Increased AMP-activated Protein Kinase Phosphorylation Research Articles

Menopause is associated with decreased whole body fat oxidation during exercise

The purpose of this study was to examine if fat oxidation was affected by menopausal status and to investigate if this could be related to the oxidative capacity of skeletal muscle. Forty-one healthy women were enrolled in this cross-sectional study [premenopausal (n = 19), perimenopausal (n = 8), and postmenopausal (n = 14)]. Estimated insulin sensitivity was obtained from an oral glucose tolerance test. Body composition was measured by dual-energy X-ray absorptiometry and magnetic resonance imaging. Fat oxidation and energy expenditure were measured during an acute exercise bout of 45 min of ergometer biking at 50% of maximal oxygen consumption (Vo2 max). Muscle biopsies from the vastus lateralis of the quadriceps muscle were obtained before and immediately after the exercise bout. Postmenopausal women had 33% [confidence interval (CI) 95%: 12-55] lower whole body fat oxidation (P = 0.005) and 19% (CI 95%: 9-22) lower energy expenditure (P = 0.02) during exercise, as well as 4.28 kg lower lean body mass (LBM) than premenopausal women. Correction for LBM reduced differences in fat oxidation to 23% (P = 0.05), whereas differences in energy expenditure disappeared (P = 0.22). No differences between groups were found in mRNA [carnitine palmitoyltransferase I, β-hydroxyacyl-CoA dehydrogenase (β-HAD), peroxisome proliferator-activated receptor-α, citrate synthase (CS), pyruvate dehydrogenase kinase 4, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)], protein [phosphorylated AMP-activated protein kinase (AMPK), vascular endothelial growth factor, pyruvate dehydrogenase-1Eα, cytochrome oxidase I], or enzyme activities (β-HAD, CS) in resting skeletal muscle, except for an increased protein level of cytochrome c in the post- and perimenopausal women relative to premenopausal women. Postmenopausal women demonstrated a trend to a blunted exercise-induced increase in phosphorylation of AMPK compared with premenopausal women (P = 0.06). We conclude that reduced whole body fat oxidation after menopause is associated with reduced LBM.

백모근(白茅根) 청피(靑皮) 오수유(吳茱萸) 복합방(複合方)(YY312)의 고지방식이로 유도된 마우스와 3T3-L1 세포에서 항비만 효과

Objectives : The purpose of this study was to determine the anti-obesity effect and molecular mechanism of YY312, a herbal extract composed of Imperatae Rhizoma, Citri Unshius Pericarpium Immaturus, and Evodiae Fructus, on a high-fat diet-induced animal model and on 3T3-L1 cells. Methods : C57BL/6 mice were fed for 6 weeks with a normal diet or a high-fat diet (HFD). Then they orally administered daily with 300 mg/kg YY312 for next 10 weeks. Body weight and food consumption were recorded weekly and daily, respectively. Tissue weights, serum lipid, and glucose levels were analyzed at the end of the study. Additionally, the effects of YY312 on adipocyte differentiation in 3T3-L1 cells were examined. After differentiating 3T3-L1 cells were treated with YY312, Oil-red O staining, RT-PCR, and Western blotting were performed for lipid accumulation, mRNA expression of adipogenesis gene, and AMP-activated protein kinase (AMPK) phosphorylation, respectively. Results : YY312-administered mice showed a significant reduction of body weights and abdominal adipose tissue weights. YY312 also reduced the serum levels of triglycerides and total cholesterol, compared with the HFD group. Treatment with YY312 inhibited lipid accumulation and blocked expression of adipogenic transcription factors and lipogenesis genes, including peroxisome proliferator-activated receptor <TEX>${\gamma}$</TEX>, CCAT/enhancer binding protein <TEX>${\alpha}$</TEX> and fatty acid synthase. YY312 increased AMPK phosphorylation in 3T3-L1 adipocytes. Conclusions : This study showed that herbal extract YY312 has an anti-obesity effect in vitro and in vivo. Thus, YY312 could be developed as a supplement for reduction of body weight gain induced by an HFD.

AMP‐activated protein kinase (AMPK)–induced preconditioning in primary cortical neurons involves activation of MCL‐1

Neuronal preconditioning is a phenomenon where a previous exposure to a sub-lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP-activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling-induced preconditioning in neurons, however, the molecular basis of AMPK-mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5-aminoimidazole-4-carboxamide riboside (AICAR) in a model of NMDA-mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA-induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) in AICAR-preconditioned neurons. Interestingly, over-expression of MCL-1 protected neurons against NMDA-induced excitotoxicity while MCL-1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca²⁺ levels during NMDA excitation revealed that MCL-1 over-expressing neurons exhibited improved bioenergetics and markedly reduced Ca²⁺ elevations, suggesting a potential mechanism through which MCL-1 confers neuroprotection. This study identifies MCL-1 as a key effector of AMPK-induced preconditioning in neurons.

A role for inducible 6-phosphofructo-2-kinase in the control of neuronal glycolysis

Increased glycolysis is the result of the sensing of glucose by hypothalamic neurons. The biochemical mechanisms underlying the control of hypothalamic glycolysis, however, remain to be elucidated. Here we showed that PFKFB3, the gene that encodes for inducible 6-phosphofructo-2-kinase (iPFK2), was expressed at high abundance in both mouse hypothalami and clonal hypothalamic neurons. In response to re-feeding, PFKFB3 mRNA levels were increased by 10-fold in mouse hypothalami. In the hypothalamus, re-feeding also decreased the phosphorylation of AMP-activated protein kinase (AMPK) (Thr172) and the mRNA levels of agouti-related protein (AgRP), and increased the mRNA levels of cocaine-amphetamine-related transcript (CART). Similar results were observed in N-43/5 clonal hypothalamic neurons upon treatment with glucose and/or insulin. In addition, knockdown of PFKFB3/iPFK2 in N-43/5 neurons caused a decrease in rates of glycolysis, which was accompanied by increased AMPK phosphorylation, increased AgRP mRNA levels and decreased CART mRNA levels. In contrast, overexpression of PFKFB3/iPFK2 in N-43/5 neurons caused an increase in glycolysis, which was accompanied by decreased AMPK phosphorylation and decreased AgRP mRNA levels and increased CART mRNA levels. Together, these results suggest that PFKFB3/iPFK2 responds to re-feeding, which in turn stimulates hypothalamic glycolysis and decreases hypothalamic AMPK phosphorylation and alters neuropeptide expression in a pattern that is associated with suppression of food intake.

Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis

Salvianolic acid A (SalA) is one of the main efficacious, water-soluble constituents of Salvia miltiorrhiza Bunge. This study investigated the protective effects of SalA on peripheral nerve in diabetic rats. Administration of SalA (0.3, 1 and 3 mg/kg, ig) was started from the 5th week after strepotozotocin (STZ60 mg/kg) intraperitoneal injection and continued for 8 weeks. Paw withdrawal mechanical threshold (PWMT) and motor nerve conduction velocity (MNCV) were used to assess peripheral nerve function. The western blot methods were employed to test the expression levels of serine-threonine liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α), silent information regulator protein3 (sirtuin 3/Sirt3) and neuronal nitric oxide synthase (nNOS) in sciatic nerve. Results showed that SalA administration could increase PWMT and MNCV in diabetic rats; reduce the deterioration of sciatic nerve pathology; increase AMPK phosphorylation level, up-regulate PGC-1α, Sirt3 and nNOS expression, but had no influence on LKB1. These results suggest that SalA has protective effects against diabetic neuropathy. The beneficial effects of SalA on peripheral nerve function in diabetic rats might be attributed to improvements in glucose metabolism through regulation of the AMPK-PGC1α-Sirt3 axis.

Alpha-lipoic acid induces adipose triglyceride lipase expression and decreases intracellular lipid accumulation in HepG2 cells

Non-alcoholic fatty liver disease can be attributed to the imbalance between lipogenesis and lipolysis in the liver. Alpha-lipoic acid has been shown to activate the 5′-AMP-activated protein kinase (AMPK) signalling pathway and to effectively inhibit the lipogenesis pathway in liver. However, whether alpha-lipoic acid stimulates lipolysis remains unclear. Recently, adipose triglyceride lipase (ATGL) was shown to be responsible for triacylglycerol hydrolase activity in cells. In the present study, we established a fatty liver cell model by incubating HepG2 cells in a high glucose (30mM glucose) and high fat (0.1mM palmitate) medium. We found that the activation of the AMPK signalling pathway induced ATGL protein expression and enhanced lipid hydrolysis. Similarly, treatment of the fatty liver cell model with alpha-lipoic acid reduced intracellular lipid accumulation in HepG2 cells, increased AMPK phosphorylation, and induced ATGL expression. We showed that insulin phosphorylates the transcription factor forkhead box O1 (FOXO1), which regulates ATGL expression and inhibits FOXO1 translocation into the nucleus. In contrast, alpha-lipoic acid dephosphorylated FOXO1 and reversed the nuclear exclusion of FOXO1. These data suggest that alpha-lipoic acid can effectively ameliorate intracellular lipid accumulation and induce ATGL expression through the FOXO1/ATGL pathway in liver cells. Thus, alpha-lipoic acid may be a potential therapeutic agent for treating fatty liver disease.

Activation of AMP-activated Protein Kinase Stimulates Na+,K+-ATPase Activity in Skeletal Muscle Cells

Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.

Fructose-Induced Hypothalamic AMPK Activation Stimulates Hepatic PEPCK and Gluconeogenesis due to Increased Corticosterone Levels

Fructose consumption causes insulin resistance and favors hepatic gluconeogenesis through mechanisms that are not completely understood. Recent studies demonstrated that the activation of hypothalamic 5'-AMP-activated protein kinase (AMPK) controls dynamic fluctuations in hepatic glucose production. Thus, the present study was designed to investigate whether hypothalamic AMPK activation by fructose would mediate increased gluconeogenesis. Both ip and intracerebroventricular (icv) fructose treatment stimulated hypothalamic AMPK and acetyl-CoA carboxylase phosphorylation, in parallel with increased hepatic phosphoenolpyruvate carboxy kinase (PEPCK) and gluconeogenesis. An increase in AMPK phosphorylation by icv fructose was observed in the lateral hypothalamus as well as in the paraventricular nucleus and the arcuate nucleus. These effects were mimicked by icv 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment. Hypothalamic AMPK inhibition with icv injection of compound C or with injection of a small interfering RNA targeted to AMPKα2 in the mediobasal hypothalamus (MBH) suppressed the hepatic effects of ip fructose. We also found that fructose increased corticosterone levels through a mechanism that is dependent on hypothalamic AMPK activation. Concomitantly, fructose-stimulated gluconeogenesis, hepatic PEPCK expression, and glucocorticoid receptor binding to the PEPCK gene were suppressed by pharmacological glucocorticoid receptor blockage. Altogether the data presented herein support the hypothesis that fructose-induced hypothalamic AMPK activation stimulates hepatic gluconeogenesis by increasing corticosterone levels.

AMP‐activated protein kinase mediates erythropoietin‐induced activation of endothelial nitric oxide synthase

We investigated whether AMP-activated protein kinase (AMPK), a multi-functional regulator of energy homeostasis, participates in the regulation of erythropoietin (EPO)-mediated activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) and mice. In ECs, treatment with EPO increased the phosphorylation of AMPK, acetyl-CoA carboxylase (ACC), and eNOS, as revealed by Western blot analysis. Inhibition of AMPK activation by compound C or dominant-negative AMPK mutant abrogated the EPO-induced increase in the phosphorylation of AMPK, ACC, and eNOS, as well as nitric oxide (NO) production. Additionally, suppression of AMPK activation abolished EPO-induced EC proliferation, migration and tube formation. Immunoprecipitation analysis demonstrated that AMPK mediated the EPO-induced increase in the phosphorylation of β common receptor (βCR) and the formation of a βCR-AMPK-eNOS complex. In mice, inhibition of AMPK activation by compound C markedly decreased EPO-elicited angiogenesis in Matrigel plugs. Furthermore, the phosphorylation of AMPK and eNOS was significantly higher in aortas from EPO transgenic mice than wild-type mice. Moreover, treatment with EPO neutralizing antibody greatly reduced the exercise training-induced increase in phosphorylation of AMPK and eNOS in aortas of wild-type mice. Taken together, EPO may trigger AMPK-dependent signaling, which leads to enhanced phosphorylation of βCR and eNOS, increased βCR-AMPK-eNOS complex formation, NO production, and, ultimately, angiogenesis.

Ataxia telangiectasia mutated (ATM) influences AICAR‐stimulated glucose transport

Literature reports suggest that ATM can activate the AMP‐activated protein kinase (AMPK), a protein that can stimulate glucose transport in skeletal muscle. We hypothesized that AICAR, an AMPK activator, would increase AMPK phosphorylation and glucose transport in an ATM‐dependent manner. Extensor digitorum longus (EDL) muscles from wild‐type mice were exposed to 2 mM AICAR for one hour in the absence or presence of KU55933, a specific inhibitor of ATM. In separate procedures, wild‐type and ATM‐deficient (ATM−/−) EDL were exposed to AICAR. AICAR‐stimulated glucose transport was prevented by the ATM inhibitor despite no effect on AICAR‐stimulated phosphorylation of AMPK. AICAR increased glucose transport in EDL from wild‐type but not ATM−/− mice despite normal AICAR‐stimulated phosphorylation of AMPK in ATM−/− mice. Preliminary work (n=2–3) suggests that AICAR‐stimulated phosphorylation of TBC1D1 at S237 is prevented by inhibition of ATM. Our results suggest that ATM plays a role in AICAR‐stimulated glucose transport. However, contrary to our original hypothesis, ATM's influence occurs independent of AMPK phosphorylation, perhaps at the level of TBC1D1. Supported by USPHS award R15DK080437.

Implication of AMP-Activated Protein Kinase in Transient Receptor Potential Vanilloid Type 1-Mediated Activation of Endothelial Nitric Oxide Synthase

We investigated whether AMP-activated protein kinase (AMPK), a multifunctional regulator of energy homeostasis, is involved in transient receptor potential vanilloid type 1 (TRPV1)-mediated activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) and mice. In ECs, treatment with evodiamine, the activator of TRPV1, increased the phosphorylation of AMPK, acetyl-CoA carboxylase (ACC) and eNOS, as revealed by Western blot analysis. Inhibition of AMPK activation by compound C or dominant-negative AMPK mutant abrogated the evodiamine-induced increase in phosphorylation of AMPK and eNOS and NO bioavailability, as well as tube formation in ECs. Immunoprecipitation and two-hybrid analysis demonstrated that AMPK mediated the evodiamine-induced increase in the formation of a TRPVl-eNOS complex. Additionally, TRPV1 activation by evodiamine increased the phosphorylation of AMPK and eNOS in aortas of wild-type mice but did not activate eNOS in aortas of TRPV1-deficient mice. In mice, inhibition of AMPK activation by compound C markedly decreased evodiamine-evoked angiogenesis in matrigel plugs and in a hind-limb ischemia model. Moreover, evodiamine-induced phosphorylation of AMPK and eNOS in aortas of apolipoprotein E-deficient (ApoE−/−) mice was abrogated in TRPVl-deficient ApoE−/− mice. In conclusion, TRPV1 activation may trigger AMPK-dependent signaling, which leads to enhanced activation of AMPK and eNOS and retarded development of atherosclerosis.

The Outcome of Renal Ischemia-Reperfusion Injury Is Unchanged in AMPK-β1 Deficient Mice

AimActivation of the master energy-regulator AMP-activated protein kinase (AMPK) in the heart reduces the severity of ischemia-reperfusion injury (IRI) but the role of AMPK in renal IRI is not known. The aim of this study was to determine whether activation of AMPK by acute renal ischemia influences the severity of renal IRI.MethodsAMPK expression and activation and the severity of renal IRI was studied in mice lacking the AMPK β1 subunit and compared to wild type (WT) mice.ResultsBasal expression of activated AMPK, phosphorylayed at αThr172, was markedly reduced by 96% in AMPK-β1−/− mice. Acute renal ischaemia caused a 3.2-fold increase in α1-AMPK activity and a 2.5-fold increase in α2-AMPK activity (P<0.001) that was associated with an increase in AMPK phosphorylation of the AMPK-α subunit at Thr172 and Ser485, and increased inhibitory phosphorylation of the AMPK substrate acetyl-CoA carboxylase. After acute renal ischemia AMPK activity was reduced by 66% in AMPK-β1−/− mice compared with WT. There was no difference, however, in the severity of renal IRI at 24-hours between AMPK-β1−/− and WT mice, as measured by serum urea and creatinine and histological injury score. In the heart, macrophage migration inhibitory factor (MIF) released during IRI contributes to AMPK activation and protects from injury. In the kidney, however, no difference in AMPK activation by acute ischemia was observed between MIF−/− and WT mice. Compared with the heart, expression of the MIF receptor CD74 was found to be reduced in the kidney.ConclusionThe failure of AMPK activation to influence the outcome of IRI in the kidney contrasts with what is reported in the heart. This difference might be due to a lack of effect of MIF on AMPK activation and lower CD74 expression in the kidney.

Berberine-induced AMPK activation inhibits the metastatic potential of melanoma cells via reduction of ERK activity and COX-2 protein expression

Berberine is clinically important natural isoquinoline alkaloid that affects various biological functions, such as cell proliferation, migration and survival. The activation of AMP-activated protein kinase (AMPK) regulates tumor cell migration. However, the specific role of AMPK on the metastatic potential of cancer cells remains largely unknown. The present study investigated whether berberine induces AMPK activation and whether this induction directly affects mouse melanoma cell migration, adhesion and invasion. Berberine strongly increased AMPK phosphorylation via reactive oxygen species (ROS) production. 5-Aminoimidazole-4-carboxamide-1-β- d-ribofuranoside (AICAR), a well-known AMPK activator, also inhibited tumor cell adhesion and invasion and reduced the expression of epithelial to mesenchymal transition (EMT)-related genes. Knockdown of AMPKα subunits using siRNAs significantly abated the berberine-induced inhibition of tumor cell invasion. Furthermore, berberine inhibited the metastatic potential of melanoma cells through a decrease in ERK activity and protein levels of cyclooxygenase-2 (COX-2) by a berberine-induced AMPK activation. These data were confirmed using specific MEK inhibitor, PD98059, and a COX-2 inhibitor, celecoxib. Berberine- and AICAR-treated groups demonstrated significantly decreased lung metastases in the pulmonary metastasis model in vivo. Treatment with berberine also decreased the metastatic potential of A375 human melanoma cells. Collectively, our results suggest that berberine-induced AMPK activation inhibits the metastatic potential of tumor cells through a reduction in the activity of the ERK signaling pathway and COX-2 protein levels.

Suppression of 5′-Nucleotidase Enzymes Promotes AMP-activated Protein Kinase (AMPK) Phosphorylation and Metabolism in Human and Mouse Skeletal Muscle

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

Arctigenin Efficiently Enhanced Sedentary Mice Treadmill Endurance

Physical inactivity is considered as one of the potential risk factors for the development of type 2 diabetes and other metabolic diseases, while endurance exercise training could enhance fat oxidation that is associated with insulin sensitivity improvement in obesity. AMP-activated protein kinase (AMPK) as an energy sensor plays pivotal roles in the regulation of energy homeostasis, and its activation could improve glucose uptake, promote mitochondrial biogenesis and increase glycolysis. Recent research has even suggested that AMPK activation contributed to endurance enhancement without exercise. Here we report that the natural product arctigenin from the traditional herb Arctium lappa L. (Compositae) strongly increased AMPK phosphorylation and subsequently up-regulated its downstream pathway in both H9C2 and C2C12 cells. It was discovered that arctigenin phosphorylated AMPK via calmodulin-dependent protein kinase kinase (CaMKK) and serine/threonine kinase 11(LKB1)-dependent pathways. Mice treadmill based in vivo assay further indicated that administration of arctigenin improved efficiently mice endurance as reflected by the increased fatigue time and distance, and potently enhanced mitochondrial biogenesis and fatty acid oxidation (FAO) related genes expression in muscle tissues. Our results thus suggested that arctigenin might be used as a potential lead compound for the discovery of the agents with mimic exercise training effects to treat metabolic diseases.

Activation of AMP-activated Protein Kinase Is Essential for Lysophosphatidic Acid-induced Cell Migration in Ovarian Cancer Cells

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-β3 (PLC-β3) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPKα1, PLC-β3, or (CaMKKβ) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPKα1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.

Chronic high cytosolic calcium decreases AICAR-induced AMPK activity via calcium/calmodulin activated protein kinase II signaling cascade

Calcium is important for muscle contraction and controls many cellular processes. Although there is evidence that calcium-mediated signals regulate AMP-activated protein kinase (AMPK) activity, the molecular mechanisms by which calcium regulates AMPK are poorly understood. To compare the function of sustained vs. intermittent calcium oscillations on AMPK activity and define specific signals in this pathway, we administered mice with aminoimidazole-carboxamide-ribonucleotide (AICAR) and caffeine with or without dantrolene. AMPK activity was increased by 10 d AICAR treatment ( P < 0.01). Ten day caffeine treatment decreased AICAR-induced AMPK activity to control level. This repressed AMPK activity was blocked by dantrolene. Different calcium frequencies were simulated in C2C12 myotubes by alternating media containing caffeine and dantrolene. Intermittent calcium oscillation increased AMPK activity compared to control ( P < 0.05), whereas sustained calcium oscillation decreases AICAR-induced AMPK activity to control level. This result suggests a biphasic control of AMPK activity by calcium. Knockdown of CaMKII expression by short-hairpin RNA resulted in increased AMPK phosphorylation by AICAR even in the presence of caffeine. These data show different calcium oscillations elicit distinct responses in muscle cells suggesting that the negative effects of chronic calcium treatment on AMPK activity is partly mediated through the CaMKII signals.

AMP-activated protein kinase plays a role in initiating metabolic rate suppression in goldfish hepatocytes

In the present study, we test the hypothesis that AMP-activated protein kinase (AMPK) initiates metabolic rate suppression in isolated goldfish hepatocytes. To accomplish this, we attempted to pharmacologically activate AMPK in goldfish hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the thienopyridone, A769662, to examine the effects of AMPK activation on eukaryotic elongation factor-2 (eEF2), protein synthesis, and cellular oxygen consumption rate ([Formula: see text]). Goldfish hepatocytes treated with 1mM AICAR under normoxic conditions (>200μMO(2)) showed a modest but significant 1.1-fold increase in AMPK phosphorylation, a 7.5-fold increase in AMPK activity, a 1.4-fold increase in eEF2 phosphorylation, and a 24% decrease in [Formula: see text]. At physiologically relevant [O(2)] (<40μMO(2)), the addition of 1mM AICAR resulted in only a 13% decrease in cellular [Formula: see text] with no change in sensitivity to [O(2)] as assessed by estimates of cellular P(50) and P(90) values. The addition of compound C, a general protein kinase inhibitor, after AICAR incubation did not reverse the effects of AICAR on [Formula: see text] in normoxia. Treatment of hepatocytes with ≤200μM A769662 did not affect AMPK activity, AMPK phosphorylation, eEF2 phosphorylation, or cellular [Formula: see text]. These data suggest that A769662 is not an activator of AMPK in goldfish hepatocytes. Although our study provides support for the hypothesis that AMPK plays a role in initiating metabolic rate suppression in goldfish hepatocytes, this support must be viewed cautiously because of the known off-target effects of the pharmacological agents used.

CaM Kinase Kinase β-Mediated Activation of the Growth Regulatory Kinase AMPK Is Required for Androgen-Dependent Migration of Prostate Cancer Cells

While patients with advanced prostate cancer initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1-2 years. Although hormone-refractory disease is unresponsive to androgen-deprivation, androgen receptor (AR)-regulated signaling pathways remain active and are necessary for cancer progression. Thus, both AR itself and the processes downstream of the receptor remain viable targets for therapeutic intervention. Microarray analysis of multiple clinical cohorts showed that the serine/threonine kinase Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) is both highly expressed in the prostate and further elevated in prostate cancers. Using cellular models of prostate cancer, we have determined that androgens (a) directly increase the expression of a CaMKKβ splice variant and (b) increase functional CaMKKβ protein levels as determined by the phosphorylation of both CaMKI and AMP-activated protein kinase (AMPK), two of CaMKKβ's primary substrates. Importantly, inhibition of the CaMKKβ-AMPK, but not CaMKI, signaling axis in prostate cancer cells by pharmacological inhibitors or siRNA-mediated knockdown blocks androgen-mediated migration and invasion. Conversely, overexpression of CaMKKβ alone leads to both increased AMPK phosphorylation and cell migration. Given the key roles of CaMKKβ and AMPK in the biology of prostate cancer cells, we propose that these enzymes are potential therapeutic targets in prostate cancer.

Abnormal metabolism flexibility in response to high palmitate concentrations in myotubes derived from obese type 2 diabetic patients

Insulin resistance in type 2 diabetes (T2D) is associated with intramuscular lipid (IMCL) accumulation. To determine whether impaired lipid oxidation is involved in IMCL accumulation, we measured expression of genes involved in mitochondrial oxidative metabolism or biogenesis, mitochondrial content and palmitate beta-oxidation before and after palmitate overload (600μM for 16h), in myotubes derived from healthy subjects and obese T2D patients. Mitochondrial gene expression, content and network were not different between groups. Basal palmitate beta-oxidation was not affected in T2D myotubes, whereas after 16h of palmitate pre-treatment, T2D myotubes in contrast to control myotubes, showed an inability to increase palmitate beta-oxidation (p<0.05). Interestingly, acetyl-CoA carboxylase (ACC) phosphorylation was increased with a tendency for statistical significance after palmitate pre-treatment in control myotubes (p=0.06) but not in T2D myotubes which can explain their inability to increase palmitate beta-oxidation after palmitate overload. To determine whether the activation of the AMP activated protein kinase (AMPK)-ACC pathway was able to decrease lipid content in T2D myotubes, cells were treated with AICAR and metformin. These AMPK activators had no effect on ACC and AMPK phosphorylation in T2D myotubes as well as on lipid content, whereas AICAR, but not metformin, increased AMPK phosphorylation in control myotubes. Interestingly, metformin treatment and mitochondrial inhibition by antimycin induced increased lipid content in control myotubes. We conclude that T2D myotubes display an impaired capacity to respond to metabolic stimuli.