5'-AMP-activated protein kinase (AMPK) regulates progesterone receptor transcriptional activity in breast cancer cells

The steroid hormone progesterone is an essential regulator of the cellular processes that are required for the development and maintenance of reproductive function. The diverse effects of progesterone are mediated by the progesterone receptor (PR). The functions of the PR are regulated not only by ligands but also by modulators of various cell signaling pathways. However, it is not clear which energy state regulates PR activity. AMP-activated protein kinase (AMPK), a serine/threonine protein kinase, is a key modulator of energy homeostasis. Once activated by an increasing cellular AMP:ATP ratio, AMPK switches off ATP-consuming processes and switches on ATP-producing processes. We found that both 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin, traditional pharmacological activators of AMPK, inhibited the PR pathway, as evidenced by progesterone response element (PRE)-driven luciferase activity and PR target gene expression. Compound C, an inhibitor of AMPK, partly but significantly reversed the anti-PR effects of AICAR and metformin. The downregulation of endogenous AMPK by small interfering RNAs (siRNAs) stimulated PR activity. AMPK activation by AICAR decreased the progesterone-induced phosphorylation of PR at serine 294 and inhibited the recruitment of PR to an endogenous PRE. Taken together, our data suggest that AMPK, an energy sensor, is involved in the regulation of PR signaling.     

5'-AMP-activated protein kinase is inactivated by adrenergic signalling in adult cardiac myocytes

In adult rat cardiac myocytes adrenaline decreased AMPK (AMP-activated protein kinase) activity with a half-time of approximately 4?min, decreased phosphorylation of AMPK (α-Thr172) and decreased phosphorylation of ACC (acetyl-CoA carboxylase). Inactivation of AMPK by adrenaline was through both α1- and β-ARs (adrenergic receptors), but did not involve cAMP or calcium signalling, was not blocked by the PKC (protein kinase C) inhibitor BIM I (bisindoylmaleimide I), by the ERK (extracellular-signal-regulated kinase) cascade inhibitor U0126 or by PTX (pertussis toxin). Adrenaline caused no measurable change in LKB1 activity. Adrenaline decreased AMPK activity through a process that was distinct from AMPK inactivation in response to insulin or PMA. Neither adrenaline nor PMA altered the myocyte AMP:ATP ratio although the adrenaline effect was attenuated by oligomycin and by AICAR (5-amino-4-imidazolecarboxamide-1-β-D-ribofuranoside), agents that mimic 'metabolic stress'. Inactivation of AMPK by adrenaline was abolished by 1?μM okadaic acid suggesting that activation of PP2A (phosphoprotein phosphatase 2A) might mediate the adrenaline effect. However, no change in PP2A activity was detected in myocyte extracts. Adrenaline increased phosphorylation of the AMPK β-subunit in vitro but there was no detectable change in vivo in phosphorylation of previously identified AMPK sites (β-Ser24, β-Ser108 or β-Ser182) suggesting that another site(s) is targeted.     

p38 mitogen-activated protein kinase mediates adenosine-induced alterations in myocardial glucose utilization via 5'-AMP-activated protein kinase

Adenosine-induced acceleration of glycolysis in hearts stressed by transient ischemia is accompanied by suppression of glycogen synthesis and by increases in activity of adenosine 5'-monophosphate-activated protein kinase (AMPK). Because p38 mitogen-activated protein kinase (MAPK) may regulate glucose metabolism and may be activated downstream of AMPK, this study determined the effects of the p38 MAPK inhibitors SB202190 and SB203580 on adenosine-induced alterations in glucose utilization and AMPK activity. Studies were performed in working rat hearts perfused aerobically following stressing by transient ischemia (2 x 10-min ischemia followed by 5-min reperfusion). Phosphorylation of AMPK and p38 MAPK each were increased fourfold by adenosine, and these effects were inhibited by either SB202190 or SB203580. Neither of these inhibitors directly affected AMPK activity. Attenuation of the adenosine-induced increase in AMPK and p38 MAPK phosphorylation by SB202190 and SB203580 occurred independently of any change in tissue ATP-to-AMP ratio and did not alter glucose uptake, but it was accompanied by an increase in glycogen synthesis and glycogen content and by inhibition of glycolysis and proton production. There was a significant inverse correlation between the rate of glycogen synthesis and AMPK activity and between AMPK activity and glycogen content. These data demonstrate that AMPK is likely downstream of p38 MAPK in mediating the effects of adenosine on glucose utilization in hearts stressed by transient ischemia. The ability of p38 MAPK inhibitors to relieve the inhibition of glycogen synthesis and to inhibit glycolysis and proton production suggests that these agents may restore adenosine-induced cardioprotection in stressed hearts.     

