Search

The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the alpha subunit of AMPK in a dose- and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviral-mediated expression of a constitutively active AMPKalpha or a kinase-inactive AMPKalpha, respectively. An insulin-resistant state was induced by exposing cells to 30 mm glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3alpha/beta. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPKalpha. The kinase-inactive AMPKalpha increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPKalpha negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance.

Leptin has been shown to activate AMP-activated protein kinase (AMPK), an enzyme that regulates the activities of key enzymes of lipid synthesis and metabolism. We assess here (i) whether AMPK activity is diminished in rodents deficient in leptin or the leptin receptor, and (ii) the effects of treating the diabetes-prone, leptin-receptor-deficient Zucker Diabetic Fatty (ZDF) rat with an AMPK activator.AMPK activity and related parameters were measured in muscle and or liver of fa/fa and ZDF rats and ob/ob mice. We also explored the effect of treatment with the AMPK activator 5-aminoimidazole 4-carboxamide 1-beta-D ribofuranoside (AICAR) (7.4 mmol/l, on Monday, Wednesday and Friday for 15 weeks, beginning at 7 weeks of age) on the phenotype of the ZDF rat.AMPK activity was diminished in muscle and/or liver of fa/fa (leptin-receptor-deficient, non-diabetic) and ZDF (leptin-receptor-deficient, diabetes-prone) rats and ob/ob mice (leptin-deficient). ZDF rats that had free access to food became hyperglycaemic (22.2 mmol/l) and hyperphagic after 2 to 5 weeks and remained so during the remainder of the study. Treatment of ZDF rats with AICAR prevented the development of diabetes, as well as increases of triglyceride content in liver, muscle and the pancreatic islets. It also attenuated the morphological abnormalities observed in the islets of untreated rats. Rats diet-matched with the AICAR-treated animals developed diabetes of intermediate severity and showed decreases in triglyceride content in the islets, but not in liver or muscle.The results indicate that a deficiency of leptin or the leptin receptor is associated with a decrease in AMPK activity in muscle and/or liver. They also suggest that treatment with an AMPK activator prevents the development of diabetes and ectopic lipid accumulation in the ZDF rat.

Following exercise, AMP-activated protein kinase (AMPK) activity is increased several fold in rat liver and adipose tissue as well as muscle; however, the mechanism by which this occurs is not known. Interleukin-6 (IL-6) is released from muscle in large amounts during and after sustained physical activity resulting in up to 100-fold increases in its plasma concentration, from 1-2 ng/ml to 50-100 ng/ml. We report here that incubation with IL-6 (30-120 ng/ml) increases the phosphorylation of AMPK (an indicator of its activation) and that of its target molecule, acetyl CoA carboxylase (ACC), in both extensor digitorum longus muscle and cultured F422a adipocytes. To assess more directly whether IL-6 regulates AMPK in vivo during exercise, measurements were carried out in skeletal muscle, liver, and adipose tissue of 3-month-old IL-6 knockout (IL-6(-/-)) and C57 black control mice. In agreement with previous studies in the rat, in control mice P-AMPK and P-ACC abundance was increased by 30-150% in the three tissues in response to exercise with the greatest increases in skeletal muscle. In contrast, in IL-6(-/-) mice, we found that the abundance of both P-AMPK and P-ACC was lower (60-90%) in muscle and adipose tissue at rest. Also the absolute increases in P-AMPK caused by exercise were diminished compared to those in control mice, although percentage increases were similar. In liver, decreases in P-AMPK and P-ACC in the IL-6(-/-) mice were more modest and the increases in their abundance caused by exercise were indistinguishable from those of control mice. The results indicate that IL-6 can activate AMPK in muscle and adipose tissue, and that this contributes to, but does not fully account for, the increase in AMPK activity in these tissues in response to exercise. They also suggest that a genetic lack of IL-6 is associated with a decrease in AMPK activity.

From the perspective of a muscle physiologist, adipose tissue has long been perceived predominantly as a fuel reservoir that provides muscle and other tissues with NEFA when exogenous nutrients are insufficient for their energy needs. Recently, studies have established that adipose tissue is also an endocrine organ. Among the hormones it releases are adiponectin and leptin, both of which can activate AMP-activated protein kinase and increase fatty acid oxidation in skeletal muscle and probably other tissues. Deficiencies of leptin or leptin receptor, adiponectin and IL-6 are associated with obesity, insulin resistance and a propensity to type 2 diabetes. In addition, a lack of adiponectin has been linked to atherosclerosis. Whether this pathology reflects a deficient activation of AMP-activated protein kinase in peripheral tissues remains to be determined. Finally, recent studies have suggested that skeletal muscle may also function as an endocrine organ when it releases the cytokine IL-6 into the circulation during sustained exercise. Interestingly, one of the apparent effects of IL-6 is to stimulate lipolysis, causing the release of NEFA from the adipocyte. Thus, hormonal communications exist between the adipocyte and muscle that could enable them to talk to each other. The physiological relevance of this cross talk clearly warrants further study.

Patients with the metabolic syndrome are characterized by insulin resistance, obesity and a predisposition to hypertension, dyslipidaemia, pancreatic β-cell dysfunction, type 2 diabetes and premature atherosclerosis. Here we review the hypothesis that a common feature linking these multiple abnormalities is dysregulation of the AMP-activated protein kinase (AMPK)/malonyl-CoA fuel-sensing and signalling network. It is proposed that such dysregulation leads to alterations in cellular fatty-acid metabolism that in turn cause ectopic lipid accumulation, cellular dysfunction and ultimately disease. Evidence is also presented that factors that activate AMP kinase and/or reduce malonyl-CoA levels might reverse these abnormalities or prevent them from occurring.

The malonyl-CoA/long-chain acyl-CoA (LC-CoA) model of glucose-induced insulin secretion (GIIS) predicts that malonyl-CoA derived from glucose metabolism inhibits fatty acid oxidation, thereby increasing the availability of LC-CoA for lipid signaling to cellular processes involved in exocytosis. For directly testing the model, INSr3 cell clones overexpressing malonyl-CoA decarboxylase in the cytosol (MCDc) in a tetracycline regulatable manner were generated, and INS(832/13) and rat islets were infected with MCDc-expressing adenoviruses. MCD activity was increased more than fivefold, and the malonyl-CoA content was markedly diminished. This was associated with enhanced fat oxidation at high glucose, a suppression of the glucose-induced increase in cellular free fatty acid (FFA) content, and reduced partitioning at elevated glucose of exogenous palmitate into lipid esterification products. MCDc overexpression, in the presence of exogenous FFAs but not in their absence, reduced GIIS in all β-cell lines and in rat islets. It also markedly curtailed the stimulation of insulin secretion by other fuel and nonfuel secretagogues. In the absence of MCDc overexpression, the secretory responses to all types of secretagogues were amplified by the provision of exogenous fatty acids. In the presence of exogenous FFAs, the fatty acyl-CoA synthetase inhibitor triacsin C reduced secretion in response to glucose and nonfuel stimuli. The data show the existence of important links between the metabolic coupling factor malonyl-CoA, the partitioning of fