMPK signaling pathway.

    PubMed

    Yang, Hui; Sun, Wanqing; Quan, Nanhu; Wang, Lin; Chu, Dongyang; Cates, Courtney; Liu, Quan; Zheng, Yang; Li, Ji

    2016-05-15

    AMP-activated protein kinase (AMPK) signaling pathway plays a pivotal role in intracellular adaptation to energy stress during myocardial ischemia. Notch1 signaling in the adult myocardium is also activated in response to ischemic stress. However, the relationship between Notch1 and AMPK signaling pathways during ischemia remains unclear. We hypothesize that Notch1 as an adaptive signaling pathway protects the heart from ischemic injury via modulating the cardioprotective AMPK signaling pathway. C57BL/6J mice were subjected to an in vivo ligation of left anterior descending coronary artery and the hearts from C57BL/6J mice were subjected to an ex vivo globe ischemia and reperfusion in the Langendorff perfusion system. The Notch1 signaling was activated during myocardial ischemia. A Notch1 γ-secretase inhibitor, dibenzazepine (DBZ), was intraperitoneally injected into mice to inhibit Notch1 signaling pathway by ischemia. The inhibition of Notch1 signaling by DBZ significantly augmented cardiac dysfunctions caused by myocardial infarction. Intriguingly, DBZ treatment also significantly blunted the activation of AMPK signaling pathway. The immunoprecipitation experiments demonstrated that an interaction between Notch1 and liver kinase beta1 (LKB1) modulated AMPK activation during myocardial ischemia. Furthermore, a ligand of Notch1 Jagged1 can significantly reduce cardiac damage caused by ischemia via activation of AMPK signaling pathway and modulation of glucose oxidation and fatty acid oxidation during ischemia and reperfusion. But Jagged1 did not have any cardioprotections on AMPK kinase dead transgenic hearts. Taken together, the results indicate that the cardioprotective effect of Notch1 against ischemic damage is mediated by AMPK signaling via an interaction with upstream LKB1.

  4. AICAR-Induced Activation of AMPK Inhibits TSH/SREBP-2/HMGCR Pathway in Liver

    PubMed Central

    Liu, Shudong; Jing, Fei; Yu, Chunxiao; Gao, Ling; Qin, Yejun; Zhao, Jiajun

    2015-01-01

    Our previous study found that thyroid-stimulating hormone promoted sterol regulatory element-binding protein-2 (SREBP-2) expression and suppressed AMP-activated protein kinase (AMPK) activity in the liver, but it was unclear whether there was a direct link between TSH, AMPK and SREBP-2. Here, we demonstrate that the 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR)-induced activation of AMPK directly inhibited the expression of SREBP-2 and its target genes HMGCR and HMGCS, which are key enzymes in cholesterol biosynthesis, and suppressed the TSH-stimulated up-regulation of SREBP-2 in HepG2 cells; similar results were obtained in TSH receptor knockout mice. Furthermore, AMPK, an evolutionally conserved serine/threonine kinase, phosphorylated threonine residues in the precursor and nuclear forms of SREBP-2, and TSH interacted with AMPK to influence SREBP-2 phosphorylation. These findings may represent a molecular mechanism by which AMPK ameliorates the hepatic steatosis and hypercholesterolemia associated with high TSH levels in patients with subclinical hypothyroidism (SCH). PMID:25933205

  5. Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC

    SciTech Connect

    Murakami, Hisashi; Murakami, Ryuichiro . E-mail: ryuichi@med.nagoya-u.ac.jp; Kambe, Fukushi; Cao, Xia; Takahashi, Ryotaro; Asai, Toru; Hirai, Toshihisa; Numaguchi, Yasushi; Okumura, Kenji; Seo, Hisao; Murohara, Toyoaki

    2006-03-24

    Fenofibrate improves endothelial function by lipid-lowering and anti-inflammatory effects. Additionally, fenofibrate has been demonstrated to upregulate endothelial nitric oxide synthase (eNOS). AMP-activated protein kinase (AMPK) has been reported to phosphorylate eNOS at Ser-1177 and stimulate vascular endothelium-derived nitric oxide (NO) production. We report here that fenofibrate activates AMPK and increases eNOS phosphorylation and NO production in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with fenofibrate increased the phosphorylation of AMPK and acetyl-CoA carboxylase. Fenofibrate simultaneously increased eNOS phosphorylation and NO production. Inhibitors of protein kinase A and phosphatidylinositol 3-kinase failed to suppress the fenofibrate-induced eNOS phosphorylation. Neither bezafibrate nor WY-14643 activated AMPK in HUVEC. Furthermore, fenofibrate activated AMPK without requiring any transcriptional activities. These results indicate that fenofibrate stimulates eNOS phosphorylation and NO production through AMPK activation, which is suggested to be a novel characteristic of this agonist and unrelated to its effects on peroxisome proliferator-activated receptor {alpha}.

  6. Activation of AMPK Prevents Monocrotaline-Induced Extracellular Matrix Remodeling of Pulmonary Artery

    PubMed Central

    Li, Shaojun; Han, Dong; Zhang, Yonghong; Xie, Xinming; Ke, Rui; Zhu, Yanting; Liu, Lu; Song, Yang; Yang, Lan; Li, Manxiang

    2016-01-01

    Background The current study was performed to investigate the effect of adenosine monophosphate (AMP) – activated protein kinase (AMPK) activation on the extracellular matrix (ECM) remodeling of pulmonary arteries in pulmonary arterial hypertension (PAH) and to address its potential mechanisms. Material/Methods PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) into Sprague-Dawley rats. Metformin (MET) was administered to activate AMPK. Immunoblotting was used to determine the phosphorylation and expression of AMPK and expression of tissue inhibitor of metalloproteinase-1 (TIMP-1). Gelatin zymography was performed to determine the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9. Results Activation of AMPK by MET significantly reduced the right ventricle systolic pressure and the right ventricular hypertrophy in MCT-induced rat PAH model, and partially inhibited the ECM remodeling of pulmonary arteries. These effects were coupled with the decrease of MMP-2/9 activity and TIMP-1 expression. Conclusions This study suggests that activation of AMPK benefits PAH by inhibiting ECM remodeling of pulmonary arteries. Enhancing AMPK activity might have potential value in clinical treatment of PAH. PMID:26978596

  7. AMPK is a negative regulator of the Warburg Effect and suppresses tumor growth in vivo

    PubMed Central

    Faubert, Brandon; Boily, Gino; Izreig, Said; Griss, Takla; Samborska, Bozena; Dong, Zhifeng; Dupuy, Fanny; Chambers, Christopher; Fuerth, Benjamin J.; Viollet, Benoit; Mamer, Orval A.; Avizonis, Daina; DeBerardinis, Ralph J.; Siegel, Peter M.; Jones, Russell G.

    2012-01-01

    Summary AMPK is a metabolic sensor that helps maintain cellular energy homeostasis. Despite evidence linking AMPK with tumor suppressor functions, the role of AMPK in tumorigenesis and tumor metabolism is unknown. Here we show that AMPK negatively regulates aerobic glycolysis (the Warburg effect) in cancer cells, and suppresses tumor growth in vivo. Genetic ablation of the α1 catalytic subunit of AMPK accelerates Myc-induced lymphomagenesis. Inactivation of AMPKα in both transformed and non-transformed cells promotes a metabolic shift to aerobic glycolysis, increased allocation of glucose carbon into lipids, and biomass accumulation. These metabolic effects require normoxic stabilization of the hypoxia-inducible factor-1α (HIF-1α), as silencing HIF-1α reverses the shift to aerobic glycolysis and the biosynthetic and proliferative advantages conferred by reduced AMPKα signaling. Together our findings suggest that AMPK activity opposes tumor development, and its loss fosters tumor progression in part by regulating cellular metabolic pathways that support cell growth and proliferation. PMID:23274086

  8. AMPK activation regulates apoptosis, adipogenesis, and lipolysis by eIF2{alpha} in adipocytes

    SciTech Connect

    Dagon, Yossi; Avraham, Yosefa; Berry, Elliot M. . E-mail: Berry@md.huji.ac.il

    2006-02-03

    AMP-activated protein kinase (AMPK) is a metabolic master switch regulating glucose and lipid metabolism. Recently, AMPK has been implicated in the control of adipose tissue content. Yet, the nature of this action is controversial. We examined the effect on F442a adipocytes of the AMPK activator-AICAR. Activation of AMPK induced dose-dependent apoptotic cell death, inhibition of lipolysis, and downregulatation key adipogenic genes, such as peroxisome proliferator-activated receptor (PPAR{gamma}) and CCAAT/enhancer-binding protein alpha (C/EBP{alpha}). We have identified the {alpha}-subunit of the eukaryotic initiation factor-2 (eIF2{alpha}) as a target gene which is phosphorylated following AICAR treatment. Such phosphorylation is one of the best-characterized mechanisms for downregulating protein synthesis. 2-Aminopurine (2-AP), an inhibitor of eIF2{alpha} kinases, could overcome the apoptotic effect of AICAR, abolishing the reduction of PPAR{gamma} and C/EBP{alpha} and the lipolytic properties of AMPK. Thus, AMPK may diminish adiposity via reduction of fat cell number through eIF2{alpha}-dependent translation shutdown.

  9. An evolutionary perspective of AMPK-TOR signaling in the three domains of life.

    PubMed

    Roustan, Valentin; Jain, Arpit; Teige, Markus; Ebersberger, Ingo; Weckwerth, Wolfram

    2016-06-01

    AMPK and TOR protein kinases are the major control points of energy signaling in eukaryotic cells and organisms. They form the core of a complex regulatory network to co-ordinate metabolic activities in the cytosol with those in the mitochondria and plastids. Despite its relevance, it is still unclear when and how this regulatory pathway was formed during evolution, and to what extent its representations in the major eukaryotic lineages resemble each other. Here we have traced 153 essential proteins forming the human AMPK-TOR pathways across 412 species representing all three domains of life-prokaryotes (bacteria, archaea) and eukaryotes-and reconstructed their evolutionary history. The resulting phylogenetic profiles indicate the presence of primordial core pathways including seven proto-kinases in the last eukaryotic common ancestor. The evolutionary origins of the oldest components of the AMPK pathway, however, extend into the pre-eukaryotic era, and descendants of these ancient proteins can still be found in contemporary prokaryotes. The TOR complex in turn appears as a eukaryotic invention, possibly to aid in retrograde signaling between the mitochondria and the remainder of the cell. Within the eukaryotes, AMPK/TOR showed both a highly conserved core structure and a considerable plasticity. Most notably, KING1, a protein originally assigned as the γ subunit of AMPK in plants, is more closely related to the yeast SDS23 gene family than to the γ subunits in animals or fungi. This suggests its functional difference from a canonical AMPK γ subunit.

  10. Berberine regulates neurite outgrowth through AMPK-dependent pathways by lowering energy status

    SciTech Connect

    Lu, Jiaqi; Cao, Yuanzhao; Cheng, Kuoyuan; Xu, Bo; Wang, Tianchang; Yang, Qi; Yang, Qin; Feng, Xudong; Xia, Qing

    2015-06-10

    As a widely used anti-bacterial agent and a metabolic inhibitor as well as AMP-activated protein kinase (AMPK) activator, berberine (BBR) has been shown to cross the blood–brain barrier. Its efficacy has been investigated in various disease models of the central nervous system. Neurite outgrowth is critical for nervous system development and is a highly energy-dependent process regulated by AMPK-related pathways. In the present study, we aimed to investigate the effects of BBR on AMPK activation and neurite outgrowth in neurons. The neurite outgrowth of primary rat cortical neurons at different stages of polarization was monitored after exposure of BBR. Intracellular energy level, AMPK activation and polarity-related pathways were also inspected. The results showed that BBR suppressed neurite outgrowth and affected cytoskeleton stability in the early stages of neuronal polarization, which was mediated by lowered energy status and AMPK activation. Liver kinase B1 and PI3K–Akt–GSK3β signaling pathways were also involved. In addition, mitochondrial dysfunction and endoplasmic reticulum stress contributed to the lowered energy status induced by BBR. This study highlighted the knowledge of the complex activities of BBR in neurons and corroborated the significance of energy status during the neuronal polarization. - Highlights: • BBR inhibited neurite outgrowth in early stages of neuronal development. • Lowered neuronal energy status was induced by BBR treatment. • Neuronal energy stress induced by BBR activated AMPK-related pathways. • BBR induced mitochondrial dysfunction and endoplasmic reticulum stress.

  11. Myeloid Deletion of α1AMPK Exacerbates Atherosclerosis in LDL Receptor Knockout (LDLRKO) Mice.

    PubMed

    Cao, Qiang; Cui, Xin; Wu, Rui; Zha, Lin; Wang, Xianfeng; Parks, John S; Yu, Liqing; Shi, Hang; Xue, Bingzhong

    2016-06-01

    Macrophage inflammation marks all stages of atherogenesis, and AMPK is a regulator of macrophage inflammation. We therefore generated myeloid α1AMPK knockout (MAKO) mice on the LDL receptor knockout (LDLRKO) background to investigate whether myeloid deletion of α1AMPK exacerbates atherosclerosis. When fed an atherogenic diet, MAKO/LDLRKO mice displayed exacerbated atherosclerosis compared with LDLRKO mice. To determine the underlying pathophysiological pathways, we characterized macrophage inflammation/chemotaxis and lipid/cholesterol metabolism in MAKO/LDLRKO mice. Myeloid deletion of α1AMPK increased macrophage inflammatory gene expression and enhanced macrophage migration and adhesion to endothelial cells. Remarkably, MAKO/LDLRKO mice also displayed higher composition of circulating chemotaxically active Ly-6C(high) monocytes, enhanced atherosclerotic plaque chemokine expression, and monocyte recruitment into plaques, leading to increased atherosclerotic plaque macrophage content and inflammation. MAKO/LDLRKO mice also exhibited higher plasma LDL and VLDL cholesterol content, increased circulating apolipoprotein B (apoB) levels, and higher liver apoB expression. We conclude that macrophage α1AMPK deficiency promotes atherogenesis in LDLRKO mice and is associated with enhanced macrophage inflammation and hypercholesterolemia and that macrophage α1AMPK may serve as a therapeutic target for prevention and treatment of atherosclerosis. PMID:26822081

  12. LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress.

    PubMed

    Liu, Xiaowen; Xiao, Zhen-Dong; Han, Leng; Zhang, Jiexin; Lee, Szu-Wei; Wang, Wenqi; Lee, Hyemin; Zhuang, Li; Chen, Junjie; Lin, Hui-Kuan; Wang, Jing; Liang, Han; Gan, Boyi

    2016-04-01

    Long non-coding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signalling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that the lncRNA NBR2 (neighbour of BRCA1 gene 2) is induced by the LKB1-AMPK pathway under energy stress. On energy stress, NBR2 in turn interacts with AMPK and promotes AMPK kinase activity, thus forming a feed-forward loop to potentiate AMPK activation during energy stress. Depletion of NBR2 attenuates energy-stress-induced AMPK activation, resulting in unchecked cell cycling, altered apoptosis/autophagy response, and increased tumour development in vivo. NBR2 is downregulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, the results of our study uncover a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to understand further the regulation of kinase signalling by lncRNAs. PMID:26999735

  13. An evolutionary perspective of AMPK-TOR signaling in the three domains of life.

    PubMed

    Roustan, Valentin; Jain, Arpit; Teige, Markus; Ebersberger, Ingo; Weckwerth, Wolfram

    2016-06-01

    AMPK and TOR protein kinases are the major control points of energy signaling in eukaryotic cells and organisms. They form the core of a complex regulatory network to co-ordinate metabolic activities in the cytosol with those in the mitochondria and plastids. Despite its relevance, it is still unclear when and how this regulatory pathway was formed during evolution, and to what extent its representations in the major eukaryotic lineages resemble each other. Here we have traced 153 essential proteins forming the human AMPK-TOR pathways across 412 species representing all three domains of life-prokaryotes (bacteria, archaea) and eukaryotes-and reconstructed their evolutionary history. The resulting phylogenetic profiles indicate the presence of primordial core pathways including seven proto-kinases in the last eukaryotic common ancestor. The evolutionary origins of the oldest components of the AMPK pathway, however, extend into the pre-eukaryotic era, and descendants of these ancient proteins can still be found in contemporary prokaryotes. The TOR complex in turn appears as a eukaryotic invention, possibly to aid in retrograde signaling between the mitochondria and the remainder of the cell. Within the eukaryotes, AMPK/TOR showed both a highly conserved core structure and a considerable plasticity. Most notably, KING1, a protein originally assigned as the γ subunit of AMPK in plants, is more closely related to the yeast SDS23 gene family than to the γ subunits in animals or fungi. This suggests its functional difference from a canonical AMPK γ subunit. PMID:27270999

  14. AMPK over-activation leads to accumulation of α-synuclein oligomers and decrease of neurites

    PubMed Central

    Jiang, Peizhou; Gan, Ming; Ebrahim, Abdul Shukkur; Castanedes-Casey, Monica; Dickson, Dennis W.; Yen, Shu-Hui C.

    2012-01-01

    Neuronal inclusions of α-synuclein (α-syn), termed Lewy bodies, are a hallmark of Parkinson disease (PD). Increased α-syn levels can occur in brains of aging human and neurotoxin treated mice. Since previous studies have shown increased brain lactate levels in aging brains, in PD affected subjects when compared to age-matched controls, and in mice treated with MPTP, we tested the effects of lactate exposure on α-syn in a cell based-study. We demonstrated that (i) lactate treatment led to α-syn accumulation and oligomerization in a time- and concentration-dependent manner, (ii) such alterations were mediated via adenosine-monophosphate activated protein kinase (AMPK) and associated with increasing cytoplasmic phosphorylated AMPK levels, (iii) AMPK activation facilitated α-syn accumulation and phosphorylation, (iv) lactate treatment or overexpression of active form of AMPK decreased α-syn turnover and neurite outgrowth and (v) Lewy body-bearing neurons displayed abnormal cytoplasmic distribution of phosphorylated AMPK, which normally is located in nuclei. Together, our results suggest that chronic neuronal accumulation of α-syn induced by lactate-triggered AMPK activation in aging brains may be a novel mechanism underlying α-synucleionpathies in PD and related disorders. PMID:23200460

  15. Nutrient Excess and AMPK Downregulation in Incubated Skeletal Muscle and Muscle of Glucose Infused Rats

    PubMed Central

    Valentine, Rudy J.; Petrocelli, Robert; Schultz, Vera; Brandon, Amanda; Cooney, Gregory J.; Kraegen, Edward W.; Ruderman, Neil B.; Saha, Asish K.

    2015-01-01

    We have previously shown that incubation for 1h with excess glucose or leucine causes insulin resistance in rat extensor digitorum longus (EDL) muscle by inhibiting AMP-activated protein kinase (AMPK). To examine the events that precede and follow these changes, studies were performed in rat EDL incubated with elevated levels of glucose or leucine for 30min-2h. Incubation in high glucose (25mM) or leucine (100μM) significantly diminished AMPK activity by 50% within 30min, with further decreases occurring at 1 and 2h. The initial decrease in activity at 30min coincided with a significant increase in muscle glycogen. The subsequent decreases at 1h were accompanied by phosphorylation of αAMPK at Ser485/491, and at 2h by decreased SIRT1 expression and increased PP2A activity, all of which have previously been shown to diminish AMPK activity. Glucose infusion in vivo, which caused several fold increases in plasma glucose and insulin, produced similar changes but with different timing. Thus, the initial decrease in AMPK activity observed at 3h was associated with changes in Ser485/491 phosphorylation and SIRT1 expression and increased PP2A activity was a later event. These findings suggest that both ex vivo and in vivo, multiple factors contribute to fuel-induced decreases in AMPK activity in skeletal muscle and the insulin resistance that accompanies it. PMID:25996822

  16. Interleukin-18 activates skeletal muscle AMPK and reduces weight gain and insulin resistance in mice.

    PubMed

    Lindegaard, Birgitte; Matthews, Vance B; Brandt, Claus; Hojman, Pernille; Allen, Tamara L; Estevez, Emma; Watt, Matthew J; Bruce, Clinton R; Mortensen, Ole H; Syberg, Susanne; Rudnicka, Caroline; Abildgaard, Julie; Pilegaard, Henriette; Hidalgo, Juan; Ditlevsen, Susanne; Alsted, Thomas J; Madsen, Andreas N; Pedersen, Bente K; Febbraio, Mark A

    2013-09-01

    Circulating interleukin (IL)-18 is elevated in obesity, but paradoxically causes hypophagia. We hypothesized that IL-18 may attenuate high-fat diet (HFD)-induced insulin resistance by activating AMP-activated protein kinase (AMPK). We studied mice with a global deletion of the α-isoform of the IL-18 receptor (IL-18R(-/-)) fed a standard chow or HFD. We next performed gain-of-function experiments in skeletal muscle, in vitro, ex vivo, and in vivo. We show that IL-18 is implicated in metabolic homeostasis, inflammation, and insulin resistance via mechanisms involving the activation of AMPK in skeletal muscle. IL-18R(-/-) mice display increased weight gain, ectopic lipid deposition, inflammation, and reduced AMPK signaling in skeletal muscle. Treating myotubes or skeletal muscle strips with IL-18 activated AMPK and increased fat oxidation. Moreover, in vivo electroporation of IL-18 into skeletal muscle activated AMPK and concomitantly inhibited HFD-induced weight gain. In summary, IL-18 enhances AMPK signaling and lipid oxidation in skeletal muscle implicating IL-18 in metabolic homeostasis.

  17. Leptin promotes K(ATP) channel trafficking by AMPK signaling in pancreatic β-cells.

    PubMed

    Park, Sun-Hyun; Ryu, Shin-Young; Yu, Weon-Jin; Han, Young Eun; Ji, Young-Sun; Oh, Keunhee; Sohn, Jong-Woo; Lim, Ajin; Jeon, Jae-Pyo; Lee, Hyunsu; Lee, Kyu-Hee; Lee, Suk-Ho; Berggren, Per-Olof; Jeon, Ju-Hong; Ho, Won-Kyung

    2013-07-30

    Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic β-cells. In this study, we provide evidence that leptin modulates pancreatic β-cell functions by promoting K(ATP) channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K(ATP) channels were localized mostly to intracellular compartments of pancreatic β-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase β. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K(ATP) channel trafficking and hyperpolarization of pancreatic β-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and β-cell membrane potentials, suggesting that AMPK-dependent K(ATP) channel trafficking is a key mechanism for regulating β-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating β-cell excitability.

  18. Macropinocytosis is decreased in diabetic mouse macrophages and is regulated by AMPK

    PubMed Central

    Guest, Christopher B; Chakour, Kenneth S; Freund, Gregory G

    2008-01-01

    Background Macrophages (MΦs) utilize macropinocytosis to integrate immune and metabolic signals in order to initiate an effective immune response. Diabetes is characterized by metabolic abnormalities and altered immune function. Here we examine the influence of diabetes on macropinocytosis in primary mouse macrophages and in an in vitro diabetes model. Results The data demonstrate that peritoneal MΦs from diabetic (db/db) mice had reduced macropinocytosis when compared to MΦs from non-diabetic (db/+) mice. Additionally, MΦs cultured in hyperglycemic conditions were less adept at macropinocytosis than those cultured in low glucose. Notably, AMP-activated protein kinase (AMPK) activity was decreased in MΦs cultured in hyperglycemic conditions. Activation of AMPK with leptin or 5-aminoimidazole-4-carboxamide-1-β-riboside (AICAR) increased macropinocytosis and inhibition of AMPK with compound C decreased macropinocytosis. Conclusion Taken together, these findings indicate that MΦs from diabetic mice have decreased macropinocytosis. This decrease appears dependent on reduced AMPK activity. These results demonstrate a previously unrealized role for AMPK in MΦs and suggest that increasing AMPK activity in diabetic MΦs could improve innate immunity and decrease susceptibility to infection. PMID:18667079

  19. Co-activation of AMPK and mTORC1 as a new therapeutic option for acute myeloid leukemia

    PubMed Central

    Sujobert, Pierre; Tamburini, Jerome

    2016-01-01

    ABSTRACT We report the therapeutic potential of GSK621, an AMP-activated protein kinase (AMPK) agonist, in acute myeloid leukemia (AML). GSK621-induced cytotoxicity is restricted to AML cells compared to normal hematopoietic progenitors due to a unique synthetic lethal interaction of co-activation of AMPK and mammalian target of rapamycin complex 1 (mTORC1) that involves the stress response pathway. AMPK activation thus represents an attractive perspective for cancer therapy. PMID:27652311

  20. Sex-specific regulation of AMP-activated protein kinase (AMPK) in the Pacific oyster Crassostrea gigas.

    PubMed

    Guévélou, Eric; Huvet, Arnaud; Galindo-Sánchez, Clara E; Milan, Massimo; Quillien, Virgile; Daniel, Jean-Yves; Quéré, Claudie; Boudry, Pierre; Corporeau, Charlotte

    2013-10-01

    The hermaphrodite Pacific oyster Crassostrea gigas displays a high energy allocation to reproduction. We studied the expression of AMP-activated protein kinase (AMPK) during gametogenesis in the gonad and characterized the mRNA sequences of the AMPK subunits: the AMPK alpha mRNA sequence was previously characterized; we identified AMPK beta, AMPK gamma, and mRNAs of putative AMPK-related targets following bioinformatics mining on existing genomic resources. We analyzed the mRNA expression of the AMPK alpha, beta, and gamma subunits in the gonads of male and female oysters through a reproductive cycle, and we quantified the mRNA expression of genes belonging to fatty acid and glucose metabolism. AMPK alpha mRNA levels were more abundant in males at the first stage of gametogenesis, when mitotic activity and the differentiation of germinal cells occur, and were always more abundant in males than in females. Some targets of fatty acid and glucose metabolism appeared to be correlated with the expression of AMPK subunits at the mRNA level. We then analyzed the sex-specific AMPK activity by measuring the phosphorylation of the catalytic AMPK alpha protein and its expression at the protein level. Both the amount of AMPK alpha protein and threonine 172 phosphorylation appeared to be almost totally inhibited in mature female gonads at stage 3, at the time when accumulation of reserves in oocytes was promoted, while it remained at a high level in mature spermatozoa. Its activation might play a sex-dependent role in the management of energy during gametogenesis in oyster.

  1. Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

    SciTech Connect

    Wang, Bing Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

    2013-07-19

    Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells.

  2. Co-activation of AMPK and mTORC1 as a new therapeutic option for acute myeloid leukemia.

    PubMed

    Sujobert, Pierre; Tamburini, Jerome

    2016-07-01

    We report the therapeutic potential of GSK621, an AMP-activated protein kinase (AMPK) agonist, in acute myeloid leukemia (AML). GSK621-induced cytotoxicity is restricted to AML cells compared to normal hematopoietic progenitors due to a unique synthetic lethal interaction of co-activation of AMPK and mammalian target of rapamycin complex 1 (mTORC1) that involves the stress response pathway. AMPK activation thus represents an attractive perspective for cancer therapy. PMID:27652311

  3. Activation of AMPK inhibits cervical cancer cell growth through AKT/FOXO3a/FOXM1 signaling cascade

    PubMed Central

    2013-01-01

    Background Although advanced-stage cervical cancer can benefit from current treatments, approximately 30% patients may fail after definitive treatment eventually. Therefore, exploring alternative molecular therapeutic approaches is imperatively needed for this disease. We have recently shown that activation of AMP-activated protein kinase (AMPK), a metabolic sensor, hampers cervical cancer cell growth through blocking the Wnt/β-catenin signaling activity. Here, we report that activated AMPK (p-AMPK) also inhibits cervical cancer cell growth by counteracting FOXM1 function. Methods Effect of the activation of AMPK on FOXM1 expression was examined by hypoxia and glucose deprivation, as well as pharmacological AMPK activators such as A23187, AICAR and metformin. RT Q-PCR and Western blot analysis were employed to investigate the activities of AMPK, FOXM1 and AKT/FOXO3a signaling. Results Consistent with our previous findings, the activation of AMPK by either AMPK activators such as AICAR, A23187, metformin, glucose deprivation or hypoxia significantly inhibited the cervical cancer cell growth. Importantly, we found that activated AMPK activity was concomitantly associated with the reduction of both the mRNA and protein levels of FOXM1. Mechanistically, we showed that activated AMPK was able to reduce AKT mediated phosphorylation of p-FOXO3a (Ser253). Interestingly, activated AMPK could not cause any significant changes in FOXM1 in cervical cancer cells in which endogenous FOXO3a levels were knocked down using siRNAs, suggesting that FOXO3a is involved in the suppression of FOXM1. Conclusion Taken together, our results suggest the activated AMPK impedes cervical cancer cell growth through reducing the expression of FOXM1. PMID:23819460

  4. Atrial natriuretic peptide regulates lipid mobilization and oxygen consumption in human adipocytes by activating AMPK

    SciTech Connect

    Souza, Sandra C.; Chau, Mary D.L.; Yang, Qing; Gauthier, Marie-Soleil; Clairmont, Kevin B.; Wu, Zhidan; Gromada, Jesper; Dole, William P.

    2011-07-08

    Highlights: {yields} Treatment of differentiated human adipocytes with atrial natriuretic peptide (ANP) increased lipolysis and oxygen consumption by activating AMP-activated protein kinase (AMPK). {yields} ANP stimulated lipid mobilization by selective activation of the alpha2 subunit of AMPK and increased energy utilization through activation of both the alpha1 and alpha2 subunits of AMPK. {yields} ANP enhanced adipocyte mitochondrial oxidative capacity as evidenced by induction of oxidative mitochondrial genes and increase in oxygen consumption. {yields} Exposure of human adipocytes to fatty acids and (TNF{alpha}) induced insulin resistance and decreased expression of mitochondrial genes which was restored to normal by ANP. -- Abstract: Atrial natriuretic peptide (ANP) has been shown to regulate lipid and carbohydrate metabolism providing a possible link between cardiovascular function and metabolism by mediating the switch from carbohydrate to lipid mobilization and oxidation. ANP exerts a potent lipolytic effect via cGMP-dependent protein kinase (cGK)-I mediated-stimulation of AMP-activated protein kinase (AMPK). Activation of the ANP/cGK signaling cascade also promotes muscle mitochondrial biogenesis and fat oxidation. Here we demonstrate that ANP regulates lipid metabolism and oxygen utilization in differentiated human adipocytes by activating the alpha2 subunit of AMPK. ANP treatment increased lipolysis by seven fold and oxygen consumption by two fold, both of which were attenuated by inhibition of AMPK activity. ANP-induced lipolysis was shown to be mediated by the alpha2 subunit of AMPK as introduction of dominant-negative alpha2 subunit of AMPK attenuated ANP effects on lipolysis. ANP-induced activation of AMPK enhanced mitochondrial oxidative capacity as evidenced by a two fold increase in oxygen consumption and induction of mitochondrial genes, including carnitine palmitoyltransferase 1A (CPT1a) by 1.4-fold, cytochrome C (CytC) by 1.3-fold, and

  5. 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

  6. AMPK Suppresses Connexin43 Expression in the Bladder and Ameliorates Voiding Dysfunction in Cyclophosphamide-induced Mouse Cystitis

    PubMed Central

    Zhang, Xiling; Yao, Jian; Gao, Kun; Chi, Yuan; Mitsui, Takahiko; Ihara, Tatsuya; Sawada, Norifumi; Kamiyama, Manabu; Fan, Jianglin; Takeda, Masayuki

    2016-01-01

    Bladder voiding dysfunction is closely related to local oxidation, inflammation, and enhanced channel activities. Given that the AMP-activated protein kinase (AMPK) has anti-oxidative, anti-inflammatory and channel-inhibiting properties, we examined whether and how AMPK affected bladder activity. AMPK activation in rat bladder smooth muscle cells (BSMCs) using three different AMPK agonists resulted in a decrease in connexin43 (Cx43) expression and function, which was associated with reduced CREB phosphorylation, Cx43 promoter activity and mRNA expression, but not Cx43 degradation. Downregulation of CREB with siRNA increased Cx43 expression. A functional analysis revealed that AMPK weakened BSMC contraction and bladder capacity. AMPK also counteracted the IL-1β- and TNFα-induced increase in Cx43 in BSMCs. In vivo administration of the AMPK agonist AICAR attenuated cyclophosphamide-initiated bladder oxidation, inflammation, Cx43 expression and voiding dysfunction. Further analysis comparing the responses of the wild-type (Cx43+/+) and heterozygous (Cx43+/−) Cx43 mice to cyclophosphamide revealed that the Cx43+/− mice retained a relatively normal micturition pattern compared to the Cx43+/+ mice. Taken together, our results indicate that AMPK inhibits Cx43 in BSMCs and improves bladder activity under pathological conditions. We propose that strategies that target AMPK can be developed as novel therapeutic approaches for treating bladder dysfunction. PMID:26806558

  7. mir-101-3p is a key regulator of tumor metabolism in triple negative breast cancer targeting AMPK

    PubMed Central

    Li, Xing; Tang, Hailin; Li, Shuaijie; Huang, Xiaojia; Song, Cailu; Wei, Weidong; Xie, Xiaoming

    2016-01-01

    mir-101-3p has been reported to be a tumor suppressor and a promising therapeutic target in cancer. Recently, AMPK dysfunction has been highlighted in cancers, including breast cancer. The aim of this study is to investigate the biological roles of mir-101-3p and AMPK in breast cancer. Our research demonstrated that AMPK was up-regulated in breast cancer tissues and cell lines, especially in triple negative breast cancer (TNBC). High-expression of AMPK correlated with poor outcome in both total breast cancer and TNBC patients. Ectopic expression of AMPK improved glucose uptake, glycolysis, proliferation of TNBC cells in vitro and its tumorigenicity in vivo. AMPK was predicted to be a direct target of mir-101-3p. The luciferase reporter assay was performed to certificate this prediction. The expression of AMPK was suppressed by transfection of mir-101-3p in TNBC cells. Over-expression of mir-101-3p or knock-down of AMPK inhibited glucose metabolism and proliferation of TNBC cells in vitro. Our study provides evidence that mir-101-3p- AMPK axis could be a promising therapeutic target in TNBC targeting tumor metabolism. PMID:27145268

  8. A novel AMPK activator from Chinese herb medicine and ischemia phosphorylate the cardiac transcription factor FOXO3

    PubMed Central

    Wang, Jingying; Ma, Heng; Zhang, Xiaoyu; He, Leilei; Wu, Jianming; Gao, Xiaoping; Ren, Jun; Li, Ji

    2016-01-01

    Oleanolic Acid (OA) is a nature product extracted from Chinese Herb Medicine which is traditionally used as treatment of diabetes and ischemic heart diseases. Mounting evidence showed that AMP-activated protein kinase (AMPK) has cardioprotective effect against ischemic injury and the forkhead transcription factor 3 (FOXO3) was recently identified as a downstream target of AMPK. We hypothesize that OA may protect against ischemic dysfunction of cardiomyocytes via activation of AMPK signaling pathway. Male C57BL/6 mice which were subjected to in vivo regional cardiac ischemia stimulated AMPK Thr172 phosphorylation, as well as phosphorylation of downstream FOXO3 (Ser413) and acetyl CoA carboxylase (ACC). The natural product, OA, significantly stimulated cardiac AMPK activation in cardiomyocyes in time- and dose-dependent manners. The mechanism of AMPK activation by OA may be due to the loss mitochondrial membrane potential (ΔΨm) as shown by JC-1 fluorescence assay. Intriguingly, OA as an AMPK activator also triggered FOXO3 (Ser413) phosphorylation in cardiomyocytes. Furthermore, OA treatment can protect cardiomyocytes from contractile dysfunction induced by hypoxia. Taken together, the results indicated that both ischemia and OA stimulated cardiac AMPK phosphorylation, as well downstream FOXO3 phosphorylation. The cardioprotective effect of OA maybe associated with activation of AMPK signaling pathways.

  9. Combined inhibition of glycolysis and AMPK induces synergistic breast cancer cell killing

    PubMed Central

    Wu, Yong; Sarkissyan, Marianna; Mcghee, Eva; Lee, Sangkyu

    2015-01-01

    Targeting glycolysis for cancer treatment has been investigated as a therapeutic method but has not offered a feasible chemotherapeutic strategy. Our aim was to examine whether AMP-activated protein kinase (AMPK), a conditional oncogene, rescues the energetic stress and cytotoxicity induced by 2-deoxyglucose (2-DG), a glycolytic inhibitor, and the related mechanisms. Luciferin/luciferase adenosine triphosphate (ATP) determination, Western analysis, qRT-PCR analyses, MTT growth assay, clonogenic assay, and statistical analysis were performed in this study. 2-DG decreased ATP levels and subsequently activated AMPK, which contribute to intracellular ATP recovery in MCF-7 cells thus exhibiting no apparent cytotoxicity. Compound C, an AMPK inhibitor, further potentiates 2-DG-induced decrease in ATP levels and inhibits their recovery. 2-DG, via AMPK activation, stimulated cAMP response element-binding protein (CREB) phosphorylation and activity and promoted nuclear peroxisome proliferator-activated receptor gamma coactivator-1-beta (PGC-1β) and estrogen-related receptor α (ERRα) protein expression, leading to augmented mitochondrial biogenesis and expression of fatty acid oxidation (FAO) genes including PPARα, MCAD, CPT1C, and ACO. This metabolic adaptation elicited by AMPK counteracts the ATP-depleting and cancer cell-killing effect of 2-DG. However, 2-DG in combination with AMPK antagonists or small interfering RNA caused a dramatic increase in cytotoxicity in MCF-7 but not in MCF-10A cells. Similarly, when combined with inhibition of CREB/PGC-1β/ERRα pathway, 2-DG saliently suppressed mitochondrial biogenesis and the expression of FAO genes, depleted ATP production, and enhanced cytotoxicity in cancer cells. Collectively, the combination of 2-DG and AMPK inhibition synergistically enhanced the cytotoxic potential in breast cancer cells with a relative nontoxicity to normal cells and may offer a promising, safe, and effective breast cancer therapeutic strategy

  10. Activation of the AMP-Activated Protein Kinase (AMPK) by Nitrated Lipids in Endothelial Cells

    PubMed Central

    Wu, Yong; Dong, Yunzhou; Song, Ping; Zou, Ming-Hui

    2012-01-01

    The AMP-activated protein kinase (AMPK) is an important regulator of endothelial metabolic and functional homeostasis. Here, we examined the regulation of AMPK by nitrated oleic acid (OA-NO2) and investigated the implications in endothelial function. Treatment of bovine aortic endothelial cells (BAECs) with OA-NO2 induced a significant increase in both AMPK-Thr172 phosphorylation and AMPK activity as well as upregulation of heme oxygenase (HO)-1 and hypoxia-inducible factor (HIF)-1α. Pharmacologic inhibition or genetic ablation of HO-1 or HIF-1α abolished OA-NO2-induced AMPK phosphorylation. OA-NO2 induced a dramatic increase in extracellular signal-regulated kinase (ERK)1/2 phosphorylation that was abrogated by the HO-1 inhibitor, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG). Inhibition of ERK1/2 using UO126 or PD98059 reduced but did not abolish OA-NO2-induced HIF-1α upregulation, suggesting that OA-NO2/HO-1-initiated HIF-1α induction is partially dependent on ERK1/2 activity. In addition, OA-NO2 enhanced endothelial intracellular Ca2+, an effect that was inhibited by the HIF-1α inhibitor, YC-1, and by HIF-1α siRNA. These results implicate the involvement of HIF-1α. Experiments using the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609, the selective CaMKII inhibitor KN-93, and an isoform-specific siRNA demonstrated that OA-NO2-induced AMPK phosphorylation was dependent on CaMKKβ. Together, these results demonstrate that OA-NO2 activates AMPK in endothelial cells via an HO-1–dependent mechanism that increases HIF-1α protein expression and Ca2+/CaMKKβ activation. PMID:22363546

  11. Hypoxia upregulates Malat1 expression through a CaMKK/AMPK/HIF-1α axis.

    PubMed

    Sallé-Lefort, Sandrine; Miard, Stéphanie; Nolin, Marc-André; Boivin, Louise; Paré, Marie-Ève; Debigaré, Richard; Picard, Frédéric

    2016-10-01

    Increased expression levels of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) have been associated with enhanced proliferation and metastasis of several cancer cell types. Hypoxia, a hallmark characteristic of solid tumors, has been linked to an increase in the activity of the ATP-generating AMPK protein. Since Malat1 was recently shown to be upregulated during hypoxia, the objective of this study was to determine the contribution of AMPK in the mechanistic pathways regulating Malat1 expression in low oxygen conditions. Compared to those cultured in 21% O2 conditions, HeLa cells incubated in 1.5% O2 expressed more Malat1 transcripts. This observation was mimicked in HEK293T cells using a synthetic reporter construct containing 5.6 kb of the human Malat1 promoter, suggesting that hypoxia directly impacted Malat1 gene transcription. Interestingly, pharmacological stimulation of AMPK increased Malat1 promoter transactivation in 21% O2 conditions, whereas inhibition of either AMPK or its upstream activator CaMKK completely abolished the augmentation of Malat1 under hypoxia. Pharmacological modulation of LKB1, another major regulator of AMPK, had no impact on Malat1 promoter transactivation, suggesting that calcium inputs are important in the control of Malat1 expression by AMPK. Overexpression of hypoxia-inducible factor-1α (HIF-1α) increased Malat1 expression in 21% O2 conditions, whereas pharmacological inhibition of HIF-1α blocked the impact of hypoxia on the Malat1 promoter. Taken together, these findings strongly suggest that Malat1 expression is regulated in hypoxic conditions by a CaMKK/AMPK/HIF-1α axis. More research is needed in physiological settings to test the clinical relevance of this pathway.

  12. Hypoxia upregulates Malat1 expression through a CaMKK/AMPK/HIF-1α axis.

    PubMed

    Sallé-Lefort, Sandrine; Miard, Stéphanie; Nolin, Marc-André; Boivin, Louise; Paré, Marie-Ève; Debigaré, Richard; Picard, Frédéric

    2016-10-01

    Increased expression levels of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) have been associated with enhanced proliferation and metastasis of several cancer cell types. Hypoxia, a hallmark characteristic of solid tumors, has been linked to an increase in the activity of the ATP-generating AMPK protein. Since Malat1 was recently shown to be upregulated during hypoxia, the objective of this study was to determine the contribution of AMPK in the mechanistic pathways regulating Malat1 expression in low oxygen conditions. Compared to those cultured in 21% O2 conditions, HeLa cells incubated in 1.5% O2 expressed more Malat1 transcripts. This observation was mimicked in HEK293T cells using a synthetic reporter construct containing 5.6 kb of the human Malat1 promoter, suggesting that hypoxia directly impacted Malat1 gene transcription. Interestingly, pharmacological stimulation of AMPK increased Malat1 promoter transactivation in 21% O2 conditions, whereas inhibition of either AMPK or its upstream activator CaMKK completely abolished the augmentation of Malat1 under hypoxia. Pharmacological modulation of LKB1, another major regulator of AMPK, had no impact on Malat1 promoter transactivation, suggesting that calcium inputs are important in the control of Malat1 expression by AMPK. Overexpression of hypoxia-inducible factor-1α (HIF-1α) increased Malat1 expression in 21% O2 conditions, whereas pharmacological inhibition of HIF-1α blocked the impact of hypoxia on the Malat1 promoter. Taken together, these findings strongly suggest that Malat1 expression is regulated in hypoxic conditions by a CaMKK/AMPK/HIF-1α axis. More research is needed in physiological settings to test the clinical relevance of this pathway. PMID:27499160

  13. c-Myc and AMPK Control Cellular Energy Levels by Cooperatively Regulating Mitochondrial Structure and Function

    PubMed Central

    Edmunds, Lia R.; Sharma, Lokendra; Wang, Huabo; Kang, Audry; d’Souza, Sonia; Lu, Jie; McLaughlin, Michael; Dolezal, James M.; Gao, Xiaoli; Weintraub, Susan T.; Ding, Ying; Zeng, Xuemei; Yates, Nathan; Prochownik, Edward V.

    2015-01-01

    The c-Myc (Myc) oncoprotein and AMP-activated protein kinase (AMPK) regulate glycolysis and oxidative phosphorylation (Oxphos) although often for different purposes. Because Myc over-expression depletes ATP with the resultant activation of AMPK, we explored the potential co-dependency of and cross-talk between these proteins by comparing the consequences of acute Myc induction in ampk+/+ (WT) and ampk-/- (KO) murine embryo fibroblasts (MEFs). KO MEFs showed a higher basal rate of glycolysis than WT MEFs and an appropriate increase in response to activation of a Myc-estrogen receptor (MycER) fusion protein. However, KO MEFs had a diminished ability to increase Oxphos, mitochondrial mass and reactive oxygen species in response to MycER activation. Other differences between WT and KO MEFs, either in the basal state or following MycER induction, included abnormalities in electron transport chain function, levels of TCA cycle-related oxidoreductases and cytoplasmic and mitochondrial redox states. Transcriptional profiling of pathways pertinent to glycolysis, Oxphos and mitochondrial structure and function also uncovered significant differences between WT and KO MEFs and their response to MycER activation. Finally, an unbiased mass-spectrometry (MS)-based survey capable of quantifying ~40% of all mitochondrial proteins, showed about 15% of them to be AMPK- and/or Myc-dependent in their steady state. Significant differences in the activities of the rate-limiting enzymes pyruvate kinase and pyruvate dehydrogenase, which dictate pyruvate and acetyl coenzyme A abundance, were also differentially responsive to Myc and AMPK and could account for some of the differences in basal metabolite levels that were also detected by MS. Thus, Myc and AMPK are highly co-dependent and appear to engage in significant cross-talk across numerous pathways which support metabolic and ATP-generating functions. PMID:26230505

  14. Nitrogen Regulates AMPK to Control TORC1 Signaling

    PubMed Central

    Davie, Elizabeth; Forte, Gabriella M.A.; Petersen, Janni

    2015-01-01

    Summary Background Cell growth and cell-cycle progression are tightly coordinated to enable cells to adjust their size (timing of division) to the demands of proliferation in varying nutritional environments. In fission yeast, nitrogen stress results in sustained proliferation at a reduced size. Results Here, we show that cells can sense nitrogen stress to reduce target of rapamycin complex-1 (TORC1) activity. Nitrogen-stress-induced TORC1 inhibition differs from amino-acid-dependent control of TORC1 and requires the Ssp2 (AMPKα) kinase, the Tsc1/2 complex, and Rhb1 GTPase. Importantly, the β and γ regulatory subunits of AMPK are not required to control cell division in response to nitrogen stress, providing evidence for a nitrogen-sensing mechanism that is independent of changes in intracellular ATP/AMP levels. The CaMKK homolog Ssp1 is constitutively required for phosphorylation of the AMPKαSsp2 T loop. However, we find that a second homolog CaMKKPpk34 is specifically required to stimulate AMPKαSsp2 activation in response to nitrogen stress. Finally, ammonia also controls mTORC1 activity in human cells; mTORC1 is activated upon the addition of ammonium to glutamine-starved Hep3B cancer cells. Conclusions The alternative nitrogen source ammonia can simulate TORC1 activity to support growth and division under challenging nutrient settings, a situation often seen in cancer. PMID:25639242

  15. AMPK-independent inhibition of human macrophage ER stress response by AICAR

    PubMed Central

    Boß, Marcel; Newbatt, Yvette; Gupta, Sahil; Collins, Ian; Brüne, Bernhard; Namgaladze, Dmitry

    2016-01-01

    Obesity-associated insulin resistance is driven by inflammatory processes in response to metabolic overload. Obesity-associated inflammation can be recapitulated in cell culture by exposing macrophages to saturated fatty acids (SFA), and endoplasmic reticulum (ER) stress responses essentially contribute to pro-inflammatory signalling. AMP-activated protein kinase (AMPK) is a central metabolic regulator with established anti-inflammatory actions. Whether pharmacological AMPK activation suppresses SFA-induced inflammation in a human system is unclear. In a setting of hypoxia-potentiated inflammation induced by SFA palmitate, we found that the AMP-mimetic AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) potently suppressed upregulation of ER stress marker mRNAs and pro-inflammatory cytokines. Furthermore, AICAR inhibited macrophage ER stress responses triggered by ER-stressors thapsigargin or tunicamycin. Surprisingly, AICAR acted independent of AMPK or AICAR conversion to 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl monophosphate (ZMP) while requiring intracellular uptake via the equilibrative nucleoside transporter (ENT) ENT1 or the concentrative nucleoside transporter (CNT) CNT3. AICAR did not affect the initiation of the ER stress response, but inhibited the expression of major ER stress transcriptional effectors. Furthermore, AICAR inhibited autophosphorylation of the ER stress sensor inositol-requiring enzyme 1α (IRE1α), while activating its endoribonuclease activity in vitro. Our results suggest that AMPK-independent inhibition of ER stress responses contributes to anti-inflammatory and anti-diabetic effects of AICAR. PMID:27562249

  16. AMPK-independent inhibition of human macrophage ER stress response by AICAR.

    PubMed

    Boß, Marcel; Newbatt, Yvette; Gupta, Sahil; Collins, Ian; Brüne, Bernhard; Namgaladze, Dmitry

    2016-01-01

    Obesity-associated insulin resistance is driven by inflammatory processes in response to metabolic overload. Obesity-associated inflammation can be recapitulated in cell culture by exposing macrophages to saturated fatty acids (SFA), and endoplasmic reticulum (ER) stress responses essentially contribute to pro-inflammatory signalling. AMP-activated protein kinase (AMPK) is a central metabolic regulator with established anti-inflammatory actions. Whether pharmacological AMPK activation suppresses SFA-induced inflammation in a human system is unclear. In a setting of hypoxia-potentiated inflammation induced by SFA palmitate, we found that the AMP-mimetic AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) potently suppressed upregulation of ER stress marker mRNAs and pro-inflammatory cytokines. Furthermore, AICAR inhibited macrophage ER stress responses triggered by ER-stressors thapsigargin or tunicamycin. Surprisingly, AICAR acted independent of AMPK or AICAR conversion to 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl monophosphate (ZMP) while requiring intracellular uptake via the equilibrative nucleoside transporter (ENT) ENT1 or the concentrative nucleoside transporter (CNT) CNT3. AICAR did not affect the initiation of the ER stress response, but inhibited the expression of major ER stress transcriptional effectors. Furthermore, AICAR inhibited autophosphorylation of the ER stress sensor inositol-requiring enzyme 1α (IRE1α), while activating its endoribonuclease activity in vitro. Our results suggest that AMPK-independent inhibition of ER stress responses contributes to anti-inflammatory and anti-diabetic effects of AICAR. PMID:27562249

  17. NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

    PubMed

    Thakkar, Chandni S; Kate, Abhijeet S; Desai, Dattatraya C; Ghosh, Asit Ranjan; Kulkarni-Almeida, Asha A

    2015-12-15

    NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKβ are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP.

  18. NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

    PubMed

    Thakkar, Chandni S; Kate, Abhijeet S; Desai, Dattatraya C; Ghosh, Asit Ranjan; Kulkarni-Almeida, Asha A

    2015-12-15

    NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKβ are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP. PMID:26546724

  19. Resveratrol-activated AMPK/SIRT1/autophagy in cellular models of Parkinson's disease.

    PubMed

    Wu, Yuncheng; Li, Xinqun; Zhu, Julie Xiaohong; Xie, Wenjie; Le, Weidong; Fan, Zhen; Jankovic, Joseph; Pan, Tianhong

    2011-01-01

    Excessive misfolded proteins and/or dysfunctional mitochondria, which may cause energy deficiency, have been implicated in the etiopathogenesis of Parkinson's disease (PD). Enhanced clearance of misfolded proteins or injured mitochondria via autophagy has been reported to have neuroprotective roles in PD models. The fact that resveratrol is a known compound with multiple beneficial effects similar to those associated with energy metabolism led us to explore whether neuroprotective effects of resveratrol are related to its role in autophagy regulation. We tested whether modulation of mammalian silent information regulator 2 (SIRT1) and/or metabolic energy sensor AMP-activated protein kinase (AMPK) are involved in autophagy induction by resveratrol, leading to neuronal survival. Our results showed that resveratrol protected against rotenone-induced apoptosis in SH-SY5Y cells and enhanced degradation of α-synucleins in α-synuclein-expressing PC12 cell lines via autophagy induction. We found that suppression of AMPK and/or SIRT1 caused decrease of protein level of LC3-II, indicating that AMPK and/or SIRT1 are required in resveratrol-mediated autophagy induction. Moreover, suppression of AMPK caused inhibition of SIRT1 activity and attenuated protective effects of resveratrol on rotenone-induced apoptosis, further suggesting that AMPK-SIRT1-autophagy pathway plays an important role in the neuroprotection by resveratrol on PD cellular models.

  20. The effects of hispidulin on bupivacaine-induced neurotoxicity: role of AMPK signaling pathway.

    PubMed

    Niu, Xinhuan; Chen, Jie; Wang, Ping; Zhou, Hui; Li, Song; Zhang, Mengyuan

    2014-09-01

    Bupivacaine is a sodium channel blocker, which is widely used for local infiltration nerve block, epidural and intrathecal anesthesia. However, bupivacaine could cause nerve damage. Hispidulin was shown to be able to penetrate the blood-brain barrier and possess antiepileptic activity. In this study, we investigate whether hispidulin administration could attenuate bupivacaine-induced neurotoxicity. Bupivacaine-challenged mouse neuroblastoma N2a cells were treated with hispidulin. The neuron injury was assessed by examination of cell viability and apoptosis. The levels of activation of AMP-activated protein kinase (AMPK) signaling pathway were examined along with the effect of blocking AMPK signaling on cell viability in the presence of hispidulin and bupivacaine. Our results showed that Bupivacaine treatment significantly decreased cell viability and induced apoptosis. Treatment with hispidulin significantly attenuated bupivacaine-induced cell injury. In addition, hispidulin treatment increased the levels of phospho-AMPK and phospho-GSK3β and attenuated bupivacaine-induced loss in mitochondrial membrane potential. Furthermore, we found that blocking AMPK signaling pathway significantly abolished the cytoprotective effect of hispidulin against bupivacaine-induced cell injury. Our findings suggest that treatment of neuroblastoma cells with hispidulin-protected neural cells from Bupivacaine-induced injury via the activation of the AMPK/GSK3β signaling pathway.

  1. AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

    PubMed Central

    Young, Nathan P.; Kamireddy, Anwesh; Van Nostrand, Jeanine L.; Eichner, Lillian J.; Shokhirev, Maxim Nikolaievich; Dayn, Yelena; Shaw, Reuben J.

    2016-01-01

    Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK−/− EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. PMID:26944679

  2. Geniposide Suppresses Hepatic Glucose Production via AMPK in HepG2 Cells.

    PubMed

    Guo, Lixia; Zheng, Xuxu; Liu, Jianhui; Yin, Zhongyi

    2016-01-01

    Geniposide is one of the main compounds in Gardenia jasminoides ELLIS and has many pharmacological activities, but its anti-hyperglycemic activity has not yet been fully explored. This study was designed to determine, for the first time, how geniposide from G. jasminoides regulates hepatic glucose production, and the underlying mechanisms. During in vitro study, we found the inhibitory effect of geniposide on the hepatic glucose production is partly through AMP-activated protein kinase (AMPK) activation in HepG2 cells. Geniposide significantly inhibited hepatic glucose production in a dose-dependent manner. AMPK, acetyl coenzyme A synthetase (ACC) and forkhead box class O1 (FoxO1) phosphorylation were stimulated by different concentrations of geniposide. In addition, the enzyme activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were all significantly suppressed. What is important is that these effects were partly reversed by (1) inhibition of AMPK activity by compound C, a selective AMPK inhibitor, and by (2) suppression of AMPKα expression by small interfering RNA (siRNA). In summary, geniposide potentially ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis by modulation of the AMPK-FoxO1 signaling pathway. Geniposide or geniposide-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes on gluconeogenesis. PMID:26830672

  3. AMPK, a metabolic sensor, is involved in isoeugenol-induced glucose uptake in muscle cells

    PubMed Central

    Kim, Nami; Lee, Jung Ok; Lee, Hye Jeong; Lee, Yong Woo; Kim, Hyung Ip; Kim, Su Jin; Park, Sun Hwa; Lee, Chul Su; Ryoo, Sun Woo; Hwang, Geum-Sook; Kim, Hyeon Soo

    2016-01-01

    Isoeugenol exerts various beneficial effects on human health. However, the mechanisms underlying these effects are poorly understood. In this study, we observed that isoeugenol activated AMP-activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. Isoeugenol-induced increase in intracellular calcium concentration and glucose uptake was inhibited by STO-609, an inhibitor of calcium/calmodulin-dependent protein kinase kinase (CaMKK). Isoeugenol also increased the phosphorylation of protein kinase C-α (PKCα). Chelation of calcium with BAPTA-AM blocked isoeugenol-induced AMPK phosphorylation and glucose uptake. Isoeugenol stimulated p38MAPK phosphorylation that was inhibited after pretreatment with compound C, an AMPK inhibitor. Isoeugenol also increased glucose transporter type 4 (GLUT4) expression and its translocation to the plasma membrane. GLUT4 translocation was not observed after the inhibition of AMPK and CaMKK. In addition, isoeugenol activated the Akt substrate 160 (AS160) pathway, which is downstream of the p38MAPK pathway. Knockdown of the gene encoding AS160 inhibited isoeugenol-induced glucose uptake. Together, these results indicate that isoeugenol exerts beneficial health effects by activating the AMPK/p38MAPK/AS160 pathways in skeletal muscle. PMID:26585419

  4. AMPK, a metabolic sensor, is involved in isoeugenol-induced glucose uptake in muscle cells.

    PubMed

    Kim, Nami; Lee, Jung Ok; Lee, Hye Jeong; Lee, Yong Woo; Kim, Hyung Ip; Kim, Su Jin; Park, Sun Hwa; Lee, Chul Su; Ryoo, Sun Woo; Hwang, Geum-Sook; Kim, Hyeon Soo

    2016-02-01

    Isoeugenol exerts various beneficial effects on human health. However, the mechanisms underlying these effects are poorly understood. In this study, we observed that isoeugenol activated AMP-activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. Isoeugenol-induced increase in intracellular calcium concentration and glucose uptake was inhibited by STO-609, an inhibitor of calcium/calmodulin-dependent protein kinase kinase (CaMKK). Isoeugenol also increased the phosphorylation of protein kinase C-α (PKCα). Chelation of calcium with BAPTA-AM blocked isoeugenol-induced AMPK phosphorylation and glucose uptake. Isoeugenol stimulated p38MAPK phosphorylation that was inhibited after pretreatment with compound C, an AMPK inhibitor. Isoeugenol also increased glucose transporter type 4 (GLUT4) expression and its translocation to the plasma membrane. GLUT4 translocation was not observed after the inhibition of AMPK and CaMKK. In addition, isoeugenol activated the Akt substrate 160 (AS160) pathway, which is downstream of the p38MAPK pathway. Knockdown of the gene encoding AS160 inhibited isoeugenol-induced glucose uptake. Together, these results indicate that isoeugenol exerts beneficial health effects by activating the AMPK/p38MAPK/AS160 pathways in skeletal muscle.

  5. Diacylglycerol kinase-δ regulates AMPK signaling, lipid metabolism, and skeletal muscle energetics.

    PubMed

    Jiang, Lake Q; de Castro Barbosa, Thais; Massart, Julie; Deshmukh, Atul S; Löfgren, Lars; Duque-Guimaraes, Daniella E; Ozilgen, Arda; Osler, Megan E; Chibalin, Alexander V; Zierath, Juleen R

    2016-01-01

    Decrease of AMPK-related signal transduction and insufficient lipid oxidation contributes to the pathogenesis of obesity and type 2 diabetes. Previously, we identified that diacylglycerol kinase-δ (DGKδ), an enzyme involved in triglyceride biosynthesis, is reduced in skeletal muscle from type 2 diabetic patients. Here, we tested the hypothesis that DGKδ plays a role in maintaining appropriate AMPK action in skeletal muscle and energetic aspects of contraction. Voluntary running activity was reduced in DGKδ(+/-) mice, but glycogen content and mitochondrial markers were unaltered, suggesting that DGKδ deficiency affects skeletal muscle energetics but not mitochondrial protein abundance. We next determined the role of DGKδ in AMPK-related signal transduction and lipid metabolism in isolated skeletal muscle. AMPK activation and signaling were reduced in DGKδ(+/-) mice, concomitant with impaired lipid oxidation and elevated incorporation of free fatty acids into triglycerides. Strikingly, DGKδ deficiency impaired work performance, as evident by altered force production and relaxation dynamics in response to repeated contractions. In conclusion, DGKδ deficiency impairs AMPK signaling and lipid metabolism, thereby highlighting the deleterious role of excessive lipid metabolites in the development of peripheral insulin resistance and type 2 diabetes pathogenesis. DGKδ deficiency also influences skeletal muscle energetics, which may lead to low physical activity levels in type 2 diabetes.

  6. Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB.

    PubMed

    Mair, William; Morantte, Ianessa; Rodrigues, Ana P C; Manning, Gerard; Montminy, Marc; Shaw, Reuben J; Dillin, Andrew

    2011-02-17

    Activating AMPK or inactivating calcineurin slows ageing in Caenorhabditis elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. Here we show that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. We demonstrate that CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity.

  7. Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5

    PubMed Central

    Yan, Yi; Tsukamoto, Osamu; Nakano, Atsushi; Kato, Hisakazu; Kioka, Hidetaka; Ito, Noriaki; Higo, Shuichiro; Yamazaki, Satoru; Shintani, Yasunori; Matsuoka, Ken; Liao, Yulin; Asanuma, Hiroshi; Asakura, Masanori; Takafuji, Kazuaki; Minamino, Tetsuo; Asano, Yoshihiro; Kitakaze, Masafumi; Takashima, Seiji

    2015-01-01

    Augmented AMP-activated protein kinase (AMPK) activity inhibits cell migration, possibly contributing to the clinical benefits of chemical AMPK activators in preventing atherosclerosis, vascular remodelling and cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we identify PDZ and LIM domain 5 (Pdlim5) as a novel AMPK substrate and show that it plays a critical role in the inhibition of cell migration. AMPK directly phosphorylates Pdlim5 at Ser177. Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits cell migration and attenuates lamellipodia formation. Consistent with this observation, S177D-Pdlim5 suppresses Rac1 activity at the cell periphery and displaces the Arp2/3 complex from the leading edge. Notably, S177D-Pdlim5, but not WT-Pdlim5, attenuates the association with Rac1-specific guanine nucleotide exchange factors at the cell periphery. Taken together, our findings indicate that phosphorylation of Pdlim5 on Ser177 by AMPK mediates inhibition of cell migration by suppressing the Rac1-Arp2/3 signalling pathway. PMID:25635515

  8. AMPK Mediates Glucocorticoids Stress-Induced Downregulation of the Glucocorticoid Receptor in Cultured Rat Prefrontal Cortical Astrocytes.

    PubMed

    Yuan, Shi-Ying; Liu, Jue; Zhou, Jun; Lu, Wei; Zhou, Hai-Yun; Long, Li-Hong; Hu, Zhuang-Li; Ni, Lan; Wang, Yi; Chen, Jian-Guo; Wang, Fang

    2016-01-01

    Chronic stress induces altered energy metabolism and plays important roles in the etiology of depression, in which the glucocorticoid negative feedback is disrupted due to imbalanced glucocorticoid receptor (GR) functions. The mechanism underlying the dysregulation of GR by chronic stress remains elusive. In this study, we investigated the role of AMP-activated protein kinase (AMPK), the key enzyme regulating cellular energy metabolism, and related signaling pathways in chronic stress-induced GR dysregulation. In cultured rat cortical astrocytes, glucocorticoid treatment decreased the level, which was accompanied by the decreased expression of liver kinase B1 (LKB1) and reduced phosphorylation of AMPK. Glucocorticoid-induced effects were attenuated by glucocorticoid-inducible kinase 1 (SGK1) inhibitor GSK650394, which also inhibited glucocorticoid induced phosphorylation of Forkhead box O3a (FOXO3a). Furthermore, glucocorticoid-induced down-regulation of GR was mimicked by the inhibition of AMPK and abolished by the AMPK activators or the histone deacetylase 5 (HDAC5) inhibitors. In line with the role of AMPK in GR expression, AMPK activator metformin reversed glucocorticoid-induced reduction of AMPK phosphorylation and GR expression as well as behavioral alteration of rats. Taken together, these results suggest that chronic stress activates SGK1 and suppresses the expression of LKB1 via inhibitory phosphorylation of FOXO3a. Downregulated LKB1 contributes to reduced activation of AMPK, leading to the dephosphorylation of HDAC5 and the suppression of transcription of GR. PMID:27513844

  9. α-SNAP inhibits AMPK signaling to reduce mitochondrial biogenesis and dephosphorylates Thr172 in AMPKα in vitro.

    PubMed

    Wang, Lifu; Brautigan, David L

    2013-01-01

    The AMP-activated protein kinase (AMPK) regulates metabolism in normal and pathological conditions and responds to nutrients, hormones, anti-diabetic drugs and physical exercise. AMPK is activated by the kinase LKB1 and inactivated by phosphatases whose identities remain uncertain. Here we show that AMPK associates with α-SNAP, an adapter that enables disassembly of cis-SNARE complexes formed during membrane fusion. Knockdown of α-SNAP activates AMPK to phosphorylate its endogenous substrates acetyl CoA carboxylase and Raptor, and provokes mitochondrial biogenesis. AMPK phosphorylation is rescued from α-SNAP RNA interference by LKB1 knockdown or expression of wild-type but not mutated α-SNAP. Recombinant wild-type but not mutated α-SNAP dephosphorylates pThr172 in AMPKα in vitro. Overexpression of wild-type but not mutated α-SNAP prevents AMPK activation in cells treated with agents to elevate AMP concentration. The mouse α-SNAP mutant hyh (hydrocephalus with hop gait) shows enhanced binding and inhibition of AMPK. By negatively controlling AMPK, α-SNAP therefore potentially coordinates membrane trafficking and metabolism.

  10. AMPK potentiates hypertonicity-induced apoptosis by suppressing NFκB/COX-2 in medullary interstitial cells.

    PubMed

    Han, Qifei; Zhang, Xiaoyan; Xue, Rui; Yang, Hang; Zhou, Yunfeng; Kong, Xiaomu; Zhao, Pan; Li, Jing; Yang, Jichun; Zhu, Yi; Guan, Youfei

    2011-10-01

    Cells residing in the hypertonic, hypoxic renal medulla depend on dynamic adaptation mechanisms to respond to changes in energy supply and demand. The serine/threonine kinase 5'-AMP protein kinase (AMPK) is a sensor of cellular energy status, but whether it contributes to the survival of cells in the renal medulla is unknown. Here, hypertonic conditions induced a decrease in AMPK phosphorylation within 12 hours in renal medullary interstitial cells (RMIC), followed by a gradual return to baseline levels. Activation of AMPK markedly increased hypertonicity-induced apoptosis of RMICs and suppressed both hypertonicity-induced NFκB nuclear translocation and cyclooxygenase-2 (COX-2) activation; overexpression of COX-2 significantly attenuated these effects. AMPK activation also markedly reduced generation of reactive oxygen species and nuclear expression of tonicity-responsive enhancer-binding protein, which prevented upregulation of osmoprotective genes. In vivo, pharmacologic activation of AMPK led to massive apoptosis of RMICs and renal dysfunction in the setting of water deprivation in mice. Taken together, these results identify a critical role for AMPK in the maintenance of RMIC viability and suggest that AMPK modulates the NFκB-COX-2 survival pathway in the renal medulla. Furthermore, this study raises safety concerns for the development of AMPK activators as anti-diabetic drugs, especially for patients prone to dehydration. PMID:21903993

  11. AMPK Mediates Glucocorticoids Stress-Induced Downregulation of the Glucocorticoid Receptor in Cultured Rat Prefrontal Cortical Astrocytes

    PubMed Central

    Lu, Wei; Zhou, Hai-Yun; Long, Li-Hong; Hu, Zhuang-Li; Ni, Lan; Wang, Yi; Chen, Jian-Guo; Wang, Fang

    2016-01-01

    Chronic stress induces altered energy metabolism and plays important roles in the etiology of depression, in which the glucocorticoid negative feedback is disrupted due to imbalanced glucocorticoid receptor (GR) functions. The mechanism underlying the dysregulation of GR by chronic stress remains elusive. In this study, we investigated the role of AMP-activated protein kinase (AMPK), the key enzyme regulating cellular energy metabolism, and related signaling pathways in chronic stress-induced GR dysregulation. In cultured rat cortical astrocytes, glucocorticoid treatment decreased the level, which was accompanied by the decreased expression of liver kinase B1 (LKB1) and reduced phosphorylation of AMPK. Glucocorticoid-induced effects were attenuated by glucocorticoid-inducible kinase 1 (SGK1) inhibitor GSK650394, which also inhibited glucocorticoid induced phosphorylation of Forkhead box O3a (FOXO3a). Furthermore, glucocorticoid-induced down-regulation of GR was mimicked by the inhibition of AMPK and abolished by the AMPK activators or the histone deacetylase 5 (HDAC5) inhibitors. In line with the role of AMPK in GR expression, AMPK activator metformin reversed glucocorticoid-induced reduction of AMPK phosphorylation and GR expression as well as behavioral alteration of rats. Taken together, these results suggest that chronic stress activates SGK1 and suppresses the expression of LKB1 via inhibitory phosphorylation of FOXO3a. Downregulated LKB1 contributes to reduced activation of AMPK, leading to the dephosphorylation of HDAC5 and the suppression of transcription of GR. PMID:27513844

  12. A Potent and Selective AMPK Activator That Inhibits de Novo Lipogenesis.

    PubMed

    Gómez-Galeno, Jorge E; Dang, Qun; Nguyen, Thanh H; Boyer, Serge H; Grote, Matthew P; Sun, Zhili; Chen, Mingwei; Craigo, William A; van Poelje, Paul D; MacKenna, Deidre A; Cable, Edward E; Rolzin, Paul A; Finn, Patricia D; Chi, Bert; Linemeyer, David L; Hecker, Scott J; Erion, Mark D

    2010-12-01

    AMP-activated protein kinase (AMPK) is a heterotrimeric kinase that regulates cellular energy metabolism by affecting energy-consuming pathways such as de novo lipid biosynthesis and glucose production as well as energy-producing pathways such as lipid oxidation and glucose uptake. Accordingly, compounds that activate AMPK represent potential drug candidates for the treatment of hyperlipidemia and type 2 diabetes. Screening of a proprietary library of AMP mimetics identified the phosphonic acid 2 that bears little structural resemblance to AMP but is capable of activating AMPK with high potency (EC50 = 6 nM vs AMP EC50 = 6 μM) and specificity. Phosphonate prodrugs of 2 inhibited de novo lipogenesis in cellular and animal models of hyperlipidemia.

  13. AMPK acts as a molecular trigger to coordinate glutamatergic signals and adaptive behaviours during acute starvation.

    PubMed

    Ahmadi, Moloud; Roy, Richard

    2016-01-01

    The stress associated with starvation is accompanied by compensatory behaviours that enhance foraging efficiency and increase the probability of encountering food. However, the molecular details of how hunger triggers changes in the activity of neural circuits to elicit these adaptive behavioural outcomes remains to be resolved. We show here that AMP-activated protein kinase (AMPK) regulates neuronal activity to elicit appropriate behavioural outcomes in response to acute starvation, and this effect is mediated by the coordinated modulation of glutamatergic inputs. AMPK targets both the AMPA-type glutamate receptor GLR-1 and the metabotropic glutamate receptor MGL-1 in one of the primary circuits that governs behavioural response to food availability in C. elegans. Overall, our study suggests that AMPK acts as a molecular trigger in the specific starvation-sensitive neurons to modulate glutamatergic inputs and to elicit adaptive behavioural outputs in response to acute starvation. PMID:27642785

  14. Mitochondria related peptide MOTS-c suppresses ovariectomy-induced bone loss via AMPK activation.

    PubMed

    Ming, Wei; Lu, Gan; Xin, Sha; Huanyu, Lu; Yinghao, Jiang; Xiaoying, Lei; Chengming, Xu; Banjun, Ruan; Li, Wang; Zifan, Lu

    2016-08-01

    Therapeutic targeting bone loss has been the focus of the study in osteoporosis. The present study is intended to evaluate whether MOTS-c, a novel mitochondria related 16 aa peptide, can protect mice from ovariectomy-induced osteoporosis. After ovary removal, the mice were injected with MOTS-c at a dose of 5 mg/kg once a day for 12 weeks. Our results showed that MOTS-c treatment significantly alleviated bone loss, as determined by micro-CT examination. Mechanistically, we found that the receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclast differentiation was remarkably inhibited by MOTS-c. Moreover, MOTS-c increased phosphorylated AMPK levels, and compound C, an AMPK inhibitor, could partially abrogate the effects of the MOTS-c on osteoclastogenesis. Thus, our findings provide evidence that MOTS-c may exert as an inhibitor of osteoporosis via AMPK dependent inhibition of osteoclastogenesis. PMID:27237975

  15. A Potent and Selective AMPK Activator That Inhibits de Novo Lipogenesis

    PubMed Central

    2010-01-01

    AMP-activated protein kinase (AMPK) is a heterotrimeric kinase that regulates cellular energy metabolism by affecting energy-consuming pathways such as de novo lipid biosynthesis and glucose production as well as energy-producing pathways such as lipid oxidation and glucose uptake. Accordingly, compounds that activate AMPK represent potential drug candidates for the treatment of hyperlipidemia and type 2 diabetes. Screening of a proprietary library of AMP mimetics identified the phosphonic acid 2 that bears little structural resemblance to AMP but is capable of activating AMPK with high potency (EC50 = 6 nM vs AMP EC50 = 6 μM) and specificity. Phosphonate prodrugs of 2 inhibited de novo lipogenesis in cellular and animal models of hyperlipidemia. PMID:24900234

  16. Molecular mechanisms of appetite and obesity: a role for brain AMPK.

    PubMed

    Martínez de Morentin, Pablo B; Urisarri, Adela; Couce, María L; López, Miguel

    2016-10-01

    Feeding behaviour and energy storage are both crucial aspects of survival. Thus, it is of fundamental importance to understand the molecular mechanisms regulating these basic processes. The AMP-activated protein kinase (AMPK) has been revealed as one of the key molecules modulating energy homoeostasis. Indeed, AMPK appears to be essential for translating nutritional and energy requirements into generation of an adequate neuronal response, particularly in two areas of the brain, the hypothalamus and the hindbrain. Failure of this physiological response can lead to energy imbalance, ultimately with extreme consequences, such as leanness or obesity. Here, we will review the data that put brain AMPK in the spotlight as a regulator of appetite. PMID:27555613

  17. The emerging role of AMPK in the regulation of breathing and oxygen supply

    PubMed Central

    Evans, A. Mark; Mahmoud, Amira D.; Moral-Sanz, Javier; Hartmann, Sandy

    2016-01-01

    Regulation of breathing is critical to our capacity to accommodate deficits in oxygen availability and demand during, for example, sleep and ascent to altitude. It is generally accepted that a fall in arterial oxygen increases afferent discharge from the carotid bodies to the brainstem and thus delivers increased ventilatory drive, which restores oxygen supply and protects against hypoventilation and apnoea. However, the precise molecular mechanisms involved remain unclear. We recently identified as critical to this process the AMP-activated protein kinase (AMPK), which is key to the cell-autonomous regulation of metabolic homoeostasis. This observation is significant for many reasons, not least because recent studies suggest that the gene for the AMPK-α1 catalytic subunit has been subjected to natural selection in high-altitude populations. It would appear, therefore, that evolutionary pressures have led to AMPK being utilized to regulate oxygen delivery and thus energy supply to the body in the short, medium and longer term. Contrary to current consensus, however, our findings suggest that AMPK regulates ventilation at the level of the caudal brainstem, even when afferent input responses from the carotid body are normal. We therefore hypothesize that AMPK integrates local hypoxic stress at defined loci within the brainstem respiratory network with an index of peripheral hypoxic status, namely afferent chemosensory inputs. Allied to this, AMPK is critical to the control of hypoxic pulmonary vasoconstriction and thus ventilation–perfusion matching at the lungs and may also determine oxygen supply to the foetus by, for example, modulating utero-placental blood flow. PMID:27574022

  18. The emerging role of AMPK in the regulation of breathing and oxygen supply.

    PubMed

    Evans, A Mark; Mahmoud, Amira D; Moral-Sanz, Javier; Hartmann, Sandy

    2016-09-01

    Regulation of breathing is critical to our capacity to accommodate deficits in oxygen availability and demand during, for example, sleep and ascent to altitude. It is generally accepted that a fall in arterial oxygen increases afferent discharge from the carotid bodies to the brainstem and thus delivers increased ventilatory drive, which restores oxygen supply and protects against hypoventilation and apnoea. However, the precise molecular mechanisms involved remain unclear. We recently identified as critical to this process the AMP-activated protein kinase (AMPK), which is key to the cell-autonomous regulation of metabolic homoeostasis. This observation is significant for many reasons, not least because recent studies suggest that the gene for the AMPK-α1 catalytic subunit has been subjected to natural selection in high-altitude populations. It would appear, therefore, that evolutionary pressures have led to AMPK being utilized to regulate oxygen delivery and thus energy supply to the body in the short, medium and longer term. Contrary to current consensus, however, our findings suggest that AMPK regulates ventilation at the level of the caudal brainstem, even when afferent input responses from the carotid body are normal. We therefore hypothesize that AMPK integrates local hypoxic stress at defined loci within the brainstem respiratory network with an index of peripheral hypoxic status, namely afferent chemosensory inputs. Allied to this, AMPK is critical to the control of hypoxic pulmonary vasoconstriction and thus ventilation-perfusion matching at the lungs and may also determine oxygen supply to the foetus by, for example, modulating utero-placental blood flow. PMID:27574022

  19. AMPK activation protects from neuronal dysfunction and vulnerability across nematode, cellular and mouse models of Huntington's disease

    PubMed Central

    Vázquez-Manrique, Rafael P.; Farina, Francesca; Cambon, Karine; Dolores Sequedo, María; Parker, Alex J.; Millán, José María; Weiss, Andreas; Déglon, Nicole; Neri, Christian

    2016-01-01

    The adenosine monophosphate activated kinase protein (AMPK) is an evolutionary-conserved protein important for cell survival and organismal longevity through the modulation of energy homeostasis. Several studies suggested that AMPK activation may improve energy metabolism and protein clearance in the brains of patients with vascular injury or neurodegenerative disease. However, in Huntington's disease (HD), AMPK may be activated in the striatum of HD mice at a late, post-symptomatic phase of the disease, and high-dose regiments of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide may worsen neuropathological and behavioural phenotypes. Here, we revisited the role of AMPK in HD using models that recapitulate the early features of the disease, including Caenorhabditis elegans neuron dysfunction before cell death and mouse striatal cell vulnerability. Genetic and pharmacological manipulation of aak-2/AMPKα shows that AMPK activation protects C. elegans neurons from the dysfunction induced by human exon-1 huntingtin (Htt) expression, in a daf-16/forkhead box O-dependent manner. Similarly, AMPK activation using genetic manipulation and low-dose metformin treatment protects mouse striatal cells expressing full-length mutant Htt (mHtt), counteracting their vulnerability to stress, with reduction of soluble mHtt levels by metformin and compensation of cytotoxicity by AMPKα1. Furthermore, AMPK protection is active in the mouse brain as delivery of gain-of-function AMPK-γ1 to mouse striata slows down the neurodegenerative effects of mHtt. Collectively, these data highlight the importance of considering the dynamic of HD for assessing the therapeutic potential of stress-response targets in the disease. We postulate that AMPK activation is a compensatory response and valid approach for protecting dysfunctional and vulnerable neurons in HD. PMID:26681807

  20. Human muscle fibre type-specific regulation of AMPK and downstream targets by exercise

    PubMed Central

    Kristensen, Dorte E; Albers, Peter H; Prats, Clara; Baba, Otto; Birk, Jesper B; Wojtaszewski, Jørgen F P

    2015-01-01

    AMP-activated protein kinase (AMPK) is a regulator of energy homeostasis during exercise. Studies suggest muscle fibre type-specific AMPK expression. However, fibre type-specific regulation of AMPK and downstream targets during exercise has not been demonstrated. We hypothesized that AMPK subunits are expressed in a fibre type-dependent manner and that fibre type-specific activation of AMPK and downstream targets is dependent on exercise intensity. Pools of type I and II fibres were prepared from biopsies of vastus lateralis muscle from healthy men before and after two exercise trials: (1) continuous cycling (CON) for 30 min at 69 ± 1% peak rate of O2 consumption () or (2) interval cycling (INT) for 30 min with 6 × 1.5 min high-intensity bouts peaking at 95 ± 2% . In type I vs. II fibres a higher β1 AMPK (+215%) and lower γ3 AMPK expression (−71%) was found. α1, α2, β2 and γ1 AMPK expression was similar between fibre types. In type I vs. II fibres phosphoregulation after CON was similar (AMPKThr172, ACCSer221, TBC1D1Ser231 and GS2+2a) or lower (TBC1D4Ser704). Following INT, phosphoregulation in type I vs. II fibres was lower (AMPKThr172, TBC1D1Ser231, TBC1D4Ser704 and ACCSer221) or higher (GS2+2a). Exercise-induced glycogen degradation in type I vs. II fibres was similar (CON) or lower (INT). In conclusion, a differentiated response to exercise of metabolic signalling/effector proteins in human type I and II fibres was evident during interval exercise. This could be important for exercise type-specific adaptations, i.e. insulin sensitivity and mitochondrial density, and highlights the potential for new discoveries when investigating fibre type-specific signalling. PMID:25640469

  1. Effects of feed deprivation on the AMPK signaling pathway in skeletal muscle of broiler chickens.

    PubMed

    Hu, Xiyi; Liu, Lei; Song, Zhigang; Sheikhahmadi, Ardashir; Wang, Yufeng; Buyse, Johan

    2016-01-01

    The 5'-adenosine monophosphate-activated protein kinase (AMPK) plays a key role in rapid metabolic adaptations to maintain energy homeostasis in poultry. It remains unclear if AMPK is involved in muscular energy metabolism in broiler chickens. Hence, in the present study, seven-day-old male broilers were equally divided into three groups: fed ad libitum (control); feed-deprived for 24h (S24); feed-deprived for 24h and then refed for 24h (S24R24). Compared to the control group, the plasma levels of glucose, insulin, T3 and triglycerides in the S24 group were significantly lower (P<0.05), whereas the uric acid levels were significantly higher (P<0.01). Except for glucose, refeeding for 24h reversed the fasting-induced alterations in plasma metabolite. Fasting decreased the liver kinase B1 (LKB1), AMPK alpha 2 subunit (AMPKα2), and fatty acid synthase (FAS) mRNA levels (P<0.05) in M. pectoralis major (PM). Feed deprivation did not affect the phosphorylated AKT, mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K) in PM (P>0.05), but upregulated carnitine palmitoyltransferase 1 (CPT1) gene expression and increased phosphorylated LKB1 (0.05AMPK and LKB1 of the PM muscle returned to control group levels after 24h refeeding. In M. biceps femoris (BF), the AMPKα2 and FAS mRNA levels were decreased by fasting compared to control (P<0.05), whereas CPT1 mRNA and phosphorylated LKB1 and AMPK protein levels were increased. Refeeding for 24h reversed the changes in AMPKα2 and CPT1 gene expression and phosphorylated AMPKα2 subunit. Fasting did not affect the AKT, mTOR and p70S6K in both PM and BF muscles (P>0.05). However, refeeding after 24h of fasting increased the phosphorylated mTOR level in BF muscle which was in parallel with increased plasma insulin concentration. It was likely that increased phospho-mTOR level in the BF muscle was

  2. Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction.

    PubMed

    Schaffer, Bethany E; Levin, Rebecca S; Hertz, Nicholas T; Maures, Travis J; Schoof, Michael L; Hollstein, Pablo E; Benayoun, Bérénice A; Banko, Max R; Shaw, Reuben J; Shokat, Kevan M; Brunet, Anne

    2015-11-01

    AMP-activated protein kinase (AMPK) is a central energy gauge that regulates metabolism and has been increasingly involved in non-metabolic processes and diseases. However, AMPK's direct substrates in non-metabolic contexts are largely unknown. To better understand the AMPK network, we use a chemical genetics screen coupled to a peptide capture approach in whole cells, resulting in identification of direct AMPK phosphorylation sites. Interestingly, the high-confidence AMPK substrates contain many proteins involved in cell motility, adhesion, and invasion. AMPK phosphorylation of the RHOA guanine nucleotide exchange factor NET1A inhibits extracellular matrix degradation, an early step in cell invasion. The identification of direct AMPK phosphorylation sites also facilitates large-scale prediction of AMPK substrates. We provide an AMPK motif matrix and a pipeline to predict additional AMPK substrates from quantitative phosphoproteomics datasets. As AMPK is emerging as a critical node in aging and pathological processes, our study identifies potential targets for therapeutic strategies. PMID:26456332

  3. LKB1/AMPK inhibits TGF-β1 production and the TGF-β signaling pathway in breast cancer cells.

    PubMed

    Li, Nian-Shuang; Zou, Jun-Rong; Lin, Hui; Ke, Rong; He, Xiao-Ling; Xiao, Lu; Huang, Deqiang; Luo, Lingyu; Lv, Nonghua; Luo, Zhijun

    2016-06-01

    Adenosine monophosphate-activated protein kinase (AMPK) acts as a fuel gauge that maintains energy homeostasis in both normal and cancerous cells, and has emerged as a tumor suppressor. The present study aims to delineate the functional relationship between AMPK and transforming growth factor beta (TGF-β). Our results showed that expression of liver kinase B1 (LKB1), an upstream kinase of AMPK, impeded TGF-β-induced Smad phosphorylation and their transcriptional activity in breast cancer cells, whereas knockdown of LKB1 or AMPKα1 subunit by short hairpin RNA (shRNA) enhanced the effect of TGF-β. Furthermore, AMPK activation reduced the promoter activity of TGF-β1. In accordance, type 2 diabetic patients taking metformin displayed a trend of reduction of serum TGF-β1, as compared with those without metformin. A significant reduction of serum TGF-β1 was found in mice after treatment with metformin. These results suggest that AMPK inhibits the transcription of TGF-β1, leading to reduction of its concentration in serum. Finally, metformin suppressed epithelial-to-mesenchymal transition of mammary epithelial cells. Taken together, our study demonstrates that AMPK exerts multiple actions on TGF-β signaling and supports that AMPK can serve as a therapeutic drug target for breast cancer. PMID:26718214

  4. Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates.

    PubMed

    Hoffman, Nolan J; Parker, Benjamin L; Chaudhuri, Rima; Fisher-Wellman, Kelsey H; Kleinert, Maximilian; Humphrey, Sean J; Yang, Pengyi; Holliday, Mira; Trefely, Sophie; Fazakerley, Daniel J; Stöckli, Jacqueline; Burchfield, James G; Jensen, Thomas E; Jothi, Raja; Kiens, Bente; Wojtaszewski, Jørgen F P; Richter, Erik A; James, David E

    2015-11-01

    Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPK-dependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.

  5. Activation of the AMPK-ULK1 pathway plays an important role in autophagy during prion infection

    PubMed Central

    Fan, Xue-Yu; Tian, Chan; Wang, Hui; Xu, Yin; Ren, Ke; Zhang, Bao-Yun; Gao, Chen; Shi, Qi; Meng, Ge; Zhang, Lu-Bin; Zhao, Yang-Jing; Shao, Qi-Xiang; Dong, Xiao-Ping

    2015-01-01

    AMPK is a serine/threonine protein kinase that acts as a positive regulator of autophagy, by phosphorylating ULK1 at specific sites. A previous study demonstrated activation of the macroautophagic system in scrapie-infected experimental rodents and in certain human prion diseases, in which the essential negative regulator mTOR is severely inhibited. In this study, AMPK and ULK1 in the brains of hamsters infected with scrapie strain 263 K and in the scrapie-infected cell line SMB-S15 were analysed. The results showed an up-regulated trend of AMPK and AMPK-Thr172, ULK1 and ULK1-Ser555. Increases in brain AMPK and ULK1 occurred at an early stage of agent 263 K infection. The level of phosphorylated ULK1-Ser757 decreased during mid-infection and was only negligibly present at the terminal stage, a pattern that suggested a close relationship of the phosphorylated protein with altered endogenous mTOR. In addition, the level of LKB1 associated with AMPK activation was selectively increased at the early and middle stages of infection. Knockdown of endogenous ULK1 in SMB-S15 cells inhibited LC3 lipidation. These results showed that, in addition to the abolishment of the mTOR regulatory pathway, activation of the AMPK-ULK1 pathway during prion infection contributes to autophagy activation in prion-infected brain tissues. PMID:26423766

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

    PubMed Central

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

    2015-01-01

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

  7. Resveratrol attenuates lipopolysaccharide-induced dysfunction of blood-brain barrier in endothelial cells via AMPK activation

    PubMed Central

    2016-01-01

    Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. The blood-brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. The aim of this study was to elucidate the effects of resveratrol and the role of AMPK in BBB dysfunction induced by lipopolysaccharide (LPS). Exposure of human brain microvascular endothelial cells (HBMECs) to LPS (1 µg/ml) for 4 to 24 hours week dramatically increased the permeability of the BBB in parallel with lowered expression levels of occluding and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P) H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by resveratrol. Finally, upregulation of AMPK by either AMPK-CA or resveratrol abolished the levels of LPS-enhanced NAD(P)H oxidase subunits protein expressions. We conclude that AMPK activation by resveratrol improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs. PMID:27382348

  8. LKB1/AMPK inhibits TGF-β1 production and the TGF-β signaling pathway in breast cancer cells.

    PubMed

    Li, Nian-Shuang; Zou, Jun-Rong; Lin, Hui; Ke, Rong; He, Xiao-Ling; Xiao, Lu; Huang, Deqiang; Luo, Lingyu; Lv, Nonghua; Luo, Zhijun

    2016-06-01

    Adenosine monophosphate-activated protein kinase (AMPK) acts as a fuel gauge that maintains energy homeostasis in both normal and cancerous cells, and has emerged as a tumor suppressor. The present study aims to delineate the functional relationship between AMPK and transforming growth factor beta (TGF-β). Our results showed that expression of liver kinase B1 (LKB1), an upstream kinase of AMPK, impeded TGF-β-induced Smad phosphorylation and their transcriptional activity in breast cancer cells, whereas knockdown of LKB1 or AMPKα1 subunit by short hairpin RNA (shRNA) enhanced the effect of TGF-β. Furthermore, AMPK activation reduced the promoter activity of TGF-β1. In accordance, type 2 diabetic patients taking metformin displayed a trend of reduction of serum TGF-β1, as compared with those without metformin. A significant reduction of serum TGF-β1 was found in mice after treatment with metformin. These results suggest that AMPK inhibits the transcription of TGF-β1, leading to reduction of its concentration in serum. Finally, metformin suppressed epithelial-to-mesenchymal transition of mammary epithelial cells. Taken together, our study demonstrates that AMPK exerts multiple actions on TGF-β signaling and supports that AMPK can serve as a therapeutic drug target for breast cancer.

  9. Compound C inhibits macrophage chemotaxis through an AMPK-independent mechanism.

    PubMed

    Lee, Youngyi; Park, Byung-Hyun; Bae, Eun Ju

    2016-01-15

    Macrophage infiltration in adipose tissue is a well-established cause of obesity-linked insulin resistance. AMP-activated protein kinase (AMPK) activation in peripheral tissues such as adipose tissue has beneficial effects on the protection against obesity-induced insulin resistance, which is mainly mediated by prevention of adipose tissue macrophage infiltration and inflammation. In examining the role of AMPK on adipose tissue inflammation, we unexpectedly found that compound C (CC), despite its inhibition of AMPK, robustly inhibited macrophage chemotaxis in RAW 264.7 cells when adipocyte conditioned medium (CM) was used as a chemoattractant. Here, we report that CC inhibition of macrophage migration occurred independently of AMPK. Mechanistically, this inhibitory effect of cell migration by CC was mediated by inhibition of the focal adhesion kinase, AKT, nuclear factor κB pathways. Moreover, the expression of chemokine monocyte chemoattractant protein-1 and pro-inflammatory genes such as tumor necrosis factor α and inducible nitric oxide synthase were prevented by CC treatment in RAW 264.7 cells stimulated with either adipocyte CM or lipopolysaccharide. Lastly, in accord with the findings of the anti-inflammatory effect of CC, we demonstrated that CC functioned as a repressor of macrophage CM-mediated insulin resistance in adipocytes. Taken together, our results suggest that CC serves as a useful inhibitory molecule against macrophage chemotaxis into adipose tissue and thus might have therapeutic potential for the treatment of obesity-linked adipose inflammation.

  10. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases.

    PubMed

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems. PMID:24904600

  11. Phloridzin improves lipoprotein lipase activity in stress-loaded mice via AMPK phosphorylation.

    PubMed

    Wang, Guo-En; Li, Yi-Fang; Wu, Yan-Ping; Tsoi, Bun; Zhang, Shi-Jie; Cao, Ling-Fang; Kurihara, Hiroshi; He, Rong-Rong

    2014-11-01

    Long-term stress exposure can lead to disturbed homeostasis and cause many life-style diseases. Phloridzin possesses various bioactivities, but the understanding of the effects of phloridzin on stress-related lipid metabolism disorder is limited. Our results demonstrate that phloridzin improved plasma lipoprotein lipase (LPL) activity and triglyceride metabolism in restrained mice. A decrease of angiopoietin-like protein 4 (ANGPTL4) mRNA expression and an increase of AMP-activated protein kinase (AMPK) phosphorylation were observed after phloridzin treatment. After inhibiting AMPK phosphorylation, the effects of phloridzin on the amelioration of plasma LPL activity and suppression of ANGPTL4 expression were blocked. In addition, cardiac AMPK phosphorylation, plasma LPL activity and ANGPTL4 expression were also affected by phloridzin, even if the glucocorticoid receptor was blocked. Taken together, the down-regulation of ANGPTL4 expression by phloridzin was probably via a direct activation of AMPK pathway. This discovery can provide a biochemical and nutritional basis for the use of phloridzin-containing food and beverage in daily life. PMID:24932810

  12. Irisin Inhibits Hepatic Cholesterol Synthesis via AMPK-SREBP2 Signaling

    PubMed Central

    Tang, Hong; Yu, Ruili; Liu, Shiying; Huwatibieke, Bahetiyaer; Li, Ziru; Zhang, Weizhen

    2016-01-01

    Irisin, a myokine released during exercise, promotes browning of subcutaneous adipose tissue and regulates energy homeostasis. Although exercise constantly reduces blood cholesterol, whether irisin is involved in the regulation of cholesterol remains largely unknown. In the present study, subcutaneous infusion of irisin for 2 weeks induced a reduction in plasma and hepatic cholesterol in high fat diet-induced obese (DIO) mice. These alterations were associated with an activation of 5′ AMP-activated protein kinase (AMPK) and inhibition of sterol regulatory element-binding transcription factor 2 (SREBP2) transcription and nuclear translocation. In primary hepatocytes from either lean or DIO mice, irisin significantly decreased cholesterol content via sequential activation of AMPK and inhibition of SREBP2. Suppression of AMPK by compound C or AMPKα1 siRNA blocked irisin-induced alterations in cholesterol contents and SREBP2. In conclusion, irisin could suppress hepatic cholesterol production via a mechanism dependent of AMPK and SREBP2 signaling. These findings suggest that irisin is a promising therapeutic target for treatment of hypercholesterolemia. PMID:27211556

  13. Irisin Inhibits Hepatic Cholesterol Synthesis via AMPK-SREBP2 Signaling.

    PubMed

    Tang, Hong; Yu, Ruili; Liu, Shiying; Huwatibieke, Bahetiyaer; Li, Ziru; Zhang, Weizhen

    2016-04-01

    Irisin, a myokine released during exercise, promotes browning of subcutaneous adipose tissue and regulates energy homeostasis. Although exercise constantly reduces blood cholesterol, whether irisin is involved in the regulation of cholesterol remains largely unknown. In the present study, subcutaneous infusion of irisin for 2weeks induced a reduction in plasma and hepatic cholesterol in high fat diet-induced obese (DIO) mice. These alterations were associated with an activation of 5' AMP-activated protein kinase (AMPK) and inhibition of sterol regulatory element-binding transcription factor 2 (SREBP2) transcription and nuclear translocation. In primary hepatocytes from either lean or DIO mice, irisin significantly decreased cholesterol content via sequential activation of AMPK and inhibition of SREBP2. Suppression of AMPK by compound C or AMPKα1 siRNA blocked irisin-induced alterations in cholesterol contents and SREBP2. In conclusion, irisin could suppress hepatic cholesterol production via a mechanism dependent of AMPK and SREBP2 signaling. These findings suggest that irisin is a promising therapeutic target for treatment of hypercholesterolemia.

  14. Ginkgolide C Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK Signaling Pathway.

    PubMed

    Liou, Chian-Jiun; Lai, Xuan-Yu; Chen, Ya-Ling; Wang, Chia-Ling; Wei, Ciao-Han; Huang, Wen-Chung

    2015-01-01

    Ginkgolide C, isolated from Ginkgo biloba leaves, is a flavone reported to have multiple biological functions, from decreased platelet aggregation to ameliorating Alzheimer disease. The study aim was to evaluate the antiadipogenic effect of ginkgolide C in 3T3-L1 adipocytes. Ginkgolide C was used to treat differentiated 3T3-L1 cells. Cell supernatant was collected to assay glycerol release, and cells were lysed to measure protein and gene expression related to adipogenesis and lipolysis by western blot and real-time PCR, respectively. Ginkgolide C significantly suppressed lipid accumulation in differentiated adipocytes. It also decreased adipogenesis-related transcription factor expression, including peroxisome proliferator-activated receptor and CCAAT/enhancer-binding protein. Furthermore, ginkgolide C enhanced adipose triglyceride lipase and hormone-sensitive lipase production for lipolysis and increased phosphorylation of AMP-activated protein kinase (AMPK), resulting in decreased activity of acetyl-CoA carboxylase for fatty acid synthesis. In coculture with an AMPK inhibitor (compound C), ginkgolide C also improved activation of sirtuin 1 and phosphorylation of AMPK in differentiated 3T3-L1 cells. The results suggest that ginkgolide C is an effective flavone for increasing lipolysis and inhibiting adipogenesis in adipocytes through the activated AMPK pathway.

  15. Ginkgolide C Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK Signaling Pathway

    PubMed Central

    Liou, Chian-Jiun; Lai, Xuan-Yu; Chen, Ya-Ling; Wang, Chia-Ling; Wei, Ciao-Han; Huang, Wen-Chung

    2015-01-01

    Ginkgolide C, isolated from Ginkgo biloba leaves, is a flavone reported to have multiple biological functions, from decreased platelet aggregation to ameliorating Alzheimer disease. The study aim was to evaluate the antiadipogenic effect of ginkgolide C in 3T3-L1 adipocytes. Ginkgolide C was used to treat differentiated 3T3-L1 cells. Cell supernatant was collected to assay glycerol release, and cells were lysed to measure protein and gene expression related to adipogenesis and lipolysis by western blot and real-time PCR, respectively. Ginkgolide C significantly suppressed lipid accumulation in differentiated adipocytes. It also decreased adipogenesis-related transcription factor expression, including peroxisome proliferator-activated receptor and CCAAT/enhancer-binding protein. Furthermore, ginkgolide C enhanced adipose triglyceride lipase and hormone-sensitive lipase production for lipolysis and increased phosphorylation of AMP-activated protein kinase (AMPK), resulting in decreased activity of acetyl-CoA carboxylase for fatty acid synthesis. In coculture with an AMPK inhibitor (compound C), ginkgolide C also improved activation of sirtuin 1 and phosphorylation of AMPK in differentiated 3T3-L1 cells. The results suggest that ginkgolide C is an effective flavone for increasing lipolysis and inhibiting adipogenesis in adipocytes through the activated AMPK pathway. PMID:26413119

  16. Tiliroside-derivatives enhance GLUT4 translocation via AMPK in muscle cells.

    PubMed

    Shi, Lihuan; Qin, Nan; Hu, Lijuan; Liu, Linjuan; Duan, Hongquan; Niu, Wenyan

    2011-05-01

    Tiliroside isolated from Chinese herb Potentilla chinensis showed therapeutic activities in diabetes. We synthesized 7 tiliroside-derivatives and examined their effects on surface GLUT4myc levels in muscle cells. Derivatives 2a and 3 increased surface GLUT4myc levels, and derivative 3 has the greatest potential. AMPK may be involved in tiliroside-derivatives-regulated GLUT4myc traffic.

  17. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases.

    PubMed

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems.

  18. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases

    PubMed Central

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A.; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems. PMID:24904600

  19. AMPK Agonist AICAR Improves Cognition and Motor Coordination in Young and Aged Mice

    ERIC Educational Resources Information Center

    Kobilo, Tali; Guerrieri, Davide; Zhang, Yongqing; Collica, Sarah C.; Becker, Kevin G.; van Praag, Henriette

    2014-01-01

    Normal aging can result in a decline of memory and muscle function. Exercise may prevent or delay these changes. However, aging-associated frailty can preclude physical activity. In young sedentary animals, pharmacological activation of AMP-activated protein kinase (AMPK), a transcriptional regulator important for muscle physiology, enhanced…

  20. FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores.

    PubMed

    Possik, Elite; Ajisebutu, Andrew; Manteghi, Sanaz; Gingras, Marie-Claude; Vijayaraghavan, Tarika; Flamand, Mathieu; Coull, Barry; Schmeisser, Kathrin; Duchaine, Thomas; van Steensel, Maurice; Hall, David H; Pause, Arnim

    2015-10-01

    Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis.

  1. Features of an altered AMPK metabolic pathway in Gilbert's Syndrome, and its role in metabolic health.

    PubMed

    Mölzer, Christine; Wallner, Marlies; Kern, Carina; Tosevska, Anela; Schwarz, Ursula; Zadnikar, Rene; Doberer, Daniel; Marculescu, Rodrig; Wagner, Karl-Heinz

    2016-01-01

    Energy metabolism, involving the ATP-dependent AMPK-PgC-Ppar pathway impacts metabolic health immensely, in that its impairment can lead to obesity, giving rise to disease. Based on observations that individuals with Gilbert's syndrome (GS; UGT1A1(*)28 promoter mutation) are generally lighter, leaner and healthier than controls, specific inter-group differences in the AMPK pathway regulation were explored. Therefore, a case-control study involving 120 fasted, healthy, age- and gender matched subjects with/without GS, was conducted. By utilising intra-cellular flow cytometry (next to assessing AMPKα1 gene expression), levels of functioning proteins (phospho-AMPK α1/α2, PgC 1 α, Ppar α and γ) were measured in PBMCs (peripheral blood mononucleated cells). In GS individuals, rates of phospho-AMPK α1/α2, -Ppar α/γ and of PgC 1α were significantly higher, attesting to a boosted fasting response in this condition. In line with this finding, AMPKα1 gene expression was equal between the groups, possibly stressing the post-translational importance of boosted fasting effects in GS. In reflection of an apparently improved health status, GS individuals had significantly lower BMI, glucose, insulin, C-peptide and triglyceride levels. Herewith, we propose a new theory to explain why individuals having GS are leaner and healthier, and are therefore less likely to contract metabolic diseases or die prematurely thereof. PMID:27444220

  2. FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores

    PubMed Central

    Possik, Elite; Ajisebutu, Andrew; Manteghi, Sanaz; Gingras, Marie-Claude; Vijayaraghavan, Tarika; Flamand, Mathieu; Coull, Barry; Schmeisser, Kathrin; Duchaine, Thomas; van Steensel, Maurice; Hall, David H.; Pause, Arnim

    2015-01-01

    Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5’-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis. PMID:26439621

  3. Architectural plasticity of AMPK revealed by electron microscopy and X-ray crystallography.

    PubMed

    Ouyang, Yan; Zhu, Li; Li, Yifang; Guo, Miaomiao; Liu, Yang; Cheng, Jin; Zhao, Jing; Wu, Yi

    2016-01-01

    Mammalian AMP-activated protein kinase (AMPK) acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio. The overall architecture of AMPK has been determined in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively. Accordingly proposed models have consistently revealed a key role of the α subunit linker in sensing adenosine nucleoside binding on the γ subunit and mediating allosteric regulation of kinase domain (KD) activity, whereas there are vital differences in orienting N-terminus of α subunit and locating carbohydrate-binding module (CBM) of β subunit. Given that Mg(2+), an indispensable cofactor of AMPK was present in the EM sample preparation buffer however absent when forming crystals, here we carried out further reconstructions without Mg(2+) to expectably inspect if this ion may contribute to this difference. However, no essential alteration has been found in this study compared to our early work. Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode, which might ulteriorly reveal an architectural complication of AMPK. PMID:27063142

  4. Folic acid supplementation during high-fat diet feeding restores AMPK activation via an AMP-LKB1-dependent mechanism.

    PubMed

    Sid, Victoria; Wu, Nan; Sarna, Lindsei K; Siow, Yaw L; House, James D; O, Karmin

    2015-11-15

    AMPK is an endogenous energy sensor that regulates lipid and carbohydrate metabolism. Nonalcoholic fatty liver disease (NAFLD) is regarded as a hepatic manifestation of metabolic syndrome with impaired lipid and glucose metabolism and increased oxidative stress. Our recent study showed that folic acid supplementation attenuated hepatic oxidative stress and lipid accumulation in high-fat diet-fed mice. The aim of the present study was to investigate the effect of folic acid on hepatic AMPK during high-fat diet feeding and the mechanisms involved. Male C57BL/6J mice were fed a control diet (10% kcal fat), a high-fat diet (60% kcal fat), or a high-fat diet supplemented with folic acid (26 mg/kg diet) for 5 wk. Mice fed a high-fat diet exhibited hyperglycemia, hepatic cholesterol accumulation, and reduced hepatic AMPK phosphorylation. Folic acid supplementation restored AMPK phosphorylation (activation) and reduced blood glucose and hepatic cholesterol levels. Activation of AMPK by folic acid was mediated through an elevation of its allosteric activator AMP and activation of its upstream kinase, namely, liver kinase B1 (LKB1) in the liver. Consistent with in vivo findings, 5-methyltetrahydrofolate (bioactive form of folate) restored phosphorylation (activation) of both AMPK and LKB1 in palmitic acid-treated HepG2 cells. Activation of AMPK by folic acid might be responsible for AMPK-dependent phosphorylation of HMG-CoA reductase, leading to reduced hepatic cholesterol synthesis during high-fat diet feeding. These results suggest that folic acid supplementation may improve cholesterol and glucose metabolism by restoration of AMPK activation in the liver.

  5. AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function

    PubMed Central

    Taniane, Caitlin; Farrell, Geoffrey; Arias, Irwin M.; Lippincott-Schwartz, Jennifer; Fu, Dong

    2016-01-01

    Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury. PMID:27792760

  6. Two weeks of metformin treatment enhances mitochondrial respiration in skeletal muscle of AMPK kinase dead but not wild type mice.

    PubMed

    Kristensen, Jonas M; Larsen, Steen; Helge, Jørn W; Dela, Flemming; Wojtaszewski, Jørgen F P

    2013-01-01

    Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5'AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α(2) (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice.We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.

  7. Specific deletion of AMP-activated protein kinase (α1AMPK) in mouse Sertoli cells modifies germ cell quality.

    PubMed

    Bertoldo, Michael J; Guibert, Edith; Faure, Melanie; Guillou, Florian; Ramé, Christelle; Nadal-Desbarats, Lydie; Foretz, Marc; Viollet, Benoit; Dupont, Joëlle; Froment, Pascal

    2016-03-01

    The AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis which plays a role in fertility. Complete disruption of the AMPK catalytic subunit α1 gene (α1AMPK KO) in male mice results in a decrease in litter size which is associated with the production of altered sperm morphology and motility. Because of the importance of Sertoli cells in the formation of germ cells, we have chosen to selectively disrupt α1AMPK only in the Sertoli cells in mice (Sc-α1AMPK-KO mice). Specific deletion of the α1AMPK gene in Sertoli cells resulted in a 25% reduction in male fertility associated with abnormal spermatozoa with a thin head. No clear alterations in testis morphology or modification in the number of Sertoli cells in vivo were observed, but a dysregulation in energy metabolism in Sertoli cells occurred. We have reported an increase in lactate production, in lipid droplets, and a reduction in ATP production in Sc-α1AMPK-KO Sertoli cells. These perturbations were associated with lower expression of mitochondrial markers (cytochrome c and PGC1-α). In addition another metabolic sensor, the deacetylase SIRT1, had a reduction in expression which is correlated with a decline in deacetylase activity. Finally, expression and localization of junctions forming the blood-testis barrier between Sertoli cells themselves and with germ cells were deregulated in Sc-α1AMPK-KO. In conclusion, these results suggest that dysregulation of the energy sensing machinery exclusively through disruption of α1AMPK in Sertoli cells translates to a reduction in the quality of germ cells and fertility. PMID:26772142

  8. Shizukaol D Isolated from Chloranthus japonicas Inhibits AMPK-Dependent Lipid Content in Hepatic Cells by Inducing Mitochondrial Dysfunction

    PubMed Central

    Hu, Rongkuan; Yan, Huan; Hao, Xiaojiang; Liu, Haiyang; Wu, Jiarui

    2013-01-01

    This study is the first to demonstrate that shizukaol D, a natural compound isolated from Chloranthusjaponicus, can activate AMP- activated protein kinase (AMPK), a key sensor and regulator of intracellular energy metabolism, leading to a decrease in triglyceride and cholesterol levels in HepG2 cells. Furthermore, we found that shizukaol D induces mitochondrial dysfunction by depolarizing the mitochondrial membrane and suppressing energy production, which may result in AMPK activation. Our results provide a possible link between mitochondrial dysfunction and AMPK activation and suggest that shizukaol D might be used to treat metabolic syndrome. PMID:23967345

  9. Gallic acid regulates body weight and glucose homeostasis through AMPK activation.

    PubMed

    Doan, Khanh V; Ko, Chang Mann; Kinyua, Ann W; Yang, Dong Joo; Choi, Yun-Hee; Oh, In Young; Nguyen, Nguyen Minh; Ko, Ara; Choi, Jae Won; Jeong, Yangsik; Jung, Min Ho; Cho, Won Gil; Xu, Shanhua; Park, Kyu Sang; Park, Woo Jin; Choi, Soo Yong; Kim, Hyoung Shik; Moh, Sang Hyun; Kim, Ki Woo

    2015-01-01

    Gallic acid [3,4,5-trihydroxybenzoic acid (GA)], a natural phytochemical, is known to have a variety of cellular functions including beneficial effects on metabolic syndromes. However, the molecular mechanism by which GA exerts its beneficial effects is not known. Here we report that GA plays its role through the activation of AMP-activated protein kinase (AMPK) and by regulating mitochondrial function via the activation of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Sirtuin 1 (Sirt1) knockdown significantly blunted GA's effect on PGC1α activation and downstream genes, suggesting a critical role of the AMPK/Sirt1/PGC1α pathway in GA's action. Moreover, diet-induced obese mice treated with GA showed significantly improved glucose and insulin homeostasis. In addition, the administration of GA protected diet-induced body weight gain without a change in food intake. Biochemical analyses revealed a marked activation of AMPK in the liver, muscle, and interscapular brown adipose tissue of the GA-treated mice. Moreover, uncoupling protein 1 together with other genes related to energy expenditure was significantly elevated in the interscapular brown adipose tissue. Taken together, these results indicate that GA plays its beneficial metabolic roles by activating the AMPK/Sirt1/PGC1α pathway and by changing the interscapular brown adipose tissue genes related to thermogenesis. Our study points out that targeting the activation of the AMPK/Sirt1/PGC1α pathway by GA or its derivatives might be a potential therapeutic intervention for insulin resistance in metabolic diseases.

  10. Hydrogen sulfide reduces serum triglyceride by activating liver autophagy via the AMPK-mTOR pathway.

    PubMed

    Sun, Li; Zhang, Song; Yu, Chengyuan; Pan, Zhenwei; Liu, Yang; Zhao, Jing; Wang, Xiaoyu; Yun, Fengxiang; Zhao, Hongwei; Yan, Sen; Yuan, Yue; Wang, Dingyu; Ding, Xue; Liu, Guangzhong; Li, Wenpeng; Zhao, Xuezhu; Liu, Zhaorui; Li, Yue

    2015-12-01

    Autophagy plays an important role in liver triglyceride (TG) metabolism. Inhibition of autophagy could reduce the clearance of TG in the liver. Hydrogen sulfide (H2S) is a potent stimulator of autophagic flux. Recent studies showed H2S is protective against hypertriglyceridemia (HTG) and noalcoholic fatty liver disease (NAFLD), while the mechanism remains to be explored. Here, we tested the hypothesis that H2S reduces serum TG level and ameliorates NAFLD by stimulating liver autophagic flux by the AMPK-mTOR pathway. The level of serum H2S in patients with HTG was lower than that of control subjects. Sodium hydrosulfide (NaHS, H2S donor) markedly reduced serum TG levels of male C57BL/6 mice fed a high-fat diet (HFD), which was abolished by coadministration of chloroquine (CQ), an inhibitor of autophagic flux. In HFD mice, administration of NaSH increased the LC3BII-to-LC3BI ratio and decreased the p62 protein level. Meanwhile, NaSH increased the phosphorylation of AMPK and thus reduced the phosphorylation of mTOR in a Western blot study. In cultured LO2 cells, high-fat treatment reduced the ratio of LC3BII to LC3BI and the phosphorylation of AMPK, which were reversed by the coadministration of NaSH. Knockdown of AMPK by siRNA in LO2 cells blocked the autophagic enhancing effects of NaSH. The same qualitative effect was observed in AMPKα2(-/-) mice. These results for the first time demonstrated that H2S could reduce serum TG level and ameliorate NAFLD by activating liver autophagy via the AMPK-mTOR pathway.

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

    PubMed

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

    2010-01-01

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

  12. Na,K-ATPase activity in mouse muscle is regulated by AMPK and PGC-1α.

    PubMed

    Ingwersen, Maria S; Kristensen, Michael; Pilegaard, Henriette; Wojtaszewski, Jørgen F P; Richter, Erik A; Juel, Carsten

    2011-07-01

    Na,K-ATPase activity, which is crucial for skeletal muscle function, undergoes acute and long-term regulation in response to muscle activity. The aim of the present study was to test the hypothesis that AMP kinase (AMPK) and the transcriptional coactivator PGC-1α are underlying factors in long-term regulation of Na,K-ATPase isoform (α,β and PLM) abundance and Na(+) affinity. Repeated treatment of mice with the AMPK activator AICAR decreased total PLM protein content but increased PLM phosphorylation, whereas the number of α- and β-subunits remained unchanged. The K(m) for Na(+) stimulation of Na,K-ATPase was reduced (higher affinity) after AICAR treatment. PLM abundance was increased in AMPK kinase-dead mice compared with control mice, but PLM phosphorylation and Na,K-ATPase Na(+) affinity remained unchanged. Na,K-ATPase activity and subunit distribution were also measured in mice with different degrees of PGC-1α expression. Protein abundances of α1 and α2 were reduced in PGC-1α +/- and -/- mice, and the β(1)/β(2) ratio was increased with PGC-1α overexpression (TG mice). PLM protein abundance was decreased in TG mice, but phosphorylation status was unchanged. Na,K-ATPase V (max) was decreased in PCG-1α TG and KO mice. Experimentally in vitro induced phosphorylation of PLM increased Na,K-ATPase Na(+) affinity, confirming that PLM phosphorylation is important for Na,K-ATPase function. In conclusion, both AMPK and PGC-1α regulate PLM abundance, AMPK regulates PLM phosphorylation and PGC-1α expression influences Na,K-ATPase α(1) and α(2) content and β(1)/β(2) isoform ratio. Phosphorylation of the Na,K-ATPase subunit PLM is an important regulatory mechanism.

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

    PubMed Central

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

    2012-01-01

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

  14. Beta-adrenergic stimulation of skeletal muscle HSL can be overridden by AMPK signaling.

    PubMed

    Watt, Matthew J; Steinberg, Gregory R; Chan, Stanley; Garnham, Andrew; Kemp, Bruce E; Febbraio, Mark A

    2004-09-01

    Hormone-sensitive lipase (HSL), an important regulatory enzyme for triacylglycerol hydrolysis within skeletal muscle, is controlled by beta-adrenergic signaling as well as intrinsic factors related to contraction and energy turnover. In the current study, we tested the capacity of 5'AMP-activated protein kinase (AMPK) to suppress beta-adrenergic stimulation of HSL activity. Eight male subjects completed 60 min of cycle exercise at 70% VO2 peak on two occasions: either with normal (CON) or low (LG) pre-exercise muscle glycogen content, which is known to enhance exercise-induced AMPK activity. Muscle samples were obtained before and immediately after exercise. Pre-exercise glycogen averaged 375 +/- 35 and 163 +/- 27 mmol x kg(-1) dm for CON and LG, respectively. AMPK alpha-2 was not different between trials at rest and was increased (3.7-fold, P<0.05) by exercise during LG only. HSL activity did not differ between trials at rest and increased (0 min: 1.67 +/- 0.13; 60 min: 2.60 +/- 0.26 mmol x min(-1) x kg(-1) dm) in CON. The exercise-induced increase in HSL activity was attenuated by AMPK alpha-2 activation in LG. The attenuated HSL activity during LG occurred despite higher plasma epinephrine levels (60 min: CON, 1.96 +/- 0.29 vs LG, 4.25 +/- 0.60 nM, P<0.05) compared with CON. Despite the attenuated HSL activity in LG, IMTG was decreased by exercise (0 min: 27.1 +/- 2.0; 60 min: 22.5 +/- 2.0 mmol x kg(-1) dm, P<0.05), whereas no net reduction occurred in CON. To confirm the apparent effect of AMPK on HSL activity, we performed experiments in muscle cell culture. The epineprine-induced increase in HSL activity was totally attenuated (P<0.05) by AICAR administration in L6 myotubes. These data provide new evidence indicating that AMPK is a major regulator of skeletal muscle HSL activity that can override beta-adrenergic stimulation. However, the increased IMTG degradation in LG suggests factors other than HSL activity are important for IMTG degradation.

  15. Isoquercitrin activates the AMP–activated protein kinase (AMPK) signal pathway in rat H4IIE cells

    PubMed Central

    2014-01-01

    Background Isoquercitrin, a flavonoid compound that is widely distributed in medicinal and dietary plants, possesses many biological activities, including inhibition of adipocyte differentiation. In this study, we investigated the effect of isoquercitrin on lipid accumulation and its molecular mechanisms in rat hepatoma H4IIE cells. Methods To investigate the effect of isoquercitrin on lipid accumulation, H4IIE cells were induced by FFA and the total lipid levels were detected by Oil Red O staining. Furthermore, The protein levels of AMPK and acetyl-CoA carboxylase (ACC), the gene expressions of transcriptional factor, lipogenic genes, and adiponectin receptor 1 (AdipoR1) were analyzed by Western blotting and quantitative real-time PCR. To further confirm the pathway of isoquercitrin-mediated hepatic lipid metabolism, H4IIE cells were treated with an AMPK inhibitor and AdipoR1 siRNA. Results Isoquercitrin significantly enhances AMPK phosphorylation, downregulates sterol regulatory element binding protein transcription factor 1 (SREBP-1) and fatty acid synthase (FAS) gene expressions. Pretreatment with AMPK inhibitor, significantly decreased the AMPK phosphorylation and increased FAS expression stimulated by isoquercitrin. Isoquercitrin might also upregulate the expression of AdipoR1 dose-dependently via AMPK in the presence of an AMPK inhibitor and AdipoR1 siRNA. Conclusions Isoquercitrin appears to regulate AMPK activation, thereby enhancing AdipoR1 expression, suppressing SREBP-1 and FAS expressions, and resulting in the regulation of lipid accumulation. These results suggest that isoquercitrin is a novel dietary compound that can be potentially be used to prevent lipid metabolic disorder and nonalcoholic fatty liver disease. PMID:24490657

  16. Hindbrain lactostasis regulates hypothalamic AMPK activity and metabolic neurotransmitter mRNA and protein responses to hypoglycemia.

    PubMed

    Gujar, Amit D; Ibrahim, Baher A; Tamrakar, Pratistha; Cherian, Ajeesh Koshy; Briski, Karen P

    2014-04-01

    Nerve cell metabolic activity is monitored in multiple brain regions, including the hypothalamus and hindbrain dorsal vagal complex (DVC), but it is unclear if individual metabolosensory loci operate autonomously or interact to coordinate central nervous system (CNS) reactivity to energy imbalance. This research addressed the hypothesis that hypoglycemia-associated DVC lactoprivation stimulates hypothalamic AMPK activity and metabolic neurotransmitter expression. As DVC catecholaminergic neurons express biomarkers for metabolic monitoring, we investigated whether these cells are a source of lactate deficit signaling to the hypothalamus. Caudal fourth ventricle (CV4) infusion of the glucose metabolite l-lactate during insulin-induced hypoglycemia reversed changes in DVC A2 noradrenergic, arcuate neuropeptide Y (NPY) and pro-opiomelanocortin (POMC), and lateral hypothalamic orexin-A (ORX) neuronal AMPK activity, coincident with exacerbation of hypoglycemia. Hindbrain lactate repletion also blunted hypoglycemic upregulation of arcuate NPY mRNA and protein. This treatment did not alter hypoglycemic paraventricular oxytocin (OT) and lateral hypothalamic ORX mRNA profiles, but exacerbated or reversed adjustments in OT and ORX neuropeptide synthesis, respectively. CV4 delivery of the monocarboxylate transporter inhibitor, 4-CIN, increased A2 phosphoAMPK (pAMPK), elevated circulating glucose, and stimulated feeding, responses that were attenuated by 6-hydroxydopamine pretreatment. 4-CIN-infused rats exhibited increased (NPY, ORX neurons) or decreased (POMC neurons) pAMPK concurrent with hyperglycemia. These data show that hindbrain lactoprivic signaling regulates hypothalamic AMPK and key effector neurotransmitter responses to hypoglycemia. Evidence that A2 AMPK activity is lactate-dependent, and that DVC catecholamine cells are critical for lactoprivic control of glucose, feeding, and hypothalamic AMPK, implies A2 derivation of this metabolic regulatory stimulus.

  17. AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation

    PubMed Central

    Ferretti, Anabela C.; Tonucci, Facundo M.; Hidalgo, Florencia; Almada, Evangelina; Larocca, Maria C.; Favre, Cristián

    2016-01-01

    The signaling pathways that govern survival response in hepatic cancer cells subjected to nutritional restriction have not been clarified yet. In this study we showed that liver cancer cells undergoing glucose deprivation both arrested in G0/G1 and died mainly by apoptosis. Treatment with the AMPK activator AICAR phenocopied the effect of glucose deprivation on cell survival, whereas AMPK silencing in HepG2/C3A, HuH-7 or SK-Hep-1 cells blocked the cell cycle arrest and the increase in apoptotic death induced by glucose starvation. Both AMPK and PKA were promptly activated after glucose withdrawal. PKA signaling had a dual role during glucose starvation: whereas it elicited an early decreased in cell viability, it later improved this parameter. We detected AMPK phosphorylation (AMPKα(Ser173)) by PKA, which was increased in glucose starved cells and was associated with diminution of AMPK activation. To better explore this inhibitory effect, we constructed a hepatocarcinoma derived cell line which stably expressed an AMPK mutant lacking that PKA phosphorylation site: AMPKα1(S173C). Expression of this mutant significantly decreased viability in cells undergoing glucose starvation. Furthermore, after 36 h of glucose deprivation, the index of AMPKα1(S173C) apoptotic cells doubled the apoptotic index observed in control cells. Two main remarks arise: 1. AMPK is the central signaling kinase in the scenario of cell cycle arrest and death induced by glucose starvation in hepatic cancer cells; 2. PKA phosphorylation of Ser173 comes out as a strong control point that limits the antitumor effects of AMPK in this situation. PMID:26894973

  18. The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release.

    PubMed

    Kola, Blerina; Wittman, Gábor; Bodnár, Ibolya; Amin, Faisal; Lim, Chung Thong; Oláh, Márk; Christ-Crain, Mirjam; Lolli, Francesca; van Thuijl, Hinke; Leontiou, Chrysanthia A; Füzesi, Tamás; Dalino, Paolo; Isidori, Andrea M; Harvey-White, Judith; Kunos, George; Nagy, György M; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta

    2013-12-01

    This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1-knockout (KO) and/or wild-type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1-KO or CB1 antagonist-treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1-KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1-KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin-cannabinoid-CB1-AMPK pathway.

  19. Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

    PubMed Central

    Hwang, Jin-Taek; Kwon, Dae Young; Park, Ock Jin

    2007-01-01

    Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H2O2 in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H2O2. Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H2O2-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders. PMID:18850225

  20. Enhanced amino acid utilization sustains growth of cells lacking Snf1/AMPK.

    PubMed

    Nicastro, Raffaele; Tripodi, Farida; Guzzi, Cinzia; Reghellin, Veronica; Khoomrung, Sakda; Capusoni, Claudia; Compagno, Concetta; Airoldi, Cristina; Nielsen, Jens; Alberghina, Lilia; Coccetti, Paola

    2015-07-01

    The metabolism of proliferating cells shows common features even in evolutionary distant organisms such as mammals and yeasts, for example the requirement for anabolic processes under tight control of signaling pathways. Analysis of the rewiring of metabolism, which occurs following the dysregulation of signaling pathways, provides new knowledge about the mechanisms underlying cell proliferation. The key energy regulator in yeast Snf1 and its mammalian ortholog AMPK have earlier been shown to have similar functions at glucose limited conditions and here we show that they also have analogies when grown with glucose excess. We show that loss of Snf1 in cells growing in 2% glucose induces an extensive transcriptional reprogramming, enhances glycolytic activity, fatty acid accumulation and reliance on amino acid utilization for growth. Strikingly, we demonstrate that Snf1/AMPK-deficient cells remodel their metabolism fueling mitochondria and show glucose and amino acids addiction, a typical hallmark of cancer cells.

  1. PKC and AMPK regulation of Kv1.5 potassium channels

    PubMed Central

    Andersen, Martin Nybo; Skibsbye, Lasse; Tang, Chuyi; Petersen, Frederic; MacAulay, Nanna; Rasmussen, Hanne Borger; Jespersen, Thomas

    2015-01-01

    The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K+ current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4–2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems. PMID:26043299

  2. PKC and AMPK regulation of Kv1.5 potassium channels.

    PubMed

    Andersen, Martin Nybo; Skibsbye, Lasse; Tang, Chuyi; Petersen, Frederic; MacAulay, Nanna; Rasmussen, Hanne Borger; Jespersen, Thomas

    2015-01-01

    The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K(+) current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4-2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems. PMID:26043299

  3. An Inhibited Conformation for the Protein Kinase Domain of the Saccharomyces cerevisiae AMPK Homolog Snf1

    SciTech Connect

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

    2010-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator for controlling cellular energy homeostasis. Its homolog in yeast, SNF1, is activated in response to glucose depletion and other stresses. The catalytic ({alpha}) subunit of AMPK/SNF1 in yeast (Snf1) contains a protein Ser/Thr kinase domain (KD), an auto-inhibitory domain (AID) and a region that mediates interactions with the two regulatory ({beta} and {gamma}) subunits. Here, the crystal structure of residues 41-440 of Snf1, which include the KD and AID, is reported at 2.4 {angstrom} resolution. The AID is completely disordered in the crystal. A new inhibited conformation of the KD is observed in a DFG-out conformation and with the glycine-rich loop adopting a structure that blocks ATP binding to the active site.