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

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

Stimulation of AMP-activated protein kinase (AMPK) is associated with enhancement of Glut1-mediated glucose transport

In cells expressing only the Glut1 isoform of glucose transporters, we have shown that glucose transport is markedly stimulated in response to hypoxia or inhibition of oxidative phosphorylation, conditions that would be expected to cause a stimulation of AMP-activated protein kinase (AMPK) activity. In the present study we tested the hypothesis that the stimulation of AMPK activity might be accompanied by an enhancement of Glut1-mediated glucose transport. Exposure of Clone 9 cells, 3T3-L1 preadipocytes, and C(2)C(12) myoblasts (cells that express only the Glut1 isoform) to 5-aminoimidazole-4-carboxamideribonucleoside (AICAR), an adenosine analog that stimulates AMPK activity, resulted in a marked increase in the rate of glucose transport (ranging from four- to sixfold) that was accompanied by activation of AMPK. This stimulation of AMPK activity was associated with an increase in the phosphorylation of threonine 172 on the activation loop of its alpha subunit, with the predominant change being in the alpha-2 isoform. Exposure of Clone 9 cells to 5-iodotubercidin, an inhibitor of adenosine kinase, abolished the accumulation of AICAR-5'-monophosphate (ZMP), stimulation of AMPK, and the enhancement of glucose transport in response to AICAR. There was no significant increase in the content of Glut1 in plasma membranes of Clone 9 cells exposed to AICAR. We conclude that stimulation of AMPK activity is associated with enhancement of Glut1-mediated glucose transport, and that the glucose transport response is mediated by activation of Glut1 transporters preexisting in the plasma membrane.     

Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells

The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ～20%. Consistent with this, analysis of GU in muscle cells from α1(-/-)/α2(-/-) AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ～35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.     

5'-AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle

The 5'-AMP-activated protein kinase (AMPK) is proposed to be involved in signaling pathways leading to adaptations in skeletal muscle in response to both a single exercise bout and exercise training. This study investigated the effect of endurance training on protein content of catalytic (alpha1, alpha2) and regulatory (beta1, beta2 and gamma1, gamma2, gamma3) subunit isoforms of AMPK as well as on basal AMPK activity in human skeletal muscle. Eight healthy young men performed supervised one-legged knee extensor endurance training for 3 wk. Muscle biopsies were obtained before and 15 h after training in both legs. In response to training the protein content of alpha1, beta2 and gamma1 increased in the trained leg by 41, 34, and 26%, respectively (alpha1 and beta2 P < 0.005, gamma1 P < 0.05). In contrast, the protein content of the regulatory gamma3-isoform decreased by 62% in the trained leg (P = 0.01), whereas no effect of training was seen for alpha2, beta1, and gamma2. AMPK activity associated with the alpha1- and the alpha2-isoforms increased in the trained leg by 94 and 49%, respectively (both P < 0.005). In agreement with these observations, phosphorylation of alpha-AMPK-(Thr172) and of the AMPK target acetyl-CoA carboxylase-beta(Ser221) increased by 74 and 180%, respectively (both P < 0.001). Essentially similar results were obtained in four additional subjects studied 55 h after training. This study demonstrates that protein content and basal AMPK activity in human skeletal muscle are highly susceptible to endurance exercise training. Except for the increase in gamma1 protein, all observed adaptations to training could be ascribed to local contraction-induced mechanisms, since they did not occur in the contralateral untrained muscle.     

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

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

AMP-activated protein kinase (AMPK) control of fatty acid and glucose metabolism in the ischemic heart

Myocardial ischemia is the leading cause of all cardiovascular deaths in North America. Myocardial ischemia is accompanied by profound changes in metabolism including alterations in glucose and fatty acid metabolism, increased uncoupling of glucose oxidation from glycolysis and accumulation of protons within the myocardium. These changes can contribute to a poor functional recovery of the heart. One key player in the ischemia-induced alteration in fatty acid and glucose metabolism is 5'AMP-activated protein kinase (AMPK). Accumulating evidence suggest that activation of AMPK during myocardial ischemia both increases glucose uptake and glycolysis while also increasing fatty acid oxidation during reperfusion. Gain-of-function mutations of AMPK in cardiac muscle may also be causally related to the development of hypertrophic cardiomyopathies. Therefore, a better understanding of role of AMPK in cardiac metabolism is necessary to appropriately modulate its activity as a potential therapeutic target in treating ischemia reperfusion injuries. This review attempts to update some of the recent findings that delineate various pathways through which AMPK regulates glucose and fatty acid metabolism in the ischemic myocardium.     

Myocardial ischemia differentially regulates LKB1 and an alternate 5'-AMP-activated protein kinase kinase

During myocardial ischemia, activation of 5'-AMP-activated protein kinase (AMPK) leads to the stimulation of glycolysis and fatty acid oxidation. Together these metabolic changes contribute to cardiac dysfunction. Although AMPK signaling in the ischemic heart is well characterized, the relative contribution of phosphorylation by AMPK kinase (AMPKK), and positive allosterism by the ratios of AMP:ATP and creatine (Cr):phosphocreatine (PCr), in stimulating AMPK during ischemia are unknown. In hearts subjected to severe ischemia, the ratios of AMP:ATP and Cr:PCr were significantly elevated as compared with aerobic hearts. Severe ischemia stimulated AMPK signaling, as demonstrated by an increase in both AMPK activity and acetyl-CoA carboxylase phosphorylation. Although AMPK phosphorylation was increased by severe ischemia, the protein abundance and activity of the recently identified AMPKK, LKB1, were similar between aerobic and severely ischemic hearts. However, in contrast to LKB1, the activity of AMPKK was stimulated in severely ischemic hearts. To further delineate the relative roles of positive allosterism and AMPKK in the regulation of AMPK during ischemia, hearts were subjected to mild ischemia. Although mild ischemia did not alter the ratios of AMP:ATP and Cr:PCr, mild ischemia increased AMPK activity and increased AMPK phosphorylation. Mild ischemia also stimulated the activity of AMPKK. In summary, we demonstrate that myocardial ischemia stimulates AMPK via an AMPKK other than LKB1. Additionally, we show that changes in high energy phosphates are not essential for the activation of AMPK by ischemia. Our data emphasize the critical role AMPKK plays in mediating AMPK signaling during myocardial ischemia.     

Effects of adenosine on myocardial glucose and palmitate metabolism after transient ischemia: role of 5'-AMP-activated protein kinase

Loss of cardioprotection by adenosine in hearts stressed by transient ischemia may be due to its effects on glucose metabolism. In the absence of transient ischemia, adenosine inhibits glycolysis, whereas it accelerates glycolysis after transient ischemia. Inasmuch as 5'-AMP-activated protein kinase (AMPK) is implicated as a regulator of glucose and fatty acid utilization, this study determined whether a differential alteration of AMPK activity contributes to acceleration of glycolysis by adenosine in hearts stressed by transient ischemia. Studies were performed in working rat hearts perfused aerobically under normal conditions or after transient ischemia (two 10-min periods of ischemia followed by 5 min of reperfusion). LV work was not affected by adenosine. AMPK phosphorylation was not affected by transient ischemia; however, phosphorylation and activity were increased nine- and threefold, respectively, by adenosine in stressed hearts. Phosphorylation of acetyl-CoA carboxylase and rates of palmitate oxidation were unaltered. Glycolysis and calculated proton production were increased 1.8- and 1.7-fold, respectively, in hearts with elevated AMPK activity. Elevated AMPK activity was associated with inhibition of glycogen synthesis and unchanged rates of glucose uptake and glycogenolysis. Phentolamine, an alpha-adrenoceptor antagonist, which prevents adenosine-induced activation of glycolysis in stressed hearts, prevented AMPK phosphorylation. These data demonstrate that adenosine-induced activation of AMPK after transient ischemia is not sufficient to alter palmitate oxidation or glucose uptake. Rather, activation of AMPK alters partitioning of glucose away from glycogen synthesis; the increase in glycolysis may in part contribute to loss of adenosine-induced cardioprotection in hearts subjected to transient ischemia.     

5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2% of that in sedentary subjects (not significant), whereas mRNA for gamma(3) was 61 +/- 1% of that in sedentary subjects (not significant). The level of alpha(1)-AMPK protein in trained subjects was 185 +/- 34% of that in sedentary subjects (P < 0.05), whereas the levels of the remaining subunits (alpha(2), beta(1), beta(2), gamma(1), gamma(2), gamma(3)) were similar in trained and sedentary subjects. At the end of 20 min of cycle exercise at 80% of peak O(2) uptake, the increase in phosphorylation of alpha-AMPK (Thr(172)) was blunted in the trained group (138 +/- 38% above rest) compared with the sedentary group (353 +/- 63% above rest) (P < 0.05). Acetyl CoA-carboxylase beta-phosphorylation (Ser(221)), which is a marker for in vivo AMPK activity, was increased by exercise in both groups but to a lower level in trained subjects (32 +/- 5 arbitrary units) than in sedentary controls (45 +/- 1 arbitrary units) (P < 0.01). In conclusion, trained human skeletal muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.     

Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5'-AMP-activated protein kinase.

The activity of acetyl-CoA carboxylase (ACC), a rate-limiting enzyme of fatty acid biosynthesis and malonyl-CoA production, can be regulated by several mechanisms, including multisite covalent phosphorylation, both in vitro and in intact cells. Evidence has been presented by others to indicate that a 5'-AMP-activated protein kinase (AMPK) is likely the major regulatory kinase active on ACC. While insulin is known to activate ACC in several cell types, accompanied by changes in ACC phosphorylation, the mechanism underlying this activation has been obscure. In the present study, we have examined, in Fao hepatoma cells, the effects of insulin on ACC and AMPK activity, the latter measured with a synthetic peptide corresponding to one of the phosphorylation sites on ACC for AMPK. Our results show that insulin leads to inhibition of kinase activity prior to the onset of ACC activation; the peak of maximal kinase inhibition (approximately 35% at 10 min) is seen to precede the onset of ACC activation (20 min). The inhibition of kinase activity due to insulin is observed both in the absence and presence of varying stimulating concentrations of added 5'-AMP. Both kinase inhibition and ACC activation display similar insulin sensitivity (A50 0.3 nM). Preservation of this insulin-induced kinase inhibition requires the presence of protein phosphatase inhibitors in the cell lysis buffer, suggesting that AMPK itself might be regulated by insulin-stimulated changes in kinase phosphorylation. Taken together, these data are consistent with the hypothesis that the 5'-AMP-activated protein kinase is a regulated component of the insulin signal transduction pathway and may be the major target for insulin regulation of ACC.     

Activation of the AMP-activated protein kinase (AMPK) by nitrated lipids in endothelial cells

The AMP-activated protein kinase (AMPK) is an important regulator of endothelial metabolic and functional homeostasis. Here, we examined the regulation of AMPK by nitrated oleic acid (OA-NO(2)) and investigated the implications in endothelial function. Treatment of bovine aortic endothelial cells (BAECs) with OA-NO(2) induced a significant increase in both AMPK-Thr172 phosphorylation and AMPK activity as well as upregulation of heme oxygenase (HO)-1 and hypoxia-inducible factor (HIF)-1α. Pharmacologic inhibition or genetic ablation of HO-1 or HIF-1α abolished OA-NO(2)-induced AMPK phosphorylation. OA-NO(2) induced a dramatic increase in extracellular signal-regulated kinase (ERK)1/2 phosphorylation that was abrogated by the HO-1 inhibitor, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG). Inhibition of ERK1/2 using UO126 or PD98059 reduced but did not abolish OA-NO(2)-induced HIF-1α upregulation, suggesting that OA-NO(2)/HO-1-initiated HIF-1α induction is partially dependent on ERK1/2 activity. In addition, OA-NO(2) enhanced endothelial intracellular Ca(2+), an effect that was inhibited by the HIF-1α inhibitor, YC-1, and by HIF-1α siRNA. These results implicate the involvement of HIF-1α. Experiments using the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609, the selective CaMKII inhibitor KN-93, and an isoform-specific siRNA demonstrated that OA-NO(2)-induced AMPK phosphorylation was dependent on CaMKKβ. Together, these results demonstrate that OA-NO(2) activates AMPK in endothelial cells via an HO-1-dependent mechanism that increases HIF-1α protein expression and Ca(2+)/CaMKKβ activation.     

Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5'AMP-activated protein kinase (AMPK). Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique.

Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria.     

CAPE (caffeic acid phenethyl ester) stimulates glucose uptake through AMPK (AMP-activated protein kinase) activation in skeletal muscle cells

Caffeic acid phenethyl ester (CAPE), a flavonoid-like compound, is one of the major components of honeybee propolis. In the present study, we investigated the metabolic effects of CAPE in skeletal muscle cells and found that CAPE stimulated glucose uptake in differentiated L6 rat myoblast cells and also activated AMPK (AMP-activated protein kinase). In addition, the inhibition of AMPK blocked CAPE-induced glucose uptake, and CAPE activated the Akt pathway in a PI3K (phosphoinositide 3-kinase)-dependent manner. Furthermore, CAPE enhanced both insulin-mediated Akt activation and glucose uptake. In summary, our results suggest that CAPE may have beneficial roles in glucose metabolism via stimulation of the AMPK pathway.     

Activation of 5'-AMP-activated kinase with diabetes drug metformin induces casein kinase Iepsilon (CKIepsilon)-dependent degradation of clock protein mPer2

Metformin is one of the most commonly used first line drugs for type II diabetes. Metformin lowers serum glucose levels by activating 5'-AMP-activated kinase (AMPK), which maintains energy homeostasis by directly sensing the AMP/ATP ratio. AMPK plays a central role in food intake and energy metabolism through its activities in central nervous system and peripheral tissues. Since food intake and energy metabolism is synchronized to the light-dark (LD) cycle of the environment, we investigated the possibility that AMPK may affect circadian rhythm. We discovered that the circadian period of Rat-1 fibroblasts treated with metformin was shortened by 1 h. One of the regulators of the period length is casein kinase Iepsilon (CKIepsilon), which by phosphorylating and inducing the degradation of the circadian clock component, mPer2, shortens the period length. AMPK phosphorylates Ser-389 of CKIepsilon, resulting in increased CKIepsilon activity and degradation of mPer2. In peripheral tissues, injection of metformin leads to mPer2 degradation and a phase advance in the circadian expression pattern of clock genes in wild-type mice but not in AMPK alpha2 knock-out mice. We conclude that metformin and AMPK have a previously unrecognized role in regulating the circadian rhythm.     

Adiponectin-mediated stimulation of AMP-activated protein kinase (AMPK) in pancreatic beta cells

The adipocyte-derived hormone adiponectin was recently shown to stimulate glucose-utilization and to increase fatty acid oxidation in liver and muscle. The effects were ascribed to adiponectin-receptor mediated activation of the key metabolic regulator AMP-activated protein kinase (AMPK). In pancreatic beta cells, AMPK-activation is known to affect cellular function. We therefore investigated a possible adiponectin-induced activation of AMPK in beta cells. RT-PCR analysis confirmed the expression of adiponectin receptor subtypes 1 and 2 in rat beta cells and showed their expression in insulin-secreting MIN6 cells. Culture with physiological concentrations (2.5 microg/ml) of globular adiponectin was found to increase the phosphorylation of both AMPK and acetylcoA carboxylase (ACC) in these cell types. Like the pharmacological AMPK activator 5-amino-imidazole-4-carboxamide-riboside (AICAR), adiponectin activated AMPK in beta cells and MIN6 cells. In short-term incubations of MIN6 cells with either adiponectin (2.5 microg/ml) or AICAR (1 mM), the flux of glucose-carbon to acyl CoA/cholesterol biosynthetic intermediates was reduced. We conclude that adiponectin induces an activation of AMPK in beta cells, which inhibits their cataplerosis of glucose-carbon to lipids.