AMP-activated Protein Kinase Agonist Research Articles

Why AMPK agonists not known to be stressors may surprisingly contribute to miscarriage or hinder IVF/ART.

Here we examine recent evidence suggesting that many drugs and diet supplements (DS), experimental AMP-activated protein kinase (AMPK) agonists as well as energy-depleting stress, lead to decreases in anabolism, growth or proliferation, and potency of cultured oocytes, embryos, and stem cells in an AMPK-dependent manner. Surprising data for DS and drugs that have some activity as AMPK agonists in in vitro experiments show possible toxicity. This needs to be balanced against a preponderance of evidence in vivo that these drugs and DS are beneficial for reproduction. We here discuss and analyze data that leads to two possible conclusions: First, although DS and drugs that have some of their therapeutic mechanisms mediated by AMPK activity associated with low ATP levels, some of the associated health problems in vivo and in vitro fertilization/assisted reproductive technologies (IVF/ART) may be better-treated by increasing ATP production using CoQ10 (Ben-Meir et al., Aging Cell 14:887-895, 2015). This enables high developmental trajectories simultaneous with solving stress by energy-requiring responses. In IVF/ART, it is ultimately best to maintain handling and culture of gametes and embryos in the quietest state with low metabolic activity (Leese et al., Mol Hum Reprod 14:667-672, 2008; Leese, Bioessays 24 (9):845-849, 2002) using back-to-nature or simplex algorithms to identify optima (Biggers, Reprod Biomed Online 4 Suppl 1:30-38, 2002). Stress markers, such as checkpoint proteins like TRP53 (aka p53) (Ganeshan et al., Exp Cell Res 358:227-233, 2017); Ganeshan et al., Biol Reprod 83:958-964, 2010) and a small set of kinases from the protein kinome that mediate enzymatic stress responses, can also be used to define optima. But, some gametes or embryos may have been stressed in vivo prior to IVF/ART or IVF/ART optimized for one outcome may be suboptimal for another. Increasing nutrition or adding CoQ10 to increase ATP production (Yang et al., Stem Cell Rev 13:454-464, 2017), managing stress enzyme levels with inhibitors (Xie et al., Mol Hum Reprod 12:217-224, 2006), or adding growth factors such as GM-CSF (Robertson et al., J Reprod Immunol 125:80-88, 2018); Chin et al., Hum Reprod 24:2997-3009, 2009) may increase survival and health of cultured embryos during different stress exposure contexts (Puscheck et al., Adv Exp Med Biol 843:77-128, 2015). We define "stress" as negative stimuli which decrease normal magnitude and speed of development, and these can be stress hormones, reactive oxygen species, inflammatory cytokines, or physical stimuli such as hypoxia. AMPK is normally activated by high AMP, commensurate with low ATP, but it was recently shown that if glucose is present inside the cell, AMPK activation by low ATP/high AMP is suppressed (Zhang et al., Nature 548:112-116, 2017). As we discuss in more detail below, this may also lead to greater AMPK agonist toxicity observed in two-cell embryos that do not import glucose. Stress in embryos and stem cells increases AMPK in large stimulation indexes but also direness indexes; the fastest AMPK activation occurs when stem cells are shifted from optimal oxygen to lower or high levels (Yang et al., J Reprod Dev 63:87-94, 2017). CoQ10 use may be better than risking AMPK-dependent metabolic and developmental toxicity when ATP is depleted and AMPK activated. Second, the use of AMPK agonists, DS, and drugs may best be rationalized when insulin resistance or obesity leads to aberrant hyperglycemia and hypertriglyceridemia, and obesity that negatively affect fertility. Under these conditions, beneficial effects of AMPK on increasing triglyceride and fatty acid and glucose uptake are important, as long as AMPK agonist exposures are not too high or do not occur during developmental windows of sensitivity. During these windows of sensitivity suppression of anabolism, proliferation, and stemness/potency due to AMPK activity, or overexposure may stunt or kill embryos or cause deleterious epigenetic changes.

Astragaloside IV Inhibits Triglyceride Accumulation in Insulin-Resistant HepG2 Cells via AMPK-Induced SREBP-1c Phosphorylation.

Objective: Insulin resistance (IR) is a risk factor for non-alcoholic fatty liver disease (NAFLD), which is characterized by lipid accumulation in hepatocytes. AMP-activated protein kinase (AMPK)-induced sterol regulatory element binding protein-1c (SREBP-1c) phosphorylation is crucial for proper regulation of lipid metabolism in the liver. Astragaloside IV (AST-IV) was found to decrease lipid accumulation in hepatocytes by activating AMPK, which is required to regulate lipid metabolism in liver tissue by inducing SREBP-1c phosphorylation.Method: To evaluate the direct effect of AST on lipid accumulation in hepatocytes with IR and elucidate the underlying mechanisms, we induced IR in HepG2 cells, and used compound C and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) (an AMPK inhibitor and agonist, respectively) as control substances. We evaluated glucose, triglyceride (TG), and non-esterified fatty acid (NEFA) production, as well as SREBP-1c transcription, SREBP-1c protein expression, and downstream gene expression with or without the presence of AST. We also investigated whether phosphorylation of SREBP-1c at Ser372 was required for AST function.Results: We found that AST attenuated IR and lipid accumulation in HepG2 cells. As an AMPK activator, AST promoted gene expression and activation of AMPK by increasing phosphorylation of AMPKa. AST also inhibited translocation of SREBP-1c into the nucleus of insulin-resistant HepG2 cells by inducing phosphorylation of SREBP-1c at Ser372.Conclusion: This study demonstrated that AST attenuates IR and lipid accumulation in HepG2 cells by regulating AMPK-dependent phosphorylation of SREBP-1c at Ser372, suggesting AST as a promising drug for treating hepatic steatosis.

Metformin inhibits ovarian cancer via decreasing H3K27 trimethylation.

Metformin has been used for the treatment of type II diabetes mellitus for decades. Recently, used of metformin in the therapy of diverse human cancer types has received widespread attention, while the underlying mechanisms have been not fully elucidated. In the current study, 5-ethynyl-20-de-oxyuridine assay to detect cell proliferation, flow cytometry to detect apoptosis, scratch wound healing and Transwell migration assay to detect cell migration capacity. The current study reported that metformin inhibited cell proliferation and migration, and promoted apoptosis in ovarian cancer cells, particularly under normoglycemic conditions in vitro. Metformin treatment significantly promoted the phosphorylation of AMP-activated protein kinase (AMPK), and reduced histone H3 lysine 27 trimethylation (H3K27me3) and polycomb repressor complex 2 (PRC2) levels. Additionally, overexpression of EZH2 to increase H3K27me3 abrogated the effect of metformin on the cell proliferation, migration and apoptosis in SKOV3 and ES2 cells. Similar to metformin, another AMPK agonist, 2-deoxy-D-glucose, reduced the H3K27me3 level and PRC2 expression. In cells pretreated with Compound C, an AMPK inhibitor, metformin was not able to induce AMPK phosphorylation or reduce H3K27me3. Metformin-mediated AMPK activation and H3K27me3 inhibition were more robust in cells exposed to low glucose (5.5 mM) compared with those exposed to high glucose (25 mM). These findings implicate H3K27me3 repression mediated by AMPK phosphorylation in the antitumor effect of metformin in ovarian cancer, indicating that metformin alters epigenetic modifications by targeting PRC2 and supports the use of metformin in treatment of patients with epithelial ovarian cancer without diabetes.

PFK15, a PFKFB3 antagonist, inhibits autophagy and proliferation in rhabdomyosarcoma cells.

Due to the high-level of metastatic and relapsed rates, rhabdomyosarcoma (RD) patients have a poor prognosis, and novel treatment strategies are required. Thereby, the present study evaluated the efficacy of PFK15, a PFKFB3 inhibitor, in RD cells to explore its potential underlying mechanism on the regulation of autophagy and proliferation in these cells. The effects of PFK15 on cell viability loss and cell death in different treatment groups, were evaluated by MTS assay, colony growth assay and immunoblotting, respectively. In addition, the autophagy levels were detected by electron microscopy, fluorescence microscopy and immunoblotting following PFK15 treatment, and the autophagic flux was analyzed with the addition of chloroquine diphosphate salt or by monitoring the level of p62. PFK15 was observed to evidently decrease the viability of RD cells, inhibit the colony growth and cause abnormal nuclear morphology. Furthermore, PFK15 inhibited the autophagic flux and cell proliferation, as well as induced apoptotic cell death in RD cells through downregulation of the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. An AMPK agonist rescued the inhibited cell proliferation and autophagy induced by PFK15. In conclusion, PFK15 inhibits autophagy and cell proliferation via downregulating the AMPK signaling pathway in RD cells.

AMP-activated protein kinase protects against necroptosis via regulation of Keap1-PGAM5 complex

BackgroundThe AMP-activated protein kinase (AMPK) plays critical roles in growth regulation and metabolism reprogramming. AMPK activation protects cells against apoptosis from injury in different cell and animal models. However, its function in necroptosis remains largely unclear. Methods and resultsIn the current study, we demonstrated that AMPK was activated upon necroptosis induction and protected mouse embryonic fibroblasts (MEFs) and cardiomyocytes from N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and reactive oxygen species (ROS) induced necroptosis. Activation of AMPK with chemicals A-769662, 2-deoxyglucose (2-DG), and metformin or constitutively active (CA) AMPK markedly decreased necroptosis and cytotoxicity induced by MNNG. In contrast, AMPK inhibitor compound C, dominant negative (DN) AMPK, as well as AMPK shRNAs increased necroptosis and cytotoxicity induced by MNNG. We further showed that AMPK physically associated with a protein complex containing PGAM5 and Keap1 whereby facilitating Keap1-mediated PGAM5 ubiquitination upon necroptosis induction. The AMPK agonist metformin ameliorated myocardial ischemia and reperfusion (IR) injury and reduced necroptosis through down-regulating the expression of PGAM5 in the Langendorff-perfused rat hearts. ConclusionActivation of AMPK protects against necroptosis via promoting Keap1-mediated PGAM5 degradation. Metformin may act as a valuable agent for the protection of myocardial ischemia and reperfusion injury by activating AMPK and reducing necroptosis.

Cardiomyocyte-specific deletion of Sirt1 gene sensitizes myocardium to ischaemia and reperfusion injury.

A longevity gene, Sirtuin 1 (SIRT1) and energy sensor AMP-activated protein kinase (AMPK) have common activators such as caloric restriction, oxidative stress, and exercise. The objective of this study is to characterize the role of cardiomyocyte SIRT1 in age-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults. Mice were subjected to ligation of left anterior descending coronary artery for in vivo ischemic models. The glucose and fatty acid oxidation were measured in a working heart perfusion system. The cardiac functions by echocardiography show no difference in young wild-type C57BL/6 J (WT, 4-6 months), aged WT C57BL/6 J (24-26 months), and young inducible cardiomyocyte-specific SIRT1 knockout (icSIRT1 KO) (4-6 months) mice under physiological conditions. However, after 45 mins ischaemia and 24-h reperfusion, the ejection fraction of aged WT and icSIRT1 KO mice was impaired. The aged WT and icSIRT1 KO hearts vs. young WT hearts also show an impaired post-ischemic contractile function in a Langendorff perfusion system. The infarct size of aged WT and icSIRT1 KO hearts was larger than that of young WT hearts. The immunoblotting data demonstrated that aged WT and icSIRT1 KO hearts vs. young WT hearts had impaired phosphorylation of AMPK and downstream acetyl-CoA carboxylase during ischaemia. Intriguingly, AMPK upstream LKB1 is hyper-acetylated in both aged WT and icSIRT1 KO hearts; this could blunt activation of LKB1, leading to an impaired AMPK activation. The working heart perfusion results demonstrated that SIRT1 deficiency significantly impaired substrate metabolism in the hearts; fatty acid oxidation is augmented and glucose oxidation is blunted during ischaemia and reperfusion. Adeno-associated virus (AAV9)-Sirt1 was delivered into the aged hearts via a coronary delivery approach, which significantly rescued the protein level of SIRT1 and the ischemic tolerance of aged hearts. Furthermore, AMPK agonist can rescue the tolerance of aged heart and icSIRT1 KO heart to ischemic insults. Cardiac SIRT1 mediates AMPK activation via LKB1 deacetylation, and AMPK modulates SIRT1 activity via regulation of NAD+ level during ischaemia. SIRT1 and AMPK agonists have therapeutic potential for treatment of aging-related ischemic heart disease.

AMP-activated protein kinase agonist N6-(3-hydroxyphenyl)adenosine protects against fulminant hepatitis by suppressing inflammation and apoptosis

Both AMP-activated protein kinase (AMPK) agonist and inhibitor have been reported to protect against fulminant hepatitis, implying that AMPK may play a complicated role in the development of fulminant hepatitis. In this study, we exploited whether the novel AMPK agonist N6-(3-hydroxyphenyl)adenosine (named as M1) exerted protective effects on fulminant hepatitis and whether its beneficial effects were AMPK-dependent. Results showed that intraperitoneal injection of M1 improved liver function, ameliorated liver injury and finally raised the survival rate in d-galactosamine/lipopolysaccharide (GalN/LPS)-treated mice. These beneficial effects of M1 may attribute to the suppression of pro-inflammatory cytokines production and the prevention of hepatocyte apoptosis. Furthermore, M1 pretreatment mitigated LPS-stimulated TLR4 expression and NFκB activation in murine peritoneal macrophages and prevented actinomycin D (Act D)/tumor necrosis factor α (TNFα)-induced apoptosis by promoting protective autophagy in primary hepatocytes. Additionally, M1-induced AMPK activation was responsible both for its anti-inflammatory action in macrophages and for its anti-apoptotic action in hepatocytes. To our surprise, compared with the control AMPKα1lox/lox/AMPKα2lox/lox mice, liver-specific AMPKα1 knockout (AMPKα1LS−/−) mice were more sensitive to GalN/LPS administration but not AMPKα2LS−/−mice, and the beneficial effects of M1 on acute liver failure and the production of pro-inflammatory factors were dampened in AMPKα1LS−/− mice. Therefore, our study may prove that M1 could be a promising therapeutic agent for fulminant hepatitis, and targeting AMPK may be useful therapeutically in the control of LPS-induced hepatotoxicity.

Curcumin Activates AMPK Pathway and Regulates Lipid Metabolism in Rats Following Prolonged Clozapine Exposure.

Clozapine (CLO) remains an ultimate option for patients with treatment resistant schizophrenia. However, the atypical antipsychotic is often associated with serious metabolic side effects, such as dyslipidemia. Hepatic sterol regulatory element-binding proteins (SREBPs) are central in the allosteric control of a variety of lipid biosynthetic pathways. There is emerging evidence that CLO can activate SREBP pathway and enhance downstream lipogenesis, whereas curcumin (CUR), a major active compound of Curcuma longa, contains hypolipidemic properties. Therefore, in the present study, we examined the protective effects of CUR against CLO-induced lipid disturbance and analyzed the expression of key components in hepatic lipid metabolism. Our data showed that 4-week treatment of CLO (15 mg/kg/day) markedly elevated serum lipid levels and resulted in hepatic lipid accumulation, whereas co-treatment of CUR (80 mg/kg/day) alleviated the CLO-induced dyslipidemia. We further demonstrated that CUR appears to be a novel AMP-activated protein kinase (AMPK) agonist, which enhanced AMPK phosphorylation and mitigated CLO-induced SREBP overexpression. Additionally, CUR also modulated the downstream SREBP-targeted genes involved in fatty acid synthesis and cholesterol metabolism, including fatty acid synthase (FAS) and HMG-CoA reductase (HMGCR). In summary, our study suggests that the suppressed AMPK activity and thereby enhanced SREBP-dependent lipid synthesis could be associated with the antipsychotic-stimulated dyslipidemia, whereas CUR may maintain lipid homeostasis by directly binding to AMPK, indicating that adjunctive use of CUR could be a promising preventive strategy for the drug-induced lipogenesis.

Two-cell embryos are more sensitive than blastocysts to AMPK-dependent suppression of anabolism and stemness by commonly used fertility drugs, a diet supplement, and stress.

This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos. Culture mouse zygotes to two-cell embryos and test effects after 0.5-1h AMPK agonists' (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells. Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~20-fold, and BR-DIM caused a ~30-fold increase over two-cell embryos cultured for 1h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40-85% in two-cell embryos that was ~60-90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30-50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease. Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

Hyperosmotic stress, metformin, BR-DIM cause AMPK-dependent anabolism, growth, and stemness factor decreases more strongly in 2-cell embryos than blastocysts

As a model for stress in vivo, we hypothesize that clinically relevant AMP-activated protein kinase (AMPK) agonists may cause unwanted early pregnancy complications. Agonist types include pharma such as metformin and aspirin, and diet supplements (DS) such as bromo-3,3' diindolylmethane (BR-DIM). To test whether metformin, aspirin or diet supplement (DS) BR-DIM can induce AMPK-dependent decrease in anabolism assayed by pACC ser79P, and potency loss as assayed by Cdx2 and Oct4 and whether 2-cell embryos are more sensitive than blastocyst stage embryos.

Activation of AMPK is neuroprotective in the oxidative stress by advanced glycosylation end products in human neural stem cells

Advanced glycosylation end products (AGEs) formation is correlated with the pathogenesis of diabetic neuronal damage, but its links with oxidative stress are still not well understood. Metformin, one of the most widely used anti-diabetic drugs, exerts its effects in part by activation of AMP-activated protein kinase (AMPK). Once activated, AMPK regulates many pathways central to metabolism and energy balance including, glucose uptake, glycolysis and fatty acid oxidation. AMPK is also present in neurons, but its role remains unclear. Here, we show that AGE exposure decreases cell viability of human neural stem cells (hNSCs), and that the AMPK agonist metformin reverses this effect, via AMPK-dependent downregulation of RAGE levels. Importantly, hNSCs co-treated with metformin were significantly rescued from AGE-induced oxidative stress, as reflected by the normalization in levels of reactive oxygen species. In addition, compared to AGE-treated hNSCs, metformin co-treatment significantly reversed the activity and mRNA transcript level changes ofSOD1/2 and Gpx. Furthermore, hNSCs exposed to AGEs had significantly lower mRNA levels among other components of normal cellular oxidative defenses (GSH, Catalase and HO-1), which were all rescued by co-treatment with metformin. This metformin-mediated protective effect on hNSCs for of both oxidative stress and oxidative defense genes by co-treatment with metformin was blocked by the addition of an AMPK antagonist (Compound C). These findings unveil the protective role of AMPK-dependent metformin signaling during AGE mediated oxidative stress in hNSCs, and suggests patients undergoing AGE-mediated neurodegeneration may benefit from the novel therapeutic use of metformin.

High glucose stimulates cell proliferation and Collagen IV production in rat mesangial cells through inhibiting AMPK-KATP signaling.

The present study investigated the putative mechanisms underlying effects of KATP channel on high glucose (HG)-induced mesangial cell proliferation and tissue inhibitors of metalloproteinases (TIMP)-2 and Collagen IV production. Rat mesangial cells were subjected to whole cell patch clamp to record the KATP channel currents under high glucose (HG, 30mM) condition. Cell proliferation was measured using a CCK-8 assay. The production of TIMP-2 and Collagen IV and AMP-activated protein kinase (AMPK)-signaling pathway activity was assessed by ELISA and Western blotting, respectively. AMPK agonist (AICAR) was used to analyze the role of this kinase. The expression of KATP subunit (Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B) was examined using quantitative real-time PCR (RT-PCR). We found that HG was significant decreases in the expression of Kir6.1, SUB2A and SUB2B, three subunits of KATP, TIMP-2 production, KATP channel activity and AMPK activity, while it promoted the cell proliferation and Collagen IV production in rat mesangial cells. Pretreatment with KATP selective opener (diazoxide, DZX) significantly inhibited HG-induced mesangial cell proliferation, Collagen IV production and decrease in KATP channel activity in rat mesangial cells, which were reversed by pretreatment of 5-hydroxydecanoate, a selective inhibitor of KATP. Moreover, AICAR pretreatment inhibited HG-induced decrease in KATP channel activity. Taken together, activating AMPK-KATP signaling may protect against HG-induced mesangial cell proliferation and Collagen IV production, and, thereby, provides new insights into the molecular mechanisms underlying early diabetic nephropathy (DN).

Abstract 158: A Tether Containing a UBX Domain (TUG) Mediates Glucose Transporter Translocation in the Ischemic Heart

Introduction: The adaptive metabolic regulation of glucose and fatty acid in the heart plays a critical role in limiting cardiac damage caused by ischemia and reperfusion (I/R). TUG (tether containing a UBX domain, for GLUT4) can be cleaved to mobilize glucose transporter GLUT4 from intracellular vesicles to the cell surface in skeletal muscle and adipose in response to insulin stimulation. The energy sensor AMP-activated protein kinase (AMPK) plays an important cardioprotective role in response to ischemic insults by modulating GLUT4 translocation. Hypothesis: TUG is one of the downstream targets of AMPK in the heart. TUG could be phosphorylated by ischemic AMPK and cleaved to dissociate with GLUT4 and increase GLUT4 translocation in the ischemic heart. Methods: In vivo regional ischemia by ligation of left anterior coronary artery and ex vivo isolated mouse heart perfusion Langendorff system were used to test the hypothesis. Results: Antithrombin (AT) is an endogenous AMPK agonist in the heart and used to define the role of TUG in regulating GLUT4 trafficking during ischemia and reperfusion in the heart. AT showed its cardioprotective function through recovering cardiac pumping function and activating AMPK. The results showed that AMPK activation by AT treatment was through LKB1 and Sesn2 complex. Furthermore, the ex vivo heart perfusion data demonstrated that AT administration significantly increase GLUT4 translocation, glucose uptake, glycolysis and glucose oxidation during ischemia and reperfusion (p<0.05 vs . vehicle). Moreover, AT treatment increased abundance of a TUG cleavage product (42 KD) in response to I/R. The TUG protein was clearly phosphorylated by activated AMPK in HL-1 cardiomyocytes. The in vivo myocardial ischemia results demonstrated that ischemic AMPK activation triggers TUG cleavage and significantly increases GLUT4 translocation to the cell surface. Moreover, an augmented interaction between AMPK and TUG was observed during ischemia. Conclusions: Cardiac AMPK activation stimulates TUG cleavage and causes the dissociation between TUG and GLUT4 in the intracellular vesicles. TUG is a critical mediator that modulates cardiac GLUT4 translocation to cell surface and enhances glucose uptake by AMPK signaling pathway.

Metformin ameliorates the Phenotype Transition of Peritoneal Mesothelial Cells and Peritoneal Fibrosis via a modulation of Oxidative Stress

Phenotype transition of peritoneum is an early mechanism of peritoneal fibrosis. Metformin, 5′-adenosine monophosphate-activated protein kinase (AMPK) activator, has recently received a new attention due to its preventive effect on organ fibrosis and cancer metastasis by inhibiting epithelial-to-mesenchymal transition (EMT). We investigated the effect of metformin on EMT of human peritoneal mesothelial cells (HPMC) and animal model of peritoneal dialysis (PD). TGF-β1-induced EMT in HPMC was ameliorated by metformin. Metformin alleviated NAPDH oxidase- and mitochondria-mediated ROS production with an increase in superoxide dismutase (SOD) activity and SOD2 expression. Metformin inhibited the activation of Smad2/3 and MAPK, GSK-3β phosphorylation, nuclear translocalization of β-catenin and Snail in HPMCs. Effect of metformin on TGF-β1-induced EMT was ameliorated by either AMPK inhibitor or AMPK gene silencing. Another AMPK agonist, 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide partially blocked TGF-β1-induced EMT. In animal model of PD, intraperitoneal metformin decreased the peritoneal thickness and EMT with an increase in ratio of reduced to oxidized glutathione and the expression of SOD whereas it decreased the expression of nitrotyrosine and 8-hydroxy-2′-deoxyguanosine. Therefore, a modulation of AMPK in peritoneum can be a novel tool to prevent peritoneal fibrosis by providing a favorable oxidant/anti-oxidant milieu in peritoneal cavity and ameliorating phenotype transition of peritoneal mesothelial cells.

Alloreactive T cell proliferation is suppressed by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) independent of AMP-activated protein kinase

Abstract The benefits of hematopoietic stem cell transplantation, a therapy used to treat a variety of hematologic and immunologic disorders, are often outweighed by the risks of acute graft-versus-host disease (GVHD), where donor T cells attack and destroy host tissues. Our lab has previously shown that targeting lymphocyte metabolism is a promising and novel strategy for the treatment of GVHD. More recently, we have found that AMP-activated protein kinase (AMPK), a major cellular energy sensor, plays a significant role in T-cell proliferation during disease pathogenesis. To explore this relationship further, we treated alloreactive T cells generated during a mixed leukocyte reaction (MLR) with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMP mimetic that strongly activates AMPK. AICAR treatment of MLR cultures strongly repressed T cell proliferation, with a 70% decrease in total cells by 96 hours. Interestingly this decrease in T cell proliferation was independent of AMPK, as similar results were obtained using AMPK−/− cells. Mechanistically AICAR repressed AKT signaling in stimulated T cells, with a 50% decrease in AKT phosphorylation when cells were concurrently treated with 100 μM AICAR. Cytokine driven STAT5 activation was also significantly blunted by simultaneous AICAR exposure. In summary, despite its wide use as an AMPK agonist, we found that AICAR exerts significant inhibitory effects on T cell allo-activation independent of AMPK. This inhibition occurs, at least in part, through decreased phosphorylation of AKT and down-regulation of STAT signaling, making AICAR a potential therapy to combat GVHD and similar inflammatory disorders.

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

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

AMPK activation by peri-sciatic nerve administration of ozone attenuates CCI-induced neuropathic pain in rats.

Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. Ozone is widely used as an alternative therapy for many different pain conditions, with exact mechanisms still elusive. In this study, we found that a single peri-sciatic nerve injection of ozone decreased mechanical allodynia and thermal hyperalgesia, and normalized the phosphorylation of protein kinase C γ, N-methyl-D-aspartate receptor, and extracellular signal-regulated kinase in a chronic constriction injury (CCI) model in rat sciatic nerve. Meanwhile, ozone significantly suppressed CCI-induced activation of spinal microglia. More importantly, the anti-nociceptive effect of ozone depended on the activation of 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), which was proved by the fact that the phosphorylated AMPK level increased during the ozone therapy and AMPK antagonist abolished the effect of ozone in vivo and in vitro. In addition, direct injection of AMPK agonist could replicate the anti-nociceptive effect of ozone in CCI rats. In conclusion, our observations indicate that peri-sciatic nerve injection of ozone activates AMPK to attenuate CCI-induced neuropathic pain.

AICAR activates ER stress-dependent apoptosis in gallbladder cancer cells

AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) is an AMP-activated protein kinase (AMPK) agonist, its activity in human gallbladder cancer cells was evaluated here. We show that AICAR provoked significant apoptosis in human gallbladder cancer cell lines (Mz-ChA-1, QBC939 and GBC-SD) and primary gallbladder cancer cells. AICAR-induced cytotoxicity in gallbladder cancer cells appears independent of AMPK activation. Inhibition of AMPK, via AMPKα shRNA knockdown or dominant negative mutation (T172A), failed to rescue GBC-SD cells from AICAR. Further, forced-activation of AMPK, by adding two other AMPK activators (A769662 and Compound 13), or expressing a constitutively-active mutant AMPKα (T172D), didn't induce GBC-SD cell death. Remarkably, AICAR treatment in gallbladder cancer cells induced endoplasmic reticulum (ER) stress activation, the latter was tested by caspase-12 activation, C/EBP homologous protein (CHOP) expression and IRE1/PERK phosphorylation. Contrarily, salubrinal (the ER stress inhibitor), z-ATAD-fmk (the caspase-12 inhibitor) or CHOP shRNAs significantly attenuated AICAR-induced gallbladder cancer cell apoptosis. Together, we conclude that AICAR-induced gallbladder cancer cell apoptosis requires ER stress activation, but is independent of AMPK.

Abstract 12019: Inactivation of AMP-activated Protein Kinase Sensitizes Heart to Pressure Overload-induced Hypertrophy

Introduction: Hypertrophy triggers activation of adenosine monophosphate-activated protein kinase (AMPK), an energy sensor in the heart. We have identified activated protein C (APC) is an endogenous AMPK agonist, and the non-anticoagulant APC-2Cys mutant but not the non-signaling APC-E170A mutant stimulates cardiac AMPK activation as well. Hypothesis: AMPK play a critical role in preventing heart from hypertrophy induced by pressure overload. APC as a AMPK agonist inhibits pressure overload-induced hypertrophy via APC’s cytoprotective signaling activity. Methods: Wild-type (WT) and AMPK-kinase dead (KD) transgenic mice were subjected to transverse aortic constriction (TAC) surgery. Echocardiography was performed to evaluate the heart function, and histology staining revealed the morphological changes. Real-time PCR and immunoblotting were used to detect mRNA and protein levels of signaling changes. Results: There is no phenotype difference between wild type (WT) and AMPK-kinase dead (AMPK KD) mice under normal physiological conditions. However after 4 weeks of TAC surgery, AMPK-KD mice demonstrated significantly bigger heart as compared to WT mice (p<0.05). The cardiac functions measured by echocardiography in AMPK-KD hearts were significantly impaired as compared with WT hearts (p<0.05). The immunohistochemical staining showed that the increased macrophage infiltration and reactive oxygen species (ROS) in AMPK-KD versus WT hearts after 4 weeks of TAC surgery. The immunoblotting results demonstrated that the redox status markers such as p66 shc , 4-hydroxynonenal and extracellular signal-regulated kinase (ERK) were up-regulated in the AMPK-KD hearts after 4 weeks of TAC surgery (all p<0.05 versus WT hearts). APC and APC-2Cys but not APC-E170A administration significantly attenuated hypertrophy and fibrosis caused by pressure overload. The macrophage infiltration and p66 shc activation caused by pressure overload were also inhibited by APC and APC-2Cys but not by APC-E170A. Conclusions: Cardiac AMPK inactivation aggravates pressure overload induced hypertrophy. APC-2Cys without anticoagulant activity could be a therapeutic drug for treatment of hypertension-related hypertrophy patients without bleeding risk.

Abstract 12050: Cardiomyocyte Specific Deletion of Sirt1 Gene Alters Substrate Metabolism and Sensitizes Myocardium to Ischemia and Reperfusion Injury

Introduction: A longevity gene, sirtuin 1 (SIRT1) is a conserved NAD + -dependent protein deacetylase. AMP-activated protein kinase (AMPK) is an energy sensor. AMPK and SIRT1 signaling pathways have common activators such as caloric restriction, oxidative stress and exercise. Hypothesis: Aging-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults are due to a decreased SIRT1 levels in aging. Methods: Mice were subjected to ligation of left anterior descending coronary artery for in vivo ischemic models. The glucose and fatty acid oxidation were measured in a working heart perfusion system. Results: The cardiac functions by echocardiography show no difference in young (SIRT1 flox/flox ), aged (SIRT1 flox/flox ) and young inducible cardiomyocyte specific SIRT1 knockout (icSIRT1 KO) mice under physiological conditions, but after 45 mins ischemia and 24 hours reperfusion, the ejection fraction of aged and icSIRT1 KO mice were impaired. The aged and icSIRT1 KO hearts versus young hearts also show an impaired post-ischemic contractile function in Langendorff perfusion system. The infarct size of aged and icSIRT1 KO hearts was larger than that of young hearts (p<0.05). The immunoblotting data demonstrated that aged and icSIRT1 KO hearts versus young hearts had impaired phosphorylation of AMPK and downstream acetyl-CoA carboxylase during ischemia. Intriguingly, AMPK upstream LKB1 is hyper-acetylated in both aged and icSIRT1 KO hearts that could blunt activation of LKB1, leading to an impaired AMPK activation. The working heart perfusion results demonstrated that SIRT1 deficiency significantly impair substrate metabolism in the hearts, fatty acid oxidation is augmented and glucose oxidation is blunted during ischemia and reperfusion. Aged and icSIRT1 KO hearts versus young hearts demonstrated significant cardiac fibrosis and macrophage infiltration during ischemia and reperfusion. Furthermore, AMPK agonist A769662 treatment can rescue the tolerance of aged heart but not icSIRT1 KO hearts to ischemic insults. Conclusions: Cardiac SIRT1 mediates AMPK activation via LKB1 deacetylation during ischemia. SIRT1 and AMPK agonists have therapeutic potential for treatment of aging-related ischemic heart disease.