  4. Neuronal Tsc1/2 complex controls autophagy through AMPK-dependent regulation of ULK1.

    PubMed

    Di Nardo, Alessia; Wertz, Mary H; Kwiatkowski, Erica; Tsai, Peter T; Leech, Jarrett D; Greene-Colozzi, Emily; Goto, June; Dilsiz, Pelin; Talos, Delia M; Clish, Clary B; Kwiatkowski, David J; Sahin, Mustafa

    2014-07-15

    Tuberous sclerosis complex (TSC) is a disorder arising from mutation in the TSC1 or TSC2 gene, characterized by the development of hamartomas in various organs and neurological manifestations including epilepsy, intellectual disability and autism. TSC1/2 protein complex negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) a master regulator of protein synthesis, cell growth and autophagy. Autophagy is a cellular quality-control process that sequesters cytosolic material in double membrane vesicles called autophagosomes and degrades it in autolysosomes. Previous studies in dividing cells have shown that mTORC1 blocks autophagy through inhibition of Unc-51-like-kinase1/2 (ULK1/2). Despite the fact that autophagy plays critical roles in neuronal homeostasis, little is known on the regulation of autophagy in neurons. Here we show that unlike in non-neuronal cells, Tsc2-deficient neurons have increased autolysosome accumulation and autophagic flux despite mTORC1-dependent inhibition of ULK1. Our data demonstrate that loss of Tsc2 results in autophagic activity via AMPK-dependent activation of ULK1. Thus, in Tsc2-knockdown neurons AMPK activation is the dominant regulator of autophagy. Notably, increased AMPK activity and autophagy activation are also found in the brains of Tsc1-conditional mouse models and in cortical tubers resected from TSC patients. Together, our findings indicate that neuronal Tsc1/2 complex activity is required for the coordinated regulation of autophagy by AMPK. By uncovering the autophagy dysfunction associated with Tsc2 loss in neurons, our work sheds light on a previously uncharacterized cellular mechanism that contributes to altered neuronal homeostasis in TSC disease.

  5. GLP-1 analogue improves hepatic lipid accumulation by inducing autophagy via AMPK/mTOR pathway.

    PubMed

    He, Qin; Sha, Sha; Sun, Lei; Zhang, Jing; Dong, Ming

    2016-08-01

    The incidence of nonalcoholic fatty liver disease (NAFLD) keeps rising year by year, and NAFLD is rapidly becoming the most common liver disease worldwide. Clinical studies have found that glucagon-like peptide-1 (GLP-1) analogue, liraglutide (LRG), cannot only reduce glucose levels, but also improve hepatic lipase, especially in patients also with type 2 diabetes mellitus (T2DM). In addition, enhancing autophagy decreases lipid accumulation in hepatocytes. The aim of the present study is to explore the effect of LRG on hepatocyte steatosis and the possible role of autophagy. We set up an obesity mouse model with a high-fat diet (HFD) and induced hepatocyte steatosis with free fatty acids (FFA) in human L-O2 cells. LRG and two inhibitors of autophagy, Chloroquine (CQ) and bafilomycin A1 (Baf), were added into each group, respectively. The lipid profiles and morphological modifications of each group were tested. Immunohistochemistry, immunofluorescence staining and transmission electron microscopy (TEM) were used to measure autophagy in this study. The autophagy protein expression of SQSTM1 (P62), and LC3B, along with the signaling pathway proteins of mTOR, phosphorylated mTOR (p-mTOR), AMPK, phosphorylated AMPK (p-AMPK) and Beclin1, were evaluated by western blot. Our results showed that LRG improved hepatocyte steatosis by inducing autophagy, and the AMPK/mTOR pathway is involved. These findings suggest an important mechanism for the positive effects of LRG on hepatic steatosis, and provide new evidence for clinical use of LRG in NAFLD. PMID:27208776

  6. Phyllostachys edulis extract induces apoptosis signaling in osteosarcoma cells, associated with AMPK activation

    PubMed Central

    Chou, Chi-Wen; Cheng, Ya-Wen; Tsai, Chung-Hung

    2014-01-01

    Objective Bamboo is distributed worldwide, and its different parts are used as foods or as a traditional herb. Recently, antitumoral effects of bamboo extracts on several tumors have been increasingly reported; however, antitumoral activity of bamboo extracts on osteosarcoma remains unclear. In the present study, we investigated effects of an aqueous Phyllostachys edulis leaf extract (PEE) on osteosarcoma cells and the underlying mechanism of inhibition. Methods The growth of human osteosarcoma cell lines 143B and MG-63 and lung fibroblast MRC-5 cells was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Apoptosis was demonstrated using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay and flow cytometric analysis. Phosphorylation and protein levels were determined by immunoblotting. Results After treatment with PEE, viability of 143B and MG-63 cells was dose-dependently reduced to 36.3%±1.6% of control values, which were similar to AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside) treatments. In parallel, ratios of apoptotic cells and cells in the sub-G1 phase were significantly increased. Further investigation showed that PEE treatments led to activation of caspase cascades and changes of apoptotic mediators Bcl2, Bax, and p53. Consistently, our results revealed that PEE activated adenosine monophosphate-activated protein kinase (AMPK) signaling, and the AMPK activation was associated with the induction of apoptotic signaling. Conclusion Our results indicated that PEE suppressed the growth of 143B and MG-63 cells but moderately affected MRC-5 cells. PEE-induced apoptosis may attribute to AMPK activation and the following activation of apoptotic signaling cascades. These findings revealed that PEE possesses antitumoral activity on human osteosarcoma cells by manipulating AMPK signaling, suggesting that PEE alone or combined with regular antitumor drugs may be beneficial as osteosarcoma

  7. Activation of AMPK by Metformin Improves Left Ventricular Function and Survival in Heart Failure

    PubMed Central

    Gundewar, Susheel; Calvert, John W.; Jha, Saurabh; Toedt-Pingel, Iris; Ji, Sang Yong; Nunez, Denise; Ramachandran, Arun; Anaya-Cisneros, Mauricio; Tian, Rong; Lefer, David J.

    2009-01-01

    Clinical studies have reported that the widely used anti-hyperglycemic drug metformin significantly reduces cardiac risk factors and improves clinical outcomes in patients with heart failure. The mechanisms by which metformin exerts these cardioprotective effects remain unclear and may be independent of anti-hyperglycemic effects. We tested the hypothesis that chronic activation of AMPK with low-dose metformin exerts beneficial effects on cardiac function and survival in in vivo murine models of heart failure. Mice were subjected to permanent left coronary artery (LCA) occlusion or to 60 min LCA occlusion followed by reperfusion for 4 wks. High-resolution, two-dimensional echocardiography was performed at baseline and 4 wk post myocardial infarction to assess left ventricular (LV) dimensions and function. Metformin (125 μg/kg) administered to mice at ischemia and then daily, improved survival by 47% (p < 0.05 vs. vehicle) at 4 wk following permanent LCA occlusion. Additionally, metformin given at reperfusion and then daily, preserved LV dimensions and LV ejection fraction (p < 0.01 vs. vehicle) at 4 wk. The improvement in cardiac structure and function was associated with increases in AMPK and eNOS phosphorylation as well as increased PGC-1α expression in cardiac myocytes. Furthermore, metformin significantly improved myocardial cell mitochondrial respiration and ATP synthesis compared to vehicle. The cardioprotective effects of metformin were ablated in mice lacking functional AMPK or eNOS. This study demonstrates that metformin significantly improves left ventricular function and survival via activation of AMPK and its downstream mediators, eNOS and PGC-1α in a murine model of heart failure. PMID:19096023

  8. Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

    PubMed

    Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

    2004-01-01

    AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

  9. Metformin induces apoptosis through AMPK-dependent inhibition of UPR signaling in ALL lymphoblasts.

    PubMed

    Leclerc, Gilles M; Leclerc, Guy J; Kuznetsov, Jeffim N; DeSalvo, Joanna; Barredo, Julio C

    2013-01-01

    The outcome of patients with resistant phenotypes of acute lymphoblastic leukemia (ALL) or those who relapse remains poor. We investigated the mechanism of cell death induced by metformin in Bp- and T-ALL cell models and primary cells, and show that metformin effectively induces apoptosis in ALL cells. Metformin activated AMPK, down-regulated the unfolded protein response (UPR) demonstrated by significant decrease in the main UPR regulator GRP78, and led to UPR-mediated cell death via up-regulation of the ER stress/UPR cell death mediators IRE1α and CHOP. Using shRNA, we demonstrate that metformin-induced apoptosis is AMPK-dependent since AMPK knock-down rescued ALL cells, which correlated with down-regulation of IRE1α and CHOP and restoration of the UPR/GRP78 function. Additionally rapamycin, a known inhibitor of mTOR-dependent protein synthesis, rescued cells from metformin-induced apoptosis and down-regulated CHOP expression. Finally, metformin induced PIM-2 kinase activity and co-treatment of ALL cells with a PIM-1/2 kinase inhibitor plus metformin synergistically increased cell death, suggesting a buffering role for PIM-2 in metformin's cytotoxicity. Similar synergism was seen with agents targeting Akt in combination with metformin, supporting our original postulate that AMPK and Akt exert opposite regulatory roles on UPR activity in ALL. Taken together, our data indicate that metformin induces ALL cell death by triggering ER and proteotoxic stress and simultaneously down-regulating the physiologic UPR response responsible for effectively buffering proteotoxic stress. Our findings provide evidence for a role of metformin in ALL therapy and support strategies targeting synthetic lethal interactions with Akt and PIM kinases as suitable for future consideration for clinical translation in ALL. PMID:24009772

  10. p53 coordinates decidual sestrin 2/AMPK/mTORC1 signaling to govern parturition timing

    PubMed Central

    Cha, Jeeyeon; Yuan, Jia; Haraguchi, Hirofumi; Bartos, Amanda; Bradshaw, Heather B.; Hirota, Yasushi; Dey, Sudhansu K.

    2016-01-01

    Inflammation and oxidative stress are known risk factors for preterm birth (PTB); however, the mechanisms and pathways that influence this condition are not fully described. Previously, we showed that mTORC1 signaling is increased in mice harboring a uterine-specific deletion of transformation-related protein 53 (p53d/d mice), which exhibit premature decidual senescence that triggers spontaneous and inflammation-induced PTB. Treatment with the mTORC1 inhibitor rapamycin reduced the incidence of PTB in the p53d/d mice. Decidual senescence with heightened mTORC1 signaling is also a signature of human PTB. Here, we have identified an underlying mechanism for PTB and a potential therapeutic strategy for treating the condition. Treatment of pregnant p53d/d mice with either the antidiabetic drug metformin or the antioxidant resveratrol activated AMPK signaling and inhibited mTORC1 signaling in decidual cells. Both metformin and resveratrol protected against spontaneous and inflammation-induced PTB in p53d/d females. Using multiple approaches, we determined that p53 interacts with sestrins to coordinate an inverse relationship between AMPK and mTORC1 signaling that determines parturition timing. This signature was also observed in human decidual cells. Together, these results reveal that p53-dependent coordination of AMPK and mTORC1 signaling controls parturition timing and suggest that metformin and resveratrol have therapeutic potential to prevent PTB. PMID:27454290

  11. Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers

    PubMed Central

    Law, Betty Yuen Kwan; Mok, Simon Wing Fai; Chan, Wai Kit; Xu, Su Wei; Wu, An Guo; Yao, Xiao Jun; Wang, Jing Rong; Liu, Liang; Wong, Vincent Kam Wai

    2016-01-01

    Drug resistance hinder most cancer chemotherapies and leads to disease recurrence and poor survival of patients. Resistance of cancer cells towards apoptosis is the major cause of these symptomatic behaviours. Here, we showed that isoquinoline alkaloids, including liensinine, isoliensinine, dauricine, cepharanthine and hernandezine, putatively induce cytotoxicity against a repertoire of cancer cell lines (HeLa, A549, MCF-7, PC3, HepG2, Hep3B and H1299). Proven by the use of apoptosis-resistant cellular models and autophagic assays, such isoquinoline alkaloid-induced cytotoxic effect involves energy- and autophagy-related gene 7 (Atg7)-dependent autophagy that resulted from direct activation of AMP activated protein kinase (AMPK). Hernandezine possess the highest efficacy in provoking such cell death when compared with other examined compounds. We confirmed that isoquinoline alkaloid is structurally varied from the existing direct AMPK activators. In conclusion, isoquinoline alkaloid is a new class of compound that induce autophagic cell death in drug-resistant fibroblasts or cancers by exhibiting its direct activation on AMPK. PMID:26811496

  12. Polyphenol-Rich Propolis Extracts Strengthen Intestinal Barrier Function by Activating AMPK and ERK Signaling

    PubMed Central

    Wang, Kai; Jin, Xiaolu; Chen, Yifan; Song, Zehe; Jiang, Xiasen; Hu, Fuliang; Conlon, Michael A.; Topping, David L.

    2016-01-01

    Propolis has abundant polyphenolic constituents and is used widely as a health/functional food. Here, we investigated the effects of polyphenol-rich propolis extracts (PPE) on intestinal barrier function in human intestinal epithelial Caco-2 cells, as well as in rats. In Caco-2 cells, PPE increased transepithelial electrical resistance and decreased lucifer yellow flux. PPE-treated cells showed increased expression of the tight junction (TJ) loci occludin and zona occludens (ZO)-1. Confocal microscopy showed organized expressions in proteins related to TJ assembly, i.e., occludin and ZO-1, in response to PPE. Furthermore, PPE led to the activation of AMPK, ERK1/2, p38, and Akt. Using selective inhibitors, we found that the positive effects of PPE on barrier function were abolished in cells in which AMPK and ERK1/2 signaling were inhibited. Moreover, rats fed a diet supplemented with PPE (0.3% in the diet) exhibited increased colonic epithelium ZO-1 expression. Overall, these data suggest that PPE strengthens intestinal barrier function by activating AMPK and ERK signaling and provide novel insights into the potential application of propolis for human gut health. PMID:27164138

  13. Liraglutide reduces fatty degeneration in hepatic cells via the AMPK/SREBP1 pathway

    PubMed Central

    WANG, YAN-GUI; YANG, TIAN-LUN

    2015-01-01

    Recent studies have suggested that liraglutide could have a potential function in improving non-alcoholic fatty liver disease (NAFLD); however, the underlying molecular mechanism remains unclear. The aim of the present study was to investigate the role of the AMP-activated protein kinase (AMPK)/sterol regulatory element binding protein 1 (SREBP1) pathway in mediating the effect of liraglutide in reducing fatty degeneration in an in vitro NAFLD model. To resemble the NAFLD condition in vitro, L-02 cells were treated with 0.5 mM free fatty acids (FFAs) for 24 h. Liraglutide could affect the expression of AMPKα1, phosphorylated AMPKα1 and SREBP1 in a dose-dependent manner in FFA-exposed L-02 cells, as demonstrated by western blot analysis. The intracellular lipid accumulation was significantly decreased, as shown by oil red O staining. A significant decrease in the content of triglyceride and total cholesterol was observed when the FFA-exposed L-02 cells were incubated with liraglutide. In addition, the increased expression of liver-type fatty acid-binding protein in FFA-exposed L-02 cells was suppressed by liraglutide. These effects were reversed by compound C, an AMPK inhibitor. In conclusion, this study has demonstrated that liraglutide can reduce fatty degeneration induced by FFAs in hepatocytes, and this effect may be partially mediated by the AMPK/SREBP1 pathway. PMID:26640549

  14. AMPK-dependent modulation of hepatic lipid metabolism by nesfatin-1.

    PubMed

    Yin, Yue; Li, Ziru; Gao, Ling; Li, Yin; Zhao, Jing; Zhang, Weizhen

    2015-12-01

    The aim of this study was to characterize the mechanism by which peripheral nesfatin-1 regulates hepatic lipid metabolism. Continuous peripheral infusion of nesfatin-1 reduced adiposity and plasma levels of triglyceride and cholesterol. In mice fed high fat diet, peripheral nesfatin-1 significantly decreased hepatic steatosis measured by triglyceride content and oil red staining area and diameter. These alterations were associated with a significant reduction in lipogenesis-related transcriptional factors PPARγ and SREBP1, as well as rate-limited enzyme genes such as acaca, fasn, gpam, dgat1 and dgat2. In primary hepatocytes, nesfatin-1 inhibited both basal and oleic acid stimulated triglyceride accumulation, which was accompanied by a decrement in lipogenesis-related genes and an increase in β-oxidation-related genes. In cultured hepatocytes, nesfatin-1 increased levels of AMPK phosphorylation. Inhibition of AMPK by compound C blocked the reduction of triglyceride content elicited by nesfatin-1. Our studies demonstrate that nesfatin-1 attenuates lipid accumulation in hepatocytes by an AMPK-dependent mechanism. PMID:26363221

  15. Thapsigargin sensitizes human esophageal cancer to TRAIL-induced apoptosis via AMPK activation

    PubMed Central

    Ma, Zhiqiang; Fan, Chongxi; Yang, Yang; Di, Shouyin; Hu, Wei; Li, Tian; Zhu, Yifang; Han, Jing; Xin, Zhenlong; Wu, Guiling; Zhao, Jing; Li, Xiaofei; Yan, Xiaolong

    2016-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent for esophageal squamous cell carcinoma (ESCC). Forced expression of CHOP, one of the key downstream transcription factors during endoplasmic reticulum (ER) stress, upregulates the death receptor 5 (DR5) levels and promotes oxidative stress and cell death. In this study, we show that ER stress mediated by thapsigargin promoted CHOP and DR5 synthesis thus sensitizing TRAIL treatment, which induced ESCC cells apoptosis. These effects were reversed by DR5 siRNA in vitro and CHOP siRNA both in vitro and in vivo. Besides, chemically inhibition of AMPK by Compound C and AMPK siRNA weakened the anti-cancer effect of thapsigargin and TRAIL co-treatment. Therefore, our findings suggest ER stress effectively sensitizes human ESCC to TRAIL-mediated apoptosis via the TRAIL-DR5-AMPK signaling pathway, and that activation of ER stress may be beneficial for improving the efficacy of TRAIL-based anti-cancer therapy. PMID:27731378

  16. AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest.

    PubMed

    Doménech, Elena; Maestre, Carolina; Esteban-Martínez, Lorena; Partida, David; Pascual, Rosa; Fernández-Miranda, Gonzalo; Seco, Esther; Campos-Olivas, Ramón; Pérez, Manuel; Megias, Diego; Allen, Katherine; López, Miguel; Saha, Asish K; Velasco, Guillermo; Rial, Eduardo; Méndez, Raúl; Boya, Patricia; Salazar-Roa, María; Malumbres, Marcos

    2015-10-01

    Blocking mitotic progression has been proposed as an attractive therapeutic strategy to impair proliferation of tumour cells. However, how cells survive during prolonged mitotic arrest is not well understood. We show here that survival during mitotic arrest is affected by the special energetic requirements of mitotic cells. Prolonged mitotic arrest results in mitophagy-dependent loss of mitochondria, accompanied by reduced ATP levels and the activation of AMPK. Oxidative respiration is replaced by glycolysis owing to AMPK-dependent phosphorylation of PFKFB3 and increased production of this protein as a consequence of mitotic-specific translational activation of its mRNA. Induction of autophagy or inhibition of AMPK or PFKFB3 results in enhanced cell death in mitosis and improves the anti-tumoral efficiency of microtubule poisons in breast cancer cells. Thus, survival of mitotic-arrested cells is limited by their metabolic requirements, a feature with potential implications in cancer therapies aimed to impair mitosis or metabolism in tumour cells.

  17. AMPK Promotes Aberrant PGC1β Expression To Support Human Colon Tumor Cell Survival

    PubMed Central

    Fisher, Kurt W.; Das, Binita; Kim, Hyun Seok; Clymer, Beth K.; Gehring, Drew; Smith, Deandra R.; Costanzo-Garvey, Diane L.; Fernandez, Mario R.; Brattain, Michael G.; Kelly, David L.; MacMillan, John

    2015-01-01

    A major goal of cancer research is the identification of tumor-specific vulnerabilities that can be exploited for the development of therapies that are selectively toxic to the tumor. We show here that the transcriptional coactivators peroxisome proliferator-activated receptor gamma coactivator 1β (PGC1β) and estrogen-related receptor α (ERRα) are aberrantly expressed in human colon cell lines and tumors. With kinase suppressor of Ras 1 (KSR1) depletion as a reference standard, we used functional signature ontology (FUSION) analysis to identify the γ1 subunit of AMP-activated protein kinase (AMPK) as an essential contributor to PGC1β expression and colon tumor cell survival. Subsequent analysis revealed that a subunit composition of AMPK (α2β2γ1) is preferred for colorectal cancer cell survival, at least in part, by stabilizing the tumor-specific expression of PGC1β. In contrast, PGC1β and ERRα are not detectable in nontransformed human colon epithelial cells, and depletion of the AMPKγ1 subunit has no effect on their viability. These data indicate that Ras oncogenesis relies on the aberrant activation of a PGC1β-dependent transcriptional pathway via a specific AMPK isoform. PMID:26351140

  18. Hawthorn leaf flavonoids alleviate nonalcoholic fatty liver disease by enhancing the adiponectin/AMPK pathway.

    PubMed

    Li, Zhongping; Xu, Jiaoya; Zheng, Peiyong; Xing, Lianjun; Shen, Hongyi; Yang, Lili; Zhang, Li; Ji, Guang

    2015-01-01

    Hawthorn (Crataeguspinnatifida) belongs to the genus Rosaceae family of plants. The hawthorn leaf, Crataeguspinnatifida Bunge, is used for both condiment and medicinal purposes to prevent and treat metabolic dysfunctions, such as hyperlipidemia, hypertension, and cardiovascular disease in traditional Chinese medicine. However, its effects on nonalcoholic fatty liver disease (NAFLD) remain obscure. The purpose of the present study was to investigate the protective effect of hawthorn leaf flavonoids (HLF), the dominant bioactive extracts of hawthorn leaves, on high fat diet (HFD)-induced hepatic steatosis and to elucidate its underlying mechanisms. HLF supplementation significantly lowered body weight, liver weight, liver/body weight ratio, improved serum parameters and liver dysfunction and markedly decreased hepatic lipid accumulation in HFD-fed rats. In addition, HLF intervention dramatically increased circulating adiponectin levels and up-regulated the expression of adiponectin receptors, particularly adiponectin receptor 2 (AdipoR2) in the liver. Moreover, adenosine monophosphate (AMP)-activated protein kinase (AMPK) was also activated, as well as AMPK-mediated alteration of sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor α (PPARα) and their downstream targets. Taken together, our data suggest that HLF ameliorates hepatic steatosis by enhancing the adiponectin/AMPK pathway in the liver of HFD-induced NAFLD rats. PMID:26770322

  19. p53 coordinates decidual sestrin 2/AMPK/mTORC1 signaling to govern parturition timing.

    PubMed

    Deng, Wenbo; Cha, Jeeyeon; Yuan, Jia; Haraguchi, Hirofumi; Bartos, Amanda; Leishman, Emma; Viollet, Benoit; Bradshaw, Heather B; Hirota, Yasushi; Dey, Sudhansu K

    2016-08-01

    Inflammation and oxidative stress are known risk factors for preterm birth (PTB); however, the mechanisms and pathways that influence this condition are not fully described. Previously, we showed that mTORC1 signaling is increased in mice harboring a uterine-specific deletion of transformation-related protein 53 (p53d/d mice), which exhibit premature decidual senescence that triggers spontaneous and inflammation-induced PTB. Treatment with the mTORC1 inhibitor rapamycin reduced the incidence of PTB in the p53d/d mice. Decidual senescence with heightened mTORC1 signaling is also a signature of human PTB. Here, we have identified an underlying mechanism for PTB and a potential therapeutic strategy for treating the condition. Treatment of pregnant p53d/d mice with either the antidiabetic drug metformin or the antioxidant resveratrol activated AMPK signaling and inhibited mTORC1 signaling in decidual cells. Both metformin and resveratrol protected against spontaneous and inflammation-induced PTB in p53d/d females. Using multiple approaches, we determined that p53 interacts with sestrins to coordinate an inverse relationship between AMPK and mTORC1 signaling that determines parturition timing. This signature was also observed in human decidual cells. Together, these results reveal that p53-dependent coordination of AMPK and mTORC1 signaling controls parturition timing and suggest that metformin and resveratrol have therapeutic potential to prevent PTB.

  20. A novel LKB1 isoform enhances AMPK metabolic activity and displays oncogenic properties.

    PubMed

    Dahmani, R; Just, P-A; Delay, A; Canal, F; Finzi, L; Prip-Buus, C; Lambert, M; Sujobert, P; Buchet-Poyau, K; Miller, E; Cavard, C; Marmier, S; Terris, B; Billaud, M; Perret, C

    2015-04-30

    The LKB1 tumor suppressor gene encodes a master kinase that coordinates the regulation of energetic metabolism and cell polarity. We now report the identification of a novel isoform of LKB1 (named ΔN-LKB1) that is generated through alternative transcription and internal initiation of translation of the LKB1 mRNA. The ΔN-LKB1 protein lacks the N-terminal region and a portion of the kinase domain. Although ΔN-LKB1 is catalytically inactive, it potentiates the stimulating effect of LKB1 on the AMP-activated protein kinase (AMPK) metabolic sensor through a direct interaction with the regulatory autoinhibitory domain of AMPK. In contrast, ΔN-LKB1 negatively interferes with the LKB1 polarizing activity. Finally, combining in vitro and in vivo approaches, we showed that ΔN-LKB1 has an intrinsic oncogenic property. ΔN-LKB1 is expressed solely in the lung cancer cell line, NCI-H460. Silencing of ΔN-LKB1 decreased the survival of NCI-H460 cells and inhibited their tumorigenicity when engrafted in nude mice. In conclusion, we have identified a novel LKB1 isoform that enhances the LKB1-controlled AMPK metabolic activity but inhibits LKB1-induced polarizing activity. Both the LKB1 tumor suppressor gene and the oncogene ΔN-LKB1 are expressed from the same locus and this may account for some of the paradoxical effects of LKB1 during tumorigenesis.

  1. LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

    PubMed

    Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M

    2014-01-01

    Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation. PMID:24643070

  2. AMPK acts as a molecular trigger to coordinate glutamatergic signals and adaptive behaviours during acute starvation

    PubMed Central

    Ahmadi, Moloud; Roy, Richard

    2016-01-01

    The stress associated with starvation is accompanied by compensatory behaviours that enhance foraging efficiency and increase the probability of encountering food. However, the molecular details of how hunger triggers changes in the activity of neural circuits to elicit these adaptive behavioural outcomes remains to be resolved. We show here that AMP-activated protein kinase (AMPK) regulates neuronal activity to elicit appropriate behavioural outcomes in response to acute starvation, and this effect is mediated by the coordinated modulation of glutamatergic inputs. AMPK targets both the AMPA-type glutamate receptor GLR-1 and the metabotropic glutamate receptor MGL-1 in one of the primary circuits that governs behavioural response to food availability in C. elegans. Overall, our study suggests that AMPK acts as a molecular trigger in the specific starvation-sensitive neurons to modulate glutamatergic inputs and to elicit adaptive behavioural outputs in response to acute starvation. DOI: http://dx.doi.org/10.7554/eLife.16349.001 PMID:27642785

  3. The bovine 5' AMPK gene family: mapping and single nucleotide polymorphism detection.

    PubMed

    McKay, Stephanie D; White, Stephen N; Kata, Srinivas R; Loan, Raymond; Womack, James E

    2003-12-01

    The 5'-AMP-activated protein kinase (AMPK) family is an ancient stress response system whose primary function is regulation of cellular ATP. Activation of AMPK, which is instigated by environmental and nutritional stresses, initiates energy-conserving measures that protect the cell by inhibition and phosphorylation of key enzymes in energy-consuming biochemical pathways. The seven genes that comprise the bovine AMPK family were mapped in cattle by using a radiation hybrid panel. The seven genes mapped to six different cattle chromosomes, each with a LOD score greater than 10.0. PRKAA1 mapped to BTA 20, PRKAA2 and PRKAB2 to BTA 3, PRKAB1 to BTA 17, PRKAG1 to BTA 5, PRKAG2 to BTA 4, and PRKAG3 to BTA 2. Five of the seven genes mapped to regions expected from human/cattle comparative maps. PRKAB2 and PRKAG3, however, have not been mapped in humans. We predict these genes to be located on HSA 1 and 2, respectively. Additionally, one synonymous and one non-synonymous single nucleotide polymorphism (SNP) were detected in PRKAG3 in Bos taurus cattle. In an effort to determine ancestral origins, various herds of mixed breed cattle as well as other ruminant species were characterized for sequence variation in this region of PRKAG3. Owing to the physiological importance of this gene family, we believe that its individual genes are candidate genes for conferring resistance to diseases in cattle.

  4. AICAR induces AMPK-independent programmed necrosis in prostate cancer cells.

    PubMed

    Guo, Feng; Liu, Shuang-Qing; Gao, Xing-Hua; Zhang, Long-Yang

    2016-05-27

    AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) is an AMP-activated protein kinase (AMPK) agonist, which induces cytotoxic effect to several cancer cells. Its potential activity in prostate cancer cells and the underlying signaling mechanisms have not been extensively studied. Here, we showed that AICAR primarily induced programmed necrosis, but not apoptosis, in prostate cancer cells (LNCaP, PC-3 and PC-82 lines). AICAR's cytotoxicity to prostate cancer cells was largely attenuated by the necrosis inhibitor necrostatin-1. Mitochondrial protein cyclophilin-D (CYPD) is required for AICAR-induced programmed necrosis. CYPD inhibitors (cyclosporin A and sanglifehrin A) as well as CYPD shRNAs dramatically attenuated AICAR-induced prostate cancer cell necrosis and cytotoxicity. Notably, AICAR-induced cell necrosis appeared independent of AMPK, yet requiring reactive oxygen species (ROS) production. ROS scavengers (N-acetylcysteine and MnTBAP), but not AMPKα shRNAs, largely inhibited prostate cancer cell necrosis and cytotoxicity by AICAR. In summary, the results of the present study demonstrate mechanistic evidences that AMPK-independent programmed necrosis contributes to AICAR's cytotoxicity in prostate cancer cells. PMID:27103440

  5. 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.

  6. FK866-induced NAMPT inhibition activates AMPK and downregulates mTOR signaling in hepatocarcinoma cells

    SciTech Connect

    Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Gebhardt, Rolf; Weiss, Thomas S.; Kiess, Wieland; Garten, Antje

    2015-03-06

    Background: Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. Results: FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKα activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPKα and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. Conclusion: Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC. - Highlights: • FK866 increases cell death in p53-deficient hepatocarcinoma cells. • AMPK/mTOR signaling is dysregulated in hepatocarcinoma cells. • FK866-induced NAMPT inhibition activates AMPK

  7. Ursolic Acid Inhibits Adipogenesis in 3T3-L1 Adipocytes through LKB1/AMPK Pathway

    PubMed Central

    He, Yonghan; Li, Ying; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

    2013-01-01

    Background Ursolic acid (UA) is a triterpenoid compound with multiple biological functions. This compound has recently been reported to possess an anti-obesity effect; however, the mechanisms are less understood. Objective As adipogenesis plays a critical role in obesity, the present study was conducted to investigate the effect of UA on adipogenesis and mechanisms of action in 3T3-L1 preadipocytes. Methods and Results The 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of UA for 6 days. The cells were determined for proliferation, differentiation, fat accumulation as well as the protein expressions of molecular targets that regulate or are involved in fatty acid synthesis and oxidation. The results demonstrated that ursolic acid at concentrations ranging from 2.5 µM to 10 µM dose-dependently attenuated adipogenesis, accompanied by reduced protein expression of CCAAT element binding protein β (C/EBPβ), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT element binding protein α (C/EBPα) and sterol regulatory element binding protein 1c (SREBP-1c), respectively. Ursolic acid increased the phosphorylation of acetyl-CoA carboxylase (ACC) and protein expression of carnitine palmitoyltransferase 1 (CPT1), but decreased protein expression of fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Ursolic acid increased the phosphorylation of AMP-activated protein kinase (AMPK) and protein expression of (silent mating type information regulation 2, homolog) 1 (Sirt1). Further studies demonstrated that the anti-adipogenic effect of UA was reversed by the AMPK siRNA, but not by the Sirt1 inhibitor nicotinamide. Liver kinase B1 (LKB1), the upstream kinase of AMPK, was upregulated by UA. When LKB1 was silenced with siRNA or the inhibitor radicicol, the effect of UA on AMPK activation was diminished. Conclusions Ursolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis through the LKB1/AMPK pathway

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

    PubMed Central

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

    2015-01-01

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

  9. Knockdown of GSK3β increases basal autophagy and AMPK signalling in nutrient-laden human aortic endothelial cells

    PubMed Central

    Weikel, Karen A.; Cacicedo, José M.; Ruderman, Neil B.; Ido, Yasuo

    2016-01-01

    High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3β (GSK3β). To determine whether GSK3β affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type 2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308, C249–C263]. Presently, we found that incubation of HAECs with excess nutrients (25 mM glucose and 0.4 mM palmitate) increased GSK3β activity and impaired lysosome acidification. Suppression of GSK3β in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3β attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3β increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3β inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3β may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis. PMID:27534430

  10. LPS inhibits caspase 3-dependent apoptosis in RAW264.7 macrophages induced by the AMPK activator AICAR

    SciTech Connect

    Russe, Otto Quintus Möser, Christine V. Kynast, Katharina L. King, Tanya S. Olbrich, Katrin Grösch, Sabine Geisslinger, Gerd Niederberger, Ellen

    2014-05-09

    Highlights: • AMPK-activation induces caspase 3-dependent apoptosis in macrophages. • Apoptosis is associated with decreased mTOR and increased p21 levels. • All effects can be significantly inhibited by the TLR4 agonist lipopolysaccharide. - Abstract: AMP-activated kinase is a cellular energy sensor which is activated in stages of increased ATP consumption. Its activation has been associated with a number of beneficial effects such as decreasing inflammatory processes and the disease progress of diabetes and obesity, respectively. Furthermore, AMPK activation has been linked with induction of cell cycle arrest and apoptosis in cancer and vascular cells, indicating that it might have a therapeutic impact for the treatment of cancer and atherosclerosis. However, the impact of AMPK on the proliferation of macrophages, which also play a key role in the formation of atherosclerotic plaques and in inflammatory processes, has not been focused so far. We have assessed the influence of AICAR- and metformin-induced AMPK activation on cell viability of macrophages with and without inflammatory stimulation, respectively. In cells without inflammatory stimulation, we found a strong induction of caspase 3-dependent apoptosis associated with decreased mTOR levels and increased expression of p21. Interestingly, these effects could be inhibited by co-stimulation with bacterial lipopolysaccharide (LPS) but not by other proinflammatory cytokines suggesting that AICAR induces apoptosis via AMPK in a TLR4-pathway dependent manner. In conclusion, our results revealed that AMPK activation is not only associated with positive effects but might also contribute to risk factors by disturbing important features of macrophages. The fact that LPS is able to restore AMPK-associated apoptosis might indicate an important role of TLR4 agonists in preventing unfavorable cell death of immune cells.

  11. Targeted deletion of PTEN in cardiomyocytes renders cardiac contractile dysfunction through interruption of Pink1-AMPK signaling and autophagy.

    PubMed

    Roe, Nathan D; Xu, Xihui; Kandadi, Machender R; Hu, Nan; Pang, Jiaojiao; Weiser-Evans, Mary C M; Ren, Jun

    2015-02-01

    Phosphatase and tensin homolog (PTEN) deleted from chromosome 10 has been implicated in the maintenance of cardiac homeostasis although the underlying mechanism(s) remains elusive. We generated a murine model of cardiomyocyte-specific knockout of PTEN to evaluate cardiac geometry and contractile function, as well as the effect of metformin on PTEN deficiency-induced cardiac anomalies, if any. Cardiac histology, autophagy and related signaling molecules were evaluated. Cardiomyocyte-specific PTEN deletion elicited cardiac hypertrophy and contractile anomalies (echocardiographic and cardiomyocyte contractile dysfunction) associated with compromised intracellular Ca(2+) handling. PTEN deletion-induced cardiac hypertrophy and contractile anomalies were associated with dampened phosphorylation of PTEN-inducible kinase 1 (Pink1) and AMPK. Interestingly, administration of AMPK activator metformin (200mg/kg/d, in drinking H2O for 4weeks) rescued against PTEN deletion-induced geometric and functional defects as well as interrupted autophagy and autophagic flux in the heart. Moreover, metformin administration partially although significantly attenuated PTEN deletion-induced accumulation of superoxide. RNA interference against Pink1 in H9C2 myoblasts overtly increased intracellular ATP levels and suppressed AMPK phosphorylation, confirming the role of AMPK as a downstream target for PTEN-Pink1. Further scrutiny revealed that activation of AMPK and autophagy using metformin and rapamycin, respectively, rescued against PTEN deletion-induced mechanical anomalies with little additive effect. These data demonstrated that cardiomyocyte-specific deletion of PTEN leads to the loss of Pink1-AMPK signaling, development of cardiac hypertrophy and contractile defect. Activation of AMPK rescued against PTEN deletion-induced cardiac anomalies associated with restoration of autophagy and autophagic flux. This article is part of a Special Issue entitled: Autophagy and protein quality control

  12. Chronic activation of central AMPK attenuates glucose-stimulated insulin secretion and exacerbates hepatic insulin resistance in diabetic rats.

    PubMed

    Park, Sunmin; Kim, Da Sol; Kang, Suna; Shin, Bae Keun

    2014-09-01

    We investigated the effects of chronic AMP-activated kinase (AMPK) activation in the hypothalamus on energy and glucose metabolism in 90% pancreatectomized diabetic rats. Diabetic rats fed a high fat diet were divided into 3 groups and intracerebroventricular (ICV) administered with one of the following: 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR, AMPK activator; 80 μg/day), AICAR+compound C (AMPK inhibitor; 6.2 μg/day), or an artificial cerebrospinal fluid (control) by means of osmotic pumps for 4 weeks. In the hypothalamus, central AICAR activated the phosphorylation of AMPK whereas adding compound C suppressed the activation. AICAR increased body weight and epididymal and retroperitoneal fat mass by increasing energy intake for the first 2 weeks and decreasing energy expenditure, whereas compound C reversed the AICAR effect on energy metabolism. Indirect calorimetry revealed that ICV-AICAR decreased carbohydrate oxidation, but not fat oxidation, compared to the control. During euglycemic hyperinsulinemic clamp, central AICAR increased hepatic glucose output at hyperinsulinemic states. ICV-AICAR increased expressions of hepatic genes involved in fatty acid synthesis and decreased expression of hepatic genes related to thermogenesis whereas compound C nullified the AICAR effect. Insulin secretion in the first and second phases decreased in AICAR-treated rats at hyperglycemic clamp, but compound C nullified the decrease. However, central AICAR did not alter β-cell mass via its proliferation or apoptosis. In conclusion, chronic hypothalamic AMPK activation impaired energy metabolism and glucose homeostasis by increasing food intake, increasing hepatic glucose output and decreasing insulin secretion in diabetic rats. The impairment of energy and glucose homeostasis by AMPK activation was nullified by an AMPK inhibitor.

  13. Telmisartan prevention of LPS-induced microglia activation involves M2 microglia polarization via CaMKKβ-dependent AMPK activation.

    PubMed

    Xu, Yuan; Xu, Yazhou; Wang, Yurong; Wang, Yunjie; He, Ling; Jiang, Zhenzhou; Huang, Zhangjian; Liao, Hong; Li, Jia; Saavedra, Juan M; Zhang, Luyong; Pang, Tao

    2015-11-01

    Brain inflammation plays an important role in the pathophysiology of many psychiatric and neurological diseases. During brain inflammation, microglia cells are activated, producing neurotoxic molecules and neurotrophic factors depending on their pro-inflammatory M1 and anti-inflammatory M2 phenotypes. It has been demonstrated that Angiotensin II type 1 receptor blockers (ARBs) ameliorate brain inflammation and reduce M1 microglia activation. The ARB telmisartan suppresses glutamate-induced upregulation of inflammatory genes in cultured primary neurons. We wished to clarify whether telmisartan, in addition, prevents microglia activation through polarization to an anti-inflammatory M2 phenotype. We found that telmisartan promoted M2 polarization and reduced M1 polarization in LPS-stimulated BV2 and primary microglia cells, effects partially dependent on PPARγ activation. The promoting effects of telmisartan on M2 polarization, were attenuated by an AMP-activated protein kinase (AMPK) inhibitor or AMPK knockdown, indicating that AMPK activation participates on telmisartan effects. Moreover, in LPS-stimulated BV2 cells, telmisartan enhancement of M2 gene expression was prevented by the inhibitor STO-609 and siRNA of calmodulin-dependent protein kinase kinase β (CaMKKβ), an upstream kinase of AMPK. Furthermore, telmisartan enhanced brain AMPK activation and M2 gene expression in a mouse model of LPS-induced neuroinflammation. In addition, telmisartan reduced the LPS-induced sickness behavior in this in vivo model, and this effect was prevented by prior administration of an AMPK inhibitor. Our results indicate that telmisartan can be considered as a novel AMPK activator, suppressing microglia activation by promoting M2 polarization. Telmisartan may provide a novel, safe therapeutic approach to treat brain disorders associated with enhanced inflammation.

  14. Melanocortin-induced PKA activation inhibits AMPK activity via ERK-1/2 and LKB-1 in hypothalamic GT1-7 cells.

    PubMed

    Damm, Ellen; Buech, Thomas R H; Gudermann, Thomas; Breit, Andreas

    2012-04-01

    α-Melanocyte-stimulating hormone (α-MSH)-induced activation of the melanocortin-4 receptor in hypothalamic neurons increases energy expenditure and inhibits food intake. Active hypothalamic AMP-activated protein kinase (AMPK) has recently been reported to enhance food intake, and in vivo experiments suggested that intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of AMPK activity. However, it is not clear whether α-MSH affects AMPK via direct intracellular signaling cascades or if the release of paracrine factors is involved. Here, we used a murine, hypothalamic cell line (GT1-7 cells) and monitored AMPK phosphorylation at Thr(172), which has been suggested to increase AMPK activity. We found that α-MSH dephosphorylated AMPK at Thr(172) and consequently decreased phosphorylation of the established AMPK substrate acetyl-coenzyme A-carboxylase at Ser(79). Inhibitory effects of α-MSH on AMPK were blocked by specific inhibitors of protein kinase A (PKA) or ERK-1/2, pointing to an important role of both kinases in this process. Because α-MSH-induced activation of ERK-1/2 was blunted by PKA inhibitors, we propose that ERK-1/2 serves as a link between PKA and AMPK in GT1-7 cells. Furthermore, down-regulation of liver kinase B-1, but not inhibition of calcium-calmodulin-dependent kinase kinase-β or TGFβ-activated kinase-1 decreased basal phosphorylation of AMPK and its dephosphorylation induced by α-MSH. Thus, we propose that α-MSH inhibits AMPK activity via a linear pathway, including PKA, ERK-1/2, and liver kinase B-1 in GT1-7 cells. Given the importance of the melanocortin system in the formation of adipositas, detailed knowledge about this pathway might help to develop drugs targeting obesity.

  15. The protective effect of trimetazidine on myocardial ischemia/reperfusion injury through activating AMPK and ERK signaling pathway

    PubMed Central

    Liu, Zhenling; Chen, Ji-Mei; Huang, Huanlei; Kuznicki, Michelle; Zheng, Shaoyi; Sun, Wanqing; Quan, Nanhu; Wang, Lin; Yang, Hui; Guo, Hui-Ming; Li, Ji; Zhuang, Jian; Zhu, Ping

    2016-01-01

    Introduction Trimetazidine (TMZ) is an anti-anginal drug that has been widely used in Europe and Asia. The TMZ can optimize energy metabolism via inhibition of long-chain 3-ketoacyl CoA thiolase (3-KAT) in the heart, with subsequent decrease in fatty acid oxidation and stimulation of glucose oxidation. However, the mechanism by which TMZ aids in cardioprotection against ischemic injury has not been characterized. AMP-activated protein kinase (AMPK) is an energy sensor that controls ATP supply from substrate metabolism and protects heart from energy stress. TMZ changes the cardiac AMP/ATP ratio by modulating fatty acid oxidation, thereby triggering AMPK signaling cascade that contributes to the protection of the heart from ischemia/reperfusion (I/R) injury. Methods The mouse model of in vivo regional ischemia and reperfusion by the ligation of the left anterior descending coronary artery (LAD) was used for determination of myocardial infarction. The infarct size was compared between C57BL/6J WT mice and AMPK kinase dead (KD) transgenic mice with or without TMZ treatment. The ex vivo working heart perfusion system was used to monitor the effect of TMZ on glucose oxidation and fatty acid oxidation in the heart. Results TMZ treatment significantly stimulates cardiac AMPK and extracellular signal-regulated kinase (ERK) signaling pathways (p < 0.05 vs. vehicle group). The administration of TMZ reduces myocardial infarction size in WT C57BL/6J hearts, the reduction of myocardial infarction size by TMZ in AMPK KD hearts was significantly impaired versus WT hearts (p < 0.05). Intriguingly, the administration of ERK inhibitor, PD98059, to AMPK KD mice abolished the cardioprotection of TMZ against I/R injury. The ex vivo working heart perfusion data demonstrated that TMZ treatment significantly activates AMPK signaling and modulating the substrate metabolism by shifting fatty acid oxidation to glucose oxidation during reperfusion, leading to reduction of oxidative stress in

  16. Novel mechanisms of Na+ retention in obesity: phosphorylation of NKCC2 and regulation of SPAK/OSR1 by AMPK.

    PubMed

    Davies, Matthew; Fraser, Scott A; Galic, Sandra; Choy, Suet-Wan; Katerelos, Marina; Gleich, Kurt; Kemp, Bruce E; Mount, Peter F; Power, David A

    2014-07-01

    Enhanced tubular reabsorption of salt is important in the pathogenesis of obesity-related hypertension, but the mechanisms remain poorly defined. To identify changes in the regulation of salt transporters in the kidney, C57BL/6 mice were fed a 40% fat diet [high-fat diet (HFD)] or a 12% fat diet (control diet) for 14 wk. Compared with control diet-fed mice, HFD-fed mice had significantly greater elevations in weight, blood pressure, and serum insulin and leptin levels. When we examined Na(+) transporter expression, Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) was unchanged in whole kidney and reduced in the cortex, Na(+)-Cl(-) cotransporter (NCC) and α-epithelial Na(+) channel (ENaC) and γ-ENaC were unchanged, and β-ENaC was reduced. Phosphorylation of NCC was unaltered. Activating phosphorylation of NKCC2 at S126 was increased 2.5-fold. Activation of STE-20/SPS1-related proline-alanine-rich protein kinase (SPAK)/oxidative stress responsive 1 kinase (OSR1) was increased in kidneys from HFD-fed mice, and enhanced phosphorylation of NKCC2 at T96/T101 was evident in the cortex. Increased activity of NKCC2 in vivo was confirmed with diuretic experiments. HFD-fed mice had reduced activating phosphorylation of AMP-activated protein kinase (AMPK) in the renal cortex. In vitro, activation of AMPK led to a reduction in phospho-SPAK/phospho-OSR1 in AMPK(+/+) murine embryonic fibroblasts (MEFs), but no effect was seen in AMPK(-/-) MEFs, indicating an AMPK-mediated effect. Activation of the with no lysine kinase/SPAK/OSR1 pathway with low-NaCl solution invoked a greater elevation in phospho-SPAK/phospho-OSR1 in AMPK(-/-) MEFs than in AMPK(+/+) MEFs, consistent with a negative regulatory effect of AMPK on SPAK/OSR1 phosphorylation. In conclusion, this study identifies increased phosphorylation of NKCC2 on S126 as a hitherto-unrecognized mediator of enhanced Na(+) reabsorption in obesity and identifies a new role for AMPK in regulating the activity of SPAK/OSR1.

  17. 1,3-Dichloro-2-propanol induced hyperlipidemia in C57BL/6J mice via AMPK signaling pathway.

    PubMed

    Lu, Jing; Huang, Guoren; Hu, Sizhuo; Wang, Zhenning; Guan, Shuang

    2014-02-01

    1,3-Dichloro-2-propanol (1,3-DCP) is a well-known contaminant that has been detected in a wide range of foods. Dietary intake represents the greatest source of exposure to 1,3-DCP. In the study, we first found 1,3-DCP could induce hyperlipidemia in C57BL/6J mice below 1 mg/kg/day. We investigated serum lipid profile, liver total cholesterol (TC) and triglyceride (TG), histopathology of Liver and adipose tissue. The results showed 1,3-DCP dose dependently increased serum TG, TC and low-density lipoprotein cholesterol (LDL-C), decreased serum high-density lipoprotein cholesterol (HDL-C), increased relative liver weight, liver TG and TC, relative adipose tissue weight and enlarged the size of adipose cells. Because AMPK signal pathway is important in the process of lipid metabolism, we further investigated the effects of 1,3-DCP on AMPK signaling pathway in murine models. The results showed that 1,3-DCP (0.1-1 mg/kg/day) decreased p-AMPK/tAMPK ratio, p-ACC/tACC ratio, PPARα expression, but increased FAT, SREBP1, HMGCR and FAS expression. These observations indicated that 1,3-DCP induced hyperlipidemia in C57BL/6J mice at least partially through regulating AMPK signaling pathway. PMID:24333398

  18. Demethyleneberberine attenuates non-alcoholic fatty liver disease with activation of AMPK and inhibition of oxidative stress.

    PubMed

    Qiang, Xiaoyan; Xu, Lulu; Zhang, Miao; Zhang, Pengcheng; Wang, Yinhang; Wang, Yongchen; Zhao, Zheng; Chen, Huan; Liu, Xie; Zhang, Yubin

    2016-04-15

    Non-alcoholic fatty liver disease (NAFLD) has reached an epidemic level globally, which is recognized to form non-alcoholic steatohepatitis (NASH) by the "two-hit" model, including oxidative stress and inflammation. AMP-activated protein kinase (AMPK) has long been regarded as a key regulator of energy metabolism, which is recognized as a critical target for NAFLD treatment. Here we introduce a natural product, demethyleneberberine (DMB), which potentially ameliorated NAFLD by activating AMPK pathways. Our study showed that the intraperitoneal injection of DMB (20 or 40 mg/kg body weight) decreased hepatic lipid accumulation in methionine and choline deficient (MCD) high-fat diet feeding mice and db/db mice. The further investigation demonstrated that DMB activated AMPK by increasing its phosphorylation in vitro and in vivo. Accompanied with AMPK activation, the expression of lipogenic genes were significantly reduced while genes responsible for the fatty acid β-oxidation were restored in DMB-treated NAFLD mice. In addition, the remarkable oxidative damage and inflammation induced by NAFLD were both attenuated by DMB treatment, which is reflected by decreased lipid oxidative product, malonaldehyde (MDA) and inflammatory factors, tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). Based on all above, DMB could serve as a novel AMPK activator for treating NAFLD and preventing the pathologic progression from NAFLD to NASH by inhibiting the oxidative stress and inflammation.

  19. EGCG Blocked Phenylephrin-Induced Hypertrophy in H9C2 Cardiomyocytes, by Activating AMPK-Dependent Pathway.

    PubMed

    Cai, Yi; Zhao, Li; Qin, Yuan; Wu, Xiao-Qian

    2015-05-01

    AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism. Previous studies have shown that activation of AMPK results in suppression of cardiac myocyte hypertrophy via inhibition of the p70S6 kinase (p70S6K) and eukaryotic elongation factor-2 (eEF2) signaling pathways. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea, possesses multiple protective effects on the cardiovascular system including cardiac hypertrophy. However, the molecular mechanisms has not been well investigated. In this study, we found that EGCG could significantly reduce natriuretic peptides type A (Nppa), brain natriuretic polypeptide (BNP) mRNA expression and decrease cell surface area in H9C2 cardiomyocytes stimulated with phenylephrine (PE). Moreover, we showed that AMPK is activated in H9C2 cardiomyocytes by EGCG, and AMPK-dependent pathway participates in the inhibitory effects of EGCG on cardiac hypertrophy. Taken together, our findings provide the first evidence that the effect of EGCG against cardiac hypertrophy may be attributed to its activation on AMPK-dependent signaling pathway, suggesting the therapeutic potential of EGCG on the prevention of cardiac remodeling in patients with pressure overload hypertrophy. PMID:25954124

  20. AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

    PubMed Central

    Johanns, M.; Lai, Y.-C.; Hsu, M.-F.; Jacobs, R.; Vertommen, D.; Van Sande, J.; Dumont, J. E.; Woods, A.; Carling, D.; Hue, L.; Viollet, B.; Foretz, M; Rider, M H

    2016-01-01

    Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation. PMID:26952277

  1. Activation of AMPK/MnSOD signaling mediates anti-apoptotic effect of hepatitis B virus in hepatoma cells

    PubMed Central

    Li, Lei; Hong, Hong-Hai; Chen, Shi-Ping; Ma, Cai-Qi; Liu, Han-Yan; Yao, Ya-Chao

    2016-01-01

    AIM: To investigate the anti-apoptotic capability of the hepatitis B virus (HBV) in the HepG2 hepatoma cell line and the underlying mechanisms. METHODS: Cell viability and apoptosis were measured by MTT assay and flow cytometry, respectively. Targeted knockdown of manganese superoxide dismutase (MnSOD), AMP-activated protein kinase (AMPK) and hepatitis B virus X protein (HBx) genes as well as AMPK agonist AICAR and antagonist compound C were employed to determine the correlations of expression of these genes. RESULTS: HBV markedly protected the hepatoma cells from growth suppression and cell death in the condition of serum deprivation. A decrease of superoxide anion production accompanied with an increase of MnSOD expression and activity was found in HepG2.215 cells. Moreover, AMPK activation contributed to the up-regulation of MnSOD. HBx protein was identified to induce the expression of AMPK and MnSOD. CONCLUSION: Our results suggest that HBV suppresses mitochondrial superoxide level and exerts an anti-apoptotic effect by activating AMPK/MnSOD signaling pathway, which may provide a novel pharmacological strategy to prevent HCC. PMID:27158203

  2. Activation of AMPK attenuates LPS-induced acute lung injury by upregulation of PGC1α and SOD1

    PubMed Central

    Wang, Guizuo; Song, Yang; Feng, Wei; Liu, Lu; Zhu, Yanting; Xie, Xinming; Pan, Yilin; Ke, Rui; Li, Shaojun; Li, Fangwei; Yang, Lan; Li, Manxiang

    2016-01-01

    Evidence suggests that an imbalance between oxidation and antioxidation is involved in the pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Activation of AMP-activated protein kinase (AMPK) has been shown to inhibit the occurrence of ALI/ARDS. However, it is unknown whether activation of AMPK benefits ALI/ARDS by restoration of the oxidant and antioxidant balance, and which mechanisms are responsible for this process. The present study aimed to address these issues. Lipopolysaccharide (LPS) induced pronounced pathological changes of ALI in mice; these were accompanied by elevated production of malondialdehyde (MDA) and decreased activity of superoxide dismutase (SOD) compared with control mice. Prior treatment of mice with the AMPK agonist metformin significantly suppressed the LPS-induced development of ALI, reduced the elevation of MDA and increased the activity of SOD. Further analysis indicated that activation of AMPK also stimulated the protein expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and superoxide dismutase 1 (SOD1). This study suggests that activation of AMPK by metformin inhibits oxidative stress by upregulation of PGC1α and SOD1, thereby suppressing the development of ALI/ARDS, and has potential value in the clinical treatment of such conditions. PMID:27602077

  3. AMPK activation as a strategy for reversing the endothelial lipotoxicity underlying the increased vascular risk associated with insulin resistance syndrome.

    PubMed

    McCarty, Mark F

    2005-01-01

    The endotheliopathy associated with insulin resistance syndrome appears to result largely from excessive free fatty acid (FFA) exposure that boosts endothelial production of diacylglycerol, thereby activating protein kinase C. This endothelial "lipotoxicity" can be alleviated by very-low-fat diets and by appropriate weight loss. In addition, pharmacological activation of endothelial AMP-activated kinase (AMPK), as with the drug metformin, has the potential to decrease the FFA content of endothelial cells by stimulating fat oxidation; AMPK may also suppress endothelial de novo synthesis of diacylglycerol by inhibiting glycerol-3-phosphate acyltransferase. These considerations may rationalize the superior impact of metformin therapy on the macrovascular health of diabetics. More generally, metformin - or, preferably, better tolerated activators of AMPK - may have considerable potential for promoting vascular health in the large proportion of the adult population afflicted with insulin resistance syndrome. PMID:15823720

  4. Glucose-based regulation of miR-451/AMPK signaling depends on the OCT1 transcription factor.

    PubMed

    Ansari, Khairul I; Ogawa, Daisuke; Rooj, Arun K; Lawler, Sean E; Krichevsky, Anna M; Johnson, Mark D; Chiocca, E Antonio; Bronisz, Agnieszka; Godlewski, Jakub

    2015-05-12

    In aggressive, rapidly growing solid tumors such as glioblastoma multiforme (GBM), cancer cells face frequent dynamic changes in their microenvironment, including the availability of glucose and other nutrients. These challenges require that tumor cells have the ability to adapt in order to survive periods of nutrient/energy starvation. We have identified a reciprocal negative feedback loop mechanism in which the levels of microRNA-451 (miR-451) are negatively regulated through the phosphorylation and inactivation of its direct transcriptional activator OCT1 by 5' AMP-activated protein kinase (AMPK), which is activated by glucose depletion-induced metabolic stress. Conversely, in a glucose-rich environment, unrestrained expression of miR-451 suppresses AMPK pathway activity. These findings uncover miR-451 as a major effector of glucose-regulated AMPK signaling, allowing tumor cell adaptation to variations in nutrient availability in the tumor microenvironment. PMID:25937278

  5. Environmental and Genetic Preconditioning for Long-Term Anoxia Responses Requires AMPK in Caenorhabditis elegans

    PubMed Central

    LaRue, Bobby L.; Padilla, Pamela A.

    2011-01-01

    Background Preconditioning environments or therapeutics, to suppress the cellular damage associated with severe oxygen deprivation, is of interest to our understanding of diseases associated with oxygen deprivation. Wildtype C. elegans exposed to anoxia enter into a state of suspended animation in which energy-requiring processes reversibly arrest. C. elegans at all developmental stages survive 24-hours of anoxia exposure however, the ability of adult hermaphrodites to survive three days of anoxia significantly decreases. Mutations in the insulin-like signaling receptor (daf-2) and LIN-12/Notch (glp-1) lead to an enhanced long-term anoxia survival phenotype. Methodology/Principal Findings In this study we show that the combined growth environment of 25°C and a diet of HT115 E. coli will precondition adult hermaphrodites to survive long-term anoxia; many of these survivors have normal movement after anoxia treatment. Animals fed the drug metformin, which induces a dietary-restriction like state in animals and activates AMPK in mammalian cell culture, have a higher survival rate when exposed to long-term anoxia. Mutations in genes encoding components of AMPK (aak-2, aakb-1, aakb-2, aakg-2) suppress the environmentally and genetically induced long-term anoxia survival phenotype. We further determine that there is a correlation between the animals that survive long-term anoxia and increased levels of carminic acid staining, which is a fluorescent dye that incorporates in with carbohydrates such as glycogen. Conclusions/Significance We conclude that small changes in growth conditions such as increased temperature and food source can influence the physiology of the animal thus affecting the responses to stress such as anoxia. Furthermore, this supports the idea that metformin should be further investigated as a therapeutic tool for treatment of oxygen-deprived tissues. Finally, the capacity for an animal to survive long bouts of severe oxygen deprivation is likely

  6. Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer

    PubMed Central

    Chen, Wei-Ching; Chang, Yung-Sheng; Hsu, Hui-Ping; Yen, Meng-Chi; Huang, Hau-Lun; Cho, Chien-Yu; Wang, Chih-Yang; Weng, Tzu-Yang; Lai, Po-Ting; Chen, Ching-Shih; Lin, Yih-Jyh; Lai, Ming-Derg

    2015-01-01

    CD90 is used as a marker for cancer stem cell in liver cancer. We aimed to study the mechanism by which CD90 promoted liver cancer progression and identify the new therapeutic targets on CD90 signal pathway. Ectopic expression of CD90 in liver cancer cell lines enhanced anchorage-independent growth and tumor progression. Furthermore, CD90 promoted sphere formation in vitro and upregulated the expression of the cancer stem cell marker CD133. The CD133 expression was higher in CD45-CD90+ cells in liver cancer specimen. The natural carcinogenic molecules TGF-β-1, HGF, and hepatitis B surface antigen increased the expression of CD90 and CD133. Inhibition of CD90 by either shRNA or antibody attenuated the induction of CD133 and anchorage-independent growth. Lentiviral delivery of CD133 shRNA abolished the tumorigenicity induced by CD90. Ectopic expression of CD90 induced mTOR phosphorylation and AMPK dephosphorylation. Mutation of integrin binding-RLD domain in CD90 attenuated the induction of CD133 and anchorage-independent growth. Similar results were observed after silencing β3 integrin. Signaling analyses revealed that AMPK/mTOR and β3 integrin were required for the induction of CD133 and tumor formation by CD90. Importantly, the energy restriction mimetic agent OSU-CG5 reduced the CD90 population in fresh liver tumor sample and repressed the tumor growth. In contrast, sorafenib did not decrease the CD90+ population. In conclusion, the signal axis of CD90-integrin-mTOR/AMPK-CD133 is critical for promoting liver carcinogenesis. Molecules inhibiting the signal axis, including OSU-CG5 and other inhibitors, may serve as potential novel cancer therapeutic targets in liver cancer. PMID:26556861

  7. Diosgenin regulates adipokine expression in perivascular adipose tissue and ameliorates endothelial dysfunction via regulation of AMPK.

    PubMed

    Chen, Yan; Xu, Xiaoshan; Zhang, Yuanyuan; Liu, Kang; Huang, Fang; Liu, Baolin; Kou, Junping

    2016-01-01

    Perivascular adipose tissue (PVAT) has been recognized as an active contributor to vascular function due to its paracrine effects on cells contained within vascular wall. The present study was designed to investigate the effect of diosgenin on adipokine expression in PVAT with emphasis on the regulation of endothelial function. Palmitic acid (PA) stimulation induced inflammation and dysregulation of adipokine expression in PVAT. Diosgenin treatment inhibited IKKβ phosphorylation and downregulated mRNA expressions of proinflammatory cytokines/proteins including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein (MCP-1), and inducible nitric oxide synthase (iNOS), while reduced gene expressions for adiponectin, PPARγ, and arginase 1 (Arg-1) were reversed by diosgenin treatment. Diosgenin enhanced AMPK phosphorylation under basal and inflammatory conditions in PVAT, whereas knockdown of AMPK by SiRNA diminished its modulatory effect, indicating that diosgenin inhibited inflammation in an AMPK-dependent manner. We prepared conditioned medium from PA-stimulated PVAT to induce endothelial dysfunction and found that pre-treatment of PVAT with diosgenin effectively restored the loss of ACh-induced vasodilation and increased eNOS phosphorylation in rat aorta. High-fat diet feeding in rats induced inflammation in PVAT and the impairment of endothelium-dependent vasodilation, whereas these alterations were prevented by oral administration of diosgenin at doses of 20 and 40 mg/kg. In conclusion, the obtained data showed that diosgenin ameliorated inflammation-associated adipokine dysregulation, and thereby prevented endothelial dysfunction. Our findings would shed a novel insight into the potential mechanism by which diosgenin protected endothelial function against inflammatory insult. PMID:26277096

  8. Combined pharmacological activation of AMPK and PPARδ potentiates the effects of exercise in trained mice.

    PubMed

    Manio, Mark Christian C; Inoue, Kazuo; Fujitani, Mina; Matsumura, Shigenobu; Fushiki, Tohru

    2016-03-01

    The combined activation of the cellular energy sensor AMP-activated protein kinase (AMPK) and the nuclear transcription factor peroxisome proliferator-activated receptor delta (PPARδ) has been demonstrated to improve endurance and muscle function by mimicking the effects of exercise training. However, their combined pharmacological activation with exercise training has not been explored. Balb/c mice were trained on a treadmill and administered both the AMPK activator AICAR and the PPARδ agonist GW0742 for 4 weeks. AICAR treatment potentiated endurance, but the combination of AICAR and GW0742 further potentiated endurance and increased all running parameters significantly relative to exercised and nonexercised groups (138-179% and 355% increase in running time, respectively). Despite the lack of change in basal whole-body metabolism, a significant shift to fat as the main energy source with a decline in carbohydrate utilization was observed upon indirect calorimetry analysis at the period near exhaustion. Increased energy substrates before exercise, and elevated muscle nonesterified fatty acids (NEFA) and elevated muscle glycogen at exhaustion were observed together with increased PDK4 mRNA expression. Citrate synthase activity was elevated in AICAR-treated groups, while PGC-1α protein level tended to be increased in GW0742-treated groups. At exhaustion, Pgc1a was robustly upregulated together with Pdk4, Cd36, and Lpl in the muscle. A robust upregulation of Pgc1a and a downregulation in Chrebp were observed in the liver. Our data show that combined pharmacological activation of AMPK and PPARδ potentiates endurance in trained mice by transcriptional changes in muscle and liver, increased available energy substrates, delayed hypoglycemia through glycogen sparing accompanied by increased NEFA availability, and improved substrate shift from carbohydrate to fat. PMID:26997622

  9. Cigarette smoke exposure triggers the autophagic cascade via activation of the AMPK pathway in mice.

    PubMed

    Furlong, Hayley C; Stämpfli, Martin R; Gannon, Anne M; Foster, Warren G

    2015-10-01

    We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 wk, using a whole-body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of proautophagic genes Cdkn1b, Map1lc3a, Bad, and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3, and Maplc31b expression, as well as a significant decrease in Akt1 and Mtor expression, was detected by quantitative PCR. The 5'-AMP-activated protein kinase catalytic subunit (AMPK) alpha1 + alpha2 and ATG7 protein expression was significantly increased, whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed versus control ovaries. Up-regulation of AMPK alpha1 + alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two prosurvival factors, AKT and mTOR, were decreased in expression, an outcome that favors induction of the autophagy pathway, whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of antiautophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility.

  10. Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer.

    PubMed

    Chen, Wei-Ching; Chang, Yung-Sheng; Hsu, Hui-Ping; Yen, Meng-Chi; Huang, Hau-Lun; Cho, Chien-Yu; Wang, Chih-Yang; Weng, Tzu-Yang; Lai, Po-Ting; Chen, Ching-Shih; Lin, Yih-Jyh; Lai, Ming-Derg

    2015-12-15

    CD90 is used as a marker for cancer stem cell in liver cancer. We aimed to study the mechanism by which CD90 promoted liver cancer progression and identify the new therapeutic targets on CD90 signal pathway. Ectopic expression of CD90 in liver cancer cell lines enhanced anchorage-independent growth and tumor progression. Furthermore, CD90 promoted sphere formation in vitro and upregulated the expression of the cancer stem cell marker CD133. The CD133 expression was higher in CD45-CD90+ cells in liver cancer specimen. The natural carcinogenic molecules TGF-β-1, HGF, and hepatitis B surface antigen increased the expression of CD90 and CD133. Inhibition of CD90 by either shRNA or antibody attenuated the induction of CD133 and anchorage-independent growth. Lentiviral delivery of CD133 shRNA abolished the tumorigenicity induced by CD90. Ectopic expression of CD90 induced mTOR phosphorylation and AMPK dephosphorylation. Mutation of integrin binding-RLD domain in CD90 attenuated the induction of CD133 and anchorage-independent growth. Similar results were observed after silencing β3 integrin. Signaling analyses revealed that AMPK/mTOR and β3 integrin were required for the induction of CD133 and tumor formation by CD90. Importantly, the energy restriction mimetic agent OSU-CG5 reduced the CD90 population in fresh liver tumor sample and repressed the tumor growth. In contrast, sorafenib did not decrease the CD90+ population. In conclusion, the signal axis of CD90-integrin-mTOR/AMPK-CD133 is critical for promoting liver carcinogenesis. Molecules inhibiting the signal axis, including OSU-CG5 and other inhibitors, may serve as potential novel cancer therapeutic targets in liver cancer.

  11. AMPK is involved in the differential neonatal performance of chicks hatching at different time.

    PubMed

    Wang, Yufeng; Buyse, Johan; Song, Zhigang; Decuypere, Eddy; Everaert, Nadia

    2016-03-01

    We have recently reported that the hatching time may be in relation to the distinct neonatal performance of female chicks. The present study was aimed to investigate the potential involvement of AMPK, an energy sensor which plays a pivotal role in energy homeostasis, in the distinct performance of the spread of hatching time model. As a result, hypothalamic AMPKα1 isoform gene expression was significantly higher in the late hatcher as compared to that of their early counterparts, whereas the total and phosphorylated levels of AMPKα subunit did not differ between the three hatchers. The hypothalamic orexigenic NPY and AgRP mRNA levels were higher in the late hatchers as compared to the early, and that of the middle hatchers was at an intermediate level. However, the anorexigenic POMC and CRH was also higher expressed in the late hatchers as compared to the early hatchers. In the liver, AMPKα2 mRNA level and the phosphorylation ratio of AMPKα was significantly lower in the late hatchers, as compared to their early counterparts. The hepatic phosphorylated GS levels of the late and middle hatchers were lower than that of their early counterparts. The expression of hepatic FTO gene of the late hatchers was significantly higher than that of their early and middle counterparts. Taken together, AMPK may play a significant role in the different neonatal performance of the spread of hatching time model. The central and peripheral AMPK in late hatchers exhibited a pattern of higher energy intake and lower energy expenditure, which resulted in a faster post-hatch growth.

  12. Cigarette Smoke Exposure Triggers the Autophagic Cascade via Activation of the AMPK Pathway in Mice1

    PubMed Central

    Furlong, Hayley C.; Stämpfli, Martin R.; Gannon, Anne M.; Foster, Warren G.

    2015-01-01

    We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 wk, using a whole-body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of proautophagic genes Cdkn1b, Map1lc3a, Bad, and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3, and Maplc31b expression, as well as a significant decrease in Akt1 and Mtor expression, was detected by quantitative PCR. The 5′-AMP-activated protein kinase catalytic subunit (AMPK) alpha1 + alpha2 and ATG7 protein expression was significantly increased, whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed versus control ovaries. Up-regulation of AMPK alpha1 + alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two prosurvival factors, AKT and mTOR, were decreased in expression, an outcome that favors induction of the autophagy pathway, whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of antiautophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility. PMID:26377221

  13. Disturbed adiponectin – AMPK system in skeletal muscle of patients with metabolic syndrome.

    PubMed

    Van Berendoncks, An M; Stensvold, Dorthe; Garnier, Anne; Fortin, Dominique; Sente, Tahnee; Vrints, Christiaan J; Arild, Slørdahl Stig; Ventura-Clapier, Renee; Wisløff, Ulrik; Conraads, Viviane M

    2015-02-01

    Patients with metabolic syndrome are characterized by low circulating adiponectin levels and reduced adiponectin sensitivity in skeletal muscles. Through binding on its main skeletal muscle receptor AdipoR1, adiponectin activates AMP-activated protein kinase (AMPK), a key player in energy homeostasis. Fourteen metabolic syndrome patients and seven healthy control subjects were included. Blood samples were taken to determine insulin resistance, adiponectin, lipoproteins, and C-reactive protein. Muscle biopsies (m. vastus lateralis) were obtained to assess mRNA expression of AdipoR1 and both AMPKα1 and AMPKα2 subunits, as well as downstream targets in lipid and glucose metabolism. Skeletal muscle mRNA expression of AMPKα1 and AMPKα2 was lower in metabolic syndrome patients (100 ± 6 vs. 122 ± 8 AU, p = 0.030 and 64 ± 4 vs. 85 ± 9 AU, p = 0.044, respectively), whereas the expression of AdipoR1 was upregulated (138 ± 9 vs. 105 ± 7, p = 0.012). AMPKα1 and AdipoR1 correlated positively in both the control (r = 0.964, p < 0.001) and the metabolic syndrome group (r = 0.600, p = 0.023). However, this relation was shifted upwards in metabolic syndrome patients, indicating increased AdipoR1mRNA expression for a similar AMPKα1 expression. Previously, a blunted stimulatory effect of adiponectin on AMPK activation has been shown in metabolic syndrome patients. The present data suggest that the disturbed interaction of adiponectin with AMPK is located downstream of the AdipoR1 receptor.

  14. AMPK Reverses the Mesenchymal Phenotype of Cancer Cells by Targeting the Akt-MDM2-Foxo3a Signaling Axis

    PubMed Central

    Chou, Chih-Chien; Lee, Kuen-Haur; Lai, I-Lu; Wang, Dasheng; Mo, Xiaokui; Kulp, Samuel K.; Shapiro, Charles L.; Chen, Ching-Shih

    2014-01-01

    In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacological activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis. PMID:24994714

  15. AMPK reverses the mesenchymal phenotype of cancer cells by targeting the Akt-MDM2-Foxo3a signaling axis.

    PubMed

    Chou, Chih-Chien; Lee, Kuen-Haur; Lai, I-Lu; Wang, Dasheng; Mo, Xiaokui; Kulp, Samuel K; Shapiro, Charles L; Chen, Ching-Shih

    2014-09-01

    In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacologic activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis.

  16. Ampelopsin protects endothelial cells from hyperglycemia-induced oxidative damage by inducing autophagy via the AMPK signaling pathway.

    PubMed

    Liang, Xinyu; Zhang, Ting; Shi, Linying; Kang, Chao; Wan, Jing; Zhou, Yong; Zhu, Jundong; Mi, Mantian

    2015-01-01

    Diabetic angiopathy is a major diabetes-specific complication that often begins with endothelial dysfunction induced by hyperglycemia; however, the pathological mechanisms of this progression remain unclear. Ampelopsin is a natural flavonol that has strong antioxidant activity, but little information is available regarding its antidiabetic effect. This study focused on the effect of ampelopsin on hyperglycemia-induced oxidative damage and the underlying mechanism of this effect in human umbilical vein endothelial cells (HUVECs). We found that hyperglycemia impaired autophagy in HUVECs through the inhibition of AMP-activated protein kinase (AMPK), which directly led to endothelial cell damage. Ampelopsin significantly attenuated the detrimental effect of hyperglycemia-induced cell dysfunction in a concentration-dependent manner in HUVECs. Ampelopsin significantly upregulated LC3-II, Beclin1, and Atg5 protein levels but downregulated p62 protein levels in HUVECs. Transmission electron microscopy and confocal microscopy indicated that ampelopsin notably induced autophagosomes and LC3-II dots, respectively. Additionally, the autophagy-specific inhibitor 3-MA, as well as Atg5 and Beclin1 siRNA pretreatment, markedly attenuated ampelopsin-induced autophagy, which subsequently abolished the protective effect of ampelopsin against hyperglycemia in HUVECs. Moreover, ampelopsin also increased AMPK activity and inhibited mTOR (mammalian target of rapamycin) complex activation. Ampelopsin-induced autophagy was attenuated by the AMPK antagonist compound C but strengthened by the AMPK agonist AICAR (5-minoimidazole-4-carboxamide ribonucleotide). Furthermore, AMPK siRNA transfection eliminated ampelopsin's alleviation of cell injury induced by hyperglycemia. The protective effect of ampelopsin against hyperglycemia-induced cell damage, which functions by targeting autophagy via AMPK activation, makes it a promising pharmacological treatment for type-2 diabetes.

  17. Cadmium induces autophagy through ROS-dependent activation of the LKB1-AMPK signaling in skin epidermal cells

    SciTech Connect

    Son, Young-Ok; Wang Xin; Hitron, John Andrew; Zhang Zhuo; Cheng Senping; Budhraja, Amit; Ding Songze; Lee, Jeong-Chae; Shi Xianglin

    2011-09-15

    Cadmium is a toxic heavy metal which is environmentally and occupationally relevant. The mechanisms underlying cadmium-induced autophagy are not yet completely understood. The present study shows that cadmium induces autophagy, as demonstrated by the increase of LC3-II formation and the GFP-LC3 puncta cells. The induction of autophagosomes was directly visualized by electron microscopy in cadmium-exposed skin epidermal cells. Blockage of LKB1 or AMPK by siRNA transfection suppressed cadmium-induced autophagy. Cadmium-induced autophagy was inhibited in dominant-negative AMPK-transfected cells, whereas it was accelerated in cells transfected with the constitutively active form of AMPK. mTOR signaling, a negative regulator of autophagy, was downregulated in cadmium-exposed cells. In addition, cadmium generated reactive oxygen species (ROS) at relatively low levels, and caused poly(ADP-ribose) polymerase-1 (PARP) activation and ATP depletion. Inhibition of PARP by pharmacological inhibitors or its siRNA transfection suppressed ATP reduction and autophagy in cadmium-exposed cells. Furthermore, cadmium-induced autophagy signaling was attenuated by either exogenous addition of catalase and superoxide dismutase, or by overexpression of these enzymes. Consequently, these results suggest that cadmium-mediated ROS generation causes PARP activation and energy depletion, and eventually induces autophagy through the activation of LKB1-AMPK signaling and the down-regulation of mTOR in skin epidermal cells. - Highlights: > Cadmium, a toxic heavy metal, induces autophagic cell death through ROS-dependent activation of the LKB1-AMPK signaling. > Cadmium generates intracellular ROS at low levels and this leads to severe DNA damage and PARP activation, resulting in ATP depletion, which are the upstream events of LKB1-AMPK-mediated autophagy. > This novel finding may contribute to further understanding of cadmium-mediated diseases.

  18. Nootkatone, a characteristic constituent of grapefruit, stimulates energy metabolism and prevents diet-induced obesity by activating AMPK.

    PubMed

    Murase, Takatoshi; Misawa, Koichi; Haramizu, Satoshi; Minegishi, Yoshihiko; Hase, Tadashi

    2010-08-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is implicated in the control of energy metabolism and is considered to be a molecular target for the suppression of obesity and the treatment of metabolic syndrome. Here, we identified and characterized nootkatone, a constituent of grapefruit, as a naturally occurring AMPK activator. Nootkatone induced an increase in AMPKalpha1 and -alpha2 activity along with an increase in the AMP/ATP ratio and an increase the phosphorylation of AMPKalpha and the downstream target acetyl-CoA carboxylase (ACC), in C(2)C(12) cells. Nootkatone-induced activation of AMPK was possibly mediated both by LKB1 and Ca(2+)/calmodulin-dependent protein kinase kinase. Nootkatone also upregulated PPARgamma coactivator-1alpha in C(2)C(12) cells and C57BL/6J mouse muscle. In addition, administration of nootkatone (200 mg/kg body wt) significantly enhanced AMPK activity, accompanied by LKB1, AMPK, and ACC phosphorylation in the liver and muscle of mice. Whole body energy expenditure evaluated by indirect calorimetry was also increased by nootkatone administration. Long-term intake of diets containing 0.1% to 0.3% (wt/wt) nootkatone significantly reduced high-fat and high-sucrose diet-induced body weight gain, abdominal fat accumulation, and the development of hyperglycemia, hyperinsulinemia, and hyperleptinemia in C57BL/6J mice. Furthermore, endurance capacity, evaluated as swimming time to exhaustion in BALB/c mice, was 21% longer in mice fed 0.2% nootkatone than in control mice. These findings indicate that long-term intake of nootkatone is beneficial toward preventing obesity and improving physical performance and that these effects are due, at least in part, to enhanced energy metabolism through AMPK activation in skeletal muscle and liver. PMID:20501876

  19. Rapamycin requires AMPK activity and p27 expression for promoting autophagy-dependent Tsc2-null cell survival.

    PubMed

    Campos, Tania; Ziehe, Javiera; Fuentes-Villalobos, Francisco; Riquelme, Orlando; Peña, Daniela; Troncoso, Rodrigo; Lavandero, Sergio; Morin, Violeta; Pincheira, Roxana; Castro, Ariel F

    2016-06-01

    Tuberous sclerosis complex (TSC) disease results from inactivation of the TSC1 or TSC2 gene, and is characterized by benign tumors in several organs. Because TSC tumorigenesis correlates with hyperactivation of mTORC1, current therapies focus on mTORC1 inhibition with rapamycin or its analogs. Rapamycin-induced tumor shrinkage has been reported, but tumor recurrence occurs on withdrawal from rapamycin. Autophagy has been associated with development of TSC tumors and with tumor cell survival during rapamycin treatment. mTORC1 and AMPK directly inhibit and activate autophagy, respectively. AMPK is hyperactivated in TSC cells and tumors, and drives cytoplasmic sequestration of the cell-cycle inhibitor p27KIP (p27). Whether AMPK and p27 are involved in rapamycin-induced autophagy and survival of TSC cells remain unexplored. Here, we show that inhibition of AMPK by compound C or by shRNA-mediated depletion of LKB1 reduces activation of autophagy by rapamycin in Tsc2-null cells. Similarly, shRNA-mediated depletion of p27 inhibited rapamycin-induced autophagy. In support of p27 lying downstream of AMPK on the activation of autophagy in Tsc2-null cells, a p27 mutant that preferentially localizes in the cytosol recovered the effect of rapamycin on autophagy in both p27- and LKB1-depleted cells, but a nuclear p27 mutant was inactive. Finally, we show that p27-dependent activation of autophagy is involved in Tsc2-null cell survival under rapamycin treatment. These results indicate that an AMPK/p27 axis is promoting a survival mechanism that could explain in part the relapse of TSC tumors treated with rapamycin, exposing new avenues for designing more efficient treatments for TSC patients. PMID:26975583

  20. Region-specific activation of the AMPK system by cocaine: The role of D1 and D2 receptors.

    PubMed

    Xu, Shijie; Kang, Ung Gu

    2016-01-01

    The 5' adenosine monophosphate-activated protein kinase (AMPK) functions as an intracellular energy sensor that regulates and maintains energy balance. The psychostimulant drug cocaine has profound effects on behavior that are accentuated with repeated use, which is a process termed sensitization. Thus, the present study examined whether the sensitizing effects of cocaine could be observed in the AMPK system and aimed to determine whether these effects were mediated by dopamine (DA) D1 or D2 receptors. In the first set of experiments, rats were injected daily for 5days with either cocaine (15mg/kg, intraperitoneal [IP]) or saline. On the day 6, each group was divided into two subgroups and given either cocaine or saline. In the second set of experiments, rats were pretreated with SCH23390 (0.5mg/kg, IP), haloperidol (1mg/kg, IP), or both agents in combination, followed by cocaine or saline treatment. In the drug-naïve state, acute treatment with cocaine produced an increase in locomotor activity and increased AMPK phosphorylation in the frontal cortex but decreased it in the dorsal striatum. In the drug-sensitized state (following repeated treatment), the behavioral responsiveness to cocaine was augmented and accompanied by alterations in AMPK activity. The phosphorylation levels of the upstream kinases Ser-431-LKB1 and Thr-196-CaMK4 were congruent with the changes in AMPK activity. Thr-184/187-TAK1 was phosphorylated after chronic cocaine treatment in the dorsal striatum but not in the frontal cortex. The opposite effects induced by cocaine in the AMPK system in the dorsal striatum and frontal cortex may be explained by the differential activations of DA D1 and D2 receptors in these brain regions. PMID:27132751

  1. Ampelopsin Improves Insulin Resistance by Activating PPARγ and Subsequently Up-Regulating FGF21-AMPK Signaling Pathway

    PubMed Central

    Qin, Yu; Liu, Lei; Wan, Jing; Zou, Lingyun; Zhang, Qianyong; Zhu, Jundong; Mi, Mantian

    2016-01-01

    Ampelopsin (APL), a major bioactive constituent of Ampelopsis grossedentata, exerts a number of biological effects. Here, we explored the anti-diabetic activity of APL and elucidate the underlying mechanism of this action. In palmitate-induced insulin resistance of L6 myotubes, APL treatment markedly up- regulated phosphorylated insulin receptor substrate-1 and protein kinase B, along with a corresponding increase of glucose uptake capacity. APL treatment also increased expressions of fibroblast growth factor (FGF21) and phosphorylated adenosine 5’-monophosphate -activated protein kinase (p-AMPK), however inhibiting AMPK by Compound C or AMPK siRNA, or blockage of FGF21 by FGF21 siRNA, obviously weakened APL -induced increases of FGF21 and p-AMPK as well as glucose uptake capacity in palmitate -pretreated L6 myotubes. Furthermore, APL could activate PPAR γ resulting in increases of glucose uptake capacity and expressions of FGF21 and p-AMPK in palmitate -pretreated L6 myotubes, whereas all those effects were obviously abolished by addition of GW9662, a specific inhibitor of peroxisome proliferator- activated receptor –γ (PPARγ), and PPARγsiRNA. Using molecular modeling and the luciferase reporter assays, we observed that APL could dock with the catalytic domain of PPARγ and dose-dependently up-regulate PPARγ activity. In summary, APL maybe a potential agonist of PPARγ and promotes insulin sensitization by activating PPARγ and subsequently regulating FGF21- AMPK signaling pathway. These results provide new insights into the protective health effects of APL, especially for the treatment of Type 2 diabetes mellitus. PMID:27391974

  2. AMPK reverses the mesenchymal phenotype of cancer cells by targeting the Akt-MDM2-Foxo3a signaling axis.

    PubMed

    Chou, Chih-Chien; Lee, Kuen-Haur; Lai, I-Lu; Wang, Dasheng; Mo, Xiaokui; Kulp, Samuel K; Shapiro, Charles L; Chen, Ching-Shih

    2014-09-01

    In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacologic activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis. PMID:24994714

  3. Indazole-Type Alkaloids from Nigella sativa Seeds Exhibit Antihyperglycemic Effects via AMPK Activation in Vitro

    PubMed Central

    2015-01-01

    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. PMID:25299458

  4. 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.

  5. Compound 13, an α1-selective small molecule activator of AMPK, inhibits Helicobacter pylori-induced oxidative stresses and gastric epithelial cell apoptosis

    SciTech Connect

    Zhao, Hangyong; Zhu, Huanghuang; Lin, Zhou; Lin, Gang; Lv, Guoqiang

    2015-08-07

    Half of the world's population experiences Helicobacter pylori (H. pylori) infection, which is a main cause of gastritis, duodenal and gastric ulcer, and gastric cancers. In the current study, we investigated the potential role of compound 13 (C13), a novel α1-selective small molecule activator of AMP-activated protein kinase (AMPK), against H. pylori-induced cytotoxicity in cultured gastric epithelial cells (GECs). We found that C13 induced significant AMPK activation, evidenced by phosphorylation of AMPKα1 and ACC (acetyl-CoA carboxylase), in both primary and transformed GECs. Treatment of C13 inhibited H. pylori-induced GEC apoptosis. AMPK activation was required for C13-mediated GEC protection. Inhibition of AMPK kinase activity by the AMPK inhibitor Compound C, or silencing AMPKα1 expression by targeted-shRNAs, alleviated C13-induced GEC protective activities against H. pylori. Significantly, C13 inhibited H. pylori-induced reactive oxygen species (ROS) production in GECs. C13 induced AMPK-dependent expression of anti-oxidant gene heme oxygenase (HO-1) in GECs. Zinc protoporphyrin (ZnPP) and tin protoporphyrin (SnPP), two HO-1 inhibitors, not only suppressed C13-mediated ROS scavenging activity, but also alleviated its activity in GECs against H. pylori. Together, these results indicate that C13 inhibits H. pylori-induced ROS production and GEC apoptosis through activating AMPK–HO–1 signaling. - Highlights: • We synthesized compound 13 (C13), a α1-selective small molecule AMPK activator. • C13-induced AMPK activation requires α1 subunit in gastric epithelial cells (GECs). • C13 enhances Helicobacter pylori-induced pro-survival AMPK activation to inhibit GEC apoptosis. • C13 inhibits H. pylori-induced reactive oxygen species (ROS) production in GECs. • AMPK-heme oxygenase (HO-1) activation is required for C13-mediated anti-oxidant activity.

  6. Metformin exaggerates phenylephrine-induced AMPK phosphorylation independent of CaMKKβ and attenuates contractile response in endothelium-denuded rat aorta.

    PubMed

    Pyla, Rajkumar; Osman, Islam; Pichavaram, Prahalathan; Hansen, Paul; Segar, Lakshman

    2014-11-15

    Metformin, a widely prescribed antidiabetic drug, has been shown to reduce the risk of cardiovascular disease, including hypertension. Its beneficial effect toward improved vasodilation results from its ability to activate AMPK and enhance nitric oxide formation in the endothelium. To date, metformin regulation of AMPK has not been fully studied in intact arterial smooth muscle, especially during contraction evoked by G protein-coupled receptor (GPCR) agonists. In the present study, ex vivo incubation of endothelium-denuded rat aortic rings with 3mM metformin for 2h resulted in significant accumulation of metformin (∼ 600 pmoles/mg tissue), as revealed by LC-MS/MS MRM analysis. However, metformin did not show significant increase in AMPK phosphorylation under these conditions. Exposure of aortic rings to a GPCR agonist (e.g., phenylephrine) resulted in enhanced AMPK phosphorylation by ∼ 2.5-fold. Importantly, in metformin-treated aortic rings, phenylephrine challenge showed an exaggerated increase in AMPK phosphorylation by ∼ 9.7-fold, which was associated with an increase in AMP/ATP ratio. Pretreatment with compound C (AMPK inhibitor) prevented AMPK phosphorylation induced by phenylephrine alone and also that induced by phenylephrine after metformin treatment. However, pretreatment with STO-609 (CaMKKβ inhibitor) diminished AMPK phosphorylation induced by phenylephrine alone but not that induced by phenylephrine after metformin treatment. Furthermore, attenuation of phenylephrine-induced contraction (observed after metformin treatment) was prevented by AMPK inhibition but not by CaMKKβ inhibition. Together, these findings suggest that, upon endothelial damage in the vessel wall, metformin uptake by the underlying vascular smooth muscle would accentuate AMPK phosphorylation by GPCR agonists independent of CaMKKβ to promote vasorelaxation.

  7. Effects of wintertime fasting and seasonal adaptation on AMPK and ACC in hypothalamus, adipose tissue and liver of the raccoon dog (Nyctereutes procyonoides).

    PubMed

    Kinnunen, Sanni; Mänttäri, Satu; Herzig, Karl-Heinz; Nieminen, Petteri; Mustonen, Anne-Mari; Saarela, Seppo

    2016-02-01

    The raccoon dog (Nyctereutes procyonoides) is a canid with autumnal fattening and passive wintering strategy. We examined the effects of wintertime fasting and seasonality on AMP-activated protein kinase (AMPK), a regulator of metabolism, and its target, acetyl-CoA carboxylase (ACC) on the species. Twelve farmed raccoon dogs (eleven females/one male) were divided into two groups: half were fasted for ten weeks in December-March (winter fasted) and the others were fed ad libitum (winter fed). A third group (autumn fed, eight females) was fed ad libitum and sampled in December. Total AMPK, ACC and their phosphorylated forms (pAMPK, pACC) were measured from hypothalamus, liver, intra-abdominal (iWAT) and subcutaneous white adipose tissues (sWAT). The fasted animals lost 32% and the fed 20% of their body mass. Hypothalamic AMPK expression was lower and pACC levels higher in the winter groups compared to the autumn fed group. Liver pAMPK was lower in the winter fasted group, with consistently decreased ACC and pACC. AMPK and pAMPK were down-regulated in sWAT and iWAT of both winter groups, with a parallel decline in pACC in sWAT. The responses of AMPK and ACC to fasting were dissimilar to the effects observed previously in non-seasonal mammals and hibernators. Differences between the winter fed and autumn fed groups indicate that the functions of AMPK and ACC could be regulated in a season-dependent manner. Furthermore, the distinctive effects of prolonged fasting and seasonal adaptation on AMPK-ACC pathway could contribute to the wintering strategy of the raccoon dog. PMID:26603554

  8. Higher skeletal muscle α2AMPK activation and lower energy charge and fat oxidation in men than in women during submaximal exercise

    PubMed Central

    Roepstorff, Carsten; Thiele, Maja; Hillig, Thore; Pilegaard, Henriette; Richter, Erik A; Wojtaszewski, Jørgen F P; Kiens, Bente

    2006-01-01

    5′AMP-activated protein kinase (AMPK) is an energy sensor activated by perturbed cellular energy status such as during muscle contraction. Activated AMPK is thought to regulate several key metabolic pathways. We used sex comparison to investigate whether AMPK signalling in skeletal muscle regulates fat oxidation during exercise. Moderately trained women and men completed 90 min bicycle exercise at 60% V̇O2peak. Both AMPK Thr172 phosphorylation and α2AMPK activity were increased by exercise in men (∼200%, P < 0.001) but not significantly in women. The sex difference in muscle AMPK activation with exercise was accompanied by an increase in muscle free AMP (∼164%, P < 0.01), free AMP/ATP ratio (159%, P < 0.05), and creatine (∼42%, P < 0.001) in men but not in women (NS), suggesting that lack of AMPK activation in women was due to better maintenance of muscle cellular energy balance compared with men. During exercise, fat oxidation per kg lean body mass was higher in women than in men (P < 0.05). Regression analysis revealed that a higher proportion of type 1 muscle fibres (∼23%, P < 0.01) and a higher capillarization (∼23%, P < 0.05) in women than in men could partly explain the sex difference in α2AMPK activity (r = −0.54, P < 0.05) and fat oxidation (r = 0.64, P < 0.05) during exercise. On the other hand, fat oxidation appeared not to be regulated via AMPK. In conclusion, during prolonged submaximal exercise at 60% V̇O2peak, higher fat oxidation in women cannot be explained by higher AMPK signalling but is accompanied by improved muscle cellular energy balance in women probably due to sex specific muscle morphology. PMID:16600998

  9. Effects of wintertime fasting and seasonal adaptation on AMPK and ACC in hypothalamus, adipose tissue and liver of the raccoon dog (Nyctereutes procyonoides).

    PubMed

    Kinnunen, Sanni; Mänttäri, Satu; Herzig, Karl-Heinz; Nieminen, Petteri; Mustonen, Anne-Mari; Saarela, Seppo

    2016-02-01

    The raccoon dog (Nyctereutes procyonoides) is a canid with autumnal fattening and passive wintering strategy. We examined the effects of wintertime fasting and seasonality on AMP-activated protein kinase (AMPK), a regulator of metabolism, and its target, acetyl-CoA carboxylase (ACC) on the species. Twelve farmed raccoon dogs (eleven females/one male) were divided into two groups: half were fasted for ten weeks in December-March (winter fasted) and the others were fed ad libitum (winter fed). A third group (autumn fed, eight females) was fed ad libitum and sampled in December. Total AMPK, ACC and their phosphorylated forms (pAMPK, pACC) were measured from hypothalamus, liver, intra-abdominal (iWAT) and subcutaneous white adipose tissues (sWAT). The fasted animals lost 32% and the fed 20% of their body mass. Hypothalamic AMPK expression was lower and pACC levels higher in the winter groups compared to the autumn fed group. Liver pAMPK was lower in the winter fasted group, with consistently decreased ACC and pACC. AMPK and pAMPK were down-regulated in sWAT and iWAT of both winter groups, with a parallel decline in pACC in sWAT. The responses of AMPK and ACC to fasting were dissimilar to the effects observed previously in non-seasonal mammals and hibernators. Differences between the winter fed and autumn fed groups indicate that the functions of AMPK and ACC could be regulated in a season-dependent manner. Furthermore, the distinctive effects of prolonged fasting and seasonal adaptation on AMPK-ACC pathway could contribute to the wintering strategy of the raccoon dog.

  10. Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes

    SciTech Connect

    Seo, Min Suk; Kim, Jung Hwan; Kim, Hye Jung; Chang, Ki Churl; Park, Sang Won

    2015-04-15

    Honokiol is a bioactive neolignan compound isolated from the species of Magnolia. This study was designed to elucidate the cellular mechanism by which honokiol alleviates the development of non-alcoholic steatosis. HepG2 cells were treated with honokiol for 1 h, and then exposed to 1 mM free fatty acid (FFA) for 24 h to simulate non-alcoholic steatosis in vitro. C57BL/6 mice were fed with a high-fat diet for 28 days, and honokiol (10 mg/kg/day) was daily treated. Honokiol concentration-dependently attenuated intracellular fat overloading and triglyceride (TG) accumulation in FFA-exposed HepG2 cells. These effects were blocked by pretreatment with an AMP-activated protein kinase (AMPK) inhibitor. Honokiol significantly inhibited sterol regulatory element-binding protein-1c (SREBP-1c) maturation and the induction of lipogenic proteins, stearoyl-CoA desaturase-1 (SCD-1) and fatty acid synthase (FAS) in FFA-exposed HepG2 cells, but these effects were blocked by pretreatment of an AMPK inhibitor. Honokiol induced AMPK phosphorylation and subsequent acetyl-CoA carboxylase (ACC) phosphorylation, which were inhibited by genetic deletion of liver kinase B1 (LKB1). Honokiol stimulated LKB1 phosphorylation, and genetic deletion of LKB1 blocked the effect of honokiol on SREBP-1c maturation and the induction of SCD-1 and FAS proteins in FFA-exposed HepG2 cells. Honokiol attenuated the increases in hepatic TG and lipogenic protein levels and fat accumulation in the mice fed with high-fat diet, while significantly induced LKB1 and AMPK phosphorylation. Taken together, our findings suggest that honokiol has an anti-lipogenic effect in hepatocytes, and this effect may be mediated by the LKB1–AMPK signaling pathway, which induces ACC phosphorylation and inhibits SREBP-1c maturation in hepatocytes. - Highlights: • Honokiol attenuates lipid accumulation induced by free fatty acid in hepatocyte. • Honokiol inhibits the increase in lipogenic enzyme levels induced by free fatty

  11. Cross-talk between AMPK and EGFR dependent Signaling in Non-Small Cell Lung Cancer

    PubMed Central

    Praveen, Paurush; Hülsmann, Helen; Sültmann, Holger; Kuner, Ruprecht; Fröhlich, Holger

    2016-01-01

    Lung cancers globally account for 12% of new cancer cases, 85% of these being Non Small Cell Lung Cancer (NSCLC). Therapies like erlotinib target the key player EGFR, which is mutated in about 10% of lung adenocarcinoma. However, drug insensitivity and resistance caused by second mutations in the EGFR or aberrant bypass signaling have evolved as a major challenge in controlling these tumors. Recently, AMPK activation was proposed to sensitize NSCLC cells against erlotinib treatment. However, the underlying mechanism is largely unknown. In this work we aim to unravel the interplay between 20 proteins that were previously associated with EGFR signaling and erlotinib drug sensitivity. The inferred network shows a high level of agreement with protein-protein interactions reported in STRING and HIPPIE databases. It is further experimentally validated with protein measurements. Moreover, predictions derived from our network model fairly agree with somatic mutations and gene expression data from primary lung adenocarcinoma. Altogether our results support the role of AMPK in EGFR signaling and drug sensitivity. PMID:27279498

  12. AMPK Inhibition Enhances the Neurotoxicity of Cu(II) in SH-SY5Y Cells.

    PubMed

    Lan, Ai-Ping; Xiong, Xian-Jia; Chen, Jun; Wang, Xi; Chai, Zhi-Fang; Hu, Yi

    2016-10-01

    The involvement of copper in the pathophysiology of neurodegenerative disorders has been documented but remains poorly understood. This study aimed at investigating the molecular mechanism underlying copper-induced neurotoxicity. Human neuroblastoma SH-SY5Y cells were treated with different concentrations of Cu(II) (25-800 μM). The relative levels of AMPKα, phosphorylated (p)-AMPKα were examined by western blotting. The results showed that copper reduced cell viability and enhanced apoptosis of SH-SY5Y cells. Pretreatment with N-acetyl-L-cysteine, a common ROS scavenger, decreased copper-induced cytotoxicity. Furthermore, the levels of p-AMPKα in SH-SY5Y cells were increased by a relatively low concentration of copper and decreased by a relatively high concentration of copper at 24 h. Moreover, inhibition of AMPK with compound C or RNA interference aggravated concentration-dependent cytotoxicity of Cu(II). Taken together, these results indicated that AMPK activity might be important for the neurotoxicity of Cu(II).

  13. Glioma-Initiating Cell Elimination by Metformin Activation of FOXO3 via AMPK

    PubMed Central

    Sunayama, Jun; Okada, Masashi; Watanabe, Eriko; Seino, Shizuka; Shibuya, Keita; Suzuki, Kaori; Narita, Yoshitaka; Shibui, Soichiro; Kayama, Takamasa

    2012-01-01

    Control of the cancer stem/initiating cell population is considered key to realizing the long-term survival of glioblastoma patients. Recently, we demonstrated that FOXO3 activation is sufficient to induce differentiation of glioma-initiating cells having stem-like properties and inhibit their tumor-initiating potential. Here we identified metformin, an antidiabetic agent, as a therapeutic activator of FOXO3. Metformin activated FOXO3 and promoted differentiation of such stem-like glioma-initiating cells into nontumorigenic cells. Furthermore, metformin promoted FOXO3 activation and differentiation via AMP-activated protein kinase (AMPK) activation, which was sensitive to extracellular glucose availability. Importantly, transient, systemic administration of metformin depleted the self-renewing and tumor-initiating cell population within established tumors, inhibited tumor formation by stem-like glioma-initiating cells in the brain, and provided a substantial survival benefit. Our findings demonstrate that targeting glioma-initiating cells via the AMPK-FOXO3 axis is a viable therapeutic strategy against glioblastoma, with metformin being the most clinically relevant drug ever reported for targeting of glioma-initiating cells. Our results also establish a novel, direct link between glucose metabolism and cancer stem/initiating cells. PMID:23197693

  14. Flavonoid derivative exerts an antidiabetic effect via AMPK activation in diet-induced obesity mice.

    PubMed

    Chen, Ying; Zhang, Chang; Jin, Mei-Na; Qin, Nan; Qiao, Wei; Yue, Xiao-Long; Duan, Hong-Quan; Niu, Wen-Yan

    2016-09-01

    In our previous study, a derivative of tiliroside, 3-O-[(E)-4-(4-ethoxyphenyl)-2-oxobut-3-en-1-yl]kaempferol (Fla-OEt) significantly enhanced glucose consumption in insulin resistant HepG2 cells. This article deals with the antihyperglycemic and antihyperlipidemic effects of Fla-OEt in diet-induced obesity (DIO) mice. Daily administration of Fla-OEt significantly decreased oral glucose tolerance test, intraperitoneal insulin tolerance test and serum lipids. Hyperinsulinemic-euglycemic clamp and the ratio of high-density-lipoprotein/low-density-lipoprotein with Fla-OEt treatment were increased comparing with high-fat diet (HFD) group, so lipid metabolism was improved. Histopathology examination showed that the Fla-OEt restored the damage of adipose tissues and liver in DIO mice. Moreover, compared with HFD group, Fla-OEt treatment significantly increased the phosphorylation of AMPK and ACC in adiposity tissues, liver, and muscles. The mechanism of its action might be the activation of AMPK pathway. It appears that Fla-OEt is worth further study for development as a lead compound for a potential antidiabetic agent.

  15. Exercise-stimulated interleukin-15 is controlled by AMPK and regulates skin metabolism and aging.

    PubMed

    Crane, Justin D; MacNeil, Lauren G; Lally, James S; Ford, Rebecca J; Bujak, Adam L; Brar, Ikdip K; Kemp, Bruce E; Raha, Sandeep; Steinberg, Gregory R; Tarnopolsky, Mark A

    2015-08-01

    Aging is commonly associated with a structural deterioration of skin that compromises its barrier function, healing, and susceptibility to disease. Several lines of evidence show that these changes are driven largely by impaired tissue mitochondrial metabolism. While exercise is associated with numerous health benefits, there is no evidence that it affects skin tissue or that endocrine muscle-to-skin signaling occurs. We demonstrate that endurance exercise attenuates age-associated changes to skin in humans and mice and identify exercise-induced IL-15 as a novel regulator of mitochondrial function in aging skin. We show that exercise controls IL-15 expression in part through skeletal muscle AMP-activated protein kinase (AMPK), a central regulator of metabolism, and that the elimination of muscle AMPK causes a deterioration of skin structure. Finally, we establish that daily IL-15 therapy mimics some of the anti-aging effects of exercise on muscle and skin in mice. Thus, we elucidate a mechanism by which exercise confers health benefits to skin and suggest that low-dose IL-15 therapy may prove to be a beneficial strategy to attenuate skin aging.

  16. AMPK/HuR-Driven IL-20 Post-Transcriptional Regulation in Psoriatic Skin.

    PubMed

    Garcin, Geneviève; Guiraud, Isabelle; Lacroix, Matthieu; Genthon, Clémence; Rialle, Stéphanie; Joujoux, Jean-Marie; Meunier, Laurent; Lavabre-Bertrand, Thierry; Stoebner, Pierre-Emmanuel; Le Gallic, Lionel

    2015-11-01

    IL-20 is involved in the development of skin psoriasis. The molecular mechanisms underlying IL-20 overexpression in psoriatic epidermis remain to be elucidated. We showed that IL-20 was primarily upregulated in psoriatic skin at the post-transcriptional level. The RNA-binding protein HuR relocalized to the cytoplasm of keratinocytes (KCs) of psoriatic patients, suggesting that it stabilizes numerous transcripts, as observed in the human KC cell lines used to assess IL-20 mRNA. We characterized epidermal HuR RNA targets in psoriatic skin using ribonucleoprotein immunoprecipitation analyzed via high-throughput sequencing. Numerous transcripts that are upregulated in psoriasis were targeted by HuR, supporting the participation of HuR in pathogenic processes such as morphological changes, innate and adaptive immune responses, and metabolic inflammatory responses. Finally, we identified the metabolic sensor AMP-activated protein kinase (AMPK) as being responsible for HuR cytoplasmic relocalization because its activity was severely impaired in human psoriatic epidermis, and in vivo drug-mediated AMPK inhibition in mouse epidermis promoted HuR cytoplasmic localization, IL-20 overproduction, acanthosis, and hyperkeratosis. These results provide insights into the molecular links between metabolism and post-transcriptional networks during chronic inflammation.

  17. Cannabinoids inhibit energetic metabolism and induce AMPK-dependent autophagy in pancreatic cancer cells

    PubMed Central

    Dando, I; Donadelli, M; Costanzo, C; Dalla Pozza, E; D'Alessandro, A; Zolla, L; Palmieri, M

    2013-01-01

    The anti-tumoral effects of cannabinoids have been described in different tumor systems, including pancreatic adenocarcinoma, but their mechanism of action remains unclear. We used cannabinoids specific for the CB1 (ACPA) and CB2 (GW) receptors and metabolomic analyses to unravel the potential pathways mediating cannabinoid-dependent inhibition of pancreatic cancer cell growth. Panc1 cells treated with cannabinoids show elevated AMPK activation induced by a ROS-dependent increase of AMP/ATP ratio. ROS promote nuclear translocation of GAPDH, which is further amplified by AMPK, thereby attenuating glycolysis. Furthermore, ROS determine the accumulation of NADH, suggestive of a blockage in the respiratory chain, which in turn inhibits the Krebs cycle. Concomitantly, inhibition of Akt/c-Myc pathway leads to decreased activity of both the pyruvate kinase isoform M2 (PKM2), further downregulating glycolysis, and glutamine uptake. Altogether, these alterations of pancreatic cancer cell metabolism mediated by cannabinoids result in a strong induction of autophagy and in the inhibition of cell growth. PMID:23764845

  18. Cannabinoids inhibit energetic metabolism and induce AMPK-dependent autophagy in pancreatic cancer cells.

    PubMed

    Dando, I; Donadelli, M; Costanzo, C; Dalla Pozza, E; D'Alessandro, A; Zolla, L; Palmieri, M

    2013-06-13

    The anti-tumoral effects of cannabinoids have been described in different tumor systems, including pancreatic adenocarcinoma, but their mechanism of action remains unclear. We used cannabinoids specific for the CB1 (ACPA) and CB2 (GW) receptors and metabolomic analyses to unravel the potential pathways mediating cannabinoid-dependent inhibition of pancreatic cancer cell growth. Panc1 cells treated with cannabinoids show elevated AMPK activation induced by a ROS-dependent increase of AMP/ATP ratio. ROS promote nuclear translocation of GAPDH, which is further amplified by AMPK, thereby attenuating glycolysis. Furthermore, ROS determine the accumulation of NADH, suggestive of a blockage in the respiratory chain, which in turn inhibits the Krebs cycle. Concomitantly, inhibition of Akt/c-Myc pathway leads to decreased activity of both the pyruvate kinase isoform M2 (PKM2), further downregulating glycolysis, and glutamine uptake. Altogether, these alterations of pancreatic cancer cell metabolism mediated by cannabinoids result in a strong induction of autophagy and in the inhibition of cell growth.

  19. The AMPK-related kinase SNARK regulates muscle mass and myocyte survival.

    PubMed

    Lessard, Sarah J; Rivas, Donato A; So, Kawai; Koh, Ho-Jin; Queiroz, André Lima; Hirshman, Michael F; Fielding, Roger A; Goodyear, Laurie J

    2016-02-01

    The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-related kinase (SNARK, also known as NUAK2), increased with muscle cell differentiation. SNARK expression increased in skeletal muscles from young mice exposed to metabolic stress and in muscles from healthy older human subjects. The regulation of SNARK expression in muscle with differentiation and physiological stress suggests that SNARK may function in the maintenance of muscle mass. Consistent with this hypothesis, decreased endogenous SNARK expression (using siRNA) in cultured muscle cells resulted in increased apoptosis and decreased cell survival under conditions of metabolic stress. Likewise, muscle-specific transgenic animals expressing a SNARK dominant-negative inactive mutant (SDN) had increased myonuclear apoptosis and activation of apoptotic mediators in muscle. Moreover, animals expressing SDN had severe, age-accelerated muscle atrophy and increased adiposity, consistent with sarcopenic obesity. Reduced SNARK activity, in vivo and in vitro, caused downregulation of the Rho kinase signaling pathway, a key mediator of cell survival. These findings reveal a critical role for SNARK in myocyte survival and the maintenance of muscle mass with age. PMID:26690705

  20. Cross-talk between AMPK and EGFR dependent Signaling in Non-Small Cell Lung Cancer

    NASA Astrophysics Data System (ADS)

    Praveen, Paurush; Hülsmann, Helen; Sültmann, Holger; Kuner, Ruprecht; Fröhlich, Holger

    2016-06-01

    Lung cancers globally account for 12% of new cancer cases, 85% of these being Non Small Cell Lung Cancer (NSCLC). Therapies like erlotinib target the key player EGFR, which is mutated in about 10% of lung adenocarcinoma. However, drug insensitivity and resistance caused by second mutations in the EGFR or aberrant bypass signaling have evolved as a major challenge in controlling these tumors. Recently, AMPK activation was proposed to sensitize NSCLC cells against erlotinib treatment. However, the underlying mechanism is largely unknown. In this work we aim to unravel the interplay between 20 proteins that were previously associated with EGFR signaling and erlotinib drug sensitivity. The inferred network shows a high level of agreement with protein-protein interactions reported in STRING and HIPPIE databases. It is further experimentally validated with protein measurements. Moreover, predictions derived from our network model fairly agree with somatic mutations and gene expression data from primary lung adenocarcinoma. Altogether our results support the role of AMPK in EGFR signaling and drug sensitivity.

  1. Crystal Structure of the Heterotrimer Core of Saccharomyces cerevisiae AMPK Homologue SNF1

    SciTech Connect

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

    2007-01-01

    AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals and is an attractive target for drug discovery against diabetes, obesity and other diseases. The AMPK homologue in Saccharomyces cerevisiae, known as SNF1, is essential for responses to glucose starvation as well as for other cellular processes, although SNF1 seems to be activated by a ligand other than AMP. Here we report the crystal structure at 2.6 resolution of the heterotrimer core of SNF1. The ligand-binding site in the {gamma}-subunit (Snf4) has clear structural differences from that of the Schizosaccharomyces pombe enzyme, although our crystallographic data indicate that AMP can also bind to Snf4. The glycogen-binding domain in the {beta}-subunit (Sip2) interacts with Snf4 in the heterotrimer but should still be able to bind carbohydrates. Our structure is supported by a large body of biochemical and genetic data on this complex. Most significantly, the structure reveals that part of the regulatory sequence in the {alpha}-subunit (Snf1) is sequestered by Snf4, demonstrating a direct interaction between the {alpha}- and {gamma}-subunits and indicating that our structure may represent the heterotrimer core of SNF1 in its activated state.

  2. The AMPK-related kinase SNARK regulates muscle mass and myocyte survival

    PubMed Central

    Lessard, Sarah J.; Rivas, Donato A.; So, Kawai; Koh, Ho-Jin; Queiroz, André Lima; Hirshman, Michael F.; Fielding, Roger A.; Goodyear, Laurie J.

    2015-01-01

    The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-related kinase (SNARK, also known as NUAK2), increased with muscle cell differentiation. SNARK expression increased in skeletal muscles from young mice exposed to metabolic stress and in muscles from healthy older human subjects. The regulation of SNARK expression in muscle with differentiation and physiological stress suggests that SNARK may function in the maintenance of muscle mass. Consistent with this hypothesis, decreased endogenous SNARK expression (using siRNA) in cultured muscle cells resulted in increased apoptosis and decreased cell survival under conditions of metabolic stress. Likewise, muscle-specific transgenic animals expressing a SNARK dominant-negative inactive mutant (SDN) had increased myonuclear apoptosis and activation of apoptotic mediators in muscle. Moreover, animals expressing SDN had severe, age-accelerated muscle atrophy and increased adiposity, consistent with sarcopenic obesity. Reduced SNARK activity, in vivo and in vitro, caused downregulation of the Rho kinase signaling pathway, a key mediator of cell survival. These findings reveal a critical role for SNARK in myocyte survival and the maintenance of muscle mass with age. PMID:26690705

  3. Quercetin induces bladder cancer cells apoptosis by activation of AMPK signaling pathway

    PubMed Central

    Su, Qiongli; Peng, Mei; Zhang, Yuqing; Xu, Wanjun; Darko, Kwame Oteng; Tao, Ting; Huang, Yanjun; Tao, Xiaojun; Yang, Xiaoping

    2016-01-01

    Quercetin, a natural existing polyphenol compound, has shown anticancer capacity for liver, breast, nasopharyngeal and prostate carcinoma but has not been clinically approved yet. This might be due to lack of clear mechanistic picture. Bladder cancer is one of the most common cancers of the urinary tract in the world. In China, bladder cancer has the highest rate of incidence out of all malignancies of the urinary system. The anticancer application of quercetin on bladder cancer has not been investigated either. This study was aimed to examine the mechanisms of quercetin on inhibition of bladder cancer. First, two human and one murine bladder cancer cell lines were tested in vitro for inhibitory sensitivity by MTT and cologenic assays. Second, AMPK pathway including 4E-BP1 and S6K were examined by western blot. Quercetin induces apoptosis and inhibits migration. We are the first to show that quercetin displays potent inhibition on bladder cancer cells via activation of AMPK pathway. PMID:27186419

  4. AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of autophagy.

    PubMed

    Shin, Hi-Jai R; Kim, Hyunkyung; Oh, Sungryong; Lee, Jun-Gi; Kee, Minjung; Ko, Hyun-Jeong; Kweon, Mi-Na; Won, Kyoung-Jae; Baek, Sung Hee

    2016-06-23

    Autophagy is a highly conserved self-digestion process, which is essential for maintaining homeostasis and viability in response to nutrient starvation. Although the components of autophagy in the cytoplasm have been well studied, the molecular basis for the transcriptional and epigenetic regulation of autophagy is poorly understood. Here we identify co-activator-associated arginine methyltransferase 1 (CARM1) as a crucial component of autophagy in mammals. Notably, CARM1 stability is regulated by the SKP2-containing SCF (SKP1-cullin1-F-box protein) E3 ubiquitin ligase in the nucleus, but not in the cytoplasm, under nutrient-rich conditions. Furthermore, we show that nutrient starvation results in AMP-activated protein kinase (AMPK)-dependent phosphorylation of FOXO3a in the nucleus, which in turn transcriptionally represses SKP2. This repression leads to increased levels of CARM1 protein and subsequent increases in histone H3 Arg17 dimethylation. Genome-wide analyses reveal that CARM1 exerts transcriptional co-activator function on autophagy-related and lysosomal genes through transcription factor EB (TFEB). Our findings demonstrate that CARM1-dependent histone arginine methylation is a crucial nuclear event in autophagy, and identify a new signalling axis of AMPK-SKP2-CARM1 in the regulation of autophagy induction after nutrient starvation. PMID:27309807

  5. Adaptive Benefits of Storage Strategy and Dual AMPK/TOR Signaling in Metabolic Stress Response

    PubMed Central

    Pfeuty, Benjamin; Thommen, Quentin

    2016-01-01

    Cellular metabolism must ensure that supply of nutrient meets the biosynthetic and bioenergetic needs. Cells have therefore developed sophisticated signaling and regulatory pathways in order to cope with dynamic fluctuations of both resource and demand and to regulate accordingly diverse anabolic and catabolic processes. Intriguingly, these pathways are organized around a relatively small number of regulatory hubs, such as the highly conserved AMPK and TOR kinase families in eukaryotic cells. Here, the global metabolic adaptations upon dynamic environment are investigated using a prototypical model of regulated metabolism. In this model, the optimal enzyme profiles as well as the underlying regulatory architecture are identified by combining perturbation and evolutionary methods. The results reveal the existence of distinct classes of adaptive strategies, which differ in the management of storage reserve depending on the intensity of the stress and in the regulation of ATP-producing reaction depending on the nature of the stress. The regulatory architecture that optimally implements these adaptive features is characterized by a crosstalk between two specialized signaling pathways, which bears close similarities with the sensing and regulatory properties of AMPK and TOR pathways. PMID:27505075

  6. In vivo correction of COX deficiency by activation of the AMPK/PGC-1α axis.

    PubMed

    Viscomi, Carlo; Bottani, Emanuela; Civiletto, Gabriele; Cerutti, Raffaele; Moggio, Maurizio; Fagiolari, Gigliola; Schon, Eric A; Lamperti, Costanza; Zeviani, Massimo

    2011-07-01

    Increased mitochondrial biogenesis by activation of PPAR- or AMPK/PGC-1α-dependent homeostatic pathways has been proposed as a treatment for mitochondrial disease. We tested this hypothesis on three recombinant mouse models characterized by defective cytochrome c-oxidase (COX) activity: a knockout (KO) mouse for Surf1, a knockout/knockin mouse for Sco2, and a muscle-restricted KO mouse for Cox15. First, we demonstrated that double-recombinant animals overexpressing PGC-1α in skeletal muscle on a Surf1 KO background showed robust induction of mitochondrial biogenesis and increase of mitochondrial respiratory chain activities, including COX. No such effect was obtained by treating both Surf1(-/-) and Cox15(-/-) mice with the pan-PPAR agonist bezafibrate, which instead showed adverse effects in either model. Contrariwise, treatment with the AMPK agonist AICAR led to partial correction of COX deficiency in all three models, and, importantly, significant motor improvement up to normal in the Sco2(KO/KI) mouse. These results open new perspectives for therapy of mitochondrial disease. PMID:21723506

  7. Attenuation of AMPK signaling by ROQUIN promotes T follicular helper cell formation

    PubMed Central

    Ramiscal, Roybel R; Parish, Ian A; Lee-Young, Robert S; Babon, Jeffrey J; Blagih, Julianna; Pratama, Alvin; Martin, Jaime; Hawley, Naomi; Cappello, Jean Y; Nieto, Pablo F; Ellyard, Julia I; Kershaw, Nadia J; Sweet, Rebecca A; Goodnow, Christopher C; Jones, Russell G; Febbraio, Mark A; Vinuesa, Carola G; Athanasopoulos, Vicki

    2015-01-01

    T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN–AMPK metabolic signaling nexus essential for selectively promoting Tfh responses. DOI: http://dx.doi.org/10.7554/eLife.08698.001 PMID:26496200

  8. AMPK dependent protective effects of metformin on tumor necrosis factor-induced apoptotic liver injury.

    PubMed

    Cai, Lu; Hu, Kai; Lin, Ling; Ai, Qing; Ge, Pu; Liu, Yiqing; Dai, Jie; Ye, Bin; Zhang, Li

    2015-09-25

    Tumor necrosis factor α (TNF-α)-induced cellular apoptosis represents a common pathological mechanism underlying the progression of various liver disorders. Recently studies revealed that the anti-diabetic metformin provided protective benefits in several animal models of liver injury. In the present study, the potential modulatory effects of metformin on TNF-α-dependent apoptotic liver damage was investigated in mice with TNF-α/d-galactosamine (D-Gal)-induced liver injury. The results indicated that treatment with metformin significantly suppressed the elevation of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the activation of caspase cascade and the induction of cleaved caspase-3. Morphological analysis showed that metformin alleviated histopathological abnormalities and reduced TUNEL-positive apoptotic cells. Co-administration of the AMPK inhibitor compound C completely abolished the inhibitory effects of metformin on caspase cascade activation, significantly reversed the beneficial effects of metformin on histopathological abnormalities and hepatocytes apoptosis, and partially abolished the suppressive effects of metformin on plasma ALT elevation. These data indicated that metformin effectively alleviated TNF-α/D-Gal-induced apoptotic liver injury and these beneficial effects were at least partially mediated by AMPK.

  9. Adaptive Benefits of Storage Strategy and Dual AMPK/TOR Signaling in Metabolic Stress Response.

    PubMed

    Pfeuty, Benjamin; Thommen, Quentin

    2016-01-01

    Cellular metabolism must ensure that supply of nutrient meets the biosynthetic and bioenergetic needs. Cells have therefore developed sophisticated signaling and regulatory pathways in order to cope with dynamic fluctuations of both resource and demand and to regulate accordingly diverse anabolic and catabolic processes. Intriguingly, these pathways are organized around a relatively small number of regulatory hubs, such as the highly conserved AMPK and TOR kinase families in eukaryotic cells. Here, the global metabolic adaptations upon dynamic environment are investigated using a prototypical model of regulated metabolism. In this model, the optimal enzyme profiles as well as the underlying regulatory architecture are identified by combining perturbation and evolutionary methods. The results reveal the existence of distinct classes of adaptive strategies, which differ in the management of storage reserve depending on the intensity of the stress and in the regulation of ATP-producing reaction depending on the nature of the stress. The regulatory architecture that optimally implements these adaptive features is characterized by a crosstalk between two specialized signaling pathways, which bears close similarities with the sensing and regulatory properties of AMPK and TOR pathways. PMID:27505075

  10. Glucagon-like peptide-1 prevents methylglyoxal-induced apoptosis of beta cells through improving mitochondrial function and suppressing prolonged AMPK activation

    PubMed Central

    Chang, Tien-Jyun; Tseng, Hsing-Chi; Liu, Meng-Wei; Chang, Yi-Cheng; Hsieh, Meng-Lun; Chuang, Lee-Ming

    2016-01-01

    Accumulation of methylglyoxal (MG) contributes to glucotoxicity and mediates beta cell apoptosis. The molecular mechanism by which GLP-1 protects MG-induced beta cell apoptosis remains unclear. Metformin is a first-line drug for treating type 2 diabetes associated with AMPK activation. However, whether metformin prevents MG-induced beta cell apoptosis is controversial. Here, we explored the signaling pathway involved in the anti-apoptotic effect of GLP-1, and investigated whether metformin had an anti-apoptotic effect on beta cells. MG treatment induced apoptosis of beta cells, impaired mitochondrial function, and prolonged activation of AMP-dependent protein kinase (AMPK). The MG-induced pro-apoptotic effects were abolished by an AMPK inhibitor. Pretreatment of GLP-1 reversed MG-induced apoptosis, and mitochondrial dysfunction, and suppressed prolonged AMPK activation. Pretreatment of GLP-1 reversed AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR)-induced apoptosis, and suppressed prolonged AMPK activation. However, metformin neither leads to beta cell apoptosis nor ameliorates MG-induced beta cell apoptosis. In parallel, GLP-1 also prevents MG-induced beta cell apoptosis through PKA and PI3K-dependent pathway. In conclusion, these data indicates GLP-1 but not metformin protects MG-induced beta cell apoptosis through improving mitochondrial function, and alleviating the prolonged AMPK activation. Whether adding GLP-1 to metformin provides better beta cell survival and delays disease progression remains to be validated. PMID:26997114

  11. AICAR and Metformin Exert AMPK-dependent Effects on INS-1E Pancreatic β-cell Apoptosis via Differential Downstream Mechanisms

    PubMed Central

    Dai, Yu-Lu; Huang, Su-Ling; Leng, Ying

    2015-01-01

    The role of AMP-activated protein kinase (AMPK) in pancreatic β-cell apoptosis is still controversial, and the reasons for the discrepancies have not been clarified. In the current study, we observed the effects of two well-known AMPK activators 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and metformin, on apoptosis in rat insulinoma INS-1E cells, and further explored their possible mechanisms. Both AICAR and metformin protected INS-1E cells from palmitate-induced apoptosis, as reflected by decreases in both cleaved caspase 3 protein expression and caspase 3/7 activity, and these protective effects were abrogated by AMPK inhibitor compound C. The protective action of AICAR was probably mediated by the suppression of triacylglycerol accumulation, increase in Akt phosphorylation and decrease in p38 MAPK phosphorylation, while metformin might exert its protective effect on INS-1E cells by decreases in both JNK and p38 MAPK phosphorylation. All these regulations were dependent on AMPK activation. However, under standard culture condition, AICAR increased JNK phosphorylation and promoted INS-1E cell apoptosis in an AMPK-dependent manner, whereas metformin showed no effect on apoptosis. Our study revealed that AMPK activators AICAR and metformin exhibited different effects on INS-1E cell apoptosis under different culture conditions, which might be largely attributed to different downstream mediators. Our results provided new and informative clues for better understanding of the role of AMPK in β-cell apoptosis. PMID:26435693

  12. Glucagon-like peptide-1 prevents methylglyoxal-induced apoptosis of beta cells through improving mitochondrial function and suppressing prolonged AMPK activation.

    PubMed

    Chang, Tien-Jyun; Tseng, Hsing-Chi; Liu, Meng-Wei; Chang, Yi-Cheng; Hsieh, Meng-Lun; Chuang, Lee-Ming

    2016-01-01

    Accumulation of methylglyoxal (MG) contributes to glucotoxicity and mediates beta cell apoptosis. The molecular mechanism by which GLP-1 protects MG-induced beta cell apoptosis remains unclear. Metformin is a first-line drug for treating type 2 diabetes associated with AMPK activation. However, whether metformin prevents MG-induced beta cell apoptosis is controversial. Here, we explored the signaling pathway involved in the anti-apoptotic effect of GLP-1, and investigated whether metformin had an anti-apoptotic effect on beta cells. MG treatment induced apoptosis of beta cells, impaired mitochondrial function, and prolonged activation of AMP-dependent protein kinase (AMPK). The MG-induced pro-apoptotic effects were abolished by an AMPK inhibitor. Pretreatment of GLP-1 reversed MG-induced apoptosis, and mitochondrial dysfunction, and suppressed prolonged AMPK activation. Pretreatment of GLP-1 reversed AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR)-induced apoptosis, and suppressed prolonged AMPK activation. However, metformin neither leads to beta cell apoptosis nor ameliorates MG-induced beta cell apoptosis. In parallel, GLP-1 also prevents MG-induced beta cell apoptosis through PKA and PI3K-dependent pathway. In conclusion, these data indicates GLP-1 but not metformin protects MG-induced beta cell apoptosis through improving mitochondrial function, and alleviating the prolonged AMPK activation. Whether adding GLP-1 to metformin provides better beta cell survival and delays disease progression remains to be validated. PMID:26997114

  13. The pivotal role of protein kinase C zeta (PKCzeta) in insulin- and AMP-activated protein kinase (AMPK)-mediated glucose uptake in muscle cells.

    PubMed

    Liu, Li-Zhong; Cheung, Stanley C K; Lan, Lin-Lin; Ho, Stanley K S; Chan, Juliana C N; Tong, Peter C Y

    2010-10-01

    Insulin and AMP-activated protein kinase (AMPK) signal pathways are involved in the regulation of glucose uptake. The integration of signals between these two pathways to maintain glucose homeostasis remains elusive. In this work, stimulation of insulin and berberine conferred a glucose uptake or surface glucose transporter 4 (GLUT4) translocation that was less than simple summation of their effects in insulin-sensitive muscle cells. Using specific inhibitors to key kinases of both pathways and PKCzeta small interference RNA, protein kinase C zeta (PKCzeta) was found to regulate insulin-stimulated protein kinase B (PKB) activation and inhibit AMPK activity on dorsal cell surface. In the presence of berberine, PKCzeta controlled AMPK activation and AMPK blocked PKB activity in perinuclear region. The inhibition effect of PKCzeta on AMPK activation or the arrestment of PKB activity by AMPK still existed in basal condition. These results suggest that there is antagonistic regulation between insulin and AMPK signal pathways, which is mediated by the switch roles of PKCzeta.

  14. AICAR and Metformin Exert AMPK-dependent Effects on INS-1E Pancreatic β-cell Apoptosis via Differential Downstream Mechanisms.

    PubMed

    Dai, Yu-Lu; Huang, Su-Ling; Leng, Ying

    2015-01-01

    The role of AMP-activated protein kinase (AMPK) in pancreatic β-cell apoptosis is still controversial, and the reasons for the discrepancies have not been clarified. In the current study, we observed the effects of two well-known AMPK activators 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and metformin, on apoptosis in rat insulinoma INS-1E cells, and further explored their possible mechanisms. Both AICAR and metformin protected INS-1E cells from palmitate-induced apoptosis, as reflected by decreases in both cleaved caspase 3 protein expression and caspase 3/7 activity, and these protective effects were abrogated by AMPK inhibitor compound C. The protective action of AICAR was probably mediated by the suppression of triacylglycerol accumulation, increase in Akt phosphorylation and decrease in p38 MAPK phosphorylation, while metformin might exert its protective effect on INS-1E cells by decreases in both JNK and p38 MAPK phosphorylation. All these regulations were dependent on AMPK activation. However, under standard culture condition, AICAR increased JNK phosphorylation and promoted INS-1E cell apoptosis in an AMPK-dependent manner, whereas metformin showed no effect on apoptosis. Our study revealed that AMPK activators AICAR and metformin exhibited different effects on INS-1E cell apoptosis under different culture conditions, which might be largely attributed to different downstream mediators. Our results provided new and informative clues for better understanding of the role of AMPK in β-cell apoptosis.

  15. Features of an altered AMPK metabolic pathway in Gilbert’s Syndrome, and its role in metabolic health

    PubMed Central

    Mölzer, Christine; Wallner, Marlies; Kern, Carina; Tosevska, Anela; Schwarz, Ursula; Zadnikar, Rene; Doberer, Daniel; Marculescu, Rodrig; Wagner, Karl-Heinz

    2016-01-01

    Energy metabolism, involving the ATP-dependent AMPK-PgC-Ppar pathway impacts metabolic health immensely, in that its impairment can lead to obesity, giving rise to disease. Based on observations that individuals with Gilbert’s syndrome (GS; UGT1A1*28 promoter mutation) are generally lighter, leaner and healthier than controls, specific inter-group differences in the AMPK pathway regulation were explored. Therefore, a case-control study involving 120 fasted, healthy, age- and gender matched subjects with/without GS, was conducted. By utilising intra-cellular flow cytometry (next to assessing AMPKα1 gene expression), levels of functioning proteins (phospho-AMPK α1/α2, PgC 1 α, Ppar α and γ) were measured in PBMCs (peripheral blood mononucleated cells). In GS individuals, rates of phospho-AMPK α1/α2, -Ppar α/γ and of PgC 1α were significantly higher, attesting to a boosted fasting response in this condition. In line with this finding, AMPKα1 gene expression was equal between the groups, possibly stressing the post-translational importance of boosted fasting effects in GS. In reflection of an apparently improved health status, GS individuals had significantly lower BMI, glucose, insulin, C-peptide and triglyceride levels. Herewith, we propose a new theory to explain why individuals having GS are leaner and healthier, and are therefore less likely to contract metabolic diseases or die prematurely thereof. PMID:27444220

  16. Chronic AMPK activation via loss of FLCN induces functional beige adipose tissue through PGC-1α/ERRα

    PubMed Central

    Yan, Ming; Audet-Walsh, Étienne; Manteghi, Sanaz; Rosa Dufour, Catherine; Walker, Benjamin; Baba, Masaya; St-Pierre, Julie; Giguère, Vincent; Pause, Arnim

    2016-01-01

    The tumor suppressor folliculin (FLCN) forms a repressor complex with AMP-activated protein kinase (AMPK). Given that AMPK is a master regulator of cellular energy homeostasis, we generated an adipose-specific Flcn (Adipoq-FLCN) knockout mouse model to investigate the role of FLCN in energy metabolism. We show that loss of FLCN results in a complete metabolic reprogramming of adipose tissues, resulting in enhanced oxidative metabolism. Adipoq-FLCN knockout mice exhibit increased energy expenditure and are protected from high-fat diet (HFD)-induced obesity. Importantly, FLCN ablation leads to chronic hyperactivation of AMPK, which in turns induces and activates two key transcriptional regulators of cellular metabolism, proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα). Together, the AMPK/PGC-1α/ERRα molecular axis positively modulates the expression of metabolic genes to promote mitochondrial biogenesis and activity. In addition, mitochondrial uncoupling proteins as well as other markers of brown fat are up-regulated in both white and brown FLCN-null adipose tissues, underlying the increased resistance of Adipoq-FLCN knockout mice to cold exposure. These findings identify a key role of FLCN as a negative regulator of mitochondrial function and identify a novel molecular pathway involved in the browning of white adipocytes and the activity of brown fat. PMID:27151976

  17. 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.

  18. ETC-1002 regulates immune response, leukocyte homing, and adipose tissue inflammation via LKB1-dependent activation of macrophage AMPK

    PubMed Central

    Filippov, Sergey; Pinkosky, Stephen L.; Lister, Richard J.; Pawloski, Catherine; Hanselman, Jeffrey C.; Cramer, Clay T.; Srivastava, Rai Ajit K.; Hurley, Timothy R.; Bradshaw, Cheryl D.; Spahr, Mark A.; Newton, Roger S.

    2013-01-01

    ETC-1002 is an investigational drug currently in Phase 2 development for treatment of dyslipidemia and other cardiometabolic risk factors. In dyslipidemic subjects, ETC-1002 not only reduces plasma LDL cholesterol but also significantly attenuates levels of hsCRP, a clinical biomarker of inflammation. Anti-inflammatory properties of ETC-1002 were further investigated in primary human monocyte-derived macrophages and in in vivo models of inflammation. In cells treated with ETC-1002, increased levels of AMP-activated protein kinase (AMPK) phosphorylation coincided with reduced activity of MAP kinases and decreased production of proinflammatory cytokines and chemokines. AMPK phosphorylation and inhibitory effects of ETC-1002 on soluble mediators of inflammation were significantly abrogated by siRNA-mediated silencing of macrophage liver kinase B1 (LKB1), indicating that ETC-1002 activates AMPK and exerts its anti-inflammatory effects via an LKB1-dependent mechanism. In vivo, ETC-1002 suppressed thioglycollate-induced homing of leukocytes into mouse peritoneal cavity. Similarly, in a mouse model of diet-induced obesity, ETC-1002 restored adipose AMPK activity, reduced JNK phosphorylation, and diminished expression of macrophage-specific marker 4F/80. These data were consistent with decreased epididymal fat-pad mass and interleukin (IL)-6 release by inflamed adipose tissue. Thus, ETC-1002 may provide further clinical benefits for patients with cardiometabolic risk factors by reducing systemic inflammation linked to insulin resistance and vascular complications of metabolic syndrome. PMID:23709692

  19. The energy sensor AMPK regulates Hedgehog signaling in human cells through a unique Gli1 metabolic checkpoint

    PubMed Central

    Di Magno, Laura; Basile, Alessio; Coni, Sonia; Manni, Simona; Sdruscia, Giulia; D'Amico, Davide; Antonucci, Laura; Infante, Paola; De Smaele, Enrico; Cucchi, Danilo; Ferretti, Elisabetta; Di Marcotullio, Lucia; Screpanti, Isabella; Canettieri, Gianluca

    2016-01-01

    Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs) and its aberrant activation is a leading cause of Medulloblastoma, the most frequent pediatric brain tumor. We show here that the energy sensor AMPK inhibits Hh signaling by phosphorylating a single residue of human Gli1 that is not conserved in other species. Studies with selective agonists and genetic deletion have revealed that AMPK activation inhibits canonical Hh signaling in human, but not in mouse cells. Indeed we show that AMPK phosphorylates Gli1 at the unique residue Ser408, which is conserved only in primates but not in other species. Once phosphorylated, Gli1 is targeted for proteasomal degradation. Notably, we show that selective AMPK activation inhibits Gli1-driven proliferation and that this effect is linked to Ser408 phosphorylation, which represents a key metabolic checkpoint for Hh signaling. Collectively, this data unveil a novel mechanism of inhibition of Gli1 function, which is exclusive for human cells and may be exploited for the treatment of Medulloblastoma or other Gli1 driven tumors. PMID:26843621

  20. ETC-1002 regulates immune response, leukocyte homing, and adipose tissue inflammation via LKB1-dependent activation of macrophage AMPK.

    PubMed

    Filippov, Sergey; Pinkosky, Stephen L; Lister, Richard J; Pawloski, Catherine; Hanselman, Jeffrey C; Cramer, Clay T; Srivastava, Rai Ajit K; Hurley, Timothy R; Bradshaw, Cheryl D; Spahr, Mark A; Newton, Roger S

    2013-08-01

    ETC-1002 is an investigational drug currently in Phase 2 development for treatment of dyslipidemia and other cardiometabolic risk factors. In dyslipidemic subjects, ETC-1002 not only reduces plasma LDL cholesterol but also significantly attenuates levels of hsCRP, a clinical biomarker of inflammation. Anti-inflammatory properties of ETC-1002 were further investigated in primary human monocyte-derived macrophages and in in vivo models of inflammation. In cells treated with ETC-1002, increased levels of AMP-activated protein kinase (AMPK) phosphorylation coincided with reduced activity of MAP kinases and decreased production of proinflammatory cytokines and chemokines. AMPK phosphorylation and inhibitory effects of ETC-1002 on soluble mediators of inflammation were significantly abrogated by siRNA-mediated silencing of macrophage liver kinase B1 (LKB1), indicating that ETC-1002 activates AMPK and exerts its anti-inflammatory effects via an LKB1-dependent mechanism. In vivo, ETC-1002 suppressed thioglycollate-induced homing of leukocytes into mouse peritoneal cavity. Similarly, in a mouse model of diet-induced obesity, ETC-1002 restored adipose AMPK activity, reduced JNK phosphorylation, and diminished expression of macrophage-specific marker 4F/80. These data were consistent with decreased epididymal fat-pad mass and interleukin (IL)-6 release by inflamed adipose tissue. Thus, ETC-1002 may provide further clinical benefits for patients with cardiometabolic risk factors by reducing systemic inflammation linked to insulin resistance and vascular complications of metabolic syndrome.

  1. Chronic AMPK activation via loss of FLCN induces functional beige adipose tissue through PGC-1α/ERRα.

    PubMed

    Yan, Ming; Audet-Walsh, Étienne; Manteghi, Sanaz; Rosa Dufour, Catherine; Walker, Benjamin; Baba, Masaya; St-Pierre, Julie; Giguère, Vincent; Pause, Arnim

    2016-05-01

    The tumor suppressor folliculin (FLCN) forms a repressor complex with AMP-activated protein kinase (AMPK). Given that AMPK is a master regulator of cellular energy homeostasis, we generated an adipose-specific Flcn (Adipoq-FLCN) knockout mouse model to investigate the role of FLCN in energy metabolism. We show that loss of FLCN results in a complete metabolic reprogramming of adipose tissues, resulting in enhanced oxidative metabolism. Adipoq-FLCN knockout mice exhibit increased energy expenditure and are protected from high-fat diet (HFD)-induced obesity. Importantly, FLCN ablation leads to chronic hyperactivation of AMPK, which in turns induces and activates two key transcriptional regulators of cellular metabolism, proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα). Together, the AMPK/PGC-1α/ERRα molecular axis positively modulates the expression of metabolic genes to promote mitochondrial biogenesis and activity. In addition, mitochondrial uncoupling proteins as well as other markers of brown fat are up-regulated in both white and brown FLCN-null adipose tissues, underlying the increased resistance of Adipoq-FLCN knockout mice to cold exposure. These findings identify a key role of FLCN as a negative regulator of mitochondrial function and identify a novel molecular pathway involved in the browning of white adipocytes and the activity of brown fat. PMID:27151976

  2. Deoxypodophyllotoxin suppresses tumor vasculature in HUVECs by promoting cytoskeleton remodeling through LKB1-AMPK dependent Rho A activatio.

    PubMed

    Wang, Yurong; Wang, Bin; Guerram, Mounia; Sun, Li; Shi, Wei; Tian, Chongchong; Zhu, Xiong; Jiang, Zhenzhou; Zhang, Luyong

    2015-10-01

    Angiogenesis plays a critical role in the growth and metastasis of tumors, which makes it an attractive target for anti-tumor drug development. Deoxypodophyllotoxin (DPT), a natural product isolated from Anthriscus sylvestris, inhibits cell proliferation and migration in various cancer cell types. Our previous studies indicate that DPT possesses both anti-angiogenic and vascular-disrupting activities. Although the RhoA/ RhoA kinase (ROCK) signaling pathway is implicated in DPT-stimulated cytoskeleton remodeling and tumor vasculature suppressing, the detailed mechanisms by which DPT mediates these effects are poorly understood. In the current study, we found that DPT promotes cytoskeleton remodeling in human umbilical vein endothelial cells (HUVECs) via stimulation of AMP-activated protein kinase (AMPK) and that this effect is abolished by either treatment with a selective AMPK inhibitor or knockdown. Moreover, the cellular levels of LKB1, a kinase upstream of AMPK, were enhanced following DPT exposure. DPT-induced activation of AMPK in tumor vasculature effect was also verified by transgenic zebrafish (VEGFR2:GFP), Matrigel plug assay, and xenograft model in nude mice. The present findings may lay the groundwork for a novel therapeutic approach in treating cancer. PMID:26470595

  3. Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan

    PubMed Central

    Nunes, Rodrigo Dutra; Ventura-Martins, Guilherme; Moretti, Débora Monteiro; Medeiros-Castro, Priscilla; Rocha-Santos, Carlucio; Daumas-Filho, Carlos Renato de Oliveira; Bittencourt-Cunha, Paula Rego Barros; Martins-Cardoso, Karina; Cudischevitch, Cecília Oliveira; Menna-Barreto, Rubem Figueiredo Sadok; Oliveira, José Henrique Maia; Gusmão, Desiely Silva; Alves Lemos, Francisco José; Alviano, Daniela Sales; Oliveira, Pedro Lagerblad; Lowenberger, Carl; Majerowicz, David; Oliveira, Ricardo Melo; Mesquita, Rafael Dias; Atella, Georgia Correa

    2016-01-01

    Background Mosquitoes feed on plant-derived fluids such as nectar and sap and are exposed to bioactive molecules found in this dietary source. However, the role of such molecules on mosquito vectorial capacity is unknown. Weather has been recognized as a major determinant of the spread of dengue, and plants under abiotic stress increase their production of polyphenols. Results Here, we show that including polyphenols in mosquito meals promoted the activation of AMP-dependent protein kinase (AMPK). AMPK positively regulated midgut autophagy leading to a decrease in bacterial proliferation and an increase in vector lifespan. Suppression of AMPK activity resulted in a 6-fold increase in midgut microbiota. Similarly, inhibition of polyphenol-induced autophagy induced an 8-fold increase in bacterial proliferation. Mosquitoes maintained on the polyphenol diet were readily infected by dengue virus. Conclusion The present findings uncover a new direct route by which exacerbation of autophagy through activation of the AMPK pathway leads to a more efficient control of mosquito midgut microbiota and increases the average mosquito lifespan. Our results suggest for the first time that the polyphenol content and availability of the surrounding vegetation may increase the population of mosquitoes prone to infection with arboviruses. PMID:27732590

  4. AKT and AMPK activation after high-fat and high-glucose in vitro treatment of prostate epithelial cells.

    PubMed

    Ribeiro, D L; Góes, R M; Pinto-Fochi, M E; Taboga, S R; Abrahamsson, P-A; Dizeyi, N

    2014-06-01

    Considering the increasing consumption of saturated fat and glucose in diets worldwide and its possible association to carcinogenesis, this investigation analysed the proliferation profile of nonmalignant human prostate epithelial cells after exposure to elevated levels of fat and glucose. PNT1A cells were cultured with palmitate (100 or 200 μM) and/or glucose (450 mg/dl) for 24 or 48 h. Treated cells were evaluated for viability test and cell proliferation (MTS assay). AKT and AMPK phosphorylation status were analysed by Western blotting. After 24 h of high-fat alone or associated with high-glucose treatment, there was an increase in AMPK and AKT activation associated to unchanged MTS-cell proliferation. Following 48 h of high-fat but not high-glucose alone, cells decreased AMPK activation and maintained elevated AKT levels. These data were associated to increased cell proliferation after further high-fat treatment. After longer high-fat exposure, MTS revealed that cells remained proliferating. High-glucose alone or associated to high-fat treatment was not able to increase cell proliferation and AKT activation. A high-fat medium containing 100 μM of palmitate stimulates proliferation in PNT1A cells by decreasing the activation of AMPK and increasing activation of AKT after longer exposure time. These findings improve the knowledge about the negative effect of high levels of this saturated fatty acid on proliferative disorders of prostate.

  5. Testosterone stimulates glucose uptake and GLUT4 translocation through LKB1/AMPK signaling in 3T3-L1 adipocytes.

    PubMed

    Mitsuhashi, Kazuteru; Senmaru, Takafumi; Fukuda, Takuya; Yamazaki, Masahiro; Shinomiya, Katsuhiko; Ueno, Morio; Kinoshita, Shigeru; Kitawaki, Jo; Katsuyama, Masato; Tsujikawa, Muneo; Obayashi, Hiroshi; Nakamura, Naoto; Fukui, Michiaki

    2016-01-01

    Decreases in serum testosterone concentrations in aging men are associated with metabolic disorders. Testosterone has been reported to increase GLUT4-dependent glucose uptake in skeletal muscle cells and cardiomyocytes. However, studies on glucose uptake occurring in response to testosterone stimulation in adipocytes are currently not available. This study was designed to determine the effects of testosterone on glucose uptake in adipocytes. Glucose uptake was assessed with 2-[(3)H] deoxyglucose in 3T3-L1 adipocytes. GLUT4 translocation was evaluated in plasma membrane (PM) sheets and PM fractions by immunofluorescence and immunoblotting, respectively. Activation of GLUT4 translocation-related protein kinases, including Akt, AMPK, LKB1, CaMKI, CaMKII, and Cbl was followed by immunoblotting. Expression levels of androgen receptor (AR) mRNA and AR translocation to the PM were assessed by real-time RT-PCR and immunoblotting, respectively. The results showed that both high-dose (100 nM) testosterone and testosterone-BSA increased glucose uptake and GLUT4 translocation to the PM, independently of the intracellular AR. Testosterone and testosterone-BSA stimulated the phosphorylation of AMPK, LKB1, and CaMKII. The knockdown of LKB1 by siRNA attenuated testosterone- and testosterone-BSA-stimulated AMPK phosphorylation and glucose uptake. These results indicate that high-dose testosterone and testosterone-BSA increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes by inducing the LKB1/AMPK signaling pathway.

  6. Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

    PubMed Central

    Shang, Fenqing; Zhang, Jiao; Li, Zhao; Zhang, Jin; Yin, Yanjun; Wang, Yaqiong; Marin, Traci L.; Gongol, Brendan; Xiao, Han; Zhang, You-yi; Chen, Zhen; Shyy, John Y-J; Lei, Ting

    2016-01-01

    Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction. PMID:26986624

  7. Two weeks of metformin treatment induces AMPK-dependent enhancement of insulin-stimulated glucose uptake in mouse soleus muscle

    PubMed Central

    Kristensen, Jonas Møller; Treebak, Jonas T.; Schjerling, Peter; Goodyear, Laurie

    2014-01-01

    Metformin-induced activation of the 5′-AMP-activated protein kinase (AMPK) has been associated with enhanced glucose uptake in skeletal muscle, but so far no direct causality has been examined. We hypothesized that an effect of in vivo metformin treatment on glucose uptake in mouse skeletal muscles is dependent on AMPK signaling. Oral doses of metformin or saline treatment were given to muscle-specific kinase dead (KD) AMPKα2 mice and wild-type (WT) littermates either once or chronically for 2 wk. Soleus and extensor digitorum longus muscles were used for measurements of glucose transport and Western blot analyses. Chronic treatment with metformin enhanced insulin-stimulated glucose uptake in soleus muscles of WT (∼45%, P < 0.01) but not of AMPK KD mice. Insulin signaling at the level of Akt protein expression or Thr308 and Ser473 phosphorylation was not changed by metformin treatment. Insulin signaling at the level of Akt and TBC1D4 protein expression as well as Akt Thr308/Ser473 and TBC1D4 Thr642/Ser711 phosphorylation were not changed by metformin treatment. Also, protein expressions of Rab4, GLUT4, and hexokinase II were unaltered after treatment. The acute metformin treatment did not affect glucose uptake in muscle of either of the genotypes. In conclusion, we provide novel evidence for a role of AMPK in potentiating the effect of insulin on glucose uptake in soleus muscle in response to chronic metformin treatment. PMID:24644243

  8. Olanzapine-induced hyperphagia and weight gain associate with orexigenic hypothalamic neuropeptide signaling without concomitant AMPK phosphorylation.

    PubMed

    Fernø, Johan; Varela, Luis; Skrede, Silje; Vázquez, María Jesús; Nogueiras, Rubén; Diéguez, Carlos; Vidal-Puig, Antonio; Steen, Vidar M; López, Miguel

    2011-01-01

    The success of antipsychotic drug treatment in patients with schizophrenia is limited by the propensity of these drugs to induce hyperphagia, weight gain and other metabolic disturbances, particularly evident for olanzapine and clozapine. However, the molecular mechanisms involved in antipsychotic-induced hyperphagia remain unclear. Here, we investigate the effect of olanzapine administration on the regulation of hypothalamic mechanisms controlling food intake, namely neuropeptide expression and AMP-activated protein kinase (AMPK) phosphorylation in rats. Our results show that subchronic exposure to olanzapine upregulates neuropeptide Y (NPY) and agouti related protein (AgRP) and downregulates proopiomelanocortin (POMC) in the arcuate nucleus of the hypothalamus (ARC). This effect was evident both in rats fed ad libitum and in pair-fed rats. Of note, despite weight gain and increased expression of orexigenic neuropeptides, subchronic administration of olanzapine decreased AMPK phosphorylation levels. This reduction in AMPK was not observed after acute administration of either olanzapine or clozapine. Overall, our data suggest that olanzapine-induced hyperphagia is mediated through appropriate changes in hypothalamic neuropeptides, and that this effect does not require concomitant AMPK activation. Our data shed new light on the hypothalamic mechanism underlying antipsychotic-induced hyperphagia and weight gain, and provide the basis for alternative targets to control energy balance.

  9. Proglucagon Promoter Cre-Mediated AMPK Deletion in Mice Increases Circulating GLP-1 Levels and Oral Glucose Tolerance

    PubMed Central

    Sayers, Sophie R.; Reimann, Frank; Gribble, Fiona M.; Parker, Helen; Zac-Varghese, Sagen; Bloom, Stephen R.; Foretz, Marc; Viollet, Benoit; Rutter, Guy A.

    2016-01-01

    Background Enteroendocrine L-cells synthesise and release the gut hormone glucagon-like peptide-1 (GLP-1) in response to food transit. Deletion of the tumour suppressor kinase LKB1 from proglucagon-expressing cells leads to the generation of intestinal polyps but no change in circulating GLP-1 levels. Here, we explore the role of the downstream kinase AMP-activated protein kinase (AMPK) in these cells. Method Loss of AMPK from proglucagon-expressing cells was achieved using a preproglucagon promoter-driven Cre (iGluCre) to catalyse recombination of floxed alleles of AMPKα1 and α2. Oral and intraperitoneal glucose tolerance were measured using standard protocols. L-cell mass was measured by immunocytochemistry. Hormone and peptide levels were measured by electrochemical-based luminescence detection or radioimmunoassay. Results Recombination with iGluCre led to efficient deletion of AMPK from intestinal L- and pancreatic alpha-cells. In contrast to mice rendered null for LKB1 using the same strategy, mice deleted for AMPK displayed an increase (WT: 0.05 ± 0.01, KO: 0.09±0.02%, p<0.01) in L-cell mass and elevated plasma fasting (WT: 5.62 ± 0.800 pg/ml, KO: 14.5 ± 1.870, p<0.01) and fed (WT: 15.7 ± 1.48pg/ml, KO: 22.0 ± 6.62, p<0.01) GLP-1 levels. Oral, but not intraperitoneal, glucose tolerance was significantly improved by AMPK deletion, whilst insulin and glucagon levels were unchanged despite an increase in alpha to beta cell ratio (WT: 0.23 ± 0.02, KO: 0.33 ± 0.03, p<0.01). Conclusion AMPK restricts L-cell growth and GLP-1 secretion to suppress glucose tolerance. Targeted inhibition of AMPK in L-cells may thus provide a new therapeutic strategy in some forms of type 2 diabetes. PMID:27010458

  10. AMPK activation restores the stimulation of glucose uptake in an in vitro model of insulin-resistant cardiomyocytes via the activation of protein kinase B.

    PubMed

    Bertrand, Luc; Ginion, Audrey; Beauloye, Christophe; Hebert, Alexandre D; Guigas, Bruno; Hue, Louis; Vanoverschelde, Jean-Louis

    2006-07-01

    Diabetic hearts are known to be more susceptible to ischemic disease. Biguanides, like metformin, are known antidiabetic drugs that lower blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in muscle. Part of these metabolic effects is thought to be mediated by the activation of AMP-activated protein kinase (AMPK). In this work, we studied the relationship between AMPK activation and glucose uptake stimulation by biguanides and oligomycin, another AMPK activator, in both insulin-sensitive and insulin-resistant cardiomyocytes. In insulin-sensitive cardiomyocytes, insulin, biguanides and oligomycin were able to stimulate glucose uptake with the same efficiency. Stimulation of glucose uptake by insulin or biguanides was correlated to protein kinase B (PKB) or AMPK activation, respectively, and were additive. In insulin-resistant cardiomyocytes, where insulin stimulation of glucose uptake was greatly reduced, biguanides or oligomycin, in the absence of insulin, induced a higher stimulation of glucose uptake than that obtained in insulin-sensitive cells. This stimulation was correlated with the activation of both AMPK and PKB and was sensitive to the phosphatidylinositol-3-kinase/PKB pathway inhibitors. Finally, an adenoviral-mediated expression of a constitutively active form of AMPK increased both PKB phosphorylation and glucose uptake in insulin-resistant cardiomyocytes. We concluded that AMPK activators, like biguanides and oligomycin, are able to restore glucose uptake stimulation, in the absence of insulin, in insulin-resistant cardiomyocytes via the additive activation of AMPK and PKB. Our results suggest that AMPK activation could restore normal glucose metabolism in diabetic hearts and could be a potential therapeutic approach to treat insulin resistance.

  11. Liraglutide attenuates the osteoblastic differentiation of MC3T3-E1 cells by modulating AMPK/mTOR signaling

    PubMed Central

    Hu, Xiong-Ke; Yin, Xin-Hua; Zhang, Hong-Qi; Guo, Chao-Feng; Tang, Ming-Xing

    2016-01-01

    Liraglutide, a synthetic analogue of glucagon-like peptide-1, is utilized in the treatment of type 2 diabetes and obesity. Liraglutide has been previously demonstrated to prevent osteoblastic differentiation of human vascular smooth muscle cells, resulting in the slowing of arterial calcification, however, its effect on bone formation remains unclear. The present study investigated the effect of liraglutide on osteoblastic differentiation using Alizarin Red S staining, and examined the molecular mechanisms underlying the regulatory effect by western blot analysis. The present study demonstrated that protein expression levels of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) were downregulated in MC3T3-E1 cells during osteoblastic differentiation in commercial osteogenic differentiation medium, whereas protein expression levels of transforming growth factor-β (TGF-β) and phosphorylated mammalian target of rapamycin (p-mTOR) increased. Liraglutide was subsequently demonstrated to dose-dependently attenuate the osteoblastic differentiation of MC3T3-E1 cells, to upregulate p-AMPK, and downregulate p-mTOR and TGF-β protein expression levels. Treatment with an AMPK-specific inhibitor, Compound C, eradicated the effect of liraglutide on osteoblastic differentiation, and p-mTOR and TGF-β downregulation. An mTOR activator, MHY1485, also abolished the inhibitory effect of liraglutide on osteoblastic differentiation, and resulted in p-mTOR and TGF-β downregulation, but did not attenuate the liraglutide-induced increase in p-AMPK protein expression levels. The results of the present study demonstrate that liraglutide attenuates osteoblastic differentiation of MC3T3-E1 cells via modulation of AMPK/mTOR signaling. The present study revealed a novel function of liraglutide, which contributes to the understanding of its pharmacological and physiological effects in clinical settings. PMID:27600753

  12. Cation-selective transporters are critical to the AMPK-mediated antiproliferative effects of metformin in human breast cancer cells.

    PubMed

    Cai, Hao; Zhang, Yunhui; Han, Tianxiang Kevin; Everett, Ruth S; Thakker, Dhiren R

    2016-05-01

    The antidiabetic drug metformin exerts antineoplastic effects against breast cancer and other cancers. One mechanism by which metformin is believed to exert its anticancer effect involves activation of its intracellular target, adenosine monophosphate-activated protein kinase (AMPK), which is also implicated in the antidiabetic effect of metformin. It is proposed that in cancer cells, AMPK activation leads to inhibition of the mammalian target of rapamycin (mTOR) and the downstream pS6K that regulates cell proliferation. Due to its hydrophilic and cationic nature, metformin requires cation-selective transporters to enter cells and activate AMPK. This study demonstrates that expression levels of cation-selective transporters correlate with the antiproliferative and antitumor efficacy of metformin in breast cancer. Metformin uptake and antiproliferative activity were compared between a cation-selective transporter-deficient human breast cancer cell line, BT-20, and a BT-20 cell line that was engineered to overexpress organic cation transporter 3 (OCT3), a representative of cation-selective transporters and a predominant transporter in human breast tumors. Metformin uptake was minimal in BT-20 cells, but increased by >13-fold in OCT3-BT20 cells, and its antiproliferative potency was >4-fold in OCT3-BT20 versus BT-20 cells. This increase in antiproliferative activity was associated with greater AMPK phosphorylation and decreased pS6K phosphorylation in OCT3-BT20 cells. In vitro data were corroborated by in vivo observations of significantly greater antitumor efficacy of metformin in xenograft mice bearing OCT3-overexpressing tumors versus low transporter-expressing wildtype tumors. Collectively, these findings establish a clear relationship between cation-selective transporter expression, the AMPK-mTOR-pS6K signaling cascade, and the antiproliferative activity of metformin in breast cancer. PMID:26669511

  13. Chronic stress-induced memory deficits are reversed by regular exercise via AMPK-mediated BDNF induction.

    PubMed

    Kim, D-M; Leem, Y-H

    2016-06-01

    Chronic stress has a detrimental effect on neurological insults, psychiatric deficits, and cognitive impairment. In the current study, chronic stress was shown to impair learning and memory functions, in addition to reducing in hippocampal Adenosine monophosphate-activated protein kinase (AMPK) activity. Similar reductions were also observed for brain-derived neurotrophic factor (BDNF), synaptophysin, and post-synaptic density-95 (PSD-95) levels, all of which was counter-regulated by a regime of regular and prolonged exercise. A 21-day restraint stress regimen (6 h/day) produced learning and memory deficits, including reduced alternation in the Y-maze and decreased memory retention in the water maze test. These effects were reversed post-administration by a 3-week regime of treadmill running (19 m/min, 1 h/day, 6 days/week). In hippocampal primary culture, phosphorylated-AMPK (phospho-AMPK) and BDNF levels were enhanced in a dose-dependent manner by 5-amimoimidazole-4-carboxamide riboside (AICAR) treatment, and AICAR-treated increase was blocked by Compound C. A 7-day period of AICAR intraperitoneal injections enhanced alternation in the Y-maze test and reduced escape latency in water maze test, along with enhanced phospho-AMPK and BDNF levels in the hippocampus. The intraperitoneal injection of Compound C every 4 days during exercise intervention diminished exercise-induced enhancement of memory improvement during the water maze test in chronically stressed mice. Also, chronic stress reduced hippocampal neurogenesis (lower Ki-67- and doublecortin-positive cells) and mRNA levels of BDNF, synaptophysin, and PSD-95. Our results suggest that regular and prolonged exercise can alleviate chronic stress-induced hippocampal-dependent memory deficits. Hippocampal AMPK-engaged BDNF induction is at least in part required for exercise-induced protection against chronic stress. PMID:26975895

  14. Effects of eugenol on hepatic glucose production and AMPK signaling pathway in hepatocytes and C57BL/6J mice.

    PubMed

    Jeong, Kyong Ju; Kim, Do Yeon; Quan, Hai-Yan; Jo, Hee Kyung; Kim, Go Woon; Chung, Sung Hyun

    2014-03-01

    Eugenol is a phenylpropanoid with many pharmacological activities, but its anti-hyperglycemic activity is not yet fully explored. For in vitro study, HepG2 cells and primary rat hepatocytes were used, and glucose production was induced by adding 100 nM of glucagon in the presence of gluconeogenic substrates. In animal study, hyperglycemia was induced by high fat diet (HFD) in male C57BL/6J mice, and eugenol was orally administered at 20 or 40 mg per kg (E20, E40) for 15 weeks. Eugenol significantly inhibited glucagon-induced glucose production and phosphorylated AMPK in the HepG2 and primary rat hepatocytes, and these effects were reversed in the presence of compound C (an AMPK inhibitor) or STO-609 (a CAMKK inhibitor). In addition, the protein and gene expression levels of CREB, CRTC2·CREB complex, PGC-1α, PEPCK and G6Pase were all significantly suppressed. Moreover, inhibition of AMPK by over-expression of dominant negative AMPK prevented eugenol from suppressions of gluconeogenic gene expression and hepatic glucose production. In animal study, plasma glucose and insulin levels of the E40 group were decreased by 31% and 63%, respectively, when compared to those of HFD control. In pyruvate tolerance tests, pyruvate-induced glucose excursions were decreased, indicating that the anti-hyperglycemic activity of eugenol is primarily due to the suppression of hepatic gluconeogenesis. In summary, eugenol effectively ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis via modulating CAMKK-AMPK-CREB signaling pathway. Eugenol or eugenol-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes.

  15. Improved myocardial perfusion in chronic diabetic mice by the up-regulation of pLKB1 and AMPK signaling.

    PubMed

    Kusmic, Claudia; L'abbate, Antonio; Sambuceti, Gianmario; Drummond, George; Barsanti, Cristina; Matteucci, Marco; Cao, Jian; Piccolomini, Francesco; Cheng, Jennifer; Abraham, Nader G

    2010-04-01

    Previous studies related impaired myocardial microcirculation in diabetes to oxidative stress and endothelial dysfunction. Thus, this study was aimed to determine the effect of up-regulating pAMPK-pAKT signaling on coronary microvascular reactivity in the isolated heart of diabetic mice. We measured coronary resistance in wild-type and streptozotocin (STZ)-treated mice, during perfusion pressure changes. Glucose, insulin, and adiponectin levels in plasma and superoxide formation, NOx levels and heme oxygenase (HO) activity in myocardial tissue were determined. In addition, the expression of HO-1, 3-nitrotyrosine, pLKB1, pAMPK, pAKT, and peNOS proteins in control and diabetic hearts were measured. Coronary response to changes in perfusion pressure diverged from control in a time-dependent manner following STZ administration. The responses observed at 28 weeks of diabetes (the maximum time examined) were mimicked by L-NAME administration to control animals and were associated with a decrease in serum adiponectin and myocardial pLKB1, pAMPK, pAKT, and pGSK-3 expression. Cobalt protoporphyrin treatment to induce HO-1 expression reversed the microvascular reactivity seen in diabetes towards that of controls. Up-regulation of HO-1 was associated with an increase in adiponectin, pLKB1, pAKT, pAMPK, pGSK-3, and peNOS levels and a decrease in myocardial superoxide and 3-nitrotyrosine levels. In the present study we describe the time course of microvascular functional changes during the development of diabetes and the existence of a unique relationship between the levels of serum adiponectin, pLKB1, pAKT, and pAMPK activation in diabetic hearts. The restoration of microvascular function suggests a new therapeutic approach to even advanced cardiac microvascular derangement in diabetes.

  16. Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts.

    PubMed

    Klein, Janet D; Wang, Yanhua; Blount, Mitsi A; Molina, Patrick A; LaRocque, Lauren M; Ruiz, Joseph A; Sands, Jeff M

    2016-05-15

    Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations. PMID:26962099

  17. Chronic stress-induced memory deficits are reversed by regular exercise via AMPK-mediated BDNF induction.

    PubMed

    Kim, D-M; Leem, Y-H

    2016-06-01

    Chronic stress has a detrimental effect on neurological insults, psychiatric deficits, and cognitive impairment. In the current study, chronic stress was shown to impair learning and memory functions, in addition to reducing in hippocampal Adenosine monophosphate-activated protein kinase (AMPK) activity. Similar reductions were also observed for brain-derived neurotrophic factor (BDNF), synaptophysin, and post-synaptic density-95 (PSD-95) levels, all of which was counter-regulated by a regime of regular and prolonged exercise. A 21-day restraint stress regimen (6 h/day) produced learning and memory deficits, including reduced alternation in the Y-maze and decreased memory retention in the water maze test. These effects were reversed post-administration by a 3-week regime of treadmill running (19 m/min, 1 h/day, 6 days/week). In hippocampal primary culture, phosphorylated-AMPK (phospho-AMPK) and BDNF levels were enhanced in a dose-dependent manner by 5-amimoimidazole-4-carboxamide riboside (AICAR) treatment, and AICAR-treated increase was blocked by Compound C. A 7-day period of AICAR intraperitoneal injections enhanced alternation in the Y-maze test and reduced escape latency in water maze test, along with enhanced phospho-AMPK and BDNF levels in the hippocampus. The intraperitoneal injection of Compound C every 4 days during exercise intervention diminished exercise-induced enhancement of memory improvement during the water maze test in chronically stressed mice. Also, chronic stress reduced hippocampal neurogenesis (lower Ki-67- and doublecortin-positive cells) and mRNA levels of BDNF, synaptophysin, and PSD-95. Our results suggest that regular and prolonged exercise can alleviate chronic stress-induced hippocampal-dependent memory deficits. Hippocampal AMPK-engaged BDNF induction is at least in part required for exercise-induced protection against chronic stress.

  18. Cation-selective transporters are critical to the AMPK-mediated antiproliferative effects of metformin in human breast cancer cells.

    PubMed

    Cai, Hao; Zhang, Yunhui; Han, Tianxiang Kevin; Everett, Ruth S; Thakker, Dhiren R

    2016-05-01

    The antidiabetic drug metformin exerts antineoplastic effects against breast cancer and other cancers. One mechanism by which metformin is believed to exert its anticancer effect involves activation of its intracellular target, adenosine monophosphate-activated protein kinase (AMPK), which is also implicated in the antidiabetic effect of metformin. It is proposed that in cancer cells, AMPK activation leads to inhibition of the mammalian target of rapamycin (mTOR) and the downstream pS6K that regulates cell proliferation. Due to its hydrophilic and cationic nature, metformin requires cation-selective transporters to enter cells and activate AMPK. This study demonstrates that expression levels of cation-selective transporters correlate with the antiproliferative and antitumor efficacy of metformin in breast cancer. Metformin uptake and antiproliferative activity were compared between a cation-selective transporter-deficient human breast cancer cell line, BT-20, and a BT-20 cell line that was engineered to overexpress organic cation transporter 3 (OCT3), a representative of cation-selective transporters and a predominant transporter in human breast tumors. Metformin uptake was minimal in BT-20 cells, but increased by >13-fold in OCT3-BT20 cells, and its antiproliferative potency was >4-fold in OCT3-BT20 versus BT-20 cells. This increase in antiproliferative activity was associated with greater AMPK phosphorylation and decreased pS6K phosphorylation in OCT3-BT20 cells. In vitro data were corroborated by in vivo observations of significantly greater antitumor efficacy of metformin in xenograft mice bearing OCT3-overexpressing tumors versus low transporter-expressing wildtype tumors. Collectively, these findings establish a clear relationship between cation-selective transporter expression, the AMPK-mTOR-pS6K signaling cascade, and the antiproliferative activity of metformin in breast cancer.

  19. Apoptotic Cells Activate AMP-activated Protein Kinase (AMPK) and Inhibit Epithelial Cell Growth without Change in Intracellular Energy Stores*

    PubMed Central

    Patel, Vimal A.; Massenburg, Donald; Vujicic, Snezana; Feng, Lanfei; Tang, Meiyi; Litbarg, Natalia; Antoni, Angelika; Rauch, Joyce; Lieberthal, Wilfred; Levine, Jerrold S.

    2015-01-01

    Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses. PMID:26183782

  20. mTOR, AMPK, and Sirt1: Key Players in Metabolic Stress Management.

    PubMed

    Cetrullo, Silvia; D'Adamo, Stefania; Tantini, Benedetta; Borzi, Rosa Maria; Flamigni, Flavio

    2015-01-01

    Cells adapt their metabolism and activities in response to signals from their surroundings, and this ability is essential for their survival in the face of environmental changes. In mammalian tissues a deficit of these mechanisms is commonly associated with cellular aging and degenerative diseases related to aging, such as cardiovascular disease, cancer, immune system decline, and neurological pathologies. Several proteins have been identified as able to respond directly to energy, nutrient, and growth factor levels and stress stimuli in order to mediate adaptations in the cell. Many of these proteins are enzymes that positively or negatively modulate the autophagic process. This review focuses on biochemical mechanisms involving enzymes--specifically, mTOR, AMPK, and Sirt1--that are currently considered important for these adaptive responses, providing an overview of the interactions of the main players in this process.

  1. Paeonol protects against endoplasmic reticulum stress-induced endothelial dysfunction via AMPK/PPARδ signaling pathway.

    PubMed

    Choy, Ker-Woon; Mustafa, Mohd Rais; Lau, Yeh Siang; Liu, Jian; Murugan, Dharmani; Lau, Chi Wai; Wang, Li; Zhao, Lei; Huang, Yu

    2016-09-15

    Endoplasmic reticulum (ER) stress in endothelial cells often leads to endothelial dysfunction which underlies the pathogenesis of cardiovascular diseases. Paeonol, a major phenolic component extracted from Moutan Cortex, possesses various medicinal benefits which have been used extensively in traditional Chinese medicine. The present study investigated the protective mechanism of paeonol against tunicamycin-induced ER stress in isolated mouse aortas and human umbilical vein endothelial cells (HUVECs). Vascular reactivity in aorta was measured using a wire myograph. The effects of paeonol on protein expression of ER stress markers, reactive oxygen species (ROS) production, nitric oxide (NO) bioavailability and peroxisome proliferator-activated receptor δ (PPARδ) activity in the vascular wall were assessed by Western blot, dihydroethidium fluorescence (DHE) or lucigenin enhanced-chemiluminescence, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA) and dual luciferase reporter assay, respectively. Ex vivo treatment with paeonol (0.1μM) for 16h reversed the impaired endothelium-dependent relaxations in C57BJ/6J and PPARδ wild type (WT) mouse aortas following incubation with tunicamycin (0.5μg/mL). Elevated ER stress markers, oxidative stress and reduction of NO bioavailability induced by tunicamycin in HUVECs, C57BJ/6J and PPARδ WT mouse aortas were reversed by paeonol treatment. These beneficial effects of paeonol were diminished in PPARδ knockout (KO) mouse aortas. Paeonol increased the expression of 5' adenosine monophosphate-activated protein kinase (AMPK) and PPARδ expression and activity while restoring the decreased phosphorylation of eNOS. The present study delineates that paeonol protects against tunicamycin-induced vascular endothelial dysfunction by inhibition of ER stress and oxidative stress, thus elevating NO bioavailability via the AMPK/PPARδ signaling pathway.

  2. Kinase Suppressor of Ras 2 (KSR2) Regulates Tumor Cell Transformation via AMPK

    PubMed Central

    Fernandez, Mario R.; Henry, MaLinda D.

    2012-01-01

    Kinase suppressor of Ras 1 (KSR1) and KSR2 are scaffolds that promote extracellular signal-regulated kinase (ERK) signaling but have dramatically different physiological functions. KSR2−/− mice show marked deficits in energy expenditure that cause obesity. In contrast, KSR1 disruption has inconsequential effects on development but dramatically suppresses tumor formation by activated Ras. We examined the role of KSR2 in the generation and maintenance of the transformed phenotype in KSR1−/− mouse embryo fibroblasts (MEFs) expressing activated RasV12 and in tumor cell lines MIN6 and NG108-15. KSR2 rescued ERK activation and accelerated proliferation in KSR1−/− MEFs. KSR2 expression alone induced anchorage-independent growth and synergized with the transforming effects of RasV12. Similarly, RNA interference (RNAi) of KSR2 in MIN6 and NG108-15 cells inhibited proliferation and colony formation, with concomitant defects in AMP-activated protein kinase (AMPK) signaling, nutrient metabolism, and metabolic capacity. While constitutive activation of AMPK was sufficient to complement the loss of KSR2 in metabolic signaling and anchorage-independent growth, KSR2 RNAi, MEK inhibition, and expression of a KSR2 mutant unable to interact with ERK demonstrated that mitogen-activated protein (MAP) kinase signaling is dispensable for the transformed phenotype of these cells. These data show that KSR2 is essential to tumor cell energy homeostasis and critical to the integration of mitogenic and metabolic signaling pathways. PMID:22801368

  3. Total saponins from Aralia taibaiensis protect against myocardial ischemia/reperfusion injury through AMPK pathway

    PubMed Central

    YAN, JIAJIA; DUAN, JIALIN; WU, XIAOXIAO; GUO, CHAO; YIN, YING; ZHU, YANRONG; HU, TIANXIN; WEI, GUO; WEN, AIDONG; XI, MIAOMIAO

    2015-01-01

    It was previously shown that total saponins extracted from Aralia taibaiensis (sAT) have potent antioxidant activities for treating diabetes mellitus and attenuate D-galactose-induced aging. Since diabetes mellitus and aging are closely associated with cardiac dysfunction, particularly ischemic heart disease, sAT may have potential protective activity against myocardial ischemia/reperfusion injury (MI/RI). However, the anti-MI/RI effects of sAT have yet to be examined, and the possible molecular mechanisms remain to be determined. The present study was undertaken to investigate the anti-MI/RI activities of sAT and to elucidate the mechanisms underlying these effects in rats using TUNEL and Hoechst 33258 staining. The results confirmed the cardioprotective effects in vivo and elucidated the potential molecular mechanisms of sAT in vitro. Pretreatment with sAT significantly reduced infarct size, decreased the levels of lactate dehydrogenase and creatine kinase in the serum and blocked apoptosis. In addition, sAT inhibited A/R-induced apoptosis by decreasing DNA strand breaks, caspase-3 activity and cytochrome c release in H9c2 cells. Furthermore, sAT markedly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase and elevated the Bcl2/Bcl-2-associated X protein ratio. These effects were blocked by compound C. The results suggested that sAT pretreatment exerts protective effects on myocardial cells in vitro and in vivo against MI/RI-induced apoptosis by activating AMPK pathway. PMID:26498380

  4. Oligonol promotes anti-aging pathways via modulation of SIRT1-AMPK-Autophagy Pathway

    PubMed Central

    Park, Seul-Ki; Seong, Rak-Kyun; Kim, Ji-Ae; Son, Seok-Jun; Kim, Younghoon; Yokozawa, Takako

    2016-01-01

    BACKGROUND/OBJECTIVES Oligonol, mainly found in lychee fruit, is an antioxidant polyphenolic compound which has been shown to have anti-inflammatory and anti-cancer properties. The detailed mechanisms by which oligonol may act as an anti-aging molecule have not been determined. MATERIALS/METHODS In this study, we evaluated the ability of oligonol to modulate sirtuin (SIRT) expression in human lung epithelial (A549) cells. Oligonol was added to A549 cells and reactive oxygen species production, mitochondrial superoxide formation, and p21 protein levels were measured. Signaling pathways activated upon oligonol treatment were also determined by western blotting. Furthermore, the anti-aging effect of oligonol was evaluated ex vivo in mouse splenocytes and in vivo in Caenorhabditis elegans. RESULTS Oligonol specifically induced the expression of SIRT1, whose activity is linked to gene expression, metabolic control, and healthy aging. In response to influenza virus infection of A549 cells, oligonol treatment significantly up-regulated SIRT1 expression and down-regulated viral hemagglutinin expression. Oligonol treatment also resulted in the activation of autophagy pathways and the phosphorylation of AMP-activated protein kinase (AMPK). Furthermore, oligonol-treated spleen lymphocytes from old mice showed increased cell proliferation, and mRNA levels of SIRT1 in the lungs of old mice were significantly lower than those in the lungs of young mice. Additionally, in vivo lethality assay revealed that oligonol extended the lifespan of C. elegans infected with lethal Vibrio cholerae. CONCLUSIONS These data demonstrated that oligonol may act as an anti-aging molecule by modulating SIRT1/autophagy/AMPK pathways. PMID:26865910

  5. Paeonol protects against endoplasmic reticulum stress-induced endothelial dysfunction via AMPK/PPARδ signaling pathway.

    PubMed

    Choy, Ker-Woon; Mustafa, Mohd Rais; Lau, Yeh Siang; Liu, Jian; Murugan, Dharmani; Lau, Chi Wai; Wang, Li; Zhao, Lei; Huang, Yu

    2016-09-15

    Endoplasmic reticulum (ER) stress in endothelial cells often leads to endothelial dysfunction which underlies the pathogenesis of cardiovascular diseases. Paeonol, a major phenolic component extracted from Moutan Cortex, possesses various medicinal benefits which have been used extensively in traditional Chinese medicine. The present study investigated the protective mechanism of paeonol against tunicamycin-induced ER stress in isolated mouse aortas and human umbilical vein endothelial cells (HUVECs). Vascular reactivity in aorta was measured using a wire myograph. The effects of paeonol on protein expression of ER stress markers, reactive oxygen species (ROS) production, nitric oxide (NO) bioavailability and peroxisome proliferator-activated receptor δ (PPARδ) activity in the vascular wall were assessed by Western blot, dihydroethidium fluorescence (DHE) or lucigenin enhanced-chemiluminescence, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM DA) and dual luciferase reporter assay, respectively. Ex vivo treatment with paeonol (0.1μM) for 16h reversed the impaired endothelium-dependent relaxations in C57BJ/6J and PPARδ wild type (WT) mouse aortas following incubation with tunicamycin (0.5μg/mL). Elevated ER stress markers, oxidative stress and reduction of NO bioavailability induced by tunicamycin in HUVECs, C57BJ/6J and PPARδ WT mouse aortas were reversed by paeonol treatment. These beneficial effects of paeonol were diminished in PPARδ knockout (KO) mouse aortas. Paeonol increased the expression of 5' adenosine monophosphate-activated protein kinase (AMPK) and PPARδ expression and activity while restoring the decreased phosphorylation of eNOS. The present study delineates that paeonol protects against tunicamycin-induced vascular endothelial dysfunction by inhibition of ER stress and oxidative stress, thus elevating NO bioavailability via the AMPK/PPARδ signaling pathway. PMID:27449753

  6. Palmitate activates mTOR/p70S6K through AMPK inhibition and hypophosphorylation of raptor in skeletal muscle cells: Reversal by oleate is similar to metformin.

    PubMed

    Kwon, Bumsup; Querfurth, Henry W

    2015-11-01

    Excessive saturated free fatty acids (SFFAs; e.g. palmitate) in blood are a pathogenic factor in diabetes, obesity, cardiovascular disease and liver failure. In contrast, monounsaturated free fatty acids (e.g. oleate) prevent the toxic effect of SFFAs in various types of cells. The mechanism is poorly understood and involvement of the mTOR complex is untested. In the present study, we demonstrate that oleate preconditioning, as well as coincubation, completely prevented palmitate-induced markers of inflammatory signaling, insulin resistance and cytotoxicity in C2C12 myotubes. We then examined the effect of palmitate and/or oleate on the mammalian target of rapamycin (mTOR) signal path and whether their link is mediated by AMP-activated protein kinase (AMPK). Palmitate decreased the phosphorylation of raptor and 4E-BP1 while increasing the phosphorylation of p70S6K. Palmitate also inhibited phosphorylation of AMPK, but did not change the phosphorylated levels of mTOR or rictor. Oleate completely prevented the palmitate-induced dysregulation of mTOR components and restored pAMPK whereas alone it produced no signaling changes. To understand this more, we show activation of AMPK by metformin also prevented palmitate-induced changes in the phosphorylations of raptor and p70S6K, confirming that the mTORC1/p70S6K signaling pathway is responsive to AMPK activity. By contrast, inhibition of AMPK phosphorylation by Compound C worsened palmitate-induced changes and correspondingly blocked the protective effect of oleate. Finally, metformin modestly attenuated palmitate-induced insulin resistance and cytotoxicity, as did oleate. Our findings indicate that palmitate activates mTORC1/p70S6K signaling by AMPK inhibition and phosphorylation of raptor. Oleate reverses these effects through a metformin-like facilitation of AMPK. PMID:26344902

  7. Polysaccharide from Fuzi likely protects against starvation-induced cytotoxicity in H9c2 cells by increasing autophagy through activation of the AMPK/mTOR pathway.

    PubMed

    Liao, Li-Zhen; Chen, Yan-Ling; Lu, Li-He; Zhao, Yong-Hua; Guo, Hua-Lei; Wu, Wei-Kang

    2013-01-01

    There is increasing evidence that starvation induces autophagy, which may be protective during starvation, in an AMPK-dependent manner. Polysaccharides from Fuzi (FPS) reportedly have protective effects on nutrition-limited livers. The present study was designed to determine whether FPS protected H9c2 cells against starvation-induced cytotoxicity using an AMPK/mTOR-dependent mechanism. H9c2 cells were incubated in serum and glucose starvation media for 12 hours to establish a cell injury model. 3-Methyladenine (3MA, an autophagy inhibitor) was used to identify the exact role of autophagy in starvation. Cells were incubated with different FPS concentrations, and the cell injury levels, autophagy activity and AMPK/mTOR phosphorylation were measured. Adenine 9-β-D-arabinofuranoside (Ara-A, an AMPK inhibitor) and 5-amino-4-imidazole-carboxamide riboside (AICAR, an AMPK activator) were used to identify whether the AMPK/mTOR pathway was involved in FPS-mediated cardioprotection. We demonstrated that starvation decreased cell viability in a time-dependent manner, and 3MA-induced autophagy inhibition aggravated the reduced cell viability. FPS treatment attenuated the cell viability decrement and the starvation-induced decline in the mitochondrial membrane potential (MMP), and autophagy; also, the AMPK/mTOR pathways were activated during treatment. Ara-A treatment abolished the protective effect of FPS, while AICAR treatment had a similar effect to FPS. We conclude that autophagy attenuates starvation-induced cardiomyocyte death, and FPS increases autophagy activity to protect against starvation-induced cytotoxicity in H9c2 cells, likely through AMPK/mTOR pathway activation.

  8. Genetic selection for body weight in chickens has altered responses of the brain's AMPK system to food intake regulation effect of ghrelin, but not obestatin.

    PubMed

    Xu, Pingwen; Siegel, Paul B; Denbow, D Michael

    2011-08-01

    The effects of ghrelin and obestatin regulation of food intake are different in mammals and chickens. We investigated central effects of ghrelin and obestatin in lines of chickens selected 50 generations for high (HWS) or low (LWS) body weight. We hypothesized that the effect of ghrelin and obestatin on food intake in 5-day-old chicks is mediated by the AMP-activated protein kinase (AMPK) system and selection for body weight alters the brain's response to ghrelin and obestatin by changing the neuronal AMPK system. Although intracerebroventricular (ICV) ghrelin injection decreased food intake in both lines, the threshold for the anorexigenic effect of central ghrelin was lower in LWS than HWS chicks. Obestatin caused a linear dose-dependent increase in food intake in HWS but not LWS chicks. ICV injection of 0.4 nmol ghrelin inhibited hypothalamic AMPK related gene expression and phosphorylation of AMPK α and acetyl-CoA carboxylase (ACC) with the magnitude of inhibition different in the two lines. In contrast, ICV injection of 4 nmol obestatin did not affect mRNA expression of AMPK system or phosphorylation of AMPK and ACC in either line. These data support the premise of a lower threshold for anorexigenic effect of central ghrelin in LWS than HWS chicks, and this difference may be associated with differential hypothalamic AMPK signaling. Additionally, the hypothalamic mRNA level of ghrelin was significantly higher in LWS than HWS, which may have also contributed to the different threshold response to ghrelin in these two lines. The expression of the ghrelin receptor was also higher in the LWS line, but not until 56 days of age. In summary, selection for body weight has resulted in differences in the central ghrelin and obestatin system, and an altered brain AMPK system may contribute to the different neuronal response to ghrelin, but not obestatin.

  9. Decreased spontaneous activity in AMPK α2 muscle specific kinase dead mice is not caused by changes in brain dopamine metabolism.

    PubMed

    Møller, Lisbeth L V; Sylow, Lykke; Gøtzsche, Casper R; Serup, Annette K; Christiansen, Søren H; Weikop, Pia; Kiens, Bente; Woldbye, David P D; Richter, Erik A

    2016-10-01

    It is well known that physical activity has several health benefits, yet many people do not exercise. Dopamine levels in the striatum of the brain are thought to be important for the motivation to exercise. Conversely, we hypothesized that muscle quality can affect the motivation to exercise through alterations of the brain dopamine levels specifically in the striatal region. To test this hypothesis, transgenic mice overexpressing an inactivatable dominant negative α2 AMPK construct (AMPK α2 KD) in muscles and littermate wildtype (WT) mice were tested. AMPK α2 KD mice have impaired running capacity and display reduced voluntary wheel running activity. Striatal content of dopamine and its metabolites were measured under basal physiological conditions and after cocaine-induced dopamine efflux from the ventral striatum by in vivo microdialysis. Moreover, cocaine-induced locomotor activity was tested in an open field test. Furthermore, we investigated maximal running capacity and voluntary running over a period of 19days. AMPK α2 KD mice ran 30% less in daily distance compared to WT. Furthermore, AMPK α2 KD mice showed significantly decreased locomotor activity in the open field test compared to WT when treated with saline or cocaine, respectively, but the increase induced by cocaine was similar in AMPK α2 KD and WT mice. The efflux of dopamine in ventral striatum after cocaine treatment increased similarly by 2.5-fold in the two genotypes, and basal levels of dopamine and its metabolites DOPAC and HVA were also similar between genotypes. These findings show that decreased AMPK activity in muscle leads to decreased voluntary activity which is not due to secondary abnormalities in dopamine levels in the ventral striatum or sensitivity to cocaine. Thus, decreased voluntary activity in AMPK muscle deficient mice is most likely unrelated to regulation of brain dopamine content and metabolism. PMID:27306083

  10. Systems biology network-based discovery of a small molecule activator BL-AD008 targeting AMPK/ZIPK and inducing apoptosis in cervical cancer

    PubMed Central

    Tong, Xupeng; Zhang, Jin; Zhang, Yonghui; Ouyang, Liang; Liu, Bo; Huang, Jian

    2015-01-01

    The aim of this study was to discover a small molecule activator BL-AD008 targeting AMPK/ZIPK and inducing apoptosis in cervical cancer. In this study, we systematically constructed the global protein-protein interaction (PPI) network and predicted apoptosis-related protein connections by the Naïve Bayesian model. Then, we identified some classical apoptotic PPIs and other previously unrecognized PPIs between apoptotic kinases, such as AMPK and ZIPK. Subsequently, we screened a series of candidate compounds targeting AMPK/ZIPK, synthesized some compounds and eventually discovered a novel dual-target activator (BL-AD008). Moreover, we found BL-AD008 bear remarkable anti-proliferative activities toward cervical cancer cells and could induce apoptosis by death-receptor and mitochondrial pathways. Additionally, we found that BL-AD008-induced apoptosis was affected by the combination of AMPK and ZIPK. Then, we found that BL-AD008 bear its anti-tumor activities and induced apoptosis by targeting AMPK/ZIPK in vivo. In conclusion, these results demonstrate the ability of systems biology network to identify some key apoptotic kinase targets AMPK and ZIPK; thus providing a dual-target small molecule activator (BL-AD008) as a potential new apoptosis-modulating drug in future cervical cancer therapy. PMID:25797270

  11. Resveratrol Inhibition of Rac1-Derived Reactive Oxygen Species by AMPK Decreases Blood Pressure in a Fructose-Induced Rat Model of Hypertension

    PubMed Central

    Cheng, Pei-Wen; Lee, Hui-Chieh; Lu, Pei-Jung; Chen, Hsin-Hung; Lai, Chi-Cheng; Sun, Gwo-Ching; Yeh, Tung-Chen; Hsiao, Michael; Lin, Yu-Te; Liu, Chun-Peng; Tseng, Ching-Jiunn

    2016-01-01

    Recent studies have reported that the activation of AMP-activated protein kinase (AMPK) suppressed oxidative stress. The aim of this study was to examine whether the activation of AMPK in the brain decreased Rac1-induced ROS generation, thereby reducing blood pressure (BP) in rats with fructose-induced hypertension. The inhibition of ROS by treatment with an AMPK activator (oral resveratrol, 10 mg/kg/day) for 1 week decreased the BP and increased the NO production in the rostral ventrolateral medulla (RVLM) of fructose-fed rats but not in control Wistar-Kyoto (WKY) rats. In addition, resveratrol treatment abolished the Rac1-induced increases in the activity of the NADPH oxidase subunits p22-phox and reduced the activity of SOD2, while treatment with an AMPK inhibitor (compound C, 40 μM/day) had the opposite effect, in the fructose-fed rats. Interestingly, the activation of AMPK abolished Rac1 activation and decreased BP by inducing the activities of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and ribosomal protein S6 kinase (RSK) and nNOS phosphorylation in the fructose-fed rats. We conclude that the activation of AMPK decreased BP, abolished ROS generation, and enhanced ERK1/2-RSK-nNOS pathway activity by negatively regulating Racl-induced NADPH oxidase levels in the RVLM during oxidative stress–associated hypertension. PMID:27138844

  12. AMPK-Activated Protein Kinase Suppresses Ccr2 Expression by Inhibiting the NF-κB Pathway in RAW264.7 Macrophages

    PubMed Central

    Kumase, Fumiaki; Takeuchi, Kimio; Morizane, Yuki; Suzuki, Jun; Matsumoto, Hidetaka; Kataoka, Keiko; Al-Moujahed, Ahmad; Maidana, Daniel E.; Miller, Joan W.; Vavvas, Demetrios G.

    2016-01-01

    C-C chemokine receptor 2 (Ccr2) is a key pro-inflammatory marker of classic (M1) macrophage activation. Although Ccr2 is known to be expressed both constitutively and inductively, the full regulatory mechanism of its expression remains unclear. AMP-activated protein kinase (AMPK) is not only a master regulator of energy homeostasis but also a central regulator of inflammation. In this study, we sought to assess AMPK’s role in regulating RAW264.7 macrophage Ccr2 protein levels in resting (M0) or LPS-induced M1 states. In both M0 and M1 RAW264.7 macrophages, knockdown of the AMPKα1 subunit by siRNA led to increased Ccr2 levels whereas pharmacologic (A769662) activation of AMPK, attenuated LPS-induced increases in Ccr2 expression in an AMPK dependent fashion. The increases in Ccr2 levels by AMPK downregulation were partially reversed by NF-κB inhibition whereas TNF-a inhibition had minimal effects. Our results indicate that AMPK is a negative regulator of Ccr2 expression in RAW264.7 macrophages, and that the mechanism of action of AMPK inhibition of Ccr2 is mediated, in part, through the NF-κB pathway. PMID:26799633

  13. Saponarin activates AMPK in a calcium-dependent manner and suppresses gluconeogenesis and increases glucose uptake via phosphorylation of CRTC2 and HDAC5.

    PubMed

    Seo, Woo-Duck; Lee, Ji Hae; Jia, Yaoyao; Wu, Chunyan; Lee, Sung-Joon

    2015-11-15

    This study investigated the molecular mechanism of saponarin, a flavone glucoside, in the regulation of insulin sensitivity. Saponarin suppressed the rate of gluconeogenesis and increased cellular glucose uptake in HepG2 and TE671 cells by regulating AMPK. Using an in vitro kinase assay, we showed that saponarin did not directly interact with the AMPK protein. Instead, saponarin increased intracellular calcium levels and induced AMPK phosphorylation, which was diminished by co-stimulation with STO-609, an inhibitor of CAMKKβ. Transcription of hepatic gluconeogenesis genes was upregulated by nuclear translocation of CRTC2 and HDAC5, coactivators of CREB and FoxO1 transcription factors, respectively. This nuclear translocation was inhibited by increased phosphorylation of CRTC2 and HDAC5 by saponarin-induced AMPK in HepG2 cells and suppression of CREB and FoxO1 transactivation activities in cells stimulated by saponarin. The results from a chromatin immunoprecipitation assay confirmed the reduced binding of CRTC2 on the PEPCK and G6Pase promoters. In TE671 cells, AMPK phosphorylated HDAC5, which suppressed nuclear penetration and upregulated GLUT4 transcription, leading to enhanced glucose uptake. Collectively, these results suggest that saponarin activates AMPK in a calcium-dependent manner, thus regulating gluconeogenesis and glucose uptake.

  14. Ketogenic diet delays the phase of circadian rhythms and does not affect AMP-activated protein kinase (AMPK) in mouse liver.

    PubMed

    Genzer, Yoni; Dadon, Maayan; Burg, Chen; Chapnik, Nava; Froy, Oren

    2015-12-01

    Ketogenic diet (KD) is used for weight loss or to treat epilepsy. KD leads to liver AMP-activated protein kinase (AMPK) activation, which would be expected to inhibit gluconeogenesis. However, KD leads to increased hepatic glucose output. As AMPK and its active phosphorylated form (pAMPK) show circadian oscillation, this discrepancy could stem from wrong-time-of-day sampling. The effect of KD was tested on mouse clock gene expression, AMPK, mTOR, SIRT1 and locomotor activity for 2 months and compared to low-fat diet (LFD). KD led to 1.5-fold increased levels of blood glucose and insulin. Brain pAMPK/AMPK ratio was 40% higher under KD, whereas that in liver was not affected. KD led to 40% and 20% down-regulation of the ratio of pP70S6K/P70S6K, the downstream target of mTOR, in the brain and liver, respectively. SIRT1 levels were 40% higher in the brain, but 40% lower in the liver of KD-fed mice. Clock genes showed delayed rhythms under KD. In the brain of KD-fed mice, amplitudes of clock genes were down-regulated, whereas 6-fold up-regulation was found in the liver. The metabolic state under KD indicates reduced satiety in the brain and reduced anabolism alongside increased gluconeogenesis in the liver.

  15. Combination of honokiol and magnolol inhibits hepatic steatosis through AMPK-SREBP-1 c pathway

    PubMed Central

    Lee, Ju-Hee; Jung, Ji Yun; Jang, Eun Jeong; Jegal, Kyung Hwan; Moon, Soo Young; Ku, Sae Kwang; Kang, Seung Ho; Cho, Il Je; Park, Sook Jahr; Lee, Jong Rok; Zhao, Rong Jie; Kim, Sang Chan

    2015-01-01

    Honokiol and magnolol, as pharmacological biphenolic compounds of Magnolia officinalis, have been reported to have antioxidant and anti-inflammatory properties. Sterol regulatory element binding protein-1 c (SREBP-1 c) plays an important role in the development and processing of steatosis in the liver. In the present study, we investigated the effects of a combination of honokiol and magnolol on SREBP-1 c-dependent lipogenesis in hepatocytes as well as in mice with fatty liver due to consumption of high-fat diet (HFD). Liver X receptor α (LXRα) agonists induced activation of SREBP-1 c and expression of lipogenic genes, which were blocked by co-treatment of honokiol and magnolol (HM). Moreover, a combination of HM potently increased mRNA of fatty acid oxidation genes. HM induced AMP-activated protein kinase (AMPK), an inhibitory kinase of the LXRα-SREBP-1 c pathway. The role of AMPK activation induced by HM was confirmed using an inhibitor of AMPK, Compound C, which reversed the ability of HM to both inhibit SREBP-1 c induction as well as induce genes for fatty acid oxidation. In mice, HM administration for four weeks ameliorated HFD-induced hepatic steatosis and liver dysfunction, as indicated by plasma parameters and Oil Red O staining. Taken together, our results demonstrated that a combination of HM has beneficial effects on inhibition of fatty liver and SREBP-1 c-mediated hepatic lipogenesis, and these events may be mediated by AMPK activation. PMID:25125496

  16. Autophagy through 4EBP1 and AMPK regulates oxidative stress-induced premature senescence in auditory cells

    PubMed Central

    Tsuchihashi, Nana Akagi; Hayashi, Ken; Dan, Katsuaki; Goto, Fumiyuki; Nomura, Yasuyuki; Fujioka, Masato; Kanzaki, Sho; Komune, Shizuo; Ogawa, Kaoru

    2015-01-01

    The aim of this study was to determine whether autophagy and AMPK contribute to premature senescence in auditory cells. Incubating HEI-OC1 auditory cells with 5 mM H2O2 for 1 h induced senescence, as demonstrated by senescence-associated β-galactosidase (SA-β-gal) staining. H2O2 treatment significantly delayed population-doubling time, leaving cell viability unchanged. Furthermore, the proportion of SA-β-gal-positive cells significantly increased. Autophagy-related protein expression increased, with Atg7 and LC3-II peaking 6 h and Lamp2 peaking 24 h after H2O2 treatment. The expression of these proteins decreased 48 h after treatment. Transmission electron microscopy revealed lipofuscin and aggregates within autolysosomes, which accumulated markedly in the cytoplasm of HEI-OC1 cells 48 h after treatment. Akt and P70S6 phosphorylation markedly decreased after H2O2 treatment, but 4EBP1 phosphorylation significantly increased 48 h after treatment. After RNAi-mediated knockdown (KD) of Atg7 and AMPK, H2O2-treated cells displayed dense SA-β-gal staining. Also, premature senescence was significantly induced. These suggest that a negative feedback loop may exist between autophagy and AMPK signaling pathways in HEI-OC1 cells. In our model, oxidative stress-induced premature senescence occurred due to impaired autophagy function through 4EBP1 phosphorylation. Our results also indicate that AMPK may regulate premature senescence in auditory cells in an autophagy-dependent and independent manner. PMID:25682865

  17. 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).

  18. G9a Inhibition Induces Autophagic Cell Death via AMPK/mTOR Pathway in Bladder Transitional Cell Carcinoma.

    PubMed

    Li, Feng; Zeng, Jin; Gao, Yang; Guan, Zhenfeng; Ma, Zhenkun; Shi, Qi; Du, Chong; Jia, Jing; Xu, Shan; Wang, Xinyang; Chang, Luke; He, Dalin; Guo, Peng

    2015-01-01

    G9a has been reported to highly express in bladder transitional cell carcinoma (TCC) and G9a inhibition significantly attenuates cell proliferation, but the underlying mechanism is not fully understood. The present study aimed at examining the potential role of autophagy in the anti-proliferation effect of G9a inhibition on TCC T24 and UMUC-3 cell lines in vitro. We found that both pharmaceutical and genetical G9a inhibition significantly attenuated cell proliferation by MTT assay, Brdu incorporation assay and colony formation assay. G9a inhibition induced autophagy like morphology as determined by transmission electron microscope and LC-3 fluorescence assay. In addition, autophagy flux was induced by G9a inhibition in TCC cells, as determined by p62 turnover assay and LC-3 turnover assay. The autophagy induced positively contributed to the inhibition of cell proliferation because the growth attenuation capacity of G9a inhibition was reversed by autophagy inhibitors 3-MA. Mechanically, AMPK/mTOR pathway was identified to be involved in the regulation of G9a inhibition induced autophagy. Intensively activating mTOR by Rheb overexpression attenuated autophagy and autophagic cell death induced by G9a inhibition. In addition, pre-inhibiting AMPK by Compound C attenuated autophagy together with the anti-proliferation effect induced by G9a inhibition while pre-activating AMPK by AICAR enhanced them. In conclusion, our results indicate that G9a inhibition induces autophagy through activating AMPK/mTOR pathway and the autophagy induced positively contributes to the inhibition of cell proliferation in TCC cells. These findings shed some light on the functional role of G9a in cell metabolism and suggest that G9a might be a therapeutic target in bladder TCC in the future. PMID:26397365

  19. Abnormalities of AMPK Activation and Glucose Uptake in Cultured Skeletal Muscle Cells from Individuals with Chronic Fatigue Syndrome

    PubMed Central

    Brown, Audrey E.; Jones, David E.; Walker, Mark; Newton, Julia L.

    2015-01-01

    Background Post exertional muscle fatigue is a key feature in Chronic Fatigue Syndrome (CFS). Abnormalities of skeletal muscle function have been identified in some but not all patients with CFS. To try to limit potential confounders that might contribute to this clinical heterogeneity, we developed a novel in vitro system that allows comparison of AMP kinase (AMPK) activation and metabolic responses to exercise in cultured skeletal muscle cells from CFS patients and control subjects. Methods Skeletal muscle cell cultures were established from 10 subjects with CFS and 7 age-matched controls, subjected to electrical pulse stimulation (EPS) for up to 24h and examined for changes associated with exercise. Results In the basal state, CFS cultures showed increased myogenin expression but decreased IL6 secretion during differentiation compared with control cultures. Control cultures subjected to 16h EPS showed a significant increase in both AMPK phosphorylation and glucose uptake compared with unstimulated cells. In contrast, CFS cultures showed no increase in AMPK phosphorylation or glucose uptake after 16h EPS. However, glucose uptake remained responsive to insulin in the CFS cells pointing to an exercise-related defect. IL6 secretion in response to EPS was significantly reduced in CFS compared with control cultures at all time points measured. Conclusion EPS is an effective model for eliciting muscle contraction and the metabolic changes associated with exercise in cultured skeletal muscle cells. We found four main differences in cultured skeletal muscle cells from subjects with CFS; increased myogenin expression in the basal state, impaired activation of AMPK, impaired stimulation of glucose uptake and diminished release of IL6. The retention of these differences in cultured muscle cells from CFS subjects points to a genetic/epigenetic mechanism, and provides a system to identify novel therapeutic targets. PMID:25836975

  20. A novel AMPK-dependent FoxO3A-SIRT3 intramitochondrial complex sensing glucose levels.

    PubMed

    Peserico, Alessia; Chiacchiera, Fulvio; Grossi, Valentina; Matrone, Antonio; Latorre, Dominga; Simonatto, Marta; Fusella, Aurora; Ryall, James G; Finley, Lydia W S; Haigis, Marcia C; Villani, Gaetano; Puri, Pier Lorenzo; Sartorelli, Vittorio; Simone, Cristiano

    2013-06-01

    Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.

  1. Curcumin attenuates hyperglycaemia-mediated AMPK activation and oxidative stress in cerebrum of streptozotocin-induced diabetic rat.

    PubMed

    Lakshmanan, Arun Prasath; Watanabe, Kenichi; Thandavarayan, Rajarajan A; Sari, Flori R; Meilei, Harima; Soetikno, Vivian; Arumugam, Somasundaram; Giridharan, Vijayasree V; Suzuki, Kenji; Kodama, Makoto

    2011-07-01

    Oxidative stress has been strongly implicated in the pathogenesis of diabetic encephalopathy (DE). Numerous studies have demonstrated a close relationship between oxidative stress and AMPK activation in various disorders, including diabetes-related brain disorders. Since curcumin has powerful antioxidant properties, this study investigated its effects on hyperglycaemia-mediated oxidative stress and AMPK activation in rats with DE. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ-55 mg/kg BW). The diabetic rats were then orally administered curcumin (100 mg/kg BW) or vehicle for 8 weeks. The cerebra of the diabetic rats displayed upregulated protein expression of AdipoR1, p-AMPKα1, Tak1, GLUT4, NADPH oxidase sub-units, caspase-12 and 3-NT and increased lipid peroxidation in comparison with the controls and all of these effects were significantly attenuated with curcumin treatment, except for the increase in AdipoR1 expressions. These results provide a new insight into the beneficial effects of curcumin on hyperglycaemia-mediated DE, which are produced through the down-regulation of AMPK-mediated gluconeogenesis associated with its anti-oxidant property.

  2. Arctigenin Inhibits Adipogenesis by Inducing AMPK Activation and Reduces Weight Gain in High-Fat Diet-Induced Obese Mice.

    PubMed

    Han, Yo-Han; Kee, Ji-Ye; Park, Jinbong; Kim, Hye-Lin; Jeong, Mi-Young; Kim, Dae-Seung; Jeon, Yong-Deok; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; So, Hong-Seob; Park, Raekil; Lee, Jong-Hyun; Shin, Soyoung; Kim, Su-Jin; Um, Jae-Young; Hong, Seung-Heon

    2016-09-01

    Although arctigenin (ARC) has been reported to have some pharmacological effects such as anti-inflammation, anti-cancer, and antioxidant, there have been no reports on the anti-obesity effect of ARC. The aim of this study is to investigate whether ARC has an anti-obesity effect and mediates the AMP-activated protein kinase (AMPK) pathway. We investigated the anti-adipogenic effect of ARC using 3T3-L1 pre-adipocytes and human adipose tissue-derived mesenchymal stem cells (hAMSCs). In high-fat diet (HFD)-induced obese mice, whether ARC can inhibit weight gain was investigated. We found that ARC reduced weight gain, fat pad weight, and triglycerides in HFD-induced obese mice. ARC also inhibited the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in in vitro and in vivo. Furthermore, ARC induced the AMPK activation resulting in down-modulation of adipogenesis-related factors including PPARγ, C/EBPα, fatty acid synthase, adipocyte fatty acid-binding protein, and lipoprotein lipase. This study demonstrates that ARC can reduce key adipogenic factors by activating the AMPK in vitro and in vivo and suggests a therapeutic implication of ARC for obesity treatment. J. Cell. Biochem. 117: 2067-2077, 2016. © 2016 Wiley Periodicals, Inc.

  3. Clopidogrel Protects Endothelium by Hindering TNFα-Induced VCAM-1 Expression through CaMKKβ/AMPK/Nrf2 Pathway

    PubMed Central

    Yang, Huabing; Zhao, Pengjun; Tian, Shiliu

    2016-01-01

    Clopidogrel (INN), an oral antiplatelet drug, has been revealed to have a number of biological properties, for instance, anti-inflammation and antioxidation. Oxidative stress plays an imperative role in inflammation, diabetes mellitus, atherosclerosis, and cancer. In the present study, human aortic endothelial cells (HAECs) were employed to explore the anti-inflammatory activity of INN. INN reduced TNFα-induced reactive oxygen species (ROS) generation and time-dependently prompted the expression and activity of heme oxygenase 1 (HO-1). Cellular glutathione (GSH) levels were augmented by INN. shHO-1 blocked the INN suppression of TNFα-induced HL-60 cell adhesion. The CaMKKβ/AMPK pathway and Nrf2 transcriptional factor were implicated in the induction of HO-1 by INN. Additionally, TNFα dramatically augmented VCAM-1 expression at protein and mRNA levels. INN treatment strikingly repressed TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Compound C, an AMPK inhibitor, and shNrf2 abolished TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Our data suggest that INN diminishes TNFα-stimulated VCAM-1 expression at least in part via HO-1 induction, which is CaMKKβ/AMPK pathway-dependent. PMID:26824050

  4. Coinjection of CCK and leptin reduces food intake via increased CART/TRH and reduced AMPK phosphorylation in the hypothalamus.

    PubMed

    Akieda-Asai, Sayaka; Poleni, Paul-Emile; Date, Yukari

    2014-06-01

    CCK and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important for elucidating the mechanisms by which energy balance is maintained. We found here that coadministration of subthreshold CCK and leptin, which individually have no effect on feeding, dramatically reduced food intake in rats. Phosphorylation of AMP-activated protein kinase (AMPK) in the hypothalamus significantly decreased after coinjection of CCK and leptin. In addition, coadministration of these hormones significantly increased mRNA levels of anorectic cocaine- and amphetamine-regulated transcript (CART) and thyrotropin-releasing hormone (TRH) in the hypothalamus. The interactive effect of CCK and leptin on food intake was abolished by intracerebroventricular preadministration of the AMPK activator AICAR or anti-CART/anti-TRH antibodies. These findings indicate that coinjection of CCK and leptin reduces food intake via reduced AMPK phosphorylation and increased CART/TRH in the hypothalamus. Furthermore, by using midbrain-transected rats, we investigated the role of the neural pathway from the hindbrain to the hypothalamus in the interaction of CCK and leptin to reduce food intake. Food intake reduction induced by coinjection of CCK and leptin was blocked in midbrain-transected rats. Therefore, the neural pathway from hindbrain to hypothalamus plays an important role in transmitting the anorectic signals provided by coinjection of CCK and leptin. Our findings give further insight into the mechanisms of feeding and energy balance.

  5. Lack of Adipocyte AMPK Exacerbates Insulin Resistance and Hepatic Steatosis through Brown and Beige Adipose Tissue Function.

    PubMed

    Mottillo, Emilio P; Desjardins, Eric M; Crane, Justin D; Smith, Brennan K; Green, Alex E; Ducommun, Serge; Henriksen, Tora I; Rebalka, Irena A; Razi, Aida; Sakamoto, Kei; Scheele, Camilla; Kemp, Bruce E; Hawke, Thomas J; Ortega, Joaquin; Granneman, James G; Steinberg, Gregory R

    2016-07-12

    Brown (BAT) and white (WAT) adipose tissues play distinct roles in maintaining whole-body energy homeostasis, and their dysfunction can contribute to non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes. The AMP-activated protein kinase (AMPK) is a cellular energy sensor, but its role in regulating BAT and WAT metabolism is unclear. We generated an inducible model for deletion of the two AMPK β subunits in adipocytes (iβ1β2AKO) and found that iβ1β2AKO mice were cold intolerant and resistant to β-adrenergic activation of BAT and beiging of WAT. BAT from iβ1β2AKO mice had impairments in mitochondrial structure, function, and markers of mitophagy. In response to a high-fat diet, iβ1β2AKO mice more rapidly developed liver steatosis as well as glucose and insulin intolerance. Thus, AMPK in adipocytes is vital for maintaining mitochondrial integrity, responding to pharmacological agents and thermal stress, and protecting against nutrient-overload-induced NAFLD and insulin resistance. PMID:27411013

  6. Arctigenin Inhibits Adipogenesis by Inducing AMPK Activation and Reduces Weight Gain in High-Fat Diet-Induced Obese Mice.

    PubMed

    Han, Yo-Han; Kee, Ji-Ye; Park, Jinbong; Kim, Hye-Lin; Jeong, Mi-Young; Kim, Dae-Seung; Jeon, Yong-Deok; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; So, Hong-Seob; Park, Raekil; Lee, Jong-Hyun; Shin, Soyoung; Kim, Su-Jin; Um, Jae-Young; Hong, Seung-Heon

    2016-09-01

    Although arctigenin (ARC) has been reported to have some pharmacological effects such as anti-inflammation, anti-cancer, and antioxidant, there have been no reports on the anti-obesity effect of ARC. The aim of this study is to investigate whether ARC has an anti-obesity effect and mediates the AMP-activated protein kinase (AMPK) pathway. We investigated the anti-adipogenic effect of ARC using 3T3-L1 pre-adipocytes and human adipose tissue-derived mesenchymal stem cells (hAMSCs). In high-fat diet (HFD)-induced obese mice, whether ARC can inhibit weight gain was investigated. We found that ARC reduced weight gain, fat pad weight, and triglycerides in HFD-induced obese mice. ARC also inhibited the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in in vitro and in vivo. Furthermore, ARC induced the AMPK activation resulting in down-modulation of adipogenesis-related factors including PPARγ, C/EBPα, fatty acid synthase, adipocyte fatty acid-binding protein, and lipoprotein lipase. This study demonstrates that ARC can reduce key adipogenic factors by activating the AMPK in vitro and in vivo and suggests a therapeutic implication of ARC for obesity treatment. J. Cell. Biochem. 117: 2067-2077, 2016. © 2016 Wiley Periodicals, Inc. PMID:26852013

  7. Downregulation of pyrroline-5-carboxylate reductase-2 induces the autophagy of melanoma cells via AMPK/mTOR pathway.

    PubMed

    Ou, Rongying; Zhang, Xueqi; Cai, Jianfeng; Shao, Xiaohong; Lv, Mingfen; Qiu, Wei; Xuan, Xuan; Liu, Jingjing; Li, Zhiming; Xu, Yunsheng

    2016-05-01

    Melanoma is the most aggressive form of skin cancer and causes 50,000 deaths annually worldwide. The roles of proline-dependent process and autophagy have both been reported in studies on melanoma. In the present study, we focused on the effect of pyrroline-5-carboxylate reductase-2 (PYCR2) on inducing autophagy process in melanoma. The expression of PYCR2 was regulated by an RNAi technique, and the cell proliferation of A375 cell line was determined by methyl thiazolyl tetrazolium test; the effect of PYCR2 on the apoptosis process and AMPK/mTOR pathway was evaluated by flow cytometry assay and Western blot. It was found that silence of PYCR2 resulted in the decrease of proliferative ability and activation of AMPK/mTOR-induced autophagy of A375 cells. PYCR2 silencing also activated AMPK/mTOR pathway in another melanoma cell line, CHL-1. However, the overexpression of PYCR2 seemed to make no difference to the cell viability and targeted pathway. Our results offered a preliminary illustration on the mechanism of the PYCR2-dependent autophagy and showed that PYCR2 was a potential therapeutic target of melanoma.

  8. Copper Ion from Cu2O Crystal Induces AMPK-Mediated Autophagy via Superoxide in Endothelial Cells

    PubMed Central

    Seo, Youngsik; Cho, Young-Sik; Huh, Young-Duk; Park, Heonyong

    2016-01-01

    Copper is an essential element required for a variety of functions exerted by cuproproteins. An alteration of the copper level is associated with multiple pathological conditions including chronic ischemia, atherosclerosis and cancers. Therefore, copper homeostasis, maintained by a combination of two copper ions (Cu+ and Cu2+), is critical for health. However, less is known about which of the two copper ions is more toxic or functional in endothelial cells. Cubic-shaped Cu2O and CuO crystals were prepared to test the role of the two different ions, Cu+ and Cu2+, respectively. The Cu2O crystal was found to have an effect on cell death in endothelial cells whereas CuO had no effect. The Cu2O crystals appeared to induce p62 degradation, LC3 processing and an elevation of LC3 puncta, important processes for autophagy, but had no effect on apoptosis and necrosis. Cu2O crystals promote endothelial cell death via autophagy, elevate the level of reactive oxygen species such as superoxide and nitric oxide, and subsequently activate AMP-activated protein kinase (AMPK) through superoxide rather than nitric oxide. Consistently, the AMPK inhibitor Compound C was found to inhibit Cu2O-induced AMPK activation, p62 degradation, and LC3 processing. This study provides insight on the pathophysiologic function of Cu+ ions in the vascular system, where Cu+ induces autophagy while Cu2+ has no detected effect. PMID:26743904

  9. Metformin prevents endoplasmic reticulum stress-induced apoptosis through AMPK-PI3K-c-Jun NH2 pathway

    USGS Publications Warehouse

    Jung, T.W.; Lee, M.W.; Lee, Y.-J.; Kim, S.M.

    2012-01-01

    Type 2 diabetes mellitus is thought to be partially associated with endoplasmic reticulum (ER) stress toxicity on pancreatic beta cells and the result of decreased insulin synthesis and secretion. In this study, we showed that a well-known insulin sensitizer, metformin, directly protects against dysfunction and death of ER stress-induced NIT-1 cells (a mouse pancreatic beta cell line) via AMP-activated protein kinase (AMPK) and phosphatidylinositol-3 (PI3) kinase activation. We also showed that exposure of NIT-1 cells to metformin (5mM) increases cellular resistance against ER stress-induced NIT-1 cell dysfunction and death. AMPK and PI3 kinase inhibitors abolished the effect of metformin on cell function and death. Metformin-mediated protective effects on ER stress-induced apoptosis were not a result of an unfolded protein response or the induced inhibitors of apoptotic proteins. In addition, we showed that exposure of ER stressed-induced NIT-1 cells to metformin decreases the phosphorylation of c-Jun NH(2) terminal kinase (JNK). These data suggest that metformin is an important determinant of ER stress-induced apoptosis in NIT-1 cells and may have implications for ER stress-mediated pancreatic beta cell destruction via regulation of the AMPK-PI3 kinase-JNK pathway.

  10. Sestrin 2 and AMPK Connect Hyperglycemia to Nox4-Dependent Endothelial Nitric Oxide Synthase Uncoupling and Matrix Protein Expression

    PubMed Central

    Eid, Assaad A.; Lee, Doug-Yoon; Roman, Linda J.; Khazim, Khaled

    2013-01-01

    Mesangial matrix accumulation is an early feature of glomerular pathology in diabetes. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. Here, we demonstrate that, in glomerular mesangial cells (MCs), endothelial nitric oxide synthase (eNOS) is uncoupled upon exposure to high glucose (HG), with enhanced generation of reactive oxygen species (ROS) and decreased production of nitric oxide. Peroxynitrite mediates the effects of HG on eNOS dysfunction. HG upregulates Nox4 protein, and inhibition of Nox4 abrogates the increase in ROS and peroxynitrite generation, as well as the eNOS uncoupling triggered by HG, demonstrating that Nox4 functions upstream from eNOS. Importantly, this pathway contributes to HG-induced MC fibronectin accumulation. Nox4-mediated eNOS dysfunction was confirmed in glomeruli of a rat model of type 1 diabetes. Sestrin 2-dependent AMP-activated protein kinase (AMPK) activation attenuates HG-induced MC fibronectin synthesis through blockade of Nox4-dependent ROS and peroxynitrite generation, with subsequent eNOS uncoupling. We also find that HG negatively regulates sestrin 2 and AMPK, thereby promoting Nox4-mediated eNOS dysfunction and increased fibronectin. These data identify a protective function for sestrin 2/AMPK and potential targets for intervention to prevent fibrotic injury in diabetes. PMID:23816887

  11. Clopidogrel Protects Endothelium by Hindering TNFα-Induced VCAM-1 Expression through CaMKKβ/AMPK/Nrf2 Pathway.

    PubMed

    Yang, Huabing; Zhao, Pengjun; Tian, Shiliu

    2016-01-01

    Clopidogrel (INN), an oral antiplatelet drug, has been revealed to have a number of biological properties, for instance, anti-inflammation and antioxidation. Oxidative stress plays an imperative role in inflammation, diabetes mellitus, atherosclerosis, and cancer. In the present study, human aortic endothelial cells (HAECs) were employed to explore the anti-inflammatory activity of INN. INN reduced TNFα-induced reactive oxygen species (ROS) generation and time-dependently prompted the expression and activity of heme oxygenase 1 (HO-1). Cellular glutathione (GSH) levels were augmented by INN. shHO-1 blocked the INN suppression of TNFα-induced HL-60 cell adhesion. The CaMKKβ/AMPK pathway and Nrf2 transcriptional factor were implicated in the induction of HO-1 by INN. Additionally, TNFα dramatically augmented VCAM-1 expression at protein and mRNA levels. INN treatment strikingly repressed TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Compound C, an AMPK inhibitor, and shNrf2 abolished TNFα-induced expression of VCAM-1 and HL-60 cell adhesion. Our data suggest that INN diminishes TNFα-stimulated VCAM-1 expression at least in part via HO-1 induction, which is CaMKKβ/AMPK pathway-dependent. PMID:26824050

  12. Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells

    PubMed Central

    Head, Sarah A.; Shi, Wei; Zhao, Liang; Gorshkov, Kirill; Pasunooti, Kalyan; Chen, Yue; Deng, Zhiyou; Li, Ruo-jing; Shim, Joong Sup; Tan, Wenzhi; Hartung, Thomas; Zhang, Jin; Zhao, Yingming; Colombini, Marco; Liu, Jun O.

    2015-01-01

    Itraconazole, a clinically used antifungal drug, was found to possess potent antiangiogenic and anticancer activity that is unique among the azole antifungals. Previous mechanistic studies have shown that itraconazole inhibits the mechanistic target of rapamycin (mTOR) signaling pathway, which is known to be a critical regulator of endothelial cell function and angiogenesis. However, the molecular target of itraconazole that mediates this activity has remained unknown. Here we identify the major target of itraconazole in endothelial cells as the mitochondrial protein voltage-dependent anion channel 1 (VDAC1), which regulates mitochondrial metabolism by controlling the passage of ions and small metabolites through the outer mitochondrial membrane. VDAC1 knockdown profoundly inhibits mTOR activity and cell proliferation in human umbilical vein cells (HUVEC), uncovering a previously unknown connection between VDAC1 and mTOR. Inhibition of VDAC1 by itraconazole disrupts mitochondrial metabolism, leading to an increase in the cellular AMP:ATP ratio and activation of the AMP-activated protein kinase (AMPK), an upstream regulator of mTOR. VDAC1-knockout cells are resistant to AMPK activation and mTOR inhibition by itraconazole, demonstrating that VDAC1 is the mediator of this activity. In addition, another known VDAC-targeting compound, erastin, also activates AMPK and inhibits mTOR and proliferation in HUVEC. VDAC1 thus represents a novel upstream regulator of mTOR signaling in endothelial cells and a promising target for the development of angiogenesis inhibitors. PMID:26655341

  13. Involvement of AMPK/SIRT1 pathway in anti-allodynic effect of troxerutin in CCI-induced neuropathic pain.

    PubMed

    Gui, Yulong; Li, Aiyuan; Chen, Feng; Zhou, Hong; Tang, Yan; Chen, Liang; Chen, Shuxian; Duan, Shunshan

    2015-12-15

    Neuropathic pain was regarded as a main form of chronic pain condition that remains difficult to treat. Conventional pharmacotherapy for neuropathic pain responsed vary and side effects limited their compliance. These prompted us to find new alternatives. In this study, we investigated the effect of troxerutin on treatment of CCI-induced neuropathic pain. Results showed that troxerutin significantly reversed mechanical allodynia and thermal hyperalgesia. In L4-6 spinal cord, troxerutin reduced the expression of INF-γ, IL-1β, TNF-α, and activation of NF-κB(p65). Immunofluorescence results showed that troxerutin significantly inhibited microglia activation induced by CCI surgery. Further, troxerutin treatment significantly induced AMPK activation and inhibited CCI-induced SIRT1 decrease. However, AMPK inhibitor compound C and SIRT1 inhibitor EX527 inhibited analgesic effect of troxerutin in CCI mice. This demonstrated the involvement of AMPK/SIRT1 pathway in anti-allodynic effect of troxerutin in CCI mice. Troxerutin could be developed as a potential therapeutic agent for neuropathic pain.

  14. AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation.

    PubMed

    Chang, Chunmei; Su, Hua; Zhang, Danhong; Wang, Yusha; Shen, Qiuhong; Liu, Bo; Huang, Rui; Zhou, Tianhua; Peng, Chao; Wong, Catherine C L; Shen, Han-Ming; Lippincott-Schwartz, Jennifer; Liu, Wei

    2015-12-17

    Eukaryotes initiate autophagy to cope with the lack of external nutrients, which requires the activation of the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase Sirtuin 1 (Sirt1). However, the mechanisms underlying the starvation-induced Sirt1 activation for autophagy initiation remain unclear. Here, we demonstrate that glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a conventional glycolytic enzyme, is a critical mediator of AMP-activated protein kinase (AMPK)-driven Sirt1 activation. Under glucose starvation, but not amino acid starvation, cytoplasmic GAPDH is phosphorylated on Ser122 by activated AMPK. This causes GAPDH to redistribute into the nucleus. Inside the nucleus, GAPDH interacts directly with Sirt1, displacing Sirt1's repressor and causing Sirt1 to become activated. Preventing this shift of GAPDH abolishes Sirt1 activation and autophagy, while enhancing it, through overexpression of nuclear-localized GAPDH, increases Sirt1 activation and autophagy. GAPDH is thus a pivotal and central regulator of autophagy under glucose deficiency, undergoing AMPK-dependent phosphorylation and nuclear translocation to activate Sirt1 deacetylase activity. PMID:26626483

  15. Second-hand smoke stimulates lipid accumulation in the liver by modulating AMPK and SREBP-1✩

    PubMed Central

    Yuan, Hongwei; Shyy, John Y.-J.; Martins-Green, Manuela

    2010-01-01

    Background/Aims The underlying mechanisms of steatosis, the first stage of non-alcoholic fatty liver disease (NAFLD) that is characterized by the accumulation of lipids in hepatocytes, remain unclear. Our study aimed to investigate the hypothesis that cigarette smoke is known to change circulating lipid profiles and thus may also contribute to the accumulation of lipids in the liver. Methods Mice and cultured hepatocytes were exposed to sidestream whole smoke (SSW), a major component of “second-hand” smoke and a variety of cellular and molecular approaches were used to study the effects of cigarette smoke on lipid metabolism. Results SSW increases lipid accumulation in hepatocytes by modulating the activity of 5′-AMP-activated protein kinase (AMPK) and sterol response element binding protein-1 (SREBP-1), two critical molecules involved in lipid synthesis. SSW causes dephosphorylation/ inactivation of AMPK, which contributes to increased activation of SREBP-1. These changes of activity lead to accumulation of triglycerides in hepatocytes. Conclusion These novel findings are important because they point to another risk factor of smoking, i.e., that of contributing to NAFLD. In addition, our results showing that both AMPK and SREBP are critically involved in these effects of smoke point to the potential use of these molecules as targets for treatment of cigarette smoke-induced metabolic diseases. PMID:19556020

  16. Down-regulation of HSP60 Suppresses the Proliferation of Glioblastoma Cells via the ROS/AMPK/mTOR Pathway

    PubMed Central

    Tang, Haiping; Li, Jin; Liu, Xiaohui; Wang, Guihuai; Luo, Minkui; Deng, Haiteng

    2016-01-01

    Glioblastoma is a fatal and incurable cancer with the hyper-activated mTOR pathway. HSP60, a major chaperone for maintenance of mitochondrial proteostasis, is highly expressed in glioblastoma patients. To understand the effects of HSP60 on glioblastoma tumorigenesis and progression, we characterized the HSP60-knockdowned glioblastoma cells and revealed that HSP60 silencing markedly suppressed cell proliferation and promoted cell to undergo the epithelial-mesenchymal transition (EMT). Proteomic analysis showed that ribosomal proteins were significantly downregulated whereas EMT-associated proteins were up-regulated in HSP60-knockdowned U87 cells as confirmed by a distinct enrichment pattern in newly synthesized proteins with azido-homoalanine labeling. Biochemical analysis revealed that HSP60 knockdown increased reactive oxygen species (ROS) production that led to AMPK activation, similarly to the complex I inhibitor rotenone-induced AMPK activation. Activated AMPK suppressed mTORC1 mediated S6K and 4EBP1 phosphorylation to decrease protein translation, which slowed down cell growth and proliferation. On the other hand, high levels of ROS in HSP60 knockdowned or rotenone-treated U87 cells contributed to EMT. These results indicate that HSP60 silencing deactivates the mTOR pathway to suppress glioblastoma progression, suggesting that HSP60 is a potential therapeutic target for glioblastoma treatment. PMID:27325206

  17. Anti-Tumor Activity of Yuanhuacine by Regulating AMPK/mTOR Signaling Pathway and Actin Cytoskeleton Organization in Non-Small Cell Lung Cancer Cells.

    PubMed

    Kang, Ji In; Hong, Ji-Young; Lee, Hye-Jung; Bae, Song Yi; Jung, Cholomi; Park, Hyen Joo; Lee, Sang Kook

    2015-01-01

    Yuanhuacine (YC), a daphnane diterpenoid from the flowers of Daphne genkwa, exhibited a potential growth inhibitory activity against human non-small cell lung cancer (NSCLC) cells. YC also suppressed the invasion and migration of lung cancer cells. However, the precise molecular mechanisms remain to be elucidated. In the present study, we report that YC significantly activated AMP-activated protein kinase (AMPK) signaling pathway and suppressed mTORC2-mediated downstream signaling pathway in H1993 human NSCLC cells. AMPK plays an important role in energy metabolism and cancer biology. Therefore, activators of AMPK signaling pathways can be applicable to the treatment of cancer. YC enhanced the expression of p-AMPKα. The co-treatment of YC and compound C (an AMPK inhibitor) or metformin (an AMPK activator) also confirmed that YC increases p-AMPKα. YC also suppressed the activation of the mammalian target of rapamycin (mTOR) expression, a downstream target of AMPK. Further study revealed that YC modulates mTORC2-associated downstream signaling pathways with a decreased expressions of p-Akt, p-protein kinase C alpha (PKCα), p-ras-related C3 botulinum toxin substrate 1 (Rac1) and filamentous actin (F-actin) that are known to activate cell growth and organize actin cytoskeleton. In addition, YC inhibited the tumor growth in H1993 cell-implanted xenograft nude mouse model. These data suggest the YC could be a potential candidate for cancer chemotherapeutic agents derived from natural products by regulating AMPK/mTORC2 signaling pathway and actin cytoskeleton organization. PMID:26656173

  18. Specific Deletion of AMP-Activated Protein Kinase (α1AMPK) in Murine Oocytes Alters Junctional Protein Expression and Mitochondrial Physiology

    PubMed Central

    Bertoldo, Michael J.; Guibert, Edith; Faure, Melanie; Ramé, Christelle; Foretz, Marc; Viollet, Benoit; Dupont, Joëlle; Froment, Pascal

    2015-01-01

    Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed. PMID:25767884

  19. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    SciTech Connect

    Adam, Tasneem; Opie, Lionel H.; Essop, M. Faadiel

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  20. Specific deletion of AMP-activated protein kinase (α1AMPK) in murine oocytes alters junctional protein expression and mitochondrial physiology.

    PubMed

    Bertoldo, Michael J; Guibert, Edith; Faure, Melanie; Ramé, Christelle; Foretz, Marc; Viollet, Benoit; Dupont, Joëlle; Froment, Pascal

    2015-01-01

    Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed. PMID:25767884

  1. Salsalate and Adiponectin Improve Palmitate-Induced Insulin Resistance via Inhibition of Selenoprotein P through the AMPK-FOXO1α Pathway.

    PubMed

    Jung, Tae Woo; Choi, Hae Yoon; Lee, So Young; Hong, Ho Cheol; Yang, Sae Jeong; Yoo, Hye Jin; Youn, Byung-Soo; Baik, Sei Hyun; Choi, Kyung Mook

    2013-01-01

    Selenoprotein P (SeP) was recently identified as a hepatokine that induces insulin resistance (IR) in rodents and humans. Recent clinical trials have shown that salsalate, a prodrug of salicylate, significantly lowers blood glucose levels and increases adiponectin concentrations. We examined the effects of salsalate and full length-adiponectin (fAd) on the expression of SeP under hyperlipidemic conditions and explored their regulatory mechanism on SeP. In palmitate-treated HepG2 cells as well as high fat diet (HFD)-fed male Spraque Dawley (SD) rats and male db/db mice, SeP expression and its regulatory pathway, including AMPK-FOXO1α, were evaluated after administration of salsalate and salicylate. Palmitate treatment significantly increased SeP expression and aggravated IR, while knock-down of SeP by siRNA restored these changes in HepG2 cells. Palmitate-induced SeP expression was inhibited by both salsalate and salicylate, which was mediated by AMPK activation, and was blocked by AMPK siRNA or an inhibitor of AMPK. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assay showed that salsalate suppressed SeP expression by AMPK-mediated phosphorylation of FOXO1α. Moreover, fAd also reduced palmitate-induced SeP expression through the activation of AMPK, which results in improved IR. Both salsalate and salicylate treatment significantly improved glucose intolerance and insulin sensitivity, accompanied by reduced SeP mRNA and protein expression in HFD-fed rats and db/db mice, respectively. Taken together, we found that salsalate and adiponectin ameliorated palmitate-induced IR in hepatocytes via SeP inhibition through the AMPK-FOXO1α pathway. The regulation of SeP might be a novel mechanism mediating the anti-diabetic effects of salsalate and adiponectin.

  2. The kinase triad, AMPK, mTORC1 and ULK1, maintains energy and nutrient homoeostasis.

    PubMed

    Dunlop, Elaine A; Tee, Andrew R

    2013-08-01

    In order for cells to divide in a proficient manner, they must first double their biomass, which is considered to be the main rate-limiting phase of cell proliferation. Cell growth requires an abundance of energy and biosynthetic precursors such as lipids and amino acids. Consequently, the energy and nutrient status of the cell is acutely monitored and carefully maintained. mTORC1 [mammalian (or mechanistic) target of rapamycin complex 1] is often considered to be the master regulator of cell growth that enhances cellular biomass through up-regulation of protein translation. In order for cells to control cellular homoeostasis during growth, there is close signalling interplay between mTORC1 and two other protein kinases, AMPK (AMP-activated protein kinase) and ULK1 (Unc-51-like kinase 1). This kinase triad collectively senses the energy and nutrient status of the cell and appropriately dictates whether the cell will actively favour energy- and amino-acid-consuming anabolic processes such as cellular growth, or energy- and amino-acid-generating catabolic processes such as autophagy. The present review discusses important feedback mechanisms between these three homoeostatic protein kinases that orchestrate cell growth and autophagy, with a particular focus on the mTORC1 component raptor (regulatory associated protein of mammalian target of rapamycin), as well as the autophagy-initiating kinase ULK1.

  3. Resveratrol attenuated estrogen-deficient-induced cardiac dysfunction: role of AMPK, SIRT1, and mitochondrial function

    PubMed Central

    Meng, Zijun; Jing, Hongjiang; Gan, Lu; Li, Hua; Luo, Bingde

    2016-01-01

    Large epidemiological studies suggest that there are important differences in the incidence and severity of a wide variety of cardiac diseases, between premenopausal and menopausal women. Recently, it has been demonstrated that resveratrol may has similar function as estrogen. However, whether resveratrol replacement could mimic estrogen to protect heart in ovariectomized mice remains completely unknown. Firstly, the present study has used OVX/CAL model to investigate the effect of RSV on ischemic heart. Echocardiography analysis revealed that RSV administration significantly improved cardiac contractile function in estrogen-deficient mice. RSV also significantly reduced CK and LDH release, and heart infarct size in OVX/CAL group. Secondly, mitochondrial functions, including MRC activities, MDA level, and mitochondrial swelling, were evaluated in OVX mice. It was found that supplementation with RSV could restore mitochondrial function dampened by OVX. Thirdly, these protective functions mediated by RSV were mainly attributed to the enhancement of SIRT1/AMPK activity. In summary, the results support a potential role of resveratrol in the protection of cardiac functions under estrogen depletion status. PMID:27398147

  4. An APPL1-AMPK signaling axis mediates beneficial metabolic effects of adiponectin in the heart

    PubMed Central

    Fang, Xiangping; Palanivel, Rengasamy; Cresser, Justin; Schram, Kristin; Ganguly, Riya; Thong, Farah S. L.; Tuinei, Joseph; Xu, Aimin; Abel, E. Dale

    2010-01-01

    Adiponectin promotes cardioprotection by various mechanisms, and this study used primary cardiomyocytes and the isolated working perfused heart to investigate cardiometabolic effects. We show in adult cardiomyocytes that adiponectin increased CD36 translocation and fatty acid uptake as well as insulin-stimulated glucose transport and Akt phosphorylation. Coimmunoprecipitation showed that adiponectin enhanced association of AdipoR1 with APPL1, subsequent binding of APPL1 with AMPKα2, which led to phosphorylation and inhibition of ACC and increased fatty acid oxidation. Using siRNA to effectively knockdown APPL1 in neonatal cardiomyocytes, we demonstrated an essential role for APPL1 in mediating increased fatty acid uptake and oxidation by adiponectin. Importantly, enhanced fatty acid oxidation in conjunction with AMPK and ACC phosphorylation was also observed in the isolated working heart. Despite increasing fatty acid oxidation and myocardial oxygen consumption, adiponectin increased hydraulic work and maintained cardiac efficiency. In summary, the present study documents several beneficial metabolic effects mediated by adiponectin in the heart and provides novel insight into the mechanisms behind these effects, in particular the importance of APPL1. PMID:20739511

  5. NAF-1 antagonizes starvation-induced autophagy through AMPK signaling pathway in cardiomyocytes.

    PubMed

    Du, Xiaohong; Xiao, Renjie; Xiao, Fan; Chen, Yong; Hua, Fuzhou; Yu, Shuchun; Xu, Guohai

    2015-07-01

    NAF-1 (nutrient-deprivation autophagy factor-1), an autophagy-related gene-related (ATG) protein, has been implicated in the autophagic pro-survival response. However, its role in autophagy has not been examined in the cardiomyocytes. In this study, we found that nutritional stress (NS) induced by glucose deprivation strongly induced autophagy in cultured neonatal rat cardiomyocytes, which was associated with NAF-1 down-regulation in cardiomyocytes under NS conditions. Furthermore, we demonstrate that ectopic expression of NAF-1 was sufficient to inhibit autophagy in cardiomyocytes under glucose deprivation conditions. Moreover, results of the co-immunoprecipitation assay indicate that NAF-1 antagonized autophagy by promoting the interaction between Beclin1 and Bcl-2 in NS-induced cardiomyocytes. Importantly, our results indicate that overexpression of NAF-1 significantly inhibited AMPK activity and protected cardiomyocytes from NS-induced cell death. Taken together, these data show that ectopic expression of NAF-1 antagonizes the degree of autophagy in cardiomyocytes and enhances cell survival during starvation conditions.

  6. Regulation of Yeast G Protein Signaling by the Kinases That Activate the AMPK Homolog Snf1

    PubMed Central

    Clement, Sarah T.; Dixit, Gauri; Dohlman, Henrik G.

    2014-01-01

    Extracellular signals, such as nutrients and hormones, cue intracellular pathways to produce adaptive responses. Often, cells must coordinate their responses to multiple signals to produce an appropriate outcome. We showed that components of a glucose-sensing pathway acted on components of a heterotrimeric guanine nucleotide–binding protein (G protein)–mediated pheromone signaling pathway in the yeast Saccharomyces cerevisiae. We demonstrated that the G protein α subunit Gpa1 was phosphorylated in response to conditions of reduced glucose availability and that this phosphorylation event contributed to reduced pheromone-dependent stimulation of mitogen-activated protein kinases, gene transcription, cell morphogenesis, and mating efficiency. We found that Elm1, Sak1, and Tos3, the kinases that phosphorylate Snf1, the yeast homolog of adenosine monophosphate–activated protein kinase (AMPK), in response to limited glucose availability, also phosphorylated Gpa1 and contributed to the diminished mating response. Reg1, the regulatory subunit of the phosphatase PP1 that acts on Snf1, was likewise required to reverse the phosphorylation of Gpa1 and to maintain the mating response. Thus, the same kinases and phosphatase that regulate Snf1 also regulate Gpa1. More broadly, these results indicate that the pheromone signaling and glucose-sensing pathways communicate directly to coordinate cell behavior. PMID:24003255

  7. Reduction in Neural Performance following Recovery from Anoxic Stress Is Mimicked by AMPK Pathway Activation

    PubMed Central

    Money, Tomas G. A.; Sproule, Michael K. J.; Hamour, Amr F.; Robertson, R. Meldrum

    2014-01-01

    Nervous systems are energetically expensive to operate and maintain. Both synaptic and action potential signalling require a significant investment to maintain ion homeostasis. We have investigated the tuning of neural performance following a brief period of anoxia in a well-characterized visual pathway in the locust, the LGMD/DCMD looming motion-sensitive circuit. We hypothesised that the energetic cost of signalling can be dynamically modified by cellular mechanisms in response to metabolic stress. We examined whether recovery from anoxia resulted in a decrease in excitability of the electrophysiological properties in the DCMD neuron. We further examined the effect of these modifications on behavioural output. We show that recovery from anoxia affects metabolic rate, flight steering behaviour, and action potential properties. The effects of anoxia on action potentials can be mimicked by activation of the AMPK metabolic pathway. We suggest this is evidence of a coordinated cellular mechanism to reduce neural energetic demand following an anoxic stress. Together, this represents a dynamically-regulated means to link the energetic demands of neural signaling with the environmental constraints faced by the whole animal. PMID:24533112

  8. GLP-1 agonism stimulates brown adipose tissue thermogenesis and browning through hypothalamic AMPK.

    PubMed

    Beiroa, Daniel; Imbernon, Monica; Gallego, Rosalía; Senra, Ana; Herranz, Daniel; Villarroya, Francesc; Serrano, Manuel; Fernø, Johan; Salvador, Javier; Escalada, Javier; Dieguez, Carlos; Lopez, Miguel; Frühbeck, Gema; Nogueiras, Ruben

    2014-10-01

    GLP-1 receptor (GLP-1R) is widely located throughout the brain, but the precise molecular mechanisms mediating the actions of GLP-1 and its long-acting analogs on adipose tissue as well as the brain areas responsible for these interactions remain largely unknown. We found that central injection of a clinically used GLP-1R agonist, liraglutide, in mice stimulates brown adipose tissue (BAT) thermogenesis and adipocyte browning independent of nutrient intake. The mechanism controlling these actions is located in the hypothalamic ventromedial nucleus (VMH), and the activation of AMPK in this area is sufficient to blunt both central liraglutide-induced thermogenesis and adipocyte browning. The decreased body weight caused by the central injection of liraglutide in other hypothalamic sites was sufficiently explained by the suppression of food intake. In a longitudinal study involving obese type 2 diabetic patients treated for 1 year with GLP-1R agonists, both exenatide and liraglutide increased energy expenditure. Although the results do not exclude the possibility that extrahypothalamic areas are also modulating the effects of GLP-1R agonists, the data indicate that long-acting GLP-1R agonists influence body weight by regulating either food intake or energy expenditure through various hypothalamic sites and that these mechanisms might be clinically relevant.

  9. Hypoxia Leads to Na,K-ATPase Downregulation via Ca2+ Release-Activated Ca2+ Channels and AMPK Activation ▿

    PubMed Central

    Gusarova, Galina A.; Trejo, Humberto E.; Dada, Laura A.; Briva, Arturo; Welch, Lynn C.; Hamanaka, Robert B.; Mutlu, Gökhan M.; Chandel, Navdeep S.; Prakriya, Murali; Sznajder, Jacob I.

    2011-01-01

    To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca2+ from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca2+ release-activated Ca2+ (CRAC) channels. This increase in intracellular Ca2+ induces Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca2+ concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca2+ signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction. PMID:21730292

  10. Functional Effects of a Pathogenic Mutation in Cereblon (CRBN) on the Regulation of Protein Synthesis via the AMPK-mTOR Cascade*

    PubMed Central

    Lee, Kwang Min; Yang, Seung-Joo; Choi, Ja-Hyun; Park, Chul-Seung

    2014-01-01

    Initially identified as a protein implicated in human mental deficit, cereblon (CRBN) was recently recognized as a negative regulator of adenosine monophosphate-activated protein kinase (AMPK) in vivo and in vitro. Here, we present results showing that CRBN can effectively regulate new protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway, a downstream target of AMPK. Whereas deficiency of Crbn repressed protein translation via activation of the AMPK-mTOR cascade in Crbn-knock-out mice, ectopic expression of the wild-type CRBN increased protein synthesis by inhibiting endogenous AMPK. Unlike the wild-type CRBN, a mutant CRBN found in human patients, which lacks the last 24 amino acids, failed to rescue mTOR-dependent repression of protein synthesis in Crbn-deficient mouse fibroblasts. These results provide the first evidence that Crbn can activate the protein synthesis machinery through the mTOR signaling pathway by inhibiting AMPK. In light of the fact that protein synthesis regulated by mTOR is essential for various forms of synaptic plasticity that underlie the cognitive functions of the brain, the results of this study suggest a plausible mechanism for CRBN involvement in higher brain function in humans, and they may help explain how a specific mutation in CRBN can affect the cognitive ability of patients. PMID:24993823

  11. Capsaicin suppresses the migration of cholangiocarcinoma cells by down-regulating matrix metalloproteinase-9 expression via the AMPK-NF-κB signaling pathway.

    PubMed

    Lee, Gong-Rak; Jang, Soo Hwa; Kim, Chang Jae; Kim, Ah-Ram; Yoon, Dong-Joon; Park, Neung-Hwa; Han, In-Seob

    2014-12-01

    Cholangiocarcinoma is one of the most difficult malignancies to cure. An important prognostic factor is metastasis, which precludes curative surgical resection. Recent evidence shows that capsaicin has an inhibitory effect on cancer cell migration and invasion. Here, we investigated the molecular mechanism of the capsaicin-induced anti-migration and anti-invasion effects on HuCCT1 cholangiocarcinoma cells. Migration and invasion were significantly reduced in response to capsaicin. Capsaicin also inhibited the expression of matrix metalloproteinase-9 (MMP-9). In capsaicin-treated cells, levels of phosphorylated AMPK increased, and this effect was abolished by treatment with the AMPK inhibitor, Compound C. Capsaicin enhanced the expression of SIRT1, which can activate the transcription factor NF-κB by deacetylation. This suggests that NF-κB is activated by capsaicin via the SIRT1 pathway. In addition, capsaicin-activated AMPK induced the phosphorylation of IκBα and nuclear localization of NF-κB p65. Chromatin immunoprecipitation assays demonstrated that capsaicin reduced MMP-9 transcription by inhibiting NF-κB p65 translocation and deacetylation via SIRT1. These findings provide evidence that capsaicin suppresses the migration and invasion of cholangiocarcinoma cells by inhibiting NF-κB p65 via the AMPK-SIRT1 and the AMPK-IκBα signaling pathways, leading to subsequent suppression of MMP-9 expression.

  12. Betahistine ameliorates olanzapine-induced weight gain through modulation of histaminergic, NPY and AMPK pathways.

    PubMed

    Lian, Jiamei; Huang, Xu-Feng; Pai, Nagesh; Deng, Chao

    2014-10-01

    Olanzapine is widely used to treat schizophrenia and other disorders, but causes adverse obesity and other metabolic side-effects. Both animal and clinical studies have shown that co-treatment with betahistine (a histaminergic H1 receptor agonist and H3 receptor antagonist) is effective for ameliorating olanzapine-induced weight gain/obesity. To reveal the mechanisms underlying these effects, this study investigated the effects of co-treatment of olanzapine and betahistine (O+B) on expressions of histaminergic H1 receptor (H1R), AMP-activated protein kinase (AMPK), neuropeptide Y (NPY), and proopiomelanocortin (POMC) in the hypothalamus associated with reducing olanzapine-induced weight gain. Olanzapine significantly upregulated the mRNA and protein expressions of H1R, while O+B co-treatment significantly downregulated the H1R levels, compared to the olanzapine-only treatment group. The NPY mRNA expression was significantly enhanced by olanzapine, but it was significantly reversed by O+B co-treatment. The hypothalamic H1R expression was positively correlated with total food intake, and NPY expression. Olanzapine also increased AMPKα activation measured by the AMPKα phosphorylation (pAMPKα)/AMPKα ratio compared with controls, whereas O+B co-treatment decreased the pAMPKα/AMPKα ratio, compared with olanzapine only treatment. The pAMPKα/AMPKα ratio was positively correlated with total food intake and H1R expression. Although olanzapine administration decreased the POMC mRNA level, this level was not affected by O+B co-treatment. Therefore, these results suggested that co-treatment with betahistine may reverse olanzapine-induced body weight gain via the H1R-NPY and H1R-pAMPKα pathways.

  13. p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

    SciTech Connect

    Huang, Shi-Wei; Wu, Chun-Ying; Wang, Yen-Ting; Kao, Jun-Kai; Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu; Chiu, Husan-Wen; Chang, Chuan-Hsun; Liang, Shu-Mei; Chen, Yi-Ju; Huang, Jau-Ling; Shieh, Jeng-Jer

    2013-02-15

    Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.

  14. Antioxidant catalase rescues against high fat diet-induced cardiac dysfunction via an IKKβ-AMPK-dependent regulation of autophagy.

    PubMed

    Liang, Lei; Shou, Xi-Ling; Zhao, Hai-Kang; Ren, Gu-Qun; Wang, Jian-Bang; Wang, Xi-Hui; Ai, Wen-Ting; Maris, Jackie R; Hueckstaedt, Lindsay K; Ma, Ai-Qun; Zhang, Yingmei

    2015-02-01

    Autophagy, a conservative degradation process for long-lived and damaged proteins, participates in a variety of biological processes including obesity. However, the precise mechanism of action behind obesity-induced changes in autophagy still remains elusive. This study was designed to examine the role of the antioxidant catalase in high fat diet-induced changes in cardiac geometry and function as well as the underlying mechanism of action involved with a focus on autophagy. Wild-type (WT) and transgenic mice with cardiac overexpression of catalase were fed low or high fat diet for 20 weeks prior to assessment of myocardial geometry and function. High fat diet intake triggered obesity, hyperinsulinemia, and hypertriglyceridemia, the effects of which were unaffected by catalase transgene. Myocardial geometry and function were compromised with fat diet intake as manifested by cardiac hypertrophy, enlarged left ventricular end systolic and diastolic diameters, fractional shortening, cardiomyocyte contractile capacity and intracellular Ca²⁺ mishandling, the effects of which were ameliorated by catalase. High fat diet intake promoted reactive oxygen species production and suppressed autophagy in the heart, the effects of which were attenuated by catalase. High fat diet intake dampened phosphorylation of inhibitor kappa B kinase β(IKKβ), AMP-activated protein kinase (AMPK) and tuberous sclerosis 2 (TSC2) while promoting phosphorylation of mTOR, the effects of which were ablated by catalase. In vitro study revealed that palmitic acid compromised cardiomyocyte autophagy and contractile function in a manner reminiscent of fat diet intake, the effect of which was significantly alleviated by inhibition of IKKβ, activation of AMPK and induction of autophagy. Taken together, our data revealed that the antioxidant catalase counteracts against high fat diet-induced cardiac geometric and functional anomalies possibly via an IKKβ-AMPK-dependent restoration of myocardial

  15. SREBP-1c, regulated by the insulin and AMPK signaling pathways, plays a role in nonalcoholic fatty liver disease.

    PubMed

    Kohjima, Motoyuki; Higuchi, Nobito; Kato, Masaki; Kotoh, Kazuhiro; Yoshimoto, Tsuyoshi; Fujino, Tatsuya; Yada, Masayoshi; Yada, Ryoko; Harada, Naohiko; Enjoji, Munechika; Takayanagi, Ryoichi; Nakamuta, Makoto

    2008-04-01

    Nonalcoholic fatty liver disease (NAFLD) is a common liver disease whose prevalence has increased markedly. We reported previously that fatty acid synthesis was enhanced in NAFLD with the accumulation of fatty acids. To clarify the disorder, we evaluated the expression of genes regulating fatty acid synthesis by real-time PCR using samples from NAFLD (n=22) and normal liver (control; n=10). A major regulator of fatty acids synthesis is sterol regulatory element-binding protein-1c (SREBP-1c). Its expression was significantly higher in NAFLD, nearly 5-fold greater than the controls. SREBP-1c is positively regulated by insulin signaling pathways, including insulin receptor substrate (IRS)-1 and -2. In NAFLD, IRS-1 expression was enhanced and correlated positively with SREBP-1c expression. In contrast, IRS-2 expression decreased by 50% and was not correlated with SREBP-1c. Forkhead box protein A2 (Foxa2) is a positive regulator of fatty acid oxidation and is itself negatively regulated by IRSs. Foxa2 expression increased in NAFLD and showed a negative correlation with IRS-2, but not with IRS-1, expression. It is known that SREBP-1c is negatively regulated by AMP-activated protein kinase (AMPK) but expression levels of AMPK in NAFLD were almost equal to those of the controls. These data indicate that, in NAFLD, insulin signaling via IRS-1 causes the up-regulation of SREBP1-c, leading to the increased synthesis of fatty acids by the hepatocytes; negative feedback regulation via AMPK does not occur and the activation of Foxa2, following a decrease of IRS-2, up-regulates fatty acid oxidation. PMID:18360697

  16. Resveratrol decreases fructose-induced oxidative stress, mediated by NADPH oxidase via an AMPK-dependent mechanism

    PubMed Central

    Cheng, Pei-Wen; Ho, Wen-Yu; Su, Yu-Ting; Lu, Pei-Jung; Chen, Bo-Zone; Cheng, Wen-Han; Lu, Wen-Hsien; Sun, Gwo-Ching; Yeh, Tung-Chen; Hsiao, Michael; Tseng, Ching-Jiunn

    2014-01-01

    Background and Purpose Oxidative stress is an important pathogenic factor in the development of hypertension. Resveratrol, the main antioxidant in red wine, improves NO bioavailability and prevents cardiovascular disease. The aim of this study was to examine whether resveratrol decreases the generation of reactive oxygen species (ROS), thereby reducing BP in rats with fructose-induced hypertension. Experimental Approach Rats were fed 10% fructose with or without resveratrol (10 mg·kg−1·day−1) for 1 week or for 4 weeks with resveratrol treatment beginning at week 2; systolic BP (SBP) was measured by tail-cuff method. Endogenous in vivo O2− production in the nucleus tractus solitarii (NTS) was determined with dihydroethidium. Real-time PCR and immunoblotting analyses were used to quantify RNA and protein expression levels. Key Results In fructose-fed rats, ROS levels in the NTS were higher, whereas the NO level was significantly decreased. Also, RNA and protein levels of NADPH oxidase subunits (p67, p22-phox) were elevated, superoxide dismutase 2 (SOD2) reduced and AMP-activated PK (AMPK) T172 phosphorylation levels in the NTS were lower in fructose-fed rats. Treatment with the AMPK activator resveratrol decreased levels of NADPH oxidase subunits and ROS, and increased NO and SOD2 levels in the NTS of fructose-fed rats. Administration of resveratrol, in combination with fructose at week 0 and later at week 2, significantly reduced the SBP of fructose-fed rats. Conclusions and Implications Collectively, resveratrol decreased BP through the phosphorylation of AMPK, Akt and neuronal NOS in fructose-fed rats. These novel findings suggest that resveratrol may be a potential pharmacological candidate for the treatment of hypertension. PMID:24547812

  17. Discovery of biaryl-4-carbonitriles as antihyperglycemic agents that may act through AMPK-p38 MAPK pathway.

    PubMed

    Goel, Atul; Nag, Pankaj; Rahuja, Neha; Srivastava, Rohit; Chaurasia, Sumit; Gautam, Sudeep; Chandra, Sharat; Siddiqi, Mohammad Imran; Srivastava, Arvind K

    2014-08-25

    A series of functionalized biaryl-4-carbonitriles was synthesized in three steps and evaluated for PTP-1B inhibitory activity. Among the synthesized compounds, four biaryls 6a-d showed inhibition (IC50 58-75 μM) against in vitro PTP-1B assay possibly due to interaction with amino acid residues Lys120, Tyr46 through hydrogen bonding and aromatic-aromatic interactions, respectively. Two biaryl-4-carbonitriles 6b and 6c showed improved glucose tolerance, fasting as well as postprandial blood glucose, serum total triglycerides, and increased high-density lipoprotein-cholesterol in SLM, STZ, STZ-S and C57BL/KsJ-db/db animal models. The bioanalysis of 4'-bromo-2,3-dimethyl-5-(piperidin-1-yl)biphenyl-4-carbonitrile (6b) revealed that like insulin, it increased 2-deoxyglucose uptake in skeletal muscle cells (L6 and C2C12 myotubes). The compound 6b significantly up-regulated the genes related to the insulin signaling pathways like AMPK, MAPK including glucose transporter-4 (GLUT-4) gene in muscle tissue of C57BL/KsJ-db/db mice. Furthermore, it was observed that the compound 6b up-regulated PPARα, UCP2 and HNF4α, which are key regulator of glucose, lipid, and fatty acid metabolism. Western blot analysis of the compound 6b showed that it significantly increased the phosphorylation of AMPK and p38 MAPK and ameliorated glucose uptake in C57BL/KsJ-db/db mice through the AMPK-p38 MAPK pathway.

  18. SIRT1 activation by methylene blue, a repurposed drug, leads to AMPK-mediated inhibition of steatosis and steatohepatitis.

    PubMed

    Shin, Seo Young; Kim, Tae Hyun; Wu, Hongmin; Choi, Young Hee; Kim, Sang Geon

    2014-03-15

    Sirtuins maintain energy balance. Particularly, sirtuin 1 (SIRT1) activation mimics calorie restriction and nutrient utilization. However, no medications are available for the up-regulation of SIRT1. Methylene blue (MB) had been in clinical trials for the treatment of neurological diseases. This study investigated the effect of MB on sirtuin expression in association with the treatment of steatosis and steatohepatitis, and explored the underlying basis. The effects of MB on mitochondrial function, molecular markers, pharmacokinetics, and histopathology were assessed using hepatocyte and/or mouse models. Immunoblotting, PCR and reporter assays were done for molecular experiments. After oral administration, MB was well distributed in the liver. MB treatment increased NAD(+)/NADH ratio in hepatocytes. Of the major forms, MB treatment up-regulated SIRT1, and thereby decreased PGC-1α acetylation. Consistently, hepatic mitochondrial DNA contents and oxygen consumption rates were enhanced. MB treatment also notably activated AMPK, CPT-1 and PPARα: the AMPK activation relied on SIRT1. Activation of LXRα and the induction of SREBP-1c and its target genes by T0901317 were diminished by MB. In addition, MB treatment antagonized the ability of palmitate to acetylate PGC-1α, and increase SERBP-1c, FAS, and ACC levels. In mice fed on a high-fat diet for 8 weeks, MB treatment inhibited excessive hepatic fat accumulation and steatohepatitis. The ability of MB to activate SIRT1 promotes mitochondrial biogenesis and oxygen consumption and activates AMPK, contributing to anti-lipogenesis in the liver. Our results provide new information on the potential use of MB for the treatment of steatosis and steatohepatitis.

  19. Exercise performance and peripheral vascular insufficiency improve with AMPK activation in high-fat diet-fed mice

    PubMed Central

    Baltgalvis, Kristen A.; White, Kathy; Li, Wei; Claypool, Mark D.; Lang, Wayne; Alcantara, Raniel; Singh, Baljit K.; Friera, Annabelle M.; McLaughlin, John; Hansen, Derek; McCaughey, Kelly; Nguyen, Henry; Smith, Ira J.; Godinez, Guillermo; Shaw, Simon J.; Goff, Dane; Singh, Rajinder; Markovtsov, Vadim; Sun, Tian-Qiang; Jenkins, Yonchu; Uy, Gerald; Li, Yingwu; Pan, Alison; Gururaja, Tarikere; Lau, David; Park, Gary; Hitoshi, Yasumichi; Payan, Donald G.

    2014-01-01

    Intermittent claudication is a form of exercise intolerance characterized by muscle pain during walking in patients with peripheral artery disease (PAD). Endothelial cell and muscle dysfunction are thought to be important contributors to the etiology of this disease, but a lack of preclinical models that incorporate these elements and measure exercise performance as a primary end point has slowed progress in finding new treatment options for these patients. We sought to develop an animal model of peripheral vascular insufficiency in which microvascular dysfunction and exercise intolerance were defining features. We further set out to determine if pharmacological activation of 5′-AMP-activated protein kinase (AMPK) might counteract any of these functional deficits. Mice aged on a high-fat diet demonstrate many functional and molecular characteristics of PAD, including the sequential development of peripheral vascular insufficiency, increased muscle fatigability, and progressive exercise intolerance. These changes occur gradually and are associated with alterations in nitric oxide bioavailability. Treatment of animals with an AMPK activator, R118, increased voluntary wheel running activity, decreased muscle fatigability, and prevented the progressive decrease in treadmill exercise capacity. These functional performance benefits were accompanied by improved mitochondrial function, the normalization of perfusion in exercising muscle, increased nitric oxide bioavailability, and decreased circulating levels of the endogenous endothelial nitric oxide synthase inhibitor asymmetric dimethylarginine. These data suggest that aged, obese mice represent a novel model for studying exercise intolerance associated with peripheral vascular insufficiency, and pharmacological activation of AMPK may be a suitable treatment for intermittent claudication associated with PAD. PMID:24561866

  20. AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: implication of the cell survival in mitochondrial diseases.

    PubMed

    Wu, Shi-Bei; Wei, Yau-Huei

    2012-02-01

    We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H(2)O(2). The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H(2)O(2)-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H(2)O(2) in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H(2)O(2) was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H(2)O(2)-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H(2)O(2)-treated normal skin fibroblasts to oxidative stress.

  1. An active metabolite of oltipraz (M2) increases mitochondrial fuel oxidation and inhibits lipogenesis in the liver by dually activating AMPK

    PubMed Central

    Kim, Tae Hyun; Eom, Jeong Sik; Lee, Chan Gyu; Yang, Yoon Mee; Lee, Yong Sup; Kim, Sang Geon

    2013-01-01

    Background and Purpose Oltipraz, a cancer chemopreventive agent, has an anti-steatotic effect via liver X receptor-α (LXRα) inhibition. Here we have assessed the biological activity of a major metabolite of oltipraz (M2) against liver steatosis and steatohepatitis and the underlying mechanism(s). Experimental Approach Blood biochemistry and histopathology were assessed in high-fat diet (HFD)-fed mice treated with M2. An in vitroHepG2 cell model was used to study the mechanism of action. Immunoblotting, real-time PCR and luciferase reporter assays were performed to measure target protein or gene expression levels. Key Results M2 treatment inhibited HFD-induced steatohepatitis and diminished oxidative stress in liver. It increased expression of genes encoding proteins involved in mitochondrial fuel oxidation. Mitochondrial DNA content and oxygen consumption rate were enhanced. Moreover, M2 treatment repressed activity of LXRα and induction of its target genes, indicating anti-lipogenic effects. M2 activated AMP-activated protein kinase (AMPK). Inhibition of AMPK by over-expression of dominant negative AMPK (DN-AMPK) or by Compound C prevented M2 from inducing genes for fatty acid oxidation and repressed sterol regulatory element binding protein-1c (SREBP-1c) expression. M2 activated liver kinase B1 (LKB1) and increased the AMP/ATP ratio. LKB1 knockdown failed to reverse target protein modulations or AMPK activation by M2, supporting the proposal that both LKB1 and increased AMP/ATP ratio contribute to its anti-steatotic effect. Conclusion and Implications M2 inhibited liver steatosis and steatohepatitis by enhancing mitochondrial fuel oxidation and inhibiting lipogenesis. These effects reflected activation of AMPK elicited by increases in LKB1 activity and AMP/ATP ratio. PMID:23145499

  2. Sestrin2 Silencing Exacerbates Cerebral Ischemia/Reperfusion Injury by Decreasing Mitochondrial Biogenesis through the AMPK/PGC-1α Pathway in Rats

    PubMed Central

    Li, Lingyu; Xiao, Lina; Hou, Yanghao; He, Qi; Zhu, Jin; Li, Yixin; Wu, Jingxian; Zhao, Jing; Yu, Shanshan; Zhao, Yong

    2016-01-01

    Sestrin2 (Sesn2) exerts neuroprotective properties in some neurodegenerative diseases. However, the role of Sesn2 in stroke is unclear. The AMP-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α (AMPK/PGC-1α) pathway plays an important role in regulating mitochondrial biogenesis, which helps prevent cerebral ischemia/reperfusion (I/R) injury. Here, we aimed to determine whether Sesn2 alleviated I/R damage by regulating mitochondrial biogenesis through the AMPK/PGC-1α signaling pathway. To be able to test this, Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h with Sesn2 silencing. At 24 h after reperfusion, we found that neurological deficits were exacerbated, infarct volume was enlarged, and oxidative stress and neuronal damage were greater in the Sesn2 siRNA group than in the MCAO group. To explore protective mechanisms, an AMPK activator was used. Expression levels of Sesn2, p-AMPK, PGC-1α, NRF-1, TFAM, SOD2, and UCP2 were significantly increased following cerebral I/R. However, upregulation of these proteins was prevented by Sesn2 small interfering RNA (siRNA). In contrast, activation of AMPK with 5′-aminoimidazole-4-carboxamide riboside weakened the effects of Sesn2 siRNA. These results suggest that Sesn2 silencing may suppress mitochondrial biogenesis, reduce mitochondrial biological activity, and finally aggravate cerebral I/R injury through inhibiting the AMPK/PGC-1α pathway. PMID:27453548

  3. AMPK inhibits MTDH expression via GSK3β and SIRT1 activation: potential role in triple negative breast cancer cell proliferation.

    PubMed

    Gollavilli, Paradesi Naidu; Kanugula, Anantha Koteswararao; Koyyada, Rajeswari; Karnewar, Santosh; Neeli, Praveen Kumar; Kotamraju, Srigiridhar

    2015-10-01

    Recent studies have highlighted the involvement of metadherin (MTDH), an oncogenic protein, in promoting cancer progression, metastasis and chemoresistance in many cancers including mammary carcinomas. However, the molecular regulation of MTDH is still not completely understood. In this study we document that AMP activated protein kinase (AMPK) activation-induced anti-proliferative effects are, in part, mediated by inhibiting MTDH expression in MDA-MB-231 and BT-549 triple negative breast cancer (TNBC) cells. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, caused growth arrest, inhibition of migration and invasion of TNBC cells. Intriguingly, AICAR or metformin treatment resulted in significant downregulation of MTDH expression via inhibiting c-Myc expression. In contrast, treatment of cells with compound C, an inhibitor of AMPK, increased both c-Myc and MTDH expressions in TNBC cells. Also, AMPK activation caused increased glycogen synthase kinase 3β (GSK3β) activity by inhibiting the inactive phosphorylation at Ser9, on the one hand, and activation of sirtuin1 (SIRT1) by inhibiting Ser47 phosphorylation, as evidenced by deacetylation of p53, on the other hand. Moreover, AMPK-induced GSK3β and SIRT1 activities were found to be responsible for inhibiting c-Myc-mediated upregulation of MTDH, as LiCl (an inhibitor of GSK3β) and EX-527 (an inhibitor of SIRT1) reversed AICAR-mediated downregulation of c-Myc and MTDH expressions. Similar results were observed with siSIRT1 treatment. Furthermore, AICAR and EX-527 treatments caused increased cell death under MTDH-depleted conditions. Finally, we uncovered a novel regulation of MTDH expression and showed that AMPK activation by inducing GSK3β and SIRT1 downregulates MTDH expression via inhibiting c-Myc in TNBC cells. PMID:26236947

  4. Muscle-specific AMPK β1β2-null mice display a myopathy due to loss of capillary density in nonpostural muscles.

    PubMed

    Thomas, Melissa M; Wang, David C; D'Souza, Donna M; Krause, Matthew P; Layne, Andrew S; Criswell, David S; O'Neill, Hayley M; Connor, Michael K; Anderson, Judy E; Kemp, Bruce E; Steinberg, Gregory R; Hawke, Thomas J

    2014-05-01

    AMP-activated protein kinase (AMPK) is a master regulator of metabolism. While muscle-specific AMPK β1β2 double-knockout (β1β2M-KO) mice display alterations in metabolic and mitochondrial capacity, their severe exercise intolerance suggested a secondary contributor to the observed phenotype. We find that tibialis anterior (TA), but not soleus, muscles of sedentary β1β2M-KO mice display a significant myopathy (decreased myofiber areas, increased split and necrotic myofibers, and increased centrally nucleated myofibers. A mitochondrial- and fiber-type-specific etiology to the myopathy was ruled out. However, β1β2M-KO TA muscles displayed significant (P<0.05) increases in platelet aggregation and apoptosis within myofibers and surrounding interstitium (P<0.05). These changes correlated with a 45% decrease in capillary density (P<0.05). We hypothesized that the β1β2M-KO myopathy in resting muscle resulted from impaired AMPK-nNOSμ signaling, causing increased platelet aggregation, impaired vasodilation, and, ultimately, ischemic injury. Consistent with this hypothesis, AMPK-specific phosphorylation (Ser1446) of nNOSμ was decreased in β1β2M-KO compared to wild-type (WT) mice. The AMPK-nNOSμ relationship was further demonstrated by administration of 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) to β1β2-MKO muscles and C2C12 myotubes. AICAR significantly increased nNOSμ phosphorylation and nitric oxide production (P<0.05) within minutes of administration in WT muscles and C2C12 myotubes but not in β1β2M-KO muscles. These findings highlight the importance of the AMPK-nNOSμ pathway in resting skeletal muscle.

  5. Methyl succinate antagonises biguanide-induced AMPK-activation and death of pancreatic β-cells through restoration of mitochondrial electron transfer

    PubMed Central

    Hinke, S A; Martens, G A; Cai, Y; Finsi, J; Heimberg, H; Pipeleers, D; Van de Casteele, M

    2007-01-01

    Background and purpose: Two mechanisms have been proposed to explain the insulin-sensitising properties of metformin in peripheral tissues: (a) inhibition of electron transport chain complex I, and (b) activation of the AMP activated protein kinase (AMPK). However the relationship between these mechanisms and their contribution to β-cell death and dysfunction in vitro, are currently unclear. Experimental approach: The effects of biguanides (metformin and phenformin) were tested on MIN6 β-cells and primary FACS-purified rat β-cells. Cell metabolism was assessed biochemically and by FACS analysis, and correlated with AMPK phosphorylation state and cell viability, with or without fuel substrates. Key results: In MIN6 cells, metformin reduced mitochondrial complex I activity by up to 44% and a 25% net reduction in mitochondrial reducing potential. In rat β-cells, metformin caused NAD(P)H accumulation above maximal glucose-inducible levels, mimicking the effect of rotenone. Drug exposure caused phosphorylation of AMPK on Thr172 in MIN6 cell extracts, indicative of kinase activation. Methyl succinate, a complex II substrate, appeared to bypass metformin blockade of complex I. This resulted in reduced phosphorylation of AMPK, establishing a link between biguanide-induced mitochondrial inhibition and AMPK activation. Corresponding assessment of cell death indicated that methyl succinate decreased biguanide toxicity to β-cells in vitro. Conclusions and implications: AMPK activation can partly be attributed to metformin's inhibitory action on mitochondrial complex I. Anaplerotic fuel metabolism via complex II rescued β-cells from metformin-associated toxicity. We propose that utilisation of anaplerotic nutrients may reconcile in vitro and in vivo effects of metformin on the pancreatic β-cell. PMID:17339833

  6. Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes.

    PubMed

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Weikel, Karen A; Breton, Lionel; Ruderman, Neil B

    2015-01-01

    The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H2O2)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H2O2-induced senescence, resveratrol also prevented the H2O2-induced decrease in proliferation (as indicated by 3H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol's effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by

  7. Metformin activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against Amyloid-beta-induced mitochondrial dysfunction.

    PubMed

    Chiang, Ming-Chang; Cheng, Yi-Chuan; Chen, Shiang-Jiuun; Yen, Chia-Hui; Huang, Rong-Nan

    2016-10-01

    Alzheimer's disease (AD) is the general consequence of dementia and is diagnostic neuropathology by the cumulation of amyloid-beta (Aβ) protein aggregates, which are thought to promote mitochondrial dysfunction processes leading to neurodegeneration. AMP-activated protein kinase (AMPK), a critical regulator of energy homeostasis and a major player in lipid and glucose metabolism, is potentially implied in the mitochondrial deficiency of AD. Metformin, one of the widespread used anti- metabolic disease drugs, use its actions in part by stimulation of AMPK. While the mechanisms of AD are well established, the neuronal roles for AMPK in AD are still not well understood. In the present study, human neural stem cells (hNSCs) exposed to Aβ had significantly reduced cell viability, which correlated with decreased AMPK, neuroprotective genes (Bcl-2 and CREB) and mitochondria associated genes (PGC1α, NRF-1 and Tfam) expressions, as well as increased activation of caspase 3/9 activity and cytosolic cytochrome c. Co-treatment with metformin distinct abolished the Aβ-caused actions in hNSCs. Metformin also significantly rescued hNSCs from Aβ-mediated mitochondrial deficiency (lower D-loop level, mitochondrial mass, maximal respiratory function, COX activity, and mitochondrial membrane potential). Importantly, co-treatment with metformin significantly restored fragmented mitochondria to almost normal morphology in the hNSCs with Aβ. These findings extend our understanding of the central role of AMPK in Aβ-related neuronal impairment. Thus, a better understanding of AMPK might assist in both the recognition of its critical effects and the implementation of new therapeutic strategies in the treatment of AD. PMID:27554603

  8. Sestrin2 Silencing Exacerbates Cerebral Ischemia/Reperfusion Injury by Decreasing Mitochondrial Biogenesis through the AMPK/PGC-1α Pathway in Rats.

    PubMed

    Li, Lingyu; Xiao, Lina; Hou, Yanghao; He, Qi; Zhu, Jin; Li, Yixin; Wu, Jingxian; Zhao, Jing; Yu, Shanshan; Zhao, Yong

    2016-01-01

    Sestrin2 (Sesn2) exerts neuroprotective properties in some neurodegenerative diseases. However, the role of Sesn2 in stroke is unclear. The AMP-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α (AMPK/PGC-1α) pathway plays an important role in regulating mitochondrial biogenesis, which helps prevent cerebral ischemia/reperfusion (I/R) injury. Here, we aimed to determine whether Sesn2 alleviated I/R damage by regulating mitochondrial biogenesis through the AMPK/PGC-1α signaling pathway. To be able to test this, Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h with Sesn2 silencing. At 24 h after reperfusion, we found that neurological deficits were exacerbated, infarct volume was enlarged, and oxidative stress and neuronal damage were greater in the Sesn2 siRNA group than in the MCAO group. To explore protective mechanisms, an AMPK activator was used. Expression levels of Sesn2, p-AMPK, PGC-1α, NRF-1, TFAM, SOD2, and UCP2 were significantly increased following cerebral I/R. However, upregulation of these proteins was prevented by Sesn2 small interfering RNA (siRNA). In contrast, activation of AMPK with 5'-aminoimidazole-4-carboxamide riboside weakened the effects of Sesn2 siRNA. These results suggest that Sesn2 silencing may suppress mitochondrial biogenesis, reduce mitochondrial biological activity, and finally aggravate cerebral I/R injury through inhibiting the AMPK/PGC-1α pathway. PMID:27453548

  9. AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs

    PubMed Central

    El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

    2015-01-01

    Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs. PMID:26162096

  10. Synergic chemoprevention with dietary carbohydrate restriction and supplementation of AMPK-activating phytochemicals: the role of SIRT1.

    PubMed

    Lee, Jong Doo; Choi, Min-Ah; Ro, Simon Weonsang; Yang, Woo Ick; Cho, Arthur E H; Ju, Hye-Lim; Baek, Sinhwa; Chung, Sook In; Kang, Won Jun; Yun, Mijin; Park, Jeon Han

    2016-01-01

    Calorie restriction or a low-carbohydrate diet (LCD) can increase life span in normal cells while inhibiting carcinogenesis. Various phytochemicals also have calorie restriction-mimetic anticancer properties. We investigated whether an isocaloric carbohydrate-restriction diet and AMP-activated protein kinase (AMPK)-activating phytochemicals induce synergic tumor suppression. We used a mixture of AMPK-activating phytochemical extracts including curcumin, quercetin, catechins, and resveratrol. Survival analysis was carried out in a B16F10 melanoma model fed a control diet (62.14% kcal carbohydrate, 24.65% kcal protein and 13.2% kcal fat), a control diet with multiple phytochemicals (MP), LCD (16.5, 55.2, and 28.3% kcal, respectively), LCD with multiple phytochemicals (LCDmp), a moderate-carbohydrate diet (MCD, 31.9, 62.4, and 5.7% kcal, respectively), or MCD with phytochemicals (MCDmp). Compared with the control group, MP, LCD, or MCD intervention did not produce survival benefit, but LCDmp (22.80±1.58 vs. 28.00±1.64 days, P=0.040) and MCDmp (23.80±1.08 vs. 30.13±2.29 days, P=0.008) increased the median survival time significantly. Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the AMPK/SIRT1/LKB1pathway, and NF-κB suppression were the critical tumor-suppression mechanisms. In addition, SIRT1 suppressed proliferation of the B16F10 and A375SM cells under a low-glucose condition. Alterations in histone methylation within Pten and FoxO3a were observed after the MCDmp intervention. In the transgenic liver cancer model developed by hydrodynamic transfection of the HrasG12V and shp53, MCDmp and LCDmp interventions induced significant cancer-prevention effects. Microarray analysis showed that PPARα increased with decreased IL-6 and NF-κB within the hepatocytes after an MCDmp intervention. In conclusion, an isocaloric carbohydrate-restriction diet and natural AMPK-activating agents induce synergistic anticancer effects. SIRT1 acts as a

  11. Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through β-arrestin-2 in THP-1 cell line

    SciTech Connect

    Wang, Wei; Zhang, Yuan; Xu, Ming; Zhang, You-Yi; He, Bei

    2015-06-26

    The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β{sub 2}-adrenergic receptor (β{sub 2}-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β{sub 2}-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1β (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β{sub 2}-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β{sub 2}-AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production.

  12. Procyanidin Promotes Translocation of Glucose Transporter 4 in Muscle of Mice through Activation of Insulin and AMPK Signaling Pathways

    PubMed Central

    Yamashita, Yoko; Wang, Liuqing; Nanba, Fumio; Ito, Chiaki; Toda, Toshiya; Ashida, Hitoshi

    2016-01-01

    Procyanidins are the oligomeric or polymeric forms of epicatechin and catechin. In this study, we isolated and purified dimer to tetramer procyanidins from black soybean seed coat and investigated the anti-hyperglycemic effects by focusing on glucose transporter 4 (GLUT4) translocation and the underlying molecular mechanism in skeletal muscle of mice. The anti-hyperglycemic effects of procyanidins were also compared with those of monomer (−)-epicatechin (EC) and major anthocyanin, cyanidin-3-O-β-glucoside (C3G). To investigate GLUT4 translocation and its related signaling pathways, ICR mice were orally given procyanidins, EC and C3G in water at 10 μg/kg body weight. The mice were sacrificed 60 min after the dose of polyphenols, and soleus muscle was extracted from the hind legs. The results showed that trimeric and tetrameric procyanidins activated both insulin- and AMPK-signaling pathways to induce GLUT4 translocation in muscle of ICR mice. We confirmed that procyanidins suppressed acute hyperglycemia with an oral glucose tolerance test in a dose-dependent manner. Of these beneficial effects, cinnamtannin A2, one of the tetramers, was the most effective. In conclusion, procyanidins, especially cinnamtannin A2, significantly ameliorate postprandial hyperglycemia at least in part by promoting GLUT4 translocation to the plasma membrane by activating both insulin- and AMPK-signaling pathways. PMID:27598258

  13. Dietary folic acid activates AMPK and improves insulin resistance and hepatic inflammation in dietary rodent models of the metabolic syndrome.

    PubMed

    Buettner, R; Bettermann, I; Hechtl, C; Gäbele, E; Hellerbrand, C; Schölmerich, J; Bollheimer, L C

    2010-10-01

    The AMP activated kinase plays an important role in metabolic control, and pharmacologic enhancement of AMPK activity is used to improve insulin resistance. We hypothesized that high dose of folic acid supplementation might improve insulin sensitivity and hepatic inflammation and examined this by a dietary intervention in (a) the high fat fed rat model of the metabolic syndrome, which shows sole hepatic steatosis as well as (b) in rats fed with a high cholesterol, high cholate diet inducing nonalcoholic steatohepatitis (NASH). Male Wistar rats were fed with folic acid supplemented (40 mg/kg) high fat diet [based on lard, fat content 25% (wt/wt)] or NASH inducing diet (containing 15% fat, 1.25% cholesterol, 0.5% sodium cholate). Metabolic profiling was performed by measuring the animals' visceral fat pads, fasting plasma glucose, insulin, and adipokines as well as in vivo insulin tolerance tests. Hepatic steatosis and inflammation were analyzed semiquantitatively by histological analysis. Folic acid supplementation reduced visceral obesity and improved plasma adiponectin levels. In vivo insulin sensitivity was improved, and in HF-FA rats folic acid increased activation of hepatic AMPK. Further, folic acid supplementation improved hepatic inflammation in animals fed with NASH-inducing diet. Dietary folic acid improved parameters of insulin resistance and hepatic inflammation in rodent models. This might be due to an increased AMK activation.

  14. Rewiring AMPK and mitochondrial retrograde signaling for metabolic control of aging and histone acetylation in respiratory-defective cells.

    PubMed

    Friis, R Magnus N; Glaves, John Paul; Huan, Tao; Li, Liang; Sykes, Brian D; Schultz, Michael C

    2014-04-24

    Abnormal respiratory metabolism plays a role in numerous human disorders. We find that regulation of overall histone acetylation is perturbed in respiratory-incompetent (ρ(0)) yeast. Because histone acetylation is highly sensitive to acetyl-coenzyme A (acetyl-CoA) availability, we sought interventions that suppress this ρ(0) phenotype through reprogramming metabolism. Nutritional intervention studies led to the discovery that genetic coactivation of the mitochondrion-to-nucleus retrograde (RTG) response and the AMPK (Snf1) pathway prevents abnormal histone deacetylation in ρ(0) cells. Metabolic profiling of signaling mutants uncovered links between chromatin-dependent phenotypes of ρ(0) cells and metabolism of ATP, acetyl-CoA, glutathione, branched-chain amino acids, and the storage carbohydrate trehalose. Importantly, RTG/AMPK activation reprograms energy metabolism to increase the supply of acetyl-CoA to lysine acetyltransferases and extend the chronological lifespan of ρ(0) cells. Our results strengthen the framework for rational design of nutrient supplementation schemes and drug-discovery initiatives aimed at mimicking the therapeutic benefits of dietary interventions.

  15. Procyanidin Promotes Translocation of Glucose Transporter 4 in Muscle of Mice through Activation of Insulin and AMPK Signaling Pathways.

    PubMed

    Yamashita, Yoko; Wang, Liuqing; Nanba, Fumio; Ito, Chiaki; Toda, Toshiya; Ashida, Hitoshi

    2016-01-01

    Procyanidins are the oligomeric or polymeric forms of epicatechin and catechin. In this study, we isolated and purified dimer to tetramer procyanidins from black soybean seed coat and investigated the anti-hyperglycemic effects by focusing on glucose transporter 4 (GLUT4) translocation and the underlying molecular mechanism in skeletal muscle of mice. The anti-hyperglycemic effects of procyanidins were also compared with those of monomer (-)-epicatechin (EC) and major anthocyanin, cyanidin-3-O-β-glucoside (C3G). To investigate GLUT4 translocation and its related signaling pathways, ICR mice were orally given procyanidins, EC and C3G in water at 10 μg/kg body weight. The mice were sacrificed 60 min after the dose of polyphenols, and soleus muscle was extracted from the hind legs. The results showed that trimeric and tetrameric procyanidins activated both insulin- and AMPK-signaling pathways to induce GLUT4 translocation in muscle of ICR mice. We confirmed that procyanidins suppressed acute hyperglycemia with an oral glucose tolerance test in a dose-dependent manner. Of these beneficial effects, cinnamtannin A2, one of the tetramers, was the most effective. In conclusion, procyanidins, especially cinnamtannin A2, significantly ameliorate postprandial hyperglycemia at least in part by promoting GLUT4 translocation to the plasma membrane by activating both insulin- and AMPK-signaling pathways. PMID:27598258

  16. Methyl-donor supplementation in obese mice prevents the progression of NAFLD, activates AMPK and decreases acyl-carnitine levelsa

    PubMed Central

    Dahlhoff, Christoph; Worsch, Stefanie; Sailer, Manuela; Hummel, Björn A.; Fiamoncini, Jarlei; Uebel, Kirsten; Obeid, Rima; Scherling, Christian; Geisel, Jürgen; Bader, Bernhard L.; Daniel, Hannelore

    2014-01-01

    Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced β-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid β-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation. PMID:25061561

  17. Kazinol B from Broussonetia kazinoki improves insulin sensitivity via Akt and AMPK activation in 3T3-L1 adipocytes.

    PubMed

    Lee, Hyejin; Li, Hua; Jeong, Ji Hye; Noh, Minsoo; Ryu, Jae-Ha

    2016-07-01

    In this study, we evaluated the insulin-sensitizing effect of flavans purified from Broussonetia kazinoki Siebold (BK) on 3T3-L1 adipocytes. Among the tested compounds, kazinol B enhanced intracellular lipid accumulation, gene expression of proliferator-activated receptorγ (PPARγ) and CCAAT/enhancer binding protein-alpha (C/EBPα), and consistently induced PPARγ transcriptional activation. To further investigate the insulin-sensitizing effect of kazinol B, we measured glucose analogue uptake by fully differentiated adipocytes and myotubes. Kazinol B increased 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) uptake by cells by upregulating the gene expression and translocation of glucose transporter 4 (GLUT-4) into the plasma membrane in adipocytes. Kazinol B stimulated the gene expression and secretion of adiponectin, which is associated with a low risk of types 1 and 2 diabetes mellitus. We also suggested the mechanism of the antidiabetic effect of kazinol B by assaying Akt and AMP-activated protein kinase (AMPK) phosphorylation. In conclusion, kazinol B isolated from BK improved insulin sensitivity by enhancing glucose uptake via the insulin-Akt signaling pathway and AMPK activation. These results suggest that kazinol B might be a therapeutic candidate for diabetes mellitus. PMID:27223849

  18. Functional differences between AMPK α1 and α2 subunits in osteogenesis, osteoblast-associated induction of osteoclastogenesis, and adipogenesis.

    PubMed

    Wang, Yu-Gang; Han, Xiu-Guo; Yang, Ying; Qiao, Han; Dai, Ke-Rong; Fan, Qi-Ming; Tang, Ting-Ting

    2016-01-01

    The endocrine role of the skeleton-which is impaired in human diseases including osteoporosis, obesity and diabetes-has been highlighted previously. In these diseases, the role of AMPK, a sensor and regulator of energy metabolism, is of biological and clinical importance. Since AMPK's main catalytic subunit α has two isoforms, it is unclear whether functional differences between them exist in the skeletal system. The current study overexpressed AMPKα1 and α2 in MC3T3-E1 cells, primary osteoblasts and mouse BMSCs by lentiviral transduction. Cells overexpressing AMPKα2 showed higher osteogenesis potential than AMPKα1, wherein androgen receptor (AR) and osteoactivin played important roles. RANKL and M-CSF were secreted at lower levels from cells overexpressing α2 than α1, resulting in decreased osteoblast-associated osteoclastogenesis. Adipogenesis was inhibited to a greater degree in 3T3-L1 cells overexpressing α2 than α1, which was modulated by AR. An abnormal downregulation of AMPKα2 was observed in human BMSCs exhibiting the fibrous dysplasia (FD) phenotype. Overexpression of AMPKα2 in these cells rescued the defect in osteogenesis, suggesting that AMPKα2 plays a role in FD pathogenesis. These findings highlight functional differences between AMPKα1 and α2, and provide a basis for investigating the molecular mechanisms of diseases associated with impaired functioning of the skeletal system. PMID:27600021

  19. A Functional Link between AMPK and Orexin Mediates the Effect of BMP8B on Energy Balance.

    PubMed

    Martins, Luís; Seoane-Collazo, Patricia; Contreras, Cristina; González-García, Ismael; Martínez-Sánchez, Noelia; González, Francisco; Zalvide, Juan; Gallego, Rosalía; Diéguez, Carlos; Nogueiras, Rubén; Tena-Sempere, Manuel; López, Miguel

    2016-08-23

    AMP-activated protein kinase (AMPK) in the ventromedial nucleus of the hypothalamus (VMH) and orexin (OX) in the lateral hypothalamic area (LHA) modulate brown adipose tissue (BAT) thermogenesis. However, whether these two molecular mechanisms act jointly or independently is unclear. Here, we show that the thermogenic effect of bone morphogenetic protein 8B (BMP8B) is mediated by the inhibition of AMPK in the VMH and the subsequent increase in OX signaling via the OX receptor 1 (OX1R). Accordingly, the thermogenic effect of BMP8B is totally absent in ox-null mice. BMP8B also induces browning of white adipose tissue (WAT), its thermogenic effect is sexually dimorphic (only observed in females), and its impact on OX expression and thermogenesis is abolished by the knockdown of glutamate vesicular transporter 2 (VGLUT2), implicating glutamatergic signaling. Overall, our data uncover a central network controlling energy homeostasis that may be of considerable relevance for obesity and metabolic disorders. PMID:27524625

  20. Mesenchymal stem cells promote colorectal cancer progression through AMPK/mTOR-mediated NF-κB activation

    PubMed Central

    Wu, Xiao-Bing; Liu, Yang; Wang, Gui-Hua; Xu, Xiao; Cai, Yang; Wang, Hong-Yi; Li, Yan-Qi; Meng, Hong-Fang; Dai, Fu; Jin, Ji-De

    2016-01-01

    Mesenchymal stem cells (MSCs) exert a tumor-promoting effect in a variety of human cancers. This study was designed to identify the molecular mechanisms related to the tumor-promoting effect of MSCs in colorectal cancer. In vitro analysis of colorectal cancer cell lines cultured in MSC conditioned media (MSC-CM) showed that MSC-CM significantly promoted the progression of the cancer cells by enhancing cell proliferation, migration and colony formation. The tumorigenic effect of MSC-CM was attributed to altered expression of cell cycle regulatory proteins and inhibition of apoptosis. Furthermore, MSC-CM induced high level expression of a number of pluripotency factors in the cancer cells. ELISAs revealed MSC-CM contained higher levels of IL-6 and IL-8, which are associated with the progression of cancer. Moreover, MSC-CM downregulated AMPK mRNA and protein phosphorylation, but upregulated mTOR mRNA and protein phosphorylation. The NF-κB pathway was activated after addition of MSC-CM. An in vivo model in Balb/C mice confirmed the ability of MSC-CM to promote the invasion and proliferation of colorectal cancer cells. This study indicates that MSCs promote the progression of colorectal cancer via AMPK/mTOR-mediated NF-κB activation. PMID:26892992

  1. Triptolide induces protective autophagy through activation of the CaMKKβ-AMPK signaling pathway in prostate cancer cells

    PubMed Central

    Zhang, Zhe; Mao, Lin; Han, Yangyang; Yan, Jun; Lei, Ming

    2016-01-01

    Triptolide, an active compound extracted from the Chinese herb thunder god vine (Tripterygium wilfordii Hook F.), has potent anti-tumor activity. Recently, triptolide was found to induce autophagy in cancer cells. However, the effects of triptolide on autophagy in human prostate cancer (PCa) cells have not yet been clearly elucidated. In this study, we demonstrated that triptolide induces autophagy in three PCa cell lines, PC-3, LNCaP and C4–2. Furthermore, we found that triptolide mediates intracellular accumulation of free calcium by stimulating the endoplasmic reticulum (ER) stress response. This activates the CaMKKβ-AMPK signaling pathway, which in turn inhibits mTOR and activates both ULK1 and Beclin 1, finally resulting in autophagy. Moreover, we found that treatment with autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) enhances triptolide-induced PCa cell death and growth inhibition. Using a PC-3-xenografted mouse model, we showed that blocking autophagy with CQ significantly promoted triptolide-induced tumor growth inhibition in vivo. Overall, our results show that triptolide induces protective autophagy through the CaMKKβ-AMPK pathway in PCa cells, implying that a combination of triptolide with autophagy inhibitors may potentially be an effective therapeutic strategy for PCa. PMID:26734992

  2. Tumor suppressor gene ING3 induces cardiomyocyte hypertrophy via inhibition of AMPK and activation of p38 MAPK signaling.

    PubMed

    Wang, Jiaojiao; Liu, Zhiping; Feng, Xiaojun; Gao, Si; Xu, Suowen; Liu, Peiqing

    2014-11-15

    Cardiac hypertrophy, an adaptive growth process that occurs in response to various pathophysiological stimuli, constitutes an important risk factor for the development of heart failure. However, the molecular mechanisms that regulate this cardiac growth response are not completely understood. Here we revealed that ING3 (inhibitor of growth family, member 3), a type II tumor suppressor, plays a critical role in the regulation of cardiac hypertrophy. ING3 expression was present in relatively high abundance in the heart, and was prominently upregulated in hypertrophic agonists angiotensin II (Ang II), phenylephrine (PE), or isoproterenol (ISO)-stimulated cardiomyocytes and in hearts of rat undergoing abdominal aortic constriction (AAC) surgery. In cardiomyocytes, overexpression of ING3 caused an increase in ANP, BNP and β-MHC mRNA levels and cell surface area, while depletion of ING3 attenuated PE-induced cardiomyocyte hypertrophy. Mechanistically, we have demonstrated that overexpression of ING3 could inactivate the AMPK and activate the canonical p38 MAPK signaling. Remarkably, AMPK agonist AICAR or p38 MAPK inhibitor SB203580 abrogated ING3-induced hypertrophic response in cardiomyocytes. In summary, our data disclose a novel role of ING3 as an inducer of pathological cardiac hypertrophy, suggesting that silencing of ING3 may be explored as a potential therapeutic target in preventing cardiac hypertrophy.

  3. Um Infixation and Prefixation in Toba Batak.

    ERIC Educational Resources Information Center

    Crowhurst, Megan J.

    1998-01-01

    Examines the behavior of the morpheme, um, in Toba Batak and Tagalog, which alternates as a prefix or an infix, arguing that the variation is conditioned by constraints on consonant clusters. Three patterns of variation that occur with um are described, noting that the stages involved in changing from infixed to prefixed positions over time are…

  4. Baicalein Induces Caspase-dependent Apoptosis Associated with the Generation of ROS and the Activation of AMPK in Human Lung Carcinoma A549 Cells.

    PubMed

    Kim, Hong Jae; Park, Cheol; Han, Min-Ho; Hong, Su-Hyun; Kim, Gi-Young; Hong, Sang Hoon; Kim, Nam Deuk; Choi, Yung Hyun

    2016-03-01

    Baicalein is one of the main bioactive flavonoids found in the roots of Scutellaria baicalensis Georgi. Here, we report that baicalein-induced growth inhibition was associated with the induction of apoptosis in human lung carcinoma A549 cells. Baicalein stimulated the expression of DR5, FasL, and FADD, and activated caspase-8 by reducing the levels of FLIPs (FLICE-inhibitory proteins). The apoptotic cell death was also connected with an activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase; however, a blockage of caspase activation abolished baicalein-induced apoptotic potentials. Additionally, baicalein caused a mitochondrial membrane potential (MMP), the truncation of Bid, and the translocation of pro-apoptotic Bax to the mitochondria, thereby inducing the release of cytochrome c into the cytosol. In turn, baicalein increased the generation of reactive oxygen species (ROS); however, an ROS scavenger, N-acetylcysteine, notably attenuated baicalein-mediated loss of MMP and activation of caspases. Furthermore, baicalein activated the AMP-activated protein kinase (AMPK) signaling pathway. Consequently, baicalein-triggered cell death was attenuated by an AMPK inhibitor, but increased by an AMPK activator, compound C. Overall, the results suggest that the apoptotic activity of baicalein may be associated with caspase-dependent cascade through the activation of both intrinsic and extrinsic signaling pathways connected with ROS generation and AMPK activation.

  5. Nutraceutical activators of AMPK/Sirt1 axis inhibit viral production and protect neurons from neurodegenerative events triggered during HSV-1 infection.

    PubMed

    Leyton, Luis; Hott, Melissa; Acuña, Francisca; Caroca, Jorge; Nuñez, Magdalena; Martin, Carolina; Zambrano, Angara; Concha, Margarita I; Otth, Carola

    2015-07-01

    Herpes simplex virus type-1 (HSV-1) is ubiquitous and is able to establish a lifelong persistent latent infection in neurons of infected individuals. It has been estimated that in approximately 70% of the population over 50 years old, the virus enters the brain and infects neurons, and possibly undergoes recurrent reactivation episodes during lifetime, especially in immunodepressed individuals. We previously showed that the sensors AMP-dependent kinase (AMPK) and Sirtuin 1 (Sirt1), involved in survival pathways and neuroprotection, were affected during the course of HSV-1 infection. To evaluate if natural activators of the AMPK/Sirt1 axis, such as Resveratrol and Quercetin could reduce viral propagation and/or counteract the effects of neuronal infection, we analyzed progeny virion production, neuronal viability and neurodegenerative events during HSV-1 infection. We found that the activators of AMPK/Sirt1 axis, increased the viability of infected neurons, significantly reduced the viral titer in the supernatant and the expression of viral genes. More importantly, pretreatment of neurons with Resveratrol or Quercetin significantly reduced the levels of caspase-3 cleaved- and hyperphosphorylated tau associated with HSV-1 infection. These results suggest that activators of the AMPK/Sirt1 axis could be potentially useful in reducing the risk of HSV-1 productive infection in neurons and the cellular damage associated with reactivation episodes.

  6. Baicalein Induces Caspase-dependent Apoptosis Associated with the Generation of ROS and the Activation of AMPK in Human Lung Carcinoma A549 Cells.

    PubMed

    Kim, Hong Jae; Park, Cheol; Han, Min-Ho; Hong, Su-Hyun; Kim, Gi-Young; Hong, Sang Hoon; Kim, Nam Deuk; Choi, Yung Hyun

    2016-03-01

    Baicalein is one of the main bioactive flavonoids found in the roots of Scutellaria baicalensis Georgi. Here, we report that baicalein-induced growth inhibition was associated with the induction of apoptosis in human lung carcinoma A549 cells. Baicalein stimulated the expression of DR5, FasL, and FADD, and activated caspase-8 by reducing the levels of FLIPs (FLICE-inhibitory proteins). The apoptotic cell death was also connected with an activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase; however, a blockage of caspase activation abolished baicalein-induced apoptotic potentials. Additionally, baicalein caused a mitochondrial membrane potential (MMP), the truncation of Bid, and the translocation of pro-apoptotic Bax to the mitochondria, thereby inducing the release of cytochrome c into the cytosol. In turn, baicalein increased the generation of reactive oxygen species (ROS); however, an ROS scavenger, N-acetylcysteine, notably attenuated baicalein-mediated loss of MMP and activation of caspases. Furthermore, baicalein activated the AMP-activated protein kinase (AMPK) signaling pathway. Consequently, baicalein-triggered cell death was attenuated by an AMPK inhibitor, but increased by an AMPK activator, compound C. Overall, the results suggest that the apoptotic activity of baicalein may be associated with caspase-dependent cascade through the activation of both intrinsic and extrinsic signaling pathways connected with ROS generation and AMPK activation. PMID:26971531

  7. Computational Analysis of AMPK-Mediated Neuroprotection Suggests Acute Excitotoxic Bioenergetics and Glucose Dynamics Are Regulated by a Minimal Set of Critical Reactions

    PubMed Central

    Connolly, Niamh M. C.; D’Orsi, Beatrice; Monsefi, Naser; Huber, Heinrich J.; Prehn, Jochen H. M.

    2016-01-01

    Loss of ionic homeostasis during excitotoxic stress depletes ATP levels and activates the AMP-activated protein kinase (AMPK), re-establishing energy production by increased expression of glucose transporters on the plasma membrane. Here, we develop a computational model to test whether this AMPK-mediated glucose import can rapidly restore ATP levels following a transient excitotoxic insult. We demonstrate that a highly compact model, comprising a minimal set of critical reactions, can closely resemble the rapid dynamics and cell-to-cell heterogeneity of ATP levels and AMPK activity, as confirmed by single-cell fluorescence microscopy in rat primary cerebellar neurons exposed to glutamate excitotoxicity. The model further correctly predicted an excitotoxicity-induced elevation of intracellular glucose, and well resembled the delayed recovery and cell-to-cell heterogeneity of experimentally measured glucose dynamics. The model also predicted necrotic bioenergetic collapse and altered calcium dynamics following more severe excitotoxic insults. In conclusion, our data suggest that a minimal set of critical reactions may determine the acute bioenergetic response to transient excitotoxicity and that an AMPK-mediated increase in intracellular glucose may be sufficient to rapidly recover ATP levels following an excitotoxic insult. PMID:26840769

  8. Nobiletin suppresses adipogenesis by regulating the expression of adipogenic transcription factors and the activation of AMP-activated protein kinase (AMPK).

    PubMed

    Choi, Youngmin; Kim, Younghwa; Ham, Hyeonmi; Park, Yooheon; Jeong, Heon-Sang; Lee, Junsoo

    2011-12-28

    The objective of this study was to elucidate the effect of nobiletin (5,6,7,8,3',4'-hexamethoxyflavone) on adipogenesis in 3T3-L1 cells. To determine the effect of nobiletin on adipogenesis, preadipocyte differentiation was induced in the presence or absence of nobiletin (10-100 μM) for 4 days. The results revealed that nobiletin markedly inhibited lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity and blocked the expression of adipogenic transcription factors, including peroxisome proliferator-activated receptors (PPARγ) and CCAAT/enhancer binding proteins (C/EBPα). Moreover, nobiletin significantly increased AMP-activated protein kinase (AMPK), a major regulator of cellular energy balance, phosphorylation, and intracellular reactive oxygen species (ROS) generation. This study also investigated the involvement of AMPK in the expression of a major transcription factor, PPARγ. It was found that pretreatment with compound C, a cell permeable inhibitor of AMPK, abolished the inhibitory effects of nobiletin on PPARγ expression. The results suggest that nobiletin exerts antiadipogenic effects through modulation of the PPARγ and AMPK signaling pathway and, therefore, may be a promising antiobesity agent.

  9. Possible Role of Interaction between PPARα and Cyclophilin D in Cardioprotection of AMPK against In Vivo Ischemia-Reperfusion in Rats

    PubMed Central

    Barreto-Torres, Giselle

    2016-01-01

    Activated AMPK protects the heart from cardiac ischemia-reperfusion (IR) injury and is associated with inhibition of mitochondrial permeability transition pore (PTP) opening. On the other hand, pharmacological inhibition of the PTP reduces infarct size and improves cardiac function. However, it is unclear whether beneficial effects of AMPK are mediated through the PTP and, if they are not, whether simultaneous activation of AMPK and inhibition of the PTP exert synergistic protective effects against cardiac IR injury. Here, we examined the effects of the AMPK activator, A-769662 in combination with the PTP inhibitor, sanglifehrin A (SfA) on in vivo cardiac IR. Cardiac dysfunction following IR injury was associated with decreased activity of the mitochondrial electron transport chain (ETC) and increased mitochondrial ROS and PTP opening. Administration of A-769662 or SfA individually upon reperfusion improved cardiac function, reduced infarction size, and inhibited ROS production and PTP opening. However, simultaneous administration of SfA and A-769662 did not provide synergistic improvement of postischemic recovery of cardiac and mitochondrial function, though both compounds disrupted IR-induced interaction between PPARα and CyP-D. In conclusion, A-769662 or SfA prevents PPARα interaction with CyP-D, improving cardiac outcomes and increasing mitochondrial function, and simultaneous administration of the drugs does not provide synergistic effects. PMID:27051413

  10. Sepsis and mechnaical ventilation restrain translation initiation in skeletal muscle by inducing AMPK-associated TSC[2] restriction of mTOR signaling in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In skeletal muscle, AMP-activated protein kinase (AMPK) acts as a cellular energy sensor of AMP: ATP and modulates translation by repressing mammalian target of rapamycin (mTOR) activation. Endotoxin (LPS)-induced sepsis reduces muscle protein synthesis by blunting translation initiation. We hypothe...

  11. Mild caloric restriction reduces blood pressure and activates endothelial AMPK-PI3K-Akt-eNOS pathway in obese Zucker rats.

    PubMed

    García-Prieto, C F; Pulido-Olmo, H; Ruiz-Hurtado, G; Gil-Ortega, M; Aranguez, I; Rubio, M A; Ruiz-Gayo, M; Somoza, B; Fernández-Alfonso, M S

    2015-01-01

    Genetic obesity models exhibit endothelial dysfunction associated to adenosine monophosphate-activated protein kinase (AMPK) dysregulation. This study aims to assess if mild short-term caloric restriction (CR) restores endothelial AMPK activity leading to an improvement in endothelial function. Twelve-week old Zucker lean and obese (fa/fa) male rats had access to standard chow either ad libitum (AL, n=8) or 80% of AL (CR, n=8) for two weeks. Systolic blood pressure was significantly higher in fa/fa AL rats versus lean AL animals, but was normalized by CR. Endothelium-dependent relaxation to acetylcholine (ACh, 10(-9) to 10(-4) M) was reduced in fa/fa AL compared to control lean AL rats (p<0.001), and restored by CR. The AMPK activator AICAR (10(-5) to 8·10(-3) M) elicited a lower relaxation in fa/fa AL rings that was normalized by CR (p<0.001). Inhibition of PI3K (wortmannin, 10(-7) M), Akt (triciribine, 10(-5) M), or eNOS (L-NAME, 10(-4) M) markedly reduced AICAR-induced relaxation in lean AL, but not in fa/fa AL rats. These inhibitions were restored by CR in Zucker fa/fa rings. These data show that mild short-term CR improves endothelial function and lowers blood pressure in obesity due to the activation of the AMPK-PI3K-Akt-eNOS pathway. PMID:25530153

  12. Citrus junos Tanaka Peel Extract Exerts Antidiabetic Effects via AMPK and PPAR-γ both In Vitro and In Vivo in Mice Fed a High-Fat Diet

    PubMed Central

    Kim, Sung Hee; Hur, Haeng Jeon; Yang, Hye Jeong; Kim, Hyun Jin; Kim, Min Jung; Park, Jae Ho; Sung, Mi Jeong; Kim, Myung Sunny; Kwon, Dae Young; Hwang, Jin-Taek

    2013-01-01

    The antidiabetic effect of the Citrus junos Tanaka (also known as yuja or yuzu) was examined. Ethanol extract of yuja peel (YPEE) significantly stimulated 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) uptake in C2C12 myotubes. However, ethanol extract of yuja pulp (YpEE) and water extract of yuja peel (YPWE) or pulp (YpWE) did not stimulate glucose uptake. In addition, peroxisome proliferator-activated receptor gamma (PPAR-γ) and AMP-activated protein kinase (AMPK) activities were increased by YPEE in a dose-dependent manner. Pretreatment of AMPK inhibitor decreased the glucose uptake stimulated by YPEE in C2C12 myotubes. We confirmed the anti-diabetic effect of YPEE in mice fed a high fat-diet (HFD). Compared with control mice on a normal diet (ND), these mice showed increased body weight, liver fat, insulin resistance, triacylglycerol (TG), and total cholesterol content. Addition of 5% YPEE significantly reduced the weight gain and rise in liver fat content, serum triacylglycerol (TG), total cholesterol, and insulin resistance found in mice fed a high-fat diet (HFD). Moreover, YPEE reduced the secretion of HFD-induced adipocytokines such as leptin and resistin. YPEE also resulted in increased phosphorylation of AMPK in muscle tissues. These results suggest that ethanol extract of yuja peel exerts anti-diabetic effects via AMPK and PPAR-γ in both cell culture and mouse models. PMID:23762167

  13. Dissection and integration of the autophagy signaling network initiated by bluetongue virus infection: crucial candidates ERK1/2, Akt and AMPK

    PubMed Central

    Lv, Shuang; Xu, Qing-Yuan; Sun, En-Cheng; Zhang, Ji-Kai; Wu, Dong-Lai

    2016-01-01

    Bluetongue virus (BTV), a complex double-stranded segmented RNA virus, has been found to initiate cellular autophagy for its own benefit. Here, with a view to understanding the underlying mechanisms, we first systematically dissected the exact signaling network in BTV-induced autophagy. We found that the activity of mTOR, a crucial pivot, was inhibited by BTV1 infection, subsequently leading to downstream p70S6K suppression and autophagy initiation. We then explored the upstream regulators of mTOR and analyzed their activities via a series of assays. We found BTV1-induced autophagy to be independent of the ERK1/2 signaling pathway. However, the BTV1-induced inhibition of PI3K/Akt was found to be partially responsible for mTOR inactivation and subsequent autophagy initiation. Furthermore, we found unexpectedly that AMPK seemed to play a more important role in BTV1-induced autophagy. Elevated [Ca2+]cyto-mediated activation of CaMKKβ exactly managed the activation of AMPK, which then positively regulated autophagy through suppressing mTOR. We must emphasize that TSC2 is a fatal mediator between upstream Akt or AMPK and downstream mTOR through its phosphorylation. Taken together, our data suggested that the BTV1-induced inhibition of the Akt-TSC2-mTOR pathway and the upregulation of the AMPK-TSC2-mTOR pathway both contributed to autophagy initiation and further favored virus replication. PMID:26976147

  14. A SIRT3/AMPK/autophagy network orchestrates the protective effects of trans-resveratrol in stressed peritoneal macrophages and RAW 264.7 macrophages.

    PubMed

    Duan, Wen-Jun; Li, Yi-Fang; Liu, Fang-Lan; Deng, Jie; Wu, Yan-Ping; Yuan, Wei-Lin; Tsoi, Bun; Chen, Jun-Li; Wang, Qi; Cai, Shao-Hui; Kurihara, Hiroshi; He, Rong-Rong

    2016-06-01

    Resveratrol gains a great interest for its strong antioxidant properties, while the molecular mechanisms underlie the beneficial effects on psychosocial stress remain controversial. In this study, we demonstrated that resveratrol protected peritoneal macrophages and RAW 264.7 cells from stress-induced decrease in the total cell count, phagocytic capability, reactive oxygen species generation, monodansylcadaverine and mitochondrial membrane potential in stressed mice. Resveratrol promoted stress-induced autophagy in both models. Modulation of autophagy by rapamycin or 3-methyladenine regulated the protective effect of resveratrol, suggesting a role of autophagy in the protective mechanisms of resveratrol. The comparison studies revealed that distinct mechanisms were implicated in the protective effect of resveratrol and other antioxidants (vitamin C and edaravone). Resveratrol promoted autophagy via upregulating SIRT3 expression and phosphorylation of AMP-activated protein kinase (AMPK). Knockdown of SIRT3 resulted in decreased autophagy and abolished protective effect of resveratrol. SIRT1 was also involved in the protective mechanism of resveratrol, although its effect on autophagy was unnoticeable. Pharmacological manipulation of autophagy modulated the effects of resveratrol on SIRT3 and AMPK, revealing the engagement of a positive feedback loop. In sharp contrast, vitamin C and edaravone effectively protected macrophages from stress-induced cytotoxicity, accompanied by downregulated SIRT3 expression and AMPK phosphorylation, and decreased level of autophagy response. Taken together, we conclude that a SIRT3/AMPK/autophagy network orchestrates in the protective effect of resveratrol in macrophages.

  15. Insulin and AMPK regulate FA translocase/CD36 plasma membrane recruitment in cardiomyocytes via Rab GAP AS160 and Rab8a Rab GTPase.

    PubMed

    Samovski, Dmitri; Su, Xiong; Xu, Yingcheng; Abumrad, Nada A; Stahl, Philip D

    2012-04-01

    The FA translocase cluster of differentiation 36 (CD36) facilitates FA uptake by the myocardium, and its surface recruitment in cardiomyocytes is induced by insulin, AMP-dependent protein kinase (AMPK), or contraction. Dysfunction of CD36 trafficking contributes to disordered cardiac FA utilization and promotes progression to disease. The Akt substrate 160 (AS160) Rab GTPase-activating protein (GAP) is a key regulator of vesicular trafficking, and its activity is modulated via phosphorylation. Our study documents that AS160 mediates insulin or AMPK-stimulated surface translocation of CD36 in cardiomyocytes. Knock-down of AS160 redistributes CD36 to the surface and abrogates its translocation by insulin or the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). Conversely, overexpression of a phosphorylation-deficient AS160 mutant (AS160 4P) suppresses the stimulated membrane recruitment of CD36. The AS160 substrate Rab8a GTPase is shown via overexpression and knock-down studies to be specifically involved in insulin/AICAR-induced CD36 membrane recruitment. Our findings directly demonstrate AS160 regulation of CD36 trafficking. In myocytes, the AS160 pathway also mediates the effect of insulin, AMPK, or contraction on surface recruitment of the glucose transporter GLUT4. Thus, AS160 constitutes a point of convergence for coordinating physiological regulation of CD36 and GLUT4 membrane recruitment.

  16. Mild Electrical Stimulation Increases Stress Resistance and Suppresses Fat Accumulation via Activation of LKB1-AMPK Signaling Pathway in C. elegans

    PubMed Central

    Matsuyama, Shingo; Moriuchi, Masataka; Suico, Mary Ann; Yano, Shuichiro; Morino-Koga, Saori; Shuto, Tsuyoshi; Yamanaka, Kunitoshi; Kondo, Tatsuya; Araki, Eiichi; Kai, Hirofumi

    2014-01-01

    Electrical current at physiological strength has been applied as a therapeutic approach for various diseases. Several of our works showed that mild electrical stimulation (MES) at 0.1-ms pulse width has positive impact on organisms. But despite the growing evidence of the beneficial effects of MES, its effects on individual animals and the molecular underpinnings are poorly understood and rarely studied. Here, we examined the effects of MES on individual animal and its mechanisms by mainly using Caenorhabditis elegans, a powerful genetic model organism. Interestingly, MES increased stress resistance and suppressed excess fat accumulation in wild-type N2 worms but not in AMPK/AAK-2 and LKB1/PAR-4 mutant worms. MES promoted the nuclear localization of transcription factors DAF-16 and SKN-1 and consequently increased the expression of anti-stress genes, whereas MES inhibited the nuclear localization of SBP-1 and suppressed the expression of lipogenic genes. Moreover, we found that MES induced the activation of LKB1/PAR4-AMPK/AAK2 pathway in C. elegans and in several mammalian cell lines. The mitochondrial membrane potential and cellular ATP level were slightly and transiently decreased by MES leading to the activation of LKB1-AMPK signaling pathway. Together, we firstly and genetically demonstrated that MES exerts beneficial effects such as stress resistance and suppression of excess fat accumulation, via activation of LKB1-AMPK signaling pathway. PMID:25490091

  17. AMPK-HDAC5 pathway facilitates nuclear accumulation of HIF-1α and functional activation of HIF-1 by deacetylating Hsp70 in the cytosol

    PubMed Central

    Chen, Shuyang; Yin, Chengqian; Lao, Taotao; Liang, Dongming; He, Dan; Wang, Chenguang; Sang, Nianli

    2015-01-01

    Hypoxia-inducible factor 1 (HIF-1) transcriptionally promotes production of adenosine triphosphate (ATP) whereas AMPK senses and regulates cellular energy homeostasis. A histone deacetylase (HDAC) activity has been proven to be critical for HIF-1 activation but the underlying mechanism and its role in energy homesostasis remain unclear. Here, we demonstrate that HIF-1 activation depends on a cytosolic, enzymatically active HDAC5. HDAC5 knockdown impairs hypoxia-induced HIF-1α accumulation and HIF-1 transactivation, whereas HDAC5 overexpression enhances HIF-1α stabilization and nuclear translocation. Mechanistically, we show that Hsp70 is a cytosolic substrate of HDAC5; and hyperacetylation renders Hsp70 higher affinity for HIF-1α binding, which correlates with accelerated degradation and attenuated nuclear accumulation of HIF-1α. Physiologically, AMPK-triggered cytosolic shuttling of HDAC5 is critical; inhibition of either AMPK or HDAC5 impairs HIF-1α nuclear accumulation under hypoxia or low glucose conditions. Finally, we show specifically suppressing HDAC5 is sufficient to inhibit tumor cell proliferation under hypoxic conditions. Our data delineate a novel link between AMPK, the energy sensor, and HIF-1, the major driver of ATP production, indicating that specifically inhibiting HDAC5 may selectively suppress the survival and proliferation of hypoxic tumor cells. PMID:26061431

  18. Ptc1 protein phosphatase 2C contributes to glucose regulation of SNF1/AMP-activated protein kinase (AMPK) in Saccharomyces cerevisiae.

    PubMed

    Ruiz, Amparo; Xu, Xinjing; Carlson, Marian

    2013-10-25

    The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αβγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability.

  19. Targeting Energy Metabolic and Oncogenic Signaling Pathways in Triple-negative Breast Cancer by a Novel Adenosine Monophosphate-activated Protein Kinase (AMPK) Activator*

    PubMed Central

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

    2011-01-01

    The antitumor activities of the novel adenosine monophosphate-activated protein kinase (AMPK) activator, OSU-53, were assessed in in vitro and in vivo models of triple-negative breast cancer. OSU-53 directly stimulated recombinant AMPK kinase activity (EC50, 0.3 μm) and inhibited the viability and clonogenic growth of MDA-MB-231 and MDA-MB-468 cells with equal potency (IC50, 5 and 2 μm, respectively) despite lack of LKB1 expression in MDA-MB-231 cells. Nonmalignant MCF-10A cells, however, were unaffected. Beyond AMPK-mediated effects on mammalian target of rapamycin signaling and lipogenesis, OSU-53 also targeted multiple AMPK downstream pathways. Among these, the protein phosphatase 2A-dependent dephosphorylation of Akt is noteworthy because it circumvents the feedback activation of Akt that results from mammalian target of rapamycin inhibition. OSU-53 also modulated energy homeostasis by suppressing fatty acid biosynthesis and shifting the metabolism to oxidation by up-regulating the expression of key regulators of mitochondrial biogenesis, such as a peroxisome proliferator-activated receptor γ coactivator 1α and the transcription factor nuclear respiratory factor 1. Moreover, OSU-53 suppressed LPS-induced IL-6 production, thereby blocking subsequent Stat3 activation, and inhibited hypoxia-induced epithelial-mesenchymal transition in association with the silencing of hypoxia-inducible factor 1a and the E-cadherin repressor Snail. In MDA-MB-231 tumor-bearing mice, daily oral administration of OSU-53 (50 and 100 mg/kg) suppressed tumor growth by 47–49% and modulated relevant intratumoral biomarkers of drug activity. However, OSU-53 also induced protective autophagy that attenuated its antiproliferative potency. Accordingly, cotreatment with the autophagy inhibitor chloroquine increased the in vivo tumor-suppressive activity of OSU-53. OSU-53 is a potent, orally bioavailable AMPK activator that acts through a broad spectrum of antitumor activities. PMID

  20. Salicylate activates AMPK and synergizes with metformin to reduce the survival of prostate and lung cancer cells ex vivo through inhibition of de novo lipogenesis.

    PubMed

    O'Brien, Andrew J; Villani, Linda A; Broadfield, Lindsay A; Houde, Vanessa P; Galic, Sandra; Blandino, Giovanni; Kemp, Bruce E; Tsakiridis, Theodoros; Muti, Paola; Steinberg, Gregory R

    2015-07-15

    Aspirin, the pro-drug of salicylate, is associated with reduced incidence of death from cancers of the colon, lung and prostate and is commonly prescribed in combination with metformin in individuals with type 2 diabetes. Salicylate activates the AMP-activated protein kinase (AMPK) by binding at the A-769662 drug binding site on the AMPK β1-subunit, a mechanism that is distinct from metformin which disrupts the adenylate charge of the cell. A hallmark of many cancers is high rates of fatty acid synthesis and AMPK inhibits this pathway through phosphorylation of acetyl-CoA carboxylase (ACC). It is currently unknown whether targeting the AMPK-ACC-lipogenic pathway using salicylate and/or metformin may be effective for inhibiting cancer cell survival. Salicylate suppresses clonogenic survival of prostate and lung cancer cells at therapeutic concentrations achievable following the ingestion of aspirin (<1.0 mM); effects not observed in prostate (PNT1A) and lung (MRC-5) epithelial cell lines. Salicylate concentrations of 1 mM increased the phosphorylation of ACC and suppressed de novo lipogenesis and these effects were enhanced with the addition of clinical concentrations of metformin (100 μM) and eliminated in mouse embryonic fibroblasts (MEFs) deficient in AMPK β1. Supplementation of media with fatty acids and/or cholesterol reverses the suppressive effects of salicylate and metformin on cell survival indicating the inhibition of de novo lipogenesis is probably important. Pre-clinical studies evaluating the use of salicylate based drugs alone and in combination with metformin to inhibit de novo lipogenesis and the survival of prostate and lung cancers are warranted. PMID:25940306

  1. Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells

    PubMed Central

    Ming, Ming; Sinnett-Smith, James; Wang, Jia; Soares, Heloisa P.; Young, Steven H.; Eibl, Guido; Rozengurt, Enrique

    2014-01-01

    Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3–6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways. PMID:25493642

  2. Combination of 5-fluorouracil and genistein induces apoptosis synergistically in chemo-resistant cancer cells through the modulation of AMPK and COX-2 signaling pathways

    SciTech Connect

    Hwang, Jin-Taek; Ha, Joohun; Park, Ock Jin . E-mail: ojpark@hannam.ac.kr

    2005-07-01

    5-Fluorouracil (5-FU) is one of the widely used chemotherapeutic drugs targeting various cancers, but its chemo-resistance remains as a major obstacle in clinical settings. In the present study, HT-29 colon cancer cells were markedly sensitized to apoptosis by both 5-FU and genistein compared to the 5-FU treatment alone. There is an emerging evidence that genistein, soy-derived phytoestrogen, may have potential as a chemotherapeutic agent capable of inducing apoptosis or suppressing tumor promoting proteins such as cyclooxygenase-2 (COX-2). However, the precise mechanism of cellular cytotoxicity of genistein is not known. The present study focused on the correlation of AMPK and COX-2 in combined cytotoxicity of 5-FU and genistein, since AMPK is known as a primary cellular homeostasis regulator and a possible target molecule of cancer treatment, and COX-2 as cell proliferation and anti-apoptotic molecule. Our results demonstrated that the combination of 5-FU and genistein abolished the up-regulated state of COX-2 and prostaglandin secretion caused by 5-FU treatment in HT-29 colon cancer cells. These appear to be followed by the specific activation of AMPK and the up-regulation of p53, p21, and Bax by genistein. Under same conditions, the induction of Glut-1 by 5-FU was diminished by the combination treatment with 5-FU and genistein. Furthermore, the reactive oxygen species (ROS) was found as an upstream signal for AMPK activation by genistein. These results suggested that the combination of 5-FU and genistein exert a novel chemotherapeutic effect in colon cancers, and AMPK may be a novel regulatory molecule of COX-2 expression, further implying its involvement in cytotoxicity caused by genistein.

  3. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

    SciTech Connect

    Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert; Rozengurt, Enrique

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Metformin inhibits cancer cell growth but the mechanism(s) are not understood. Black-Right-Pointing-Pointer We show that the potency of metformin is sharply dependent on glucose in the medium. Black-Right-Pointing-Pointer AMPK activation was enhanced in cancer cells incubated in physiological glucose. Black-Right-Pointing-Pointer Reciprocally, metformin potently inhibited mTORC1, DNA synthesis and proliferation. Black-Right-Pointing-Pointer Metformin, at low concentrations, inhibited DNA synthesis through AMPK. -- Abstract: Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser{sup 79} and Raptor at Ser{sup 792}, was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05-0.1 mM) that were 10-100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the {alpha}{sub 1} and {alpha}{sub 2} catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

  4. Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells by activating the APPL1-AMPK signaling pathway

    SciTech Connect

    Chen, Tong; Wu, Yu-wei; Lu, Hui; Guo, Yuan; Tang, Zhi-hui

    2015-05-29

    Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with multi-lineage differentiation potential including osteogenesis and adipogenesis. While significant progress has been made in understanding the transcriptional control of hASC fate, little is known about how hASC differentiation is regulated by the autocrine loop. The most abundant adipocytokine secreted by adipocytes, adiponectin (APN) plays a pivotal role in glucose metabolism and energy homeostasis. Growing evidence suggests a positive association between APN and bone formation yet little is known regarding the direct effects of APN on hASC osteogenesis. Therefore, this study was designed to investigate the varied osteogenic effects and regulatory mechanisms of APN in the osteogenic commitment of hASCs. We found that APN enhanced the expression of osteoblast-related genes in hASCs, such as osteocalcin, alkaline phosphatase, and runt-related transcription factor-2 (Runx2, also known as CBFa1), in a dose- and time-dependent manner. This was further confirmed by the higher expression levels of alkaline phosphatase and increased formation of mineralization nodules, along with the absence of inhibition of cell proliferation. Importantly, APN at 1 μg/ml was the optimal concentration, resulting in maximum deposition of calcium nodules, and was significant superior to bone morphogenetic protein 2. Mechanistically, we found for the first time that APN increased nuclear translocation of the leucine zipper motif (APPL)-1 as well as AMP-activated protein kinase (AMPK) phosphorylation, which were reversed by pretreatment with APPL1 siRNA. Our results indicate that APN promotes the osteogenic differentiation of hASCs by activating APPL1-AMPK signaling, suggesting that manipulation of APN is a novel therapeutic target for controlling hASC fate. - Highlights: • Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells. • The knock-down of APPL1 block the enhancement of

  5. Regulation of Torpor in the Gray Mouse Lemur: Transcriptional and Translational Controls and Role of AMPK Signaling.

    PubMed

    Zhang, Jing; Tessier, Shannon N; Biggar, Kyle K; Wu, Cheng-Wei; Pifferi, Fabien; Perret, Martine; Storey, Kenneth B

    2015-04-01

    The gray mouse lemur (Microcebus murinus) is one of few primate species that is able to enter daily torpor or prolonged hibernation in response to environmental stresses. With an emerging significance to human health research, lemurs present an optimal model for exploring molecular adaptations that regulate primate hypometabolism. A fundamental challenge is how to effectively regulate energy expensive cellular processes (e.g., transcription and translation) during transitions to/from torpor without disrupting cellular homeostasis. One such regulatory mechanism is reversible posttranslational modification of selected protein targets that offers fine cellular control without the energetic burden. This study investigates the role of phosphorylation and/or acetylation in regulating key factors involved in energy homeostasis (AMP-activated protein kinase, or AMPK, signaling pathway), mRNA translation (eukaryotic initiation factor 2α or eIF2α, eukaryotic initiation factor 4E or eIF4E, and initiation factor 4E binding protein or 4EBP), and gene transcription (histone H3) in six tissues of torpid and aroused gray mouse lemurs. Our results indicated selective tissue-specific changes of these regulatory proteins. The relative level of Thr172-phosphorylated AMPKα was significantly elevated in the heart but reduced in brown adipose tissue during daily torpor, as compared to the aroused lemurs, implicating the regulation of AMPK activity during daily torpor in these tissues. Interestingly, the levels of the phosphorylated eIFs were largely unaltered between aroused and torpid animals. Phosphorylation and acetylation of histone H3 were examined as a marker for transcriptional regulation. Compared to the aroused lemurs, level of Ser10-phosphorylated histone H3 decreased significantly in white adipose tissue during torpor, suggesting global suppression of gene transcription. However, a significant increase in acetyl-histone H3 in the heart of torpid lemurs indicated a possible

  6. Resistance to Dasatinib in primary chronic lymphocytic leukemia lymphocytes involves AMPK-mediated energetic re-programming.

    PubMed

    Martinez Marignac, Veronica L; Smith, Sarah; Toban, Nader; Bazile, Miguel; Aloyz, Raquel

    2013-12-01

    Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. Although promising new therapies for this incurable disease are being tested in clinical trials, the therapeutic relevance of metabolic rewiring in chronic lymphocytic leukemia (CLL) is poorly understood. The aim of this study was to identify targetable metabolic differences in primary CLL lymphocytes by the use of Dasatinib. Dasatinib is a multi-tyrosine kinase inhibitor used to treat chronic myelogenous leukemia (CML) and is being tested in clinical trials for several cancers including CLL. This drug has been shown to be beneficial to CML patients suffering from diabetes by reducing their glucose plasma levels. In keeping with this previous observation, we report that Dasatinib induced glucose use while reducing lactate production, suggesting that this tyrosine kinase inhibitor decreases aerobic glycolysis and shifts glucose use in primary CLL lymphocytes. Our results suggest that primary CLL lymphocytes (independently of traditional prognostic factors) can be stratified in two subsets by their sensitivity to Dasatinib in vitro. Increased glucose use induced by Dasatinib or by inhibition of mitochondrial respiration was not sufficient to sustain survival and ATP levels in CLL samples sensitive to Dasatinib. The two subsets of primary CLL lymphocytes are characterized as well by a differential dependency on mitochondrial respiration and the use of anabolic or catabolic processes to cope with induced metabolic/energetic stress. Differential metabolic reprogramming between subsets is supported by the contrasting effect on the survival of Dasatinib treated CLL lymphocytes with pharmacological inhibition of two master metabolic regulators (mTorc1 and AMPK) as well as induced autophagy. Alternative metabolic organization between subsets is further supported by the differential basal expression (freshly purified lymphocytes) of active AMPK, regulators of glucose metabolism and

  7. Regulation of Torpor in the Gray Mouse Lemur: Transcriptional and Translational Controls and Role of AMPK Signaling.

    PubMed

    Zhang, Jing; Tessier, Shannon N; Biggar, Kyle K; Wu, Cheng-Wei; Pifferi, Fabien; Perret, Martine; Storey, Kenneth B

    2015-04-01

    The gray mouse lemur (Microcebus murinus) is one of few primate species that is able to enter daily torpor or prolonged hibernation in response to environmental stresses. With an emerging significance to human health research, lemurs present an optimal model for exploring molecular adaptations that regulate primate hypometabolism. A fundamental challenge is how to effectively regulate energy expensive cellular processes (e.g., transcription and translation) during transitions to/from torpor without disrupting cellular homeostasis. One such regulatory mechanism is reversible posttranslational modification of selected protein targets that offers fine cellular control without the energetic burden. This study investigates the role of phosphorylation and/or acetylation in regulating key factors involved in energy homeostasis (AMP-activated protein kinase, or AMPK, signaling pathway), mRNA translation (eukaryotic initiation factor 2α or eIF2α, eukaryotic initiation factor 4E or eIF4E, and initiation factor 4E binding protein or 4EBP), and gene transcription (histone H3) in six tissues of torpid and aroused gray mouse lemurs. Our results indicated selective tissue-specific changes of these regulatory proteins. The relative level of Thr172-phosphorylated AMPKα was significantly elevated in the heart but reduced in brown adipose tissue during daily torpor, as compared to the aroused lemurs, implicating the regulation of AMPK activity during daily torpor in these tissues. Interestingly, the levels of the phosphorylated eIFs were largely unaltered between aroused and torpid animals. Phosphorylation and acetylation of histone H3 were examined as a marker for transcriptional regulation. Compared to the aroused lemurs, level of Ser10-phosphorylated histone H3 decreased significantly in white adipose tissue during torpor, suggesting global suppression of gene transcription. However, a significant increase in acetyl-histone H3 in the heart of torpid lemurs indicated a possible

  8. Involvement of adipokines, AMPK, PI3K and the PPAR signaling pathways in ovarian follicle development and cancer.

    PubMed

    Dupont, Joëlle; Reverchon, Maxime; Cloix, Lucie; Froment, Pascal; Ramé, Christelle

    2012-01-01

    The physiological mechanisms that control energy balance are reciprocally linked to those that control reproduction, and together, these mechanisms optimize reproductive success under fluctuating metabolic conditions. Adipose tissue plays an important role in this regulation. Indeed, it releases a variety of factors, termed adipokines that regulate energy metabolism, but also reproductive functions. This article summarizes the function and regulation of some better-characterized adipokines (leptin, adiponectin, resistin, visfatin, chemerin and apelin) involved in ovarian follicle development. The follicle appears to use various "nutrient sensing" mechanisms that may form the link between nutrient status and folliculogenesis. This review examines evidence for the presence of pathways that may sense nutrient flux from within the follicle including the PI3K/Akt pathway, adenosine monophosphate-activated kinase (AMPK), and peroxisome proliferator-activated receptors (PPARs). It also reviews current information on the role of these adipokines and signalling pathways in ovarian cancers. PMID:23417417

  9. Non-canonical Hedgehog/AMPK-Mediated Control of Polyamine Metabolism Supports Neuronal and Medulloblastoma Cell Growth.

    PubMed

    D'Amico, Davide; Antonucci, Laura; Di Magno, Laura; Coni, Sonia; Sdruscia, Giulia; Macone, Alberto; Miele, Evelina; Infante, Paola; Di Marcotullio, Lucia; De Smaele, Enrico; Ferretti, Elisabetta; Ciapponi, Laura; Giangaspero, Felice; Yates, John R; Agostinelli, Enzo; Cardinali, Beatrice; Screpanti, Isabella; Gulino, Alberto; Canettieri, Gianluca

    2015-10-12

    Developmental Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs), and its aberrant activation is a leading cause of medulloblastoma. We show here that Hedgehog promotes polyamine biosynthesis in GCPs by engaging a non-canonical axis leading to the translation of ornithine decarboxylase (ODC). This process is governed by AMPK, which phosphorylates threonine 173 of the zinc finger protein CNBP in response to Hedgehog activation. Phosphorylated CNBP increases its association with Sufu, followed by CNBP stabilization, ODC translation, and polyamine biosynthesis. Notably, CNBP, ODC, and polyamines are elevated in Hedgehog-dependent medulloblastoma, and genetic or pharmacological inhibition of this axis efficiently blocks Hedgehog-dependent proliferation of medulloblastoma cells in vitro and in vivo. Together, these data illustrate an auxiliary mechanism of metabolic control by a morphogenic pathway with relevant implications in development and cancer. PMID:26460945

  10. Metformin and gefitinib cooperate to inhibit bladder cancer growth via both AMPK and EGFR pathways joining at Akt and Erk

    PubMed Central

    Peng, Mei; Huang, Yanjun; Tao, Ting; Peng, Cai-Yun; Su, Qiongli; Xu, Wanjun; Darko, Kwame Oteng; Tao, Xiaojun; Yang, Xiaoping

    2016-01-01

    EGFR is a potential therapeutic target for treating bladder cancer, but has not been approved for clinical use yet. Metformin is a widely used antidiabetic drug and has demonstrated interesting anticancer effects on various cancer models, alone or in combination with chemotherapeutic drugs. The efficacy of gefitinib, a well-known EGFR tyrosine kinase inhibitor, combined with metformin was assessed on bladder cancer and underlying mechanisms were explored. This drug combination induced a strong anti-proliferative and anti-colony forming effect and apoptosis in bladder cancer cell lines. Gefitinib suppressed EGFR signaling and inhibited phosphorylation of ERK and Akt. Metformin amplified this inhibitory effect and enhanced gefitinib-induced activation of AMPK signaling pathway. In vivo intravesical treatment of metformin and gefitinib on syngeneic orthotopic mice confirmed the significant inhibitory effect on bladder tumor growth. These two drugs may be an excellent combination for the treatment of bladder cancer through intravesical instillation. PMID:27334428

  11. Cilostazol exerts antiplatelet and anti-inflammatory effects through AMPK activation and NF-kB inhibition on hypercholesterolemic rats.

    PubMed

    da Motta, Nadia Alice Vieira; de Brito, Fernanda Carla Ferreira

    2016-08-01

    This work presents a model of rats fed a high-cholesterol diet, receiving a long-term oral administration of cilostazol, a PDE3-inhibitor. The aim of this study was to evaluate the molecular mechanisms by which cilostazol interferes with platelets signaling pathways to avoid atherosclerosis early development. Male Wistar rats were divided into 3 groups: Control group received standard rat chow (C), hypercholesterolemic group (HCD), and HCD+CIL (cilostazol group) received hypercholesterolemic diet for 45 days. HCD+CIL group received cilostazol (30 mg/kg/p.o.) once daily in the last 15 days. Platelet aggregation, lipid profile, lipid peroxidation, and cytokine serum levels were assessed. Expression of P-selectin, CD40L, PKC-α, IkB-α, and iNOS and activation of AMPK, NF-κB, and eNOS in the platelets were assessed using Western blot analysis. Cilostazol reduced the levels of total cholesterol (361.0 ± 12.8 vs. 111.5 ± 1.6 mg/dL), triglycerides (186.9 ± 17.7 vs. 55.4 ±3.1 mg/dL), cLDL (330.9 ± 9.7 vs. 61.5 ± 3.5 mg/dL), cVLDL (45.0 ± 4.6 vs. 11.1 ± 0.6 mg/dL), and malondialdehyde (9.4 ± 0.5 vs. 3.2 ± 0.3 nmol/mL) compared to the HCD group. Cilostazol presented antiplatelet properties and decreased inflammatory markers levels. These effects seem to be related to AMPK activation, NF-kB inhibition, and eNOS activation.

  12. Role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle.

    PubMed

    Fritzen, Andreas Mæchel; Frøsig, Christian; Jeppesen, Jacob; Jensen, Thomas Elbenhardt; Lundsgaard, Anne-Marie; Serup, Annette Karen; Schjerling, Peter; Proud, Chris G; Richter, Erik A; Kiens, Bente

    2016-06-01

    During induction of the autophagosomal degradation process, LC3-I is lipidated to LC3-II and associates to the cargo isolation membrane allowing for autophagosome formation. Lipidation of LC3 results in an increased LC3-II/LC3-I ratio, and this ratio is an often used marker for autophagy in various tissues, including skeletal muscle. From cell studies AMPK has been proposed to be necessary and sufficient for LC3 lipidation. The aim of the present study was to investigate the role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle. We observed an increase in the LC3-II/LC3-I ratio in skeletal muscle of AMPKα2 kinase-dead (KD) (p<0.001) and wild type (WT) (p<0.05) mice after 12h of fasting, which was greater (p<0.05) in AMPKα2 KD mice than in WT. The fasting-induced increase in the LC3-II/LC3-I ratio in both genotypes coincided with an initial decrease (p<0.01) in plasma insulin concentration, a subsequent decrease in muscle mTORC1 signaling and increased (p<0.05) levels of the autophagy-promoting proteins, FoxO3a and ULK1. Furthermore, a higher (p<0.01) LC3-II/LC3-I ratio was observed in old compared to young mice. We were not able to detect any change in LC3 lipidation with either in vivo treadmill exercise or in situ contractions. Collectively, these findings suggest that AMPKα2 is not necessary for induction of LC3 lipidation with fasting and aging. Furthermore, LC3 lipidation is increased in muscle lacking functional AMPKα2 during fasting and aging. Moreover, LC3 lipidation seems not to be a universal response to muscle contraction in mice. PMID:26976209

  13. Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2-mediated suppression of mTORC1.

    PubMed

    Tripathi, Durga N; Chowdhury, Rajdeep; Trudel, Laura J; Tee, Andrew R; Slack, Rebecca S; Walker, Cheryl Lyn; Wogan, Gerald N

    2013-08-01

    Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosome-dependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death.

  14. 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

  15. Involvement of AMPK, IKβα-NFκB and eNOS in the sildenafil anti-inflammatory mechanism in a demyelination model.

    PubMed

    Nunes, Ana Karolina Santana; Rapôso, Catarina; Rocha, Sura Wanessa Santos; Barbosa, Karla Patrícia de Sousa; Luna, Rayana Leal de Almeida; da Cruz-Höfling, Maria Alice; Peixoto, Christina Alves

    2015-11-19

    Sildenafil (Viagra®) has recently been found to have a neuroprotective effect, which occurs through the inhibition of inflammation and demyelination in the cerebellum. However, the mechanism of action of sildenafil remains unknown. AMPK, the regulatory protein of the lipid and glucose metabolism, plays a protective role by activating the eNOS enzyme. The production of a nanomolar concentration of NO by eNOS has an anti-inflammatory effect through the cGMP signaling pathway and plays an important role in the regulation of the nuclear transcription factor (NFkB), preventing the expression of inflammatory genes. The present study investigated whether AMPK-eNOS-NO-cGMP-IКβα-NFkB is involved in the mechanism of action of sildenafil in a cuprizone-demyelination model. Neuroinflammation and demyelination induced by cuprizone in rodents have been widely used as a model of MS. In the present study, five male C57BL/6 mice (7-10 weeks old) were used. Over a four week period, the groups received: cuprizone (CPZ) 0.2% mixed in feed; CPZ in the diet, combined with the administration of sildenafil (Viagra®, Pfizer, 25mg/kg) orally in drinking water, starting concurrently (sild-T0) or 15 days (sild-T15) after the start of CPZ. Control animals received pure food and water. The cerebella of the mice were dissected and processed for immunohistochemistry, immunofluorescence (frozen), western blotting and dosage of cytokines (Elisa). CPZ induced an increase in the expression of GFAP, IL-1β TNF-α, total NFkB and inactive AMPK, and prompt microglia activation. CPZ also induced a reduction of IKβα. The administration of sildenafil reduced the expression of the pro-inflammatory cytokines IL-1β and TNF-α and increased the expression of the anti-inflammatory cytokine IL-10. In addition, the administration of sildenafil reduced expression of GFAP, NFkB, inactive AMPK and iNOS, and increased IKβα. Interestingly, sildenafil also reduced levels of NGF. In general, the sild-T0 group

  16. Metformin Inhibits Advanced Glycation End Products-Induced Inflammatory Response in Murine Macrophages Partly through AMPK Activation and RAGE/NFκB Pathway Suppression

    PubMed Central

    Zhou, Zhong'e; Tang, Yong; Chen, Chengjun; Lu, Yi; Liu, Liang

    2016-01-01

    Advanced glycation end products (AGEs) are major inflammatory mediators in diabetes, affecting atherosclerosis progression via macrophages. Metformin slows diabetic atherosclerosis progression through mechanisms that remain to be fully elucidated. The present study of murine bone marrow derived macrophages showed that (1) AGEs enhanced proinflammatory cytokines (interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α)) mRNA expression, RAGE expression, and NFκB activation; (2) metformin pretreatment inhibited AGEs effects and AGEs-induced cluster designation 86 (CD86) (M1 marker) expression, while promoting CD206 (M2 marker) surface expression and anti-inflammatory cytokine (IL-10) mRNA expression; and (3) the AMPK inhibitor, Compound C, attenuated metformin effects. In conclusion, metformin inhibits AGEs-induced inflammatory response in murine macrophages partly through AMPK activation and RAGE/NFκB pathway suppression. PMID:27761470

  17. Proteasomes, Sir2, and Hxk2 form an interconnected aging network that impinges on the AMPK/Snf1-regulated transcriptional repressor Mig1.

    PubMed

    Yao, Yanhua; Tsuchiyama, Scott; Yang, Ciyu; Bulteau, Anne Laure; He, Chong; Robison, Brett; Tsuchiya, Mitsuhiro; Miller, Delana; Briones, Valeria; Tar, Krisztina; Potrero, Anahi; Friguet, Bertrand; Kennedy, Brian K; Schmidt, Marion

    2015-01-01

    Elevated proteasome activity extends lifespan in model organisms such as yeast, worms and flies. This pro-longevity effect might be mediated by improved protein homeostasis, as this protease is an integral module of the protein homeostasis network. Proteasomes also regulate cellular processes through temporal and spatial degradation of signaling pathway components. Here we demonstrate that the regulatory function of the proteasome plays an essential role in aging cells and that the beneficial impact of elevated proteasome capacity on lifespan partially originates from deregulation of the AMPK signaling pathway. Proteasome-mediated lifespan extension activity was carbon-source dependent and cells with enhancement proteasome function exhibited increased respiratory activity and oxidative stress response. These findings suggested that the pro-aging impact of proteasome upregulation might be related to changes in the metabolic state through a premature induction of respiration. Deletion of yeast AMPK, SNF1, or its activator SNF4 abrogated proteasome-mediated lifespan extension, supporting this hypothesis as the AMPK pathway regulates metabolism. We found that the premature induction of respiration in cells with increased proteasome activity originates from enhanced turnover of Mig1, an AMPK/Snf1 regulated transcriptional repressor that prevents the induction of genes required for respiration. Increasing proteasome activity also resulted in partial relocation of Mig1 from the nucleus to the mitochondria. Collectively, the results argue for a model in which elevated proteasome activity leads to the uncoupling of Snf1-mediated Mig1 regulation, resulting in a premature activation of respiration and thus the induction of a mitohormetic response, beneficial to lifespan. In addition, we observed incorrect Mig1 localization in two other long-lived yeast aging models: cells that overexpress SIR2 or deleted for the Mig1-regulator HXK2. Finally, compromised proteasome function

  18. Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPK-mTOR-SREBP signaling pathway.

    PubMed

    Quan, Hai Yan; Kim, Do Yeon; Kim, Soo Jung; Jo, Hee Kyung; Kim, Go Woon; Chung, Sung Hyun

    2013-05-01

    Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. The discovery of food components that can ameliorate NAFLD is therefore of interest. Betulinic acid (BA) is a triterpenoid with many pharmacological activities, but the effect of BA on fatty liver is as yet unknown. To explore the possible anti-fatty liver effects and their underlying mechanisms, we used insulin-resistant HepG2 cells, primary rat hepatocytes and liver tissue from ICR mice fed a high-fat diet (HFD). Oil Red O staining revealed that BA significantly suppressed excessive triglyceride accumulation in HepG2 cells and in the livers of mice fed a HFD. Ca(+2)-calmodulin dependent protein kinase kinase (CAMKK) and AMP-activated protein kinase (AMPK) were both activated by BA treatment. In contrast, the protein levels of sterol regulatory element-binding protein 1 (SREBP1), mammalian target of rapamycin (mTOR) and S6 kinase (S6K) were all reduced when hepatocytes were treated with BA for up to 24h. We found that BA activates AMPK via phosphorylation, suppresses SREBP1 mRNA expression, nuclear translocation and repressed SREBP1 target gene expression in HepG2 cells and primary hepatocytes, leading to reduced lipogenesis and lipid accumulation. These effects were completely abolished in the presence of STO-609 (a CAMKK inhibitor) or compound C (an AMPK inhibitor), indicating that the BA-induced reduction in hepatic steatosis was mediated via the CAMKK-AMPK-SREBP1 signaling pathway. Taken together, our results suggest that BA effectively ameliorates intracellular lipid accumulation in liver cells and thus is a potential therapeutic agent for the prevention of fatty liver disease. PMID:23435355

  19. Mitochondria-targeted esculetin alleviates mitochondrial dysfunction by AMPK-mediated nitric oxide and SIRT3 regulation in endothelial cells: potential implications in atherosclerosis

    PubMed Central

    Karnewar, Santosh; Vasamsetti, Sathish Babu; Gopoju, Raja; Kanugula, Anantha Koteswararao; Ganji, Sai Krishna; Prabhakar, Sripadi; Rangaraj, Nandini; Tupperwar, Nitin; Kumar, Jerald Mahesh; Kotamraju, Srigiridhar

    2016-01-01

    Mitochondria-targeted compounds are emerging as a new class of drugs that can potentially alter the pathophysiology of those diseases where mitochondrial dysfunction plays a critical role. We have synthesized a novel mitochondria-targeted esculetin (Mito-Esc) with an aim to investigate its effect during oxidative stress-induced endothelial cell death and angiotensin (Ang)-II-induced atherosclerosis in ApoE−/− mice. Mito-Esc but not natural esculetin treatment significantly inhibited H2O2- and Ang-II-induced cell death in human aortic endothelial cells by enhancing NO production via AMPK-mediated eNOS phosphorylation. While L-NAME (NOS inhibitor) significantly abrogated Mito-Esc-mediated protective effects, Compound c (inhibitor of AMPK) significantly decreased Mito-Esc-mediated increase in NO production. Notably, Mito-Esc promoted mitochondrial biogenesis by enhancing SIRT3 expression through AMPK activation; and restored H2O2-induced inhibition of mitochondrial respiration. siSIRT3 treatment not only completely reversed Mito-Esc-mediated mitochondrial biogenetic marker expressions but also caused endothelial cell death. Furthermore, Mito-Esc administration to ApoE−/− mice greatly alleviated Ang-II-induced atheromatous plaque formation, monocyte infiltration and serum pro-inflammatory cytokines levels. We conclude that Mito-Esc is preferentially taken up by the mitochondria and preserves endothelial cell survival during oxidative stress by modulating NO generation via AMPK. Also, Mito-Esc-induced SIRT3 plays a pivotal role in mediating mitochondrial biogenesis and perhaps contributes to its anti-atherogenic effects. PMID:27063143

  20. Celastrol attenuates oxidative stress in the skeletal muscle of diabetic rats by regulating the AMPK-PGC1α-SIRT3 signaling pathway

    PubMed Central

    GUAN, YUE; CUI, ZI-JIAN; SUN, BEI; HAN, LI-PING; LI, CHUN-JUN; CHEN, LI-MING

    2016-01-01

    Oxidative stress plays a key role in the pathogenesis of diabetic myopathy. Celastrol provides a wide range of health benefits, including antioxidant, anti-inflammatory and antitumor effects. We hypothesized that celastrol may exert an antioxidant effect in the skeletal muscle of diabetic rats. In the present study, MnSOD activity was determined by spectrophotometry. The protein levels were evaluated by western blot analysis and mRNA content was quantified by RT-qPCR. We firstly found that the levels of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor coactivator 1α (PGC1α), silent mating-type information regulation 2 homolog 3 (SIRT3) and manganese superoxide dismutase (MnSOD) were all decreased in the skeletal muscle of diabetic patients. Male rats with diabetes were also treated with the vehicle or with celastrol at 1, 3 and 6 mg/kg/day for 8 weeks. The administration of celastrol at 3 and 6 mg/kg attenuated the deterioration of skeletal muscle, as shown by histological analysis, decreased the malondialdehyde (MDA) level and increased the glutathione (GSH) level assayed by enzyme-linked immunosorbent assay (ELISA) method. It also enhanced the enzyme activity and increased the expression of MnSOD, and increased the AMPK phosphorylation level, as well as PGC1α and Sirt3 expression. The findings of our study suggest that the expression of AMPK, PGC1α, SIRT3 and MnSOD are decreased in the skeletal muscle of diabetic patients. Celastrol exerted antioxidant effects on skeletal muscle partly by regulating the AMPK-PGC1α-SIRT3 signaling pathway. PMID:27049825

  1. Thymoquinone Inhibits Angiotensin II-Induced Proliferation and Migration of Vascular Smooth Muscle Cells Through the AMPK/PPARγ/PGC-1α Pathway.

    PubMed

    Pei, Xing; Li, Xiaoli; Chen, Heming; Han, Yong; Fan, Yigang

    2016-08-01

    The proliferation and migration of vascular smooth muscle cells (VSMCs) play crucial roles in the pathogenesis of diabetes and its complications. Thymoquinone (TQ) is the primary bioactive component of Nigella sativa L. seed oil, which exhibits antihyperglycemic effect in diabetic rats, but its role in VSMC proliferation and migration has not been investigated. The results of MTT assay and flow cytometry assay indicated that TQ dose-dependently inhibited angiotensin II (Ang II)-induced VSMCs' cell cycle progression, as well as cyclin D1 expression, whereas p21 expression was altered conversely. TQ dose-dependently suppressed Ang II-induced VSMC migration accompanied by reduced MMP-9 expression. In addition, we observed the elevated reactive oxygen species (ROS) generation and NADPH oxidase activity and reduced superoxide dismutase activity in Ang II-treated VSMCs, which were dose-dependently reversed by TQ. Western blot analysis indicated that TQ dose-dependently restored Ang II-inhibited expression of p-AMPK, PPARγ, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) proteins. Furthermore, adenosine monophosphate-activated protein kinase (AMPK) inhibitor Compound C and PGC-1α siRNA transfection abrogated the activation of TQ on Ang II-inhibited AMPK/PPARγ/PGC-1α signaling, but abolished the inhibitory effects of TQ on Ang II-induced VSMC proliferation and migration, as well as ROS generation. Taken together, these results demonstrated that TQ inhibited Ang II-induced VSMC proliferation and migration through the AMPK/PPARγ/PGC-1α pathway. PMID:27064837

  2. Yhhu981, a novel compound, stimulates fatty acid oxidation via the activation of AMPK and ameliorates lipid metabolism disorder in ob/ob mice

    PubMed Central

    Zeng, Hong-liang; Huang, Su-ling; Xie, Fu-chun; Zeng, Li-min; Hu, You-hong; Leng, Ying

    2015-01-01

    Aim: Defects in fatty acid metabolism contribute to the pathogenesis of insulin resistance and obesity. In this study, we investigated the effects of a novel compound yhhu981 on fatty acid metabolism in vitro and in vivo. Methods: The capacity to stimulate fatty acid oxidation was assessed in C2C12 myotubes. The fatty acid synthesis was studied in HepG2 cells using isotope tracing. The phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) was examined with Western blot analysis. For in vivo experiments, ob/ob mice were orally treated with yhhu981 acutely (300 mg/kg) or chronically (150 or 300 mg·kg−1·d−1 for 22 d). On the last day of treatment, serum and tissue samples were collected for analysis. Results: Yhhu981 (12.5–25 μmol/L) significantly increased fatty acid oxidation and the expression of related genes (Sirt1, Pgc1α and Mcad) in C2C12 myotubes, and inhibited fatty acid synthesis in HepG2 cells. Furthermore, yhhu981 dose-dependently increased the phosphorylation of AMPK and ACC in both C2C12 myotubes and HepG2 cells. Compound C, an AMPK inhibitor, blocked fatty acid oxidation in yhhu981-treated C2C12 myotubes and fatty acid synthesis decrease in yhhu981-treated HepG2 cells. Acute administration of yhhu981 decreased the respiratory exchange ratio in ob/ob mice, whereas chronic treatment with yhhu981 ameliorated the lipid abnormalities and ectopic lipid deposition in skeletal muscle and liver of ob/ob mice. Conclusion: Yhhu981 is a potent compound that stimulates fatty acid oxidation, and exerts pleiotropic effects on lipid metabolism by activating AMPK. PMID:25732571

  3. Effect of exhaustive ultra-endurance exercise in muscular glycogen and both Alpha1 and Alpha2 Ampk protein expression in trained rats.

    PubMed

    Tarini, V A F; Carnevali, L C; Arida, R M; Cunha, C A; Alves, E S; Seeleander, M C L; Schmidt, B; Faloppa, F

    2013-09-01

    Glycogen is the main store of readily energy in skeletal muscle and plays a key role in muscle function, demonstrated by the inability to sustain prolonged high-intensity exercise upon depletion of these glycogen stores. With prolonged exercise, glycogen depletion occurs and 5'-AMP-activated protein kinase (AMPK), a potent regulator of muscle metabolism and gene expression, is activated promoting molecular signalling that increases glucose uptake by muscular skeletal cells. The aim of this study was primarily to determine the effect of ultra-endurance exercise on muscle glycogen reserves and secondly to verify the influence of this type of exercise on AMPK protein expression. Twenty-four male Wistar rats, 60 days old, were divided into four experimental groups: sedentary, sedentary exhausted (SE), endurance trained (T) and endurance trained exhausted (TE). The animals ran for 10 to 90 min/day, 5 days/week, for 12 weeks to attain trained status. Rats were killed immediately after the exhaustion protocol, which consisted of running on a treadmill (at approximately 60% Vmax until exhaustion). Optical density of periodic acid-Schiff was detected and glycogen depletion observed predominantly in type I muscle fibres of the TE group and in both type I and II muscle fibres in the SE group. Plasma glucose decreased only in the TE group. Hepatic glycogen was increased in T group and significantly depleted in TE group. AMPK protein expression was significantly elevated in TE and T groups. In conclusion, acute exhaustive ultra-endurance exercise promoted muscle glycogen depletion. It seems that total AMPK protein and gene expression is more influenced by status training.

  4. 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

  5. Curcumin attenuates glutamate neurotoxicity in the hippocampus by suppression of ER stress-associated TXNIP/NLRP3 inflammasome activation in a manner dependent on AMPK

    SciTech Connect

    Li, Ying; Li, Jia; Li, Shanshan; Li, Yi; Wang, Xiangxiang; Liu, Baolin; Fu, Qiang; Ma, Shiping

    2015-07-01

    Curcumin is a natural polyphenolic compound in Curcuma longa with beneficial effects on neuronal protection. This study aims to investigate the action of curcumin in the hippocampus subjected to glutamate neurotoxicity. Glutamate stimulation induced reactive oxygen species (ROS), endoplasmic reticulum stress (ER stress) and TXNIP/NLRP3 inflammasome activation, leading to damage in the hippocampus. Curcumin treatment in the hippocampus or SH-SY5Y cells inhibited IRE1α and PERK phosphorylation with suppression of intracellular ROS production. Curcumin increased AMPK activity and knockdown of AMPKα with specific siRNA abrogated its inhibitory effects on IRE1α and PERK phosphorylation, indicating that AMPK activity was essential for the suppression of ER stress. As a result, curcumin reduced TXNIP expression and inhibited NLRP3 inflammasome activation by downregulation of NLRP3 and cleaved caspase-1 induction, and thus reduced IL-1β secretion. Specific fluorescent probe and flow cytometry analysis showed that curcumin prevented mitochondrial malfunction and protected cell survival from glutamate neurotoxicity. Moreover, oral administration of curcumin reduced brain infarct volume and attenuated neuronal damage in rats subjected to middle cerebral artery occlusion. Immunohistochemistry showed that curcumin inhibited p-IRE1α, p-PERK and NLRP3 expression in hippocampus CA1 region. Together, these results showed that curcumin attenuated glutamate neurotoxicity by inhibiting ER stress-associated TXNIP/NLRP3 inflammasome activation via the regulation of AMPK, and thereby protected the hippocampus from ischemic insult. - Highlights: • Curcumin attenuates glutamate neurotoxicity in the hippocampus. • Curcumin suppresses ER stress in glutamate-induced hippocampus slices. • Curcumin inhibits TXNIP/NLRP3 inflammasome activation. • Regulation of AMPK by curcumin contributes to suppressing ER stress.

  6. Epigallocatechin Gallate Reduces Slow-Twitch Muscle Fiber Formation and Mitochondrial Biosynthesis in C2C12 Cells by Repressing AMPK Activity and PGC-1α Expression.

    PubMed

    Wang, Lina; Wang, Zhen; Yang, Kelin; Shu, Gang; Wang, Songbo; Gao, Ping; Zhu, Xiaotong; Xi, Qianyun; Zhang, Yongliang; Jiang, Qingyan

    2016-08-31

    Epigallocatechin gallate (EGCG) is a major active compound in green tea polyphenols. EGCG acts as an antioxidant to prevent the cell damage caused by free radicals and their derivatives. In skeletal muscle, exercise causes the accumulation of intracellular reactive oxygen species (ROS) and promotes the formation of slow-type muscle fiber. To determine whether EGCG, as a ROS scavenger, has any effect on skeletal muscle fiber type, we applied different concentrations (0, 5, 25, and 50 μM) of EGCG in the culture medium of differentiated C2C12 cells for 2 days. The fiber-type composition, mitochondrial biogenesis-related gene expression, antioxidant and glucose metabolism enzyme activity, and ROS levels in C2C12 cells were then detected. According to our results, 5 μM EGCG significantly decreased the cellular activity of SDH, 25 μM EGCG significantly downregulated the MyHC I, PGC-1α, NRF-1, and p-AMPK levels and SDH activity while enhancing the CAT and GSH-Px activity and decreasing the intracellular ROS levels, and 50 μM EGCG significantly downregulated MyHC I, PGC-1α, and NRF-1 expression and HK and SDH activity while increasing LDH activity. Furthermore, 300 μM H2O2 and 0.5 mM AMPK agonist (AICAR) improved the expression of MyHC I, PGC-1α, and p-AMPK, which were all reversed by 25 μM EGCG. In conclusion, the effect of EGCG on C2C12 cells may occur through the reduction of the ROS level, thereby decreasing both AMPK activity and PGC-1α expression and eventually reducing slow-twitch muscle fiber formation and mitochondrial biosynthesis. PMID:27420899

  7. 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.

  8. N-n-butyl haloperidol iodide ameliorates hypoxia/reoxygenation injury through modulating the LKB1/AMPK/ROS pathway in cardiac microvascular endothelial cells

    PubMed Central

    Lu, Binger; Wang, Bin; Zhong, Shuping; Zhang, Yanmei; Gao, Fenfei; Chen, Yicun; Zheng, Fuchun; Shi, Ganggang

    2016-01-01

    Endothelial cells are highly sensitive to hypoxia and contribute to myocardial ischemia/reperfusion injury. We have reported that N-n-butyl haloperidol iodide (F2) can attenuate hypoxia/reoxygenation (H/R) injury in cardiac microvascular endothelial cells (CMECs). However, the molecular mechanisms remain unclear. Neonatal rat CMECs were isolated and subjected to H/R. Pretreatment of F2 leads to a reduction in H/R injury, as evidenced by increased cell viability, decreased lactate dehydrogenase (LDH) leakage and apoptosis, together with enhanced AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) phosphorylation in H/R ECs. Blockade of AMPK with compound C reversed F2-induced inhibition of H/R injury, as evidenced by decreased cell viability, increased LDH release and apoptosis. Moreover, compound C also blocked the ability of F2 to reduce H/R-induced reactive oxygen species (ROS) generation. Supplementation with the ROS scavenger N-acetyl-L-cysteine (NAC) reduced ROS levels, increased cell survival rate, and decreased both LDH release and apoptosis after H/R. In conclusion, our data indicate that F2 may mitigate H/R injury by stimulating LKB1/AMPK signaling pathway and subsequent suppression of ROS production in CMECs. PMID:27166184

  9. Curcumin enhanced cholesterol efflux by upregulating ABCA1 expression through AMPK-SIRT1-LXRα signaling in THP-1 macrophage-derived foam cells.

    PubMed

    Lin, Xiao-long; Liu, Mi-Hua; Hu, Hui-Jun; Feng, Hong-ru; Fan, Xiao-Juan; Zou, Wei-wen; Pan, Yong-quan; Hu, Xue-mei; Wang, Zuo

    2015-09-01

    Curcumin, a traditional Chinese derivative from the rhizomes of Curcuma longa, is beneficial to health by modulating lipid metabolism and suppressing atherogenesis. A key part of atherosclerosis is the failure of macrophages to restore their cellular cholesterol homeostasis and the formation of foam cells. In this study, results showed that curcumin dramatically increased the expression of ATP-binding cassette transporter 1 (ABCA1), promoted cholesterol efflux from THP-1 macrophage-derived foam cells, and reduced cellular cholesterol levels. Curcumin activated AMP-activated protein kinase (AMPK) and SIRT1, and then activated LXRα in THP-1 macrophage-derived foam cells. Inhibiting AMPK/SIRT1 activity by its specific inhibitor or by small interfering RNA could inhibit LXRα activation and abolish curcumin-induced ABCA1 expression and cholesterol efflux. Thus, curcumin enhanced cholesterol efflux by upregulating ABCA1 expression through activating AMPK-SIRT1-LXRα signaling in THP-1 macrophage-derived foam cells. This study describes a possible mechanism for understanding the antiatherogenic effects of curcumin on attenuating the progression of atherosclerosis.

  10. Metformin Induced AMPK Activation, G0/G1 Phase Cell Cycle Arrest and the Inhibition of Growth of Esophageal Squamous Cell Carcinomas In Vitro and In Vivo.

    PubMed

    Cai, Xianbin; Hu, Xi; Tan, Xiaojun; Cheng, Weijie; Wang, Qinjia; Chen, Xiaofeng; Guan, Yinghong; Chen, Chong; Jing, Xubin

    2015-01-01

    Esophageal squamous cell carcinomas (ESCC) have become a severe threat to health and the current treatments for ESCC are frequently not effective. Recent epidemiological studies suggest that the anti-hyperglycemic agent metformin may reduce the risk of developing cancer, including ESCC, among diabetic patients. However, the antitumor effects of metformin on ESCC and the mechanisms underlying its cell cycle regulation remain elusive. The findings reported herein show that the anti-proliferative action of metformin on ESCC cell lines is partially mediated by AMPK. Moreover, we observed that metformin induced G0/G1 phase arrest accompanied by the up-regulation of p21CIP1 and p27KIP1. In vivo experiments further showed that metformin inhibited tumor growth in a ESCC xenograft model. Most importantly, the up-regulation of AMPK, p53, p21CIP1, p27KIP1 and the down-regulation of cyclinD1 are involved in the anti-tumor action of metformin in vivo. In conclusion, metformin inhibits the growth of ESCC cells both in cell cultures and in an animal model. AMPK, p53, p21CIP1, p27KIP1 and cyclinD1 are involved in the inhibition of tumor growth that is induced by metformin and cell cycle arrest in ESCC. These findings indicate that metformin has the potential for use in the treatment of ESCC.

  11. C1q/Tumor Necrosis Factor-Related Protein-3 Attenuates Brain Injury after Intracerebral Hemorrhage via AMPK-Dependent Pathway in Rat

    PubMed Central

    Wang, Shaohua; Zhou, Yang; Yang, Bo; Li, Lingyu; Yu, Shanshan; Chen, Yanlin; Zhu, Jin; Zhao, Yong

    2016-01-01

    C1q/tumor necrosis factor (TNF)-related protein-3 (CTRP3) is a recently discovered adiponectin paralog with established metabolic regulatory properties. However, the role of CTRP3 in intracerebral hemorrhage (ICH) is still mostly unresolved. The aim of the present report was to explore the possible neuroprotective effect of CTRP3 in an ICH rat model and to elucidate the fundamental mechanisms. ICH was induced in rats by intracerebral infusion of autologous arterial blood. The effects of exogenous CTRP3 (recombinant or lentivirus CTRP3) on brain injury were explored on day 7. Treatment with CTRP3 reduced brain edema, protected against disruption of the blood-brain barrier (BBB), improved neurological functions and promoted angiogenesis. Furthermore, CTRP3 greatly intensified phosphorylation of AMP-activated protein kinase (AMPK) in addition to expression of hypoxia inducing factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Finally, the protective effects of CTRP3 could be blocked by either AMPK or VEGF inhibitors. Our findings give the first evidence that CTRP3 is a new proangiogenic and neuroprotective adipokine, which may exert its protective effects at least partly through an AMPK/HIF-1α/ VEGF-dependent pathway, and suggest that CTRP3 may provide a new therapeutic strategy for ICH. PMID:27807406

  12. Ajoene, a stable garlic by-product, inhibits high fat diet-induced hepatic steatosis and oxidative injury through LKB1-dependent AMPK activation.

    PubMed

    Han, Chang Yeob; Ki, Sung Hwan; Kim, Young Woo; Noh, Kyoung; Lee, Da Yeon; Kang, Bomi; Ryu, Jae-Ha; Jeon, Raok; Kim, Eun Hyun; Hwang, Se Jin; Kim, Sang Geon

    2011-01-15

    Hepatic steatosis, a hepatic component of metabolic syndrome, is common and may progress to steatohepatitis and cirrhosis. The liver X receptor-α (LXRα)-sterol regulatory element binding protein-1c (SREBP-1c) pathway plays a key role in hepatic steatosis. This study investigated the potential of ajoene, a stable garlic by-product, to inhibit high fat diet (HFD)-induced hepatic steatosis and the underlying mechanism. Ajoene treatment attenuated fat accumulation and induction of lipogenic genes in the liver of HFD-fed mice. Blood biochemical analyses and histopathologic examinations showed that ajoene prevented liver injury with the inhibition of oxidative stress, as evidenced by thiobarbituric acid reactive substances formation and nitrotyrosinylation. Moreover, ajoene treatment inhibited LXRα agonist (T0901317)-mediated SREBP-1c activation, and transactivation of the lipogenic target genes in hepatocytes. Ajoene was found to activate AMP-activated protein kinase (AMPK) via LKB1, responsible for the inhibition of p70 ribosomal S6 kinase-1 (S6K1). The ability of ajoene to repress T0901317-induced SREBP-1c expression was antagonized by inhibition of AMPK or activation of S6K1, supporting the role of these kinases in the antisteatotic effect. Our results demonstrate that ajoene has an effect of activating AMPK through LKB1 and inhibit S6K1 activity, contributing to the prevention of SREBP-1c-mediated hepatic lipogenesis via the inhibition of LXRα activity.

  13. Mg2+ Extrusion from Intestinal Epithelia by CNNM Proteins Is Essential for Gonadogenesis via AMPK-TORC1 Signaling in Caenorhabditis elegans.

    PubMed

    Ishii, Tasuku; Funato, Yosuke; Hashizume, Osamu; Yamazaki, Daisuke; Hirata, Yusuke; Nishiwaki, Kiyoji; Kono, Nozomu; Arai, Hiroyuki; Miki, Hiroaki

    2016-08-01

    Mg2+ serves as an essential cofactor for numerous enzymes and its levels are tightly regulated by various Mg2+ transporters. Here, we analyzed Caenorhabditis elegans strains carrying mutations in genes encoding cyclin M (CNNM) Mg2+ transporters. We isolated inactivating mutants for each of the five Caenorhabditis elegans cnnm family genes, cnnm-1 through cnnm-5. cnnm-1; cnnm-3 double mutant worms showed various phenotypes, among which the sterile phenotype was rescued by supplementing the media with Mg2+. This sterility was caused by a gonadogenesis defect with severely attenuated proliferation of germ cells. Using this gonadogenesis defect as an indicator, we performed genome-wide RNAi screening, to search for genes associated with this phenotype. The results revealed that RNAi-mediated inactivation of several genes restores gonad elongation, including aak-2, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK). We then generated triple mutant worms for cnnm-1; cnnm-3; aak-2 and confirmed that the aak-2 mutation also suppressed the defective gonadal elongation in cnnm-1; cnnm-3 mutant worms. AMPK is activated under low-energy conditions and plays a central role in regulating cellular metabolism to adapt to the energy status of cells. Thus, we provide genetic evidence linking Mg2+ homeostasis to energy metabolism via AMPK. PMID:27564576

  14. Resveratrol attenuated TNF-α–induced MMP-3 expression in human nucleus pulposus cells by activating autophagy via AMPK/SIRT1 signaling pathway

    PubMed Central

    Wang, Xiao-Hu; Zhu, Lei; Hong, Xin; Wang, Yun-Tao; Wang, Feng; Bao, Jun-Ping; Xie, Xin-Hui; Liu, Lei

    2016-01-01

    Resveratrol (RSV) is known to play a role of anti-TNF-α in a number of cell types. However, whether RSV modulates the effects of TNF-α on human nucleus pulposus (NP) cells is unknown. The purpose of this study is to investigate whether RSV regulates TNF-α–induced matrix metalloproteinase-3 (MMP-3) expression. Via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we found that MMP-3 expression induced by TNF-α was inhibited by RSV treatment. Depending on Western blot and qRT-PCR assay, we found that RSV induced autophagy in human NP cells, whereas inhibition of autophagy remarkably abolished the restraining role of RSV in the TNF-α–mediated up-regulation of MMP-3. Furthermore, RSV increased SIRT1 expression and SIRT1 knockdown significantly suppressed RSV-induced autophagy in NP cells. RSV also activated AMP-activated protein kinase (AMPK), while inhibition of AMPK notably abolished RSV-induced SIRT1 expression. Our data showed that RSV attenuated TNF-α–induced MMP-3 expression in human NP cells by activating autophagy via AMPK/SIRT1 signaling pathway. This new finding suggested that RSV might act as a novel preventive and therapeutic role in intervertebral disc degeneration. PMID:26946533

  15. Curcumin attenuates glutamate neurotoxicity in the hippocampus by suppression of ER stress-associated TXNIP/NLRP3 inflammasome activation in a manner dependent on AMPK.

    PubMed

    Li, Ying; Li, Jia; Li, Shanshan; Li, Yi; Wang, Xiangxiang; Liu, Baolin; Fu, Qiang; Ma, Shiping

    2015-07-01

    Curcumin is a natural polyphenolic compound in Curcuma longa with beneficial effects on neuronal protection. This study aims to investigate the action of curcumin in the hippocampus subjected to glutamate neurotoxicity. Glutamate stimulation induced reactive oxygen species (ROS), endoplasmic reticulum stress (ER stress) and TXNIP/NLRP3 inflammasome activation, leading to damage in the hippocampus. Curcumin treatment in the hippocampus or SH-SY5Y cells inhibited IRE1α and PERK phosphorylation with suppression of intracellular ROS production. Curcumin increased AMPK activity and knockdown of AMPKα with specific siRNA abrogated its inhibitory effects on IRE1α and PERK phosphorylation, indicating that AMPK activity was essential for the suppression of ER stress. As a result, curcumin reduced TXNIP expression and inhibited NLRP3 inflammasome activation by downregulation of NLRP3 and cleaved caspase-1 induction, and thus reduced IL-1β secretion. Specific fluorescent probe and flow cytometry analysis showed that curcumin prevented mitochondrial malfunction and protected cell survival from glutamate neurotoxicity. Moreover, oral administration of curcumin reduced brain infarct volume and attenuated neuronal damage in rats subjected to middle cerebral artery occlusion. Immunohistochemistry showed that curcumin inhibited p-IRE1α, p-PERK and NLRP3 expression in hippocampus CA1 region. Together, these results showed that curcumin attenuated glutamate neurotoxicity by inhibiting ER stress-associated TXNIP/NLRP3 inflammasome activation via the regulation of AMPK, and thereby protected the hippocampus from ischemic insult. PMID:25791922

  16. Mg2+ Extrusion from Intestinal Epithelia by CNNM Proteins Is Essential for Gonadogenesis via AMPK-TORC1 Signaling in Caenorhabditis elegans

    PubMed Central

    Ishii, Tasuku; Funato, Yosuke; Hashizume, Osamu; Yamazaki, Daisuke; Hirata, Yusuke; Nishiwaki, Kiyoji; Kono, Nozomu; Arai, Hiroyuki; Miki, Hiroaki

    2016-01-01

    Mg2+ serves as an essential cofactor for numerous enzymes and its levels are tightly regulated by various Mg2+ transporters. Here, we analyzed Caenorhabditis elegans strains carrying mutations in genes encoding cyclin M (CNNM) Mg2+ transporters. We isolated inactivating mutants for each of the five Caenorhabditis elegans cnnm family genes, cnnm-1 through cnnm-5. cnnm-1; cnnm-3 double mutant worms showed various phenotypes, among which the sterile phenotype was rescued by supplementing the media with Mg2+. This sterility was caused by a gonadogenesis defect with severely attenuated proliferation of germ cells. Using this gonadogenesis defect as an indicator, we performed genome-wide RNAi screening, to search for genes associated with this phenotype. The results revealed that RNAi-mediated inactivation of several genes restores gonad elongation, including aak-2, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK). We then generated triple mutant worms for cnnm-1; cnnm-3; aak-2 and confirmed that the aak-2 mutation also suppressed the defective gonadal elongation in cnnm-1; cnnm-3 mutant worms. AMPK is activated under low-energy conditions and plays a central role in regulating cellular metabolism to adapt to the energy status of cells. Thus, we provide genetic evidence linking Mg2+ homeostasis to energy metabolism via AMPK. PMID:27564576

  17. Combined Treatment of MCF-7 Cells with AICAR and Methotrexate, Arrests Cell Cycle and Reverses Warburg Metabolism through AMP-Activated Protein Kinase (AMPK) and FOXO1

    PubMed Central

    Fodor, Tamás; Szántó, Magdolna; Abdul-Rahman, Omar; Nagy, Lilla; Dér, Ádám; Kiss, Borbála; Bai, Peter

    2016-01-01

    Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK) jointly with methotrexate (MTX), a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1β or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer. PMID:26919657

  18. Insulin sensitive and resistant obesity in humans: AMPK activity, oxidative stress, and depot-specific changes in gene expression in adipose tissue[S

    PubMed Central

    Xu, X. Julia; Gauthier, Marie-Soleil; Hess, Donald T.; Apovian, Caroline M.; Cacicedo, Jose M.; Gokce, Noyan; Farb, Melissa; Valentine, Rudy J.; Ruderman, Neil B.

    2012-01-01

    We previously reported that adenosine monophosphate-activated protein kinase (AMPK) activity is lower in adipose tissue of morbidly obese individuals who are insulin resistant than in comparably obese people who are insulin sensitive. However, the number of patients and parameters studied were small. Here, we compared abdominal subcutaneous, epiploic, and omental fat from 16 morbidly obese individuals classified as insulin sensitive or insulin resistant based on the homeostatic model assessment of insulin resistance. We confirmed that AMPK activity is diminished in the insulin resistant group. A custom PCR array revealed increases in mRNA levels of a wide variety of genes associated with inflammation and decreases in PGC-1α and Nampt in omental fat of the insulin resistant group. In contrast, subcutaneous abdominal fat of the same patients showed increases in PTP-1b, VEGFa, IFNγ, PAI-1, and NOS-2 not observed in omental fat. Only angiotensinogen and CD4+ mRNA levels were increased in both depots. Surprisingly, TNFα was only increased in epiploic fat, which otherwise showed very few changes. Protein carbonyl levels, a measure of oxidative stress, were increased in all depots. Thus, adipose tissues of markedly obese insulin resistant individuals uniformly show decreased AMPK activity and increased oxidative stress compared with insulin sensitive patients. However, most changes in gene expression appear to be depot-specific. PMID:22323564

  19. α-MSH Stimulates Glucose Uptake in Mouse Muscle and Phosphorylates Rab-GTPase-Activating Protein TBC1D1 Independently of AMPK

    PubMed Central

    Enriori, Pablo J.; Jensen, Thomas Elbenhardt; Garcia-Rudaz, Cecilia; Litwak, Sara A.; Raun, Kirsten; Wojtaszewski, Jørgen; Wulff, Birgitte Schjellerup; Cowley, Michael A.

    2016-01-01

    The melanocortin system includes five G-protein coupled receptors (family A) defined as MC1R-MC5R, which are stimulated by endogenous agonists derived from proopiomelanocortin (POMC). The melanocortin system has been intensely studied for its central actions in body weight and energy expenditure regulation, which are mainly mediated by MC4R. The pituitary gland is the source of various POMC-derived hormones released to the circulation, which raises the possibility that there may be actions of the melanocortins on peripheral energy homeostasis. In this study, we examined the molecular signaling pathway involved in α-MSH-stimulated glucose uptake in differentiated L6 myotubes and mouse muscle explants. In order to examine the involvement of AMPK, we investigate α-MSH stimulation in both wild type and AMPK deficient mice. We found that α-MSH significantly induces phosphorylation of TBC1 domain (TBC1D) family member 1 (S237 and T596), which is independent of upstream PKA and AMPK. We find no evidence to support that α-MSH-stimulated glucose uptake involves TBC1D4 phosphorylation (T642 and S704) or GLUT4 translocation. PMID:27467141

  20. AMPK and mTOR: sensors and regulators of immunometabolic changes during Salmonella infection in the chicken.

    PubMed

    Kogut, Michael H; Genovese, Kenneth J; He, Haiqi; Arsenault, Ryan J

    2016-02-01

    Non-typhoidal Salmonella enterica induce an early pro-inflammatory response in chickens, but the response is short-lived, asymptomatic of clinical disease, results in a persistent colonization of the gastrointestinal (GI) tract, and can transmit infections to naïve hosts via fecal shedding of bacteria. The underlying mechanisms that facilitate this persistent colonization of the ceca of chickens by Salmonella are unknown. We have begun to concentrate on the convergence of metabolism and immune function as playing a major role in regulating the host responsiveness to infection. It is now recognized that the immune system monitors the metabolic state of tissues and responds by modulating metabolic function. The aim in this review is to summarize the literature that has defined a series of genotypic and phenotypic alterations in the regulatory host immune-metabolic signaling pathways in the local cecal microenvironment during the first 4 d following infection with Salmonella enterica serovar Enteritidis. Using chicken-specific kinomic immune-metabolism peptide arrays and quantitative real-time-PCR of cecal tissue during the early (4 to 48 h) and late stages (4 to 17 d) of a Salmonella infection in young broiler chickens, the local immunometabolic microenvironment has been ascertained. Distinct immune and metabolic pathways are altered between 2 to 4 d post-infection that dramatically changed the local immunometabolic environment. Thus, the tissue immunometabolic phenotype of the cecum plays a major role in the ability of the bacterium to establish a persistent cecal colonization. In general, our findings show that AMPK and mTOR are key players linking specific extracellular milieu and intracellular metabolism. Phenotypically, the early response (4 to 48 h) to Salmonella infection is pro-inflammatory, fueled by glycolysis and mTOR-mediated protein synthesis, whereas by the later phase (4 to 5 d), the local environment has undergone an immune-metabolic reprogramming to

  1. Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf-ERK-Nrf2 signaling and AMPK-independent pathways

    SciTech Connect

    Do, Minh Truong; Kim, Hyung Gyun; Khanal, Tilak; Choi, Jae Ho; Kim, Dong Hee; Jeong, Tae Cheon; Jeong, Hye Gwang

    2013-09-01

    Resistance to therapy is the major obstacle to more effective cancer treatment. Heme oxygenase-1 (HO-1) is often highly up-regulated in tumor tissues, and its expression is further increased in response to therapies. It has been suggested that inhibition of HO-1 expression is a potential therapeutic approach to sensitize tumors to chemotherapy and radiotherapy. In this study, we tested the hypothesis that the anti-tumor effects of metformin are mediated by suppression of HO-1 expression in cancer cells. Our results indicate that metformin strongly suppresses HO-1 mRNA and protein expression in human hepatic carcinoma HepG2, cervical cancer HeLa, and non-small-cell lung cancer A549 cells. Metformin also markedly reduced Nrf2 mRNA and protein levels in whole cell lysates and suppressed tert-butylhydroquinone (tBHQ)-induced Nrf2 protein stability and antioxidant response element (ARE)-luciferase activity in HepG2 cells. We also found that metformin regulation of Nrf2 expression is mediated by a Keap1-independent mechanism and that metformin significantly attenuated Raf-ERK signaling to suppress Nrf2 expression in cancer cells. Inhibition of Raf-ERK signaling by PD98059 decreased Nrf2 mRNA expression in HepG2 cells, confirming that the inhibition of Nrf2 expression is mediated by an attenuation of Raf-ERK signaling