Effects Of AMP-activated Protein Kinase Activation Research Articles

The mixed blessing of AMPK signaling in Cancer treatments

BackgroundNutrient acquisition and metabolism pathways are altered in cancer cells to meet bioenergetic and biosynthetic demands. A major regulator of cellular metabolism and energy homeostasis, in normal and cancer cells, is AMP-activated protein kinase (AMPK). AMPK influences cell growth via its modulation of the mechanistic target of Rapamycin (mTOR) pathway, specifically, by inhibiting mTOR complex mTORC1, which facilitates cell proliferation, and by activating mTORC2 and cell survival. Given its conflicting roles, the effects of AMPK activation in cancer can be counter intuitive. Prior to the establishment of cancer, AMPK acts as a tumor suppressor. However, following the onset of cancer, AMPK has been shown to either suppress or promote cancer, depending on cell type or state.MethodsTo unravel the controversial roles of AMPK in cancer, we developed a computational model to simulate the effects of pharmacological maneuvers that target key metabolic signalling nodes, with a specific focus on AMPK, mTORC, and their modulators. Specifically, we constructed an ordinary differential equation-based mechanistic model of AMPK-mTORC signaling, and parametrized the model based on existing experimental data.ResultsModel simulations were conducted to yield the following predictions: (i) increasing AMPK activity has opposite effects on mTORC depending on the nutrient availability; (ii) indirect inhibition of AMPK activity through inhibition of sirtuin 1 (SIRT1) only has an effect on mTORC activity under conditions of low nutrient availability; (iii) the balance between cell proliferation and survival exhibits an intricate dependence on DEP domain-containing mTOR-interacting protein (DEPTOR) abundance and AMPK activity; (iv) simultaneous direct inhibition of mTORC2 and activation of AMPK is a potential strategy for suppressing both cell survival and proliferation.ConclusionsTaken together, model simulations clarify the competing effects and the roles of key metabolic signalling pathways in tumorigenesis, which may yield insights on innovative therapeutic strategies.

AMP-activated Protein Kinase Activation Suppresses Protein Synthesis and mTORC1 Signaling in Chick Myotube Cultures.

Protein synthesis in skeletal muscle is considered one of the most energy-consuming cellular processes. AMP-activated protein kinase (AMPK) is a metabolic master switch that regulates glucose and lipid metabolism, and it is implicated in protein synthesis control in skeletal muscles. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of protein metabolism in cells. However, the effect of AMPK activation on protein synthesis and mTORC1 signaling in chicken skeletal muscle remains unclear. Therefore, in this study, we aimed to investigate the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMPK activator, on protein synthesis and mTORC1 signaling in chick myotube cultures. The incubation of chick myotubes with AICAR (1 mM) for 3 h led to a significant increase in AMPK (Thr172) phosphorylation. Nonetheless, protein synthesis, measured using the surface sensing of translation method, was significantly decreased by AICAR. In addition, the phosphorylation of p70 ribosomal S6 kinase 1 (S6K1, Thr389), S6 ribosomal protein (Ser240/244), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Thr37/46) was significantly reduced by AICAR. These results suggest that AMPK activation suppresses protein synthesis and mTORC1 signaling (through the phosphorylation of S6K1, S6 ribosomal protein, and 4E-BP1) in chick myotubes.

AICAR stimulates mitochondrial biogenesis and BCAA catabolic enzyme expression in C2C12 myotubes

Type 2 diabetes is characterized by reduced insulin sensitivity, elevated blood metabolites, and reduced mitochondrial metabolism. Insulin resistant populations often exhibit reduced expression of genes governing mitochondrial metabolism such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Interestingly, PGC-1α regulates the expression of branched-chain amino acid (BCAA) metabolism, and thus, the consistently observed increased circulating levels of BCAA in diabetics may be partially explained by reduced PGC-1α expression. Conversely, PGC-1α upregulation appears to increase BCAA catabolism. PGC-1α activity is regulated by 5′-AMP-activated protein kinase (AMPK), however, only limited experimental data exists on the effect of AMPK activation in the regulation of BCAA catabolism. The present report examined the effects of the commonly used AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on the metabolism and expression of several related targets (including BCAA catabolic enzymes) of cultured myotubes. C2C12 myotubes were treated with AICAR at 1 mM for up to 24 h. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via qRT-PCR and western blot, respectively. AICAR treatment significantly increased mitochondrial content and peak mitochondrial capacity. AICAR treatment also increased AMPK activation and mRNA expression of several regulators of mitochondrial biogenesis but reduced glycolytic metabolism and mRNA expression of several glycolytic enzymes. Interestingly, branched-chain alpha-keto acid dehydrogenase a (BCKDHa) protein was significantly increased following AICAR-treatment suggesting increased overall BCAA catabolic capacity in AICAR-treated cells. Together, these experiments demonstrate AICAR/AMPK activation can upregulate BCAA catabolic machinery in a model of skeletal muscle.

A-769662 inhibits adipocyte glucose uptake in an AMPK-independent manner.

Activation of AMP-activated protein kinase (AMPK) is considered a valid strategy for the treatment of type 2 diabetes. However, despite the importance of adipose tissue for whole-body energy homeostasis, the effect of AMPK activation in adipocytes has only been studied to a limited extent and mainly with the AMP-mimetic 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), which has limited specificity. The aim of this study was to evaluate the effect of the allosteric AMPK activators A-769662 and 991 on glucose uptake in adipocytes. For this purpose, primary rat or human adipocytes, and cultured 3T3-L1 adipocytes, were treated with either of the allosteric activators, or AICAR, and basal and insulin-stimulated glucose uptake was assessed. Additionally, the effect of AMPK activators on insulin-stimulated phosphorylation of Akt and Akt substrate of 160 kDa was assessed. Furthermore, primary adipocytes from ADaM site binding drug-resistant AMPKβ1 S108A knock-in mice were employed to investigate the specificity of the drugs for the observed effects. Our results show that insulin-stimulated adipocyte glucose uptake was significantly reduced by A-769662 but not 991, yet neither activator had any clear effects on basal or insulin-stimulated Akt/AS160 signaling. The use of AMPKβ1 S108A mutant-expressing adipocytes revealed that the observed inhibition of glucose uptake by A-769662 is most likely AMPK-independent, a finding which is supported by the rapid inhibitory effect A-769662 exerts on glucose uptake in 3T3-L1 adipocytes. These data suggest that AMPK activation per se does not inhibit glucose uptake in adipocytes and that the effects of AICAR and A-769662 are AMPK-independent.

The Mixed Blessing of AMPK Signaling in Cancer Treatments

Nutrient acquisition and metabolism pathways are altered in cancer cells to meet bioenergetic and biosynthetic demands. A major regulator of cellular metabolism and energy homeostasis, in normal and cancer cells, is AMP-activated protein kinase (AMPK). AMPK influences cell growth via its modulation of the mechanistic target of Rapamycin (mTOR) pathway, specifically, by inhibiting mTOR complex mTORC1, which facilitates cell proliferation, and by activating mTORC2 and cell survival. Given its conflicting roles, the effects of AMPK activation in cancer can be counter-intuitive. Prior to the establishment of cancer, AMPK acts as a tumor suppressor. However, following the onset of cancer, AMPK has been shown to either suppress or promote cancer, depending on cell type or state. To unravel the controversial roles of AMPK in cancer, we developed a computational model to simulate the effects of pharmacological maneuvers that target key metabolic signalling nodes, with specific focus on AMPK, mTORC, and their modulators. Model simulations clarify the competing effects and the roles of key metabolic signalling pathways in tumorigenesis, which may yield insights on innovative therapeutic strategies.

An integrative pan-cancer investigation reveals common genetic and transcriptional alterations of AMPK pathway genes as important predictors of clinical outcomes across major cancer types

BackgroundThe AMP-activated protein kinase (AMPK) is an evolutionarily conserved regulator of cellular energy homeostasis. As a nexus for transducing metabolic signals, AMPK cooperates with other energy-sensing pathways to modulate cellular responses to metabolic stressors. With metabolic reprogramming being a hallmark of cancer, the utility of agents targeting AMPK has received continued scrutiny and results have demonstrated conflicting effects of AMPK activation in tumorigenesis. Harnessing multi-omics datasets from human tumors, we seek to evaluate the seemingly pleiotropic, tissue-specific dependencies of AMPK signaling dysregulation.MethodsWe interrogated copy number variation and differential transcript expression of 92 AMPK pathway genes across 21 diverse cancers involving over 18,000 patients. Cox proportional hazards regression and receiver operating characteristic analyses were used to evaluate the prognostic significance of AMPK dysregulation on patient outcomes.ResultsA total of 24 and seven AMPK pathway genes were identified as having loss- or gain-of-function features. These genes exhibited tissue-type dependencies, where survival outcomes in glioma patients were most influenced by AMPK inactivation. Cox regression and log-rank tests revealed that the 24-AMPK-gene set could successfully stratify patients into high- and low-risk groups in glioma, sarcoma, breast and stomach cancers. The 24-AMPK-gene set could not only discriminate tumor from non-tumor samples, as confirmed by multidimensional scaling analyses, but is also independent of tumor, node and metastasis staging. AMPK inactivation is accompanied by the activation of multiple oncogenic pathways associated with cell adhesion, calcium signaling and extracellular matrix organization. Anomalous AMPK signaling converged on similar groups of transcriptional targets where a common set of transcription factors were identified to regulate these targets. We also demonstrated crosstalk between pro-catabolic AMPK signaling and two pro-anabolic pathways, mammalian target of rapamycin and peroxisome proliferator-activated receptors, where they act synergistically to influence tumor progression significantly.ConclusionGenetic and transcriptional aberrations in AMPK signaling have tissue-dependent pro- or anti-tumor impacts. Pan-cancer investigations on molecular changes of this pathway could uncover novel therapeutic targets and support risk stratification of patients in prospective trials.

Abstract P5-04-08: Timing provides context for the paradoxical effects of AMPK activation in ER+ breast cancer: Suppressing growing tumors, but promoting dormant tumor cell survival and recurrence

Abstract Precision oncology requires delivering the right drug to the right patient at the right time, but “time” is rarely studied preclinically before a drug enters a particular clinical setting. Despite showing efficacy against recurrent/metastatic solid tumors, drugs sometimes fail to prevent tumor recurrence when given (neo)adjuvantly. The biology and microenvironments of overt tumors likely differ substantially from dormant cancer cells. The development of preclinical models to investigate clinically dormant disease will increase understanding of residual tumor cell biology and enable the development of therapeutics for rational adjuvant trials. Despite treatment with adjuvant anti-estrogen therapies, ˜30% of patients with ER+ breast cancer (BC) experience recurrence. In contrast to other BC subtypes, ER+ relapses occur late, often appearing years to decades after initial diagnosis and treatment. This delay suggests that ER+ BC cells can undergo extended periods of clinical dormancy. We developed novel, clinically relevant xenograft models of dormancy in ER+ BC. Low systemic levels of estrogens in mice can be further suppressed by ovariectomy, mimicking the effects of aromatase inhibitor (AI)-induced estrogen deprivation (ED) therapy. In ovariectomized mice, luciferase-labeled MCF-7, HCC-1428, HCC-1500, and MDA-MB-415 cells, as well as the HCI-017 PDX model, formed palpable orthotopic tumors upon 17b-estradiol supplementation. ED induced rapid tumor regression and decreased bioluminescent signal. However, a small proportion of ER+ BC cells survived ED for >1 year in a clinically dormant, growth-suppressed state. This residual cell population stabilized after ˜90 days of ED, as evidenced by stabilization of bioluminescent signal. Transcriptional and immunohistochemical analyses revealed significant upregulation of AMP-activated protein kinase (AMPK)-alpha-2 levels and activity in clinically dormant tumor cells compared to estrogen-driven or acutely ED xenografts. Dormant tumor cells were dependent upon AMPK activity for survival, as short-term pharmacologic inhibition of AMPK reduced residual bioluminescent signal. Metformin is an AMPK-activating drug approved for the treatment of diabetes. Metformin is currently being tested as an anti-cancer agent in many clinical trials at various points in the disease progression of diverse cancer types. In our models of clinically dormant ER+ BC, AMPK activation via metformin slowed ED-induced tumor regression, promoted residual tumor cell survival, and caused earlier tumor regrowth. In vitro studies indicated that metformin promotes cell survival during ED by enhancing fatty acid β-oxidation. Conversely, metformin treatment slowed estrogen-driven tumor growth, in agreement with prior observations that metformin slows growth of various tumor subtypes. These findings suggest that AMPK activation may be efficacious against growing tumors, but deleterious when used in combination with drugs that suppress tumor growth and induce regression. More broadly, this work highlights the issue that the time in a disease course needs to be considered when testing potential anti-cancer agents such as AMPK modulators. Citation Format: Miller TW, Hampsch RA, McCleery CF, Wells JD, Fields JL, Dillon LM, Shee K. Timing provides context for the paradoxical effects of AMPK activation in ER+ breast cancer: Suppressing growing tumors, but promoting dormant tumor cell survival and recurrence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-08.

AMPK activation enhances the anti-atherogenic effects of high density lipoproteins in apoE−/− mice

HDL plays crucial roles at multiple stages of the pathogenesis of atherosclerosis. AMP-activated protein kinase (AMPK) is a therapeutic candidate for the treatment of cardiovascular disease. However, the effect of AMPK activation on HDL functionality has not been established in vivo. We assessed the effects of pharmacological AMPK activation using A-769662, AICAR, metformin, and IMM-H007 on the atheroprotective functions of HDL in apoE-deficient (apoE−/−) mice fed with a high-fat diet. After administration, there were no changes in serum lipid levels among the groups. However, mice treated with AMPK activators showed significantly enhanced reverse cholesterol transport in vivo and in vitro. AMPK activation also increased the expression of ABCA1 and ABCG1 in macrophages and scavenger receptor class B type I and LCAT in the liver. HDL from AMPK activation mice exhibited lower HDL inflammatory index and myeloperoxidase activity and higher paraoxonase 1 activity than HDL from untreated mice, implying superior antioxidant and anti-inflammatory capacities. Pharmacological AMPK activation also induced polarization of macrophages to the M2 state and reduced plasma lipid peroxidation, inflammatory cytokine production, and atherosclerotic plaque formation in apoE−/− mice. These observations suggest that pharmacological AMPK activation enhances the anti-atherogenic properties of HDL in vivo. This likely represents a key mechanism by which AMPK activation attenuates atherosclerosis.

Preconditioning mice with activators of AMPK ameliorates ischemic acute kidney injury in vivo.

This study had two objectives: 1) to determine whether preconditioning cultured proximal tubular cells (PTCs) with pharmacological activators of AMP-activated protein kinase (AMPK) protects these cells from apoptosis induced by metabolic stress in vitro and 2) to assess the effects of preconditioning mice with these agents on the severity of ischemic acute renal kidney injury (AKI) in vivo. We demonstrate that preconditioning PTCs with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or A-769662 reduces apoptosis of PTCs induced by subsequent stress. We also show that the reduction in cell death during metabolic stress associated with pretreatment by AMPK activators is associated with an increase in the cytosolic level of ATP, which is mediated by an increase in the rate of glycolysis. In addition, we provide evidence that the effect of AMPK activators on glycolysis is mediated, at least in part, by an increased uptake of glucose, and by the induction of hexokinase II (HK II) expression. Our data also show that the increased in HK II expression associated with preconditioning with AMPK activators is mediated by the activation (phosphorylation) of the cAMP-response element binding protein (CREB). We also provide entirely novel evidence that that A-79662 is substantially more effective than AICAR in mediating these alterations in PTCs in vitro. Finally, we demonstrate that preconditioning mice with AICAR or A-769662 substantially reduces the severity of renal dysfunction and tubular injury in a model of ischemic AKI in vivo and that the efficacy of AICAR and A-768662 in ameliorating ischemic AKI in vivo is comparable.

The Paradoxical Effects of AMPK on Insulin Gene Expression and Glucose-Induced Insulin Secretion.

The activation of AMP-activated protein kinase (AMPK) is known to repress the expression of the insulin gene and glucose-stimulated insulin secretion (GSIS). However, the mechanisms by which this occurs, as well as the effects of AMPK activation on glucolipotoxicity-induced β-cell dysfunction, have not been elucidated. To investigate the effects of 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) and peroxisome proliferator-activated receptorγ-coactivator-1α (PGC-1α) on β-cell-specific genes under glucolipotoxic conditions, we performed real-time PCR and measured insulin secretion by primary islets. To study these effects in vivo, we administered AICAR for 10 days (1 mg/g body weight) to 90% pancreatectomized hyperglycemic mice. The exposure of isolated rat and human islets to glucolipotoxic conditions and the overexpression of PGC-1α suppressed insulin and NEUROD1 mRNA expression. However, the expression of these genes was preserved by AICAR treatment and by PGC-1α inhibition. Exposure of isolated islets to glucolipotoxic conditions for 3 days decreased GSIS, which was also well maintained by AICAR treatment and by PGC-1α inhibition. The administration of AICAR to 90% pancreatectomized hyperglycemic mice improved glucose tolerance and insulin secretion. These results indicate that treatment of islets with an AMPK agonist under glucolipotoxic conditions protects against glucolipotoxicity-induced β-cell dysfunction. A better understanding of the functions of molecules such as PGC-1α and AMPK, which play key roles in intracellular fuel regulation, could herald a new era for the treatment of patients with type 2 diabetes mellitus by providing protection against glucolipotoxicity.

AMPK—friend or foe for targeted therapy?

AMP-activated protein kinase (AMPK) consists of a catalytic α subunit and regulatory β and γ subunits, and is activated in response to an increase in the AMP/ATP ratio upon metabolic stresses. Two AMPK kinases have been identified to mediate activating phosphorylation of AMPKα at Thr172, namely liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) [reviewed in 1]. Increased AMP:ATP can activate AMPK allosterically by binding to the AMPKγ subunit and inhibiting dephosphorylation of Thr172 due to constitutive LKB1 activity. In cells expressing CaMKKβ, increased intracellular Ca2+ concentrations activate AMPK independent of changes in AMP:ATP. Once activated, AMPK phosphorylates multiple downstream catabolic targets that promote ATP generation, whereas anabolic processes including fatty acid synthesis and protein translation tend to be suppressed.1 During tumorigenesis and related depletion of cellular energy, AMPK activation is thought to favor energy-producing catabolic processes. The role of AMPK in prostate cancer (PC) remains to be fully characterized, with significant discrepancies in the literature. We have recently observed that AMPK Thr172 phosphorylation was significantly elevated in human prostate cancer (PC) cells and clinical PC samples. In clinical PC, while not statistically significant, there was a trend for increasing phospho-AMPK Thr172 with increasing Gleason sum score (or increasingly less differentiated disease). The increased AMPK Thr172 phosphoryation was associated with phosphorylation of the AMPK substrate, acetyl CoA-carboxylase (ACC; p = 0.0023), suggesting enhanced AMPK and inhibited ACC activities in PC.2 These findings support earlier reports of increased phospho-ACC levels in clinical PC.3 Functional significance of the observed upregulation of AMPK/ACC status in PC progression remains unclear. In DU145 cells, an androgen receptor (AR) negative human PC line with activated Akt and upregulated glycolysis, 5-aminoimidazole-4-carboxamide riboside (AICAR)-mediated AMPK activation has been reported to suppress proliferation without evidence of increased apoptosis.4 Our recent comparative analysis of the paired PC3 and PC3M cells as models of progressively invasive (androgen-independent) PC also supported the notion of tumor suppressing effects upon activation of AMPK (Fig. 1). Treatment with AICAR or an alternative direct AMPK activator, A-769662 suppressed proliferation, migration and invasion in both cell lines, with down-regulation of mTOR and P70S6Ki levels regardless of the Akt status. Involvement of AMPK was confirmed with the AMPK inhibitor, Compound C and siRNA-mediated AMPK silencing.2 Figure 1. A schematic representation of signaling network involving AMPK and other key signaling nodes in prostate carcinogenesis. The AMPK-mediated anti-tumor effects (proliferative/migratory/invasive) are considered downstream of fatty acid and protein synthesis. ... Despite similar functional responses in PC3 and PC3M cells, AMPK activation also resulted in sustained phospho-Akt activation in PC3M cells, but not PC3 cells. However, combining LY-294002 with AICAR to simultaneously suppress phosphatidylinositol 3′-kinase (PI3K) and activate AMPK did not produce any additional anti-proliferative effects in PC3M cells. This suggests that any efforts to exploit AMPK as a potential therapeutic target may be considered independent of PI3K/Akt signaling. This is a surprising finding as there is significant evidence for cross talk between AMPK and AKT/mTOR pathways. Indeed, the PI3K signaling network exhibits extensive cross-regulatory interactions with the intermediate metabolism network, including AMPK, to fuel resistance to stress and uncontrolled growth.5 Akt has also been reported to dramatically reduce the AMP/ATP ratio and suppress AMPK activity in cells overexpressing a constitutive Akt mutant.6 Hence, while the effects of AMPK activation was not influenced by a PI3K inhibitor in our study, this may be context and cell type specific, and a detailed understanding of AMPK/PI3K/mTOR crosstalk will have important implications in cancer therapies. Popovics et al.7 have recently hypothesized that AMPK activators used in pre-clinical studies are likely to promote autophagy and apoptosis since they mediate their effects, at least in part, by an inhibitory action on the mTORC1 complex. They proposed that modulators of the CaMKKβ-AMPK pathway may be used in combination with mTOR-specific inhibitors, such as the rapamycin analogs, to maximise anti-tumor effects, thus avoiding the the potential of stimulating diverse pro-tumor processes in parallel with antitumor effects when mTOR inhibitor is used alone. Ongoing research into the role of AMPK in the followings research priorities will provide important insight into its potential for targeted therapy: (1) The in vivo impact of AMPK-mediated signaling in the context of primary and metastatic diseases; (2) The complex interplay between CAMKKβ, androgen receptor and AMPK (Fig. 1); (3) Factors that modulate AMPK-induced effects on cell fate (including survival, apoptosis and autophagy) and motility; and (4) Factors influencing the metabolic consequences of AMPK functions, including glucose and lipid metabolism.

AMPK Activation Affects Glutamate Metabolism in Astrocytes

Mammalian AMP-activated protein kinase (AMPK) functions as a metabolic switch. It is composed of 3 different subunits and its activation depends on phosphorylation of a threonine residue (Thr172) in the α-subunit. This phosphorylation can be brought about by 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) which in the cells is converted to a monophosphorylated nucleotide mimicking the effect of AMP. We show that the preparation of cultured astrocytes used for metabolic studies expresses AMPK, which could be phosphorylated by exposure of the cells to AICAR. The effect of AMPK activation on glutamate metabolism in astrocytes was studied using primary cultures of these cells from mouse cerebral cortex during incubation in media containing 2.5mM glucose and 100µM [U-(13)C]glutamate. The metabolism of glutamate including a detailed analysis of its metabolic pathways involving the tricarboxylic acid (TCA) cycle was studied using high-performance liquid chromatography analysis supplemented with gas chromatography-mass spectrometry technology. It was found that AMPK activation had profound effects on the pathways involved in glutamate metabolism since the entrance of the glutamate carbon skeleton into the TCA cycle was reduced. On the other hand, glutamate uptake into the astrocytes as well as its conversion to glutamine catalyzed by glutamine synthetase was not affected by AMPK activation. Interestingly, synthesis and release of citrate, which are hallmarks of astrocytic function, were affected by a reduction of the flux of glutamate derived carbon through the malic enzyme and pyruvate carboxylase catalyzed reactions. Finally, it was found that in the presence of glutamate as an additional substrate, glucose metabolism monitored by the use of tritiated deoxyglucose was unaffected by AMPK activation. Accordingly, the effects of AMPK activation appeared to be specific for certain key processes involved in glutamate metabolism.

Effect of AMPK activation on monocarboxylate transporter (MCT)1 and MCT4 in denervated muscle

It is now evident that exercise training leads to increases in monocarboxylate transporter (MCT)1 and MCT4, but little is known about the mechanisms of coupling muscle contraction with these changes. The aim of this study was to investigate the effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) induced activation of AMP-activated protein kinase (AMPK) on MCT1, MCT4, and GLUT4 in denervated muscle. Protein levels of MCT4 and GLUT4 after 10days of denervation were significantly decreased in mice gastrocnemius muscle, while MCT1 protein levels were not altered. AICAR treatment for 10days significantly increased MCT4, and GLUT4 protein levels in innervated muscle as shown in previous studies. We found that the MCT1 protein level was also increased in AICAR treated innervated muscle. AICAR treatment prevented the decline in MCT4 and GLUT4 protein levels in denervated muscle. Thus, the current study suggests that MCT1 and MCT4 protein expression in muscles, as well as GLUT4, may be regulated by AMPK-mediated signal pathways, and AMPK activation can prevent denervation-induced decline in MCT4 protein.

Energy Depletion of Bovine Mammary Epithelial Cells Activates AMPK and Suppresses Protein Synthesis Through Inhibition of mTORC1 Signaling

The molecular mechanisms by which cellular energy status regulates global protein synthesis in mammary epithelial cells have not been characterized. The objective of this study was to examine the effect of AMP-activated protein kinase (AMPK) activation by 2-deoxyglucose on protein synthesis and the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in bovine mammary epithelial cells. Phosphorylation of AMPK at Thr172 increased by 1.4-fold within 5 min, and remained elevated throughout a 30-min time course, in response to 2-deoxyglucose. Global rates of protein synthesis declined by 78% of control values. The decline in protein synthesis was associated with repression of mTORC1 signaling, as indicated by reduced phosphorylation of ribosomal protein S6 kinase 1 and eIF4E binding protein-1 (4E-BP1). Phosphorylation of ER-stress marker eIF2α was also increased but only at 30 min of 2-deoxyglucose exposure. 2-Deoxyglucose increased phosphorylation of tuberous sclerosis complex 2 (TSC2) on AMPK consensus sites but did not change the amount of TSC1 bound to TSC2. Activation of AMPK did not result in changes in the amount of raptor bound to mTOR. The inhibitory effects of AMPK activation on mTORC1 signaling were associated with a marked increase in Ser792 phosphorylation on raptor. Collectively, the results suggest that activation of AMPK represses global protein synthesis in mammary epithelial cells through inhibition of mTORC1 signaling.

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

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

AMPK promotes skeletal muscle autophagy through activation of forkhead FoxO3a and interaction with Ulk1

In skeletal muscle, protein levels are determined by relative rates of protein synthesis and breakdown. The balance between synthesis and degradation of intracellular components determines the overall muscle fiber size. AMP-activated protein kinase (AMPK), a sensor of cellular energy status, was recently shown to increase myofibrillar protein degradation through the expression of MAFbx and MuRF1. In the present study, the effect of AMPK activation by AICAR on autophagy was investigated in muscle cells. Our results show that FoxO3a transcription factor activation by AMPK induces the expression of the autophagy-related proteins LC3B-II, Gabarapl1, and Beclin1 in primary mouse skeletal muscle myotubes and in the Tibialis anterior (TA) muscle. Time course studies reveal that AMPK activation by AICAR leads to a transient nuclear relocalization of FoxO3a followed by an increase of its cytosolic level. Moreover, AMPK activation leads to the inhibition of mTORC1 and its subsequent dissociation of Ulk1, Atg13, and FIP200 complex. Interestingly, we identify Ulk1 as a new interacting partner of AMPK in muscle cells and we show that Ulk1 is associated with AMPK under normal conditions and dissociates from AMPK during autophagy process. Moreover, we find that AMPK phosphorylates FoxO3a and Ulk1. In conclusion, our data show that AMPK activation stimulates autophagy in skeletal muscle cells through its effects on the transcriptional function of FoxO3a and takes part in the initiation of autophagosome formation by interacting with Ulk1. Here, we present new evidences that AMPK plays a crucial role in the fine tuning of protein expression programs that control skeletal muscle mass.

Abstract A79: Evidence that the antitumor effects of AMPK activation and crosstalk with the Akt pathway are dependent on the genetic profiles of human breast cancer cell types.

Abstract The prevalence of breast cancer and drawbacks of many current therapeutic modalities underline the need for novel therapies. Those based on signal transduction have the potential for fewer side effects due to inherent redundancy of pathways. The fuel sensing enzyme, AMP-activated protein kinase (AMPK), is activated by low energy levels. We (El-Masry et al, Oncology Letters, in press), and others, have identified that AMPK is involved in cell proliferation and growth and could be a potential therapeutic target in cancer. Pharmacological activation of AMPK in MCF-7 (p53wt, ER+), MDA-MB-231 (p53−, ER−) and T47D (p53−, ER+) human breast cancer cell lines using 5-Aminoimidazole-4-carboxamide ribotide (AICA-ribonucleotide) resulted in anti-proliferative effects, with T47D cells being the most sensitive. These effects correlated with changes in apoptosis, including mitochondrial membrane potential (assayed by fluorescence activated cell sorting (FACS)) and Bax translocation to the outer mitochondrial membrane (assayed by immunofluorescence) suggesting that the apoptotic signal was p53 independent. Caspase inhibition showed that the anti-proliferative effect of AMPK activation was mediated by caspase 9 in all cell types. Execution was by caspase 6 in MCF-7 cells and caspase 3 and 6 in MDA-MB-231 cells. T47D cells were less responsive to both caspase inhibition and PARP cleavage than MCF-7 and MDA-MB-231. AMPK activation also resulted in trans-activation of p21, although this was dependent on the basal level of p21 in the different cell types. Previously we reported differential effects on the same cell lines in terms of PI3K/Akt signaling. Our current studies on possible crosstalk between the AMPK and PI3K/Akt pathways show that, whilst AMPK activation led to inhibition of Akt phosphorylation, the effect of Akt inhibition on AMPK phophorylation was cell type dependent. Akt inhibition increased phosphorylation of AMPK in T47D cells, but not in MCF-7 or MDA-MB-231 cells. Inhibition of Akt also decreased proliferation of T47D and MCF-7 cells, without effect on MDA-MB-231 cells. The adipokine leptin inhibited AMPK phosphorylation in all cell lines. Blockade of either Akt or mTOR inhibited this effect of leptin in T47D cells, suggesting that was mediated, at least in part, by activation of Akt, but did not affect MCF-7 or MDA-MB-231 cells. These data demonstrate crosstalk between metabolic and survival signaling. This interactive signaling was cell type dependent. In conclusion, the potential therapeutic effects of AMPK and its interaction with survival pathways were found to be different depending on genetics of the breast cancers investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A79.

Activation of AMP-Activated Protein Kinase Prevents Lipotoxicity in Retinal Pericytes

The recent FIELD study demonstrated that the lipid-lowering agent fenofibrate significantly reduces the development and progression of diabetic retinopathy (DR). These results suggest that lipids may play a causal role in DR. They also suggest that AMP-activated protein kinase (AMPK) activation could account for these findings given that fenofibrate is an AMPK activator. The authors previously demonstrated that free fatty acids, in addition to hyperglycemia, can induce apoptosis in retinal pericytes (PCs), the first cells lost in the diabetic retina. Incubation with the saturated fatty acid palmitate, but not the monounsaturated fatty acid oleate, elicited cytotoxicity in a manner dependent on oxidative stress, NF-κB activation, and ceramide accumulation. In this study, the authors explored whether AMPK can downregulate these pathways and, in doing so, protect PCs from apoptosis. PCs were incubated with palmitate or oleate to determine whether the factors previously linked to lipotoxicity were uniquely increased by palmitate. The effects of AMPK activation on these parameters and on apoptosis were concurrently examined. Only palmitate increased NF-κB activation, ceramide and diacylglycerol mass, and apoptosis. Activation of AMPK with AICAR or, where used, expression of a constitutively active AMPK prevented all these effects. In contrast, both palmitate and oleate markedly increased oxidative stress, and the activation of AMPK did not prevent this. AMPK activation prevents the metabolic abnormalities and apoptosis specifically caused by palmitate in cultured PCs. Pharmacologic agents that activate AMPK in the diabetic retina may warrant consideration as a therapeutic option to avert PC apoptosis and to maintain microvascular homeostasis.

Abstract 605: A novel role for AMPK as a molecular switch regulating the activation of Akt and RAS signaling in acute lymphoblastic leukemia

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related death in children. In search for novel treatment strategies, we identified AMP activated protein kinase (AMPK) as a potential target for ALL therapy due to its effects on cell proliferation and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We demonstrated that treatment of NALM6 and CCRF-CEM cells with the AMPK activators AICAR and metformin induced significant cell growth inhibition and apoptosis. Rescue experiments using the AMPK inhibitor compound-C (CC) failed to completely abrogate the cytotoxic effects induced by AICAR, attenuating AICAR's apoptosis by 42% in CCRF-CEM and 45% in NALM6. Whereas when used alone, both CC and Ara-A induced significant apoptosis in ALL cells. Using the caspase inhibitors Z-VAD and Z-IETD, we demonstrated that activation of AMPK by AICAR induced cell death via both caspase-dependent and independent mechanisms, whereas inhibition of AMPK by CC induced apoptosis mainly via the intrinsic caspase-dependent pathway. To examine the effects of AMPK activation vs. inhibition on downstream signaling, we used Western blot analysis of key signaling factors associated with the PI3K/Akt/mTOR and RAS/RAF/ERK pathways in ALL cells. We found that AICAR and CC exerted opposite effects on PI3K/Akt and RAS pathway signaling. CC decreased P-Akt (Ser473) and activated the RAS pathway, whereas AICAR increased P-Akt, suggesting that AMPK may play a role as regulator of Akt and RAS activity. We showed that activation of AMPK and Akt by AICAR down-regulated the RAS pathway via phosphorylation of cRAF at Ser621 and Ser259, respectively. Inhibition of the RAS pathway with farnesylthiosalicylic acid (FTS) and U-0126 induced significant cell growth arrest and apoptosis in ALL cells (p<0.0001) and resulted in up-regulation of P-AMPK (Thr172) and P-Akt (Thr308). The phosphorylation of Akt at Thr308 was mediated by AMPK-dependent activation of IGF-1R/IRS-1, since down-regulation of AMPK using shRNA abolished the activation of both P-Akt (Thr308) and IGF-1R (Tyr1131). Inhibition of AMPK activity with either CC or shRNA sensitized ALL cells to RAS inhibition (p<0.001). In addition, co-targeting RAS pathway signaling plus either IGF-1R or Akt pathways resulted in significant cell growth inhibition and apoptosis in ALL cells (p<0.001), confirming the functional relevance of this feedback loop mechanism. Taken together, our results indicate that activation and inhibition of AMPK induce cell death in ALL cells through alternative signaling mechanisms impinging on the expression of critical cell proliferation pathways. The data presented here support a new role for AMPK as a molecular regulatory switch coordinating cellular survival responses by activating PI3K/Akt/mTOR and RAS/RAF/ERK oncogenic pathways in ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2011-605

AMP-Activated Protein Kinase Inhibits Homocysteine-Induced Dysfunction and Apoptosis in Endothelial Progenitor Cells

Homocysteine (Hcy) has been shown to induce oxidative stress and apoptosis of endothelial progenitor cells (EPCs). AMP-activated protein kinase (AMPK) has been reported to have protective effects on endothelial function. However, effects of AMPK activation on Hcy-induced EPCs injury remain to be determined. In this study, we examined the effect of AMPK phosphorylation on Hcy-induced NO bioavailability impairment and NADPH oxidase 4 (Nox4) derived reactive oxygen species (ROS) accumulation in EPCs. EPCs were pre-treated with various concentrations of 5-amino-4-imidazolecarboxamide riboside-l-beta-D-ribofuranoside (AICAR), a pharmacological agonist of AMPK, and then incubated with Hcy for 24 h. Furthermore, we challenged EPCs with Hcy in the presence or absence of atorvastatin and AMPK-DN which expressed a dominant-negative mutant of AMPK. Migration, proliferation and apoptosis were assayed to evaluate EPCs function. NO production, expression of endothelial nitric oxide synthase (eNOS), intracellular ROS levels and Nox4 activation were determined to explore the potential mechanisms of Hcy-induced EPCs dysfunction. We observed that AICAR attenuated the inhibition effects of Hcy on EPCs migration and proliferation. The apoptosis rates of EPCs were down-regulated by AICAR compared with the group treated with Hcy only [(0.25 mmol/L AICAR: 10.48 ± 1.6%; 0.5 mmol/L AICAR: 8.70 ± 1.0%; 1 mmol/L AICAR: 5.83 ± 1.3%) vs. (500 μmol/L Hcy only: 12.60 ± 1.9%)]. We also found that NO production and eNOS expression were up-regulated by AICAR compared with the group treated with Hcy only, while ROS accumulation and Nox4 activation were inhibited. Furthermore, atorvastatin suppressed Hcy-induced dysfunction of EPCs, increased NO production and eNOS expression, and down-regulated ROS accumulation and Nox4 activation. And these effects of atorvastatin could be blunted by AMPK-DN. AMPK activation inhibits eNOS down-regulation and Nox4-derived ROS accumulation induced by Hcy in EPCs, and may contribute to the protective effects of atorvastatin on endothelial function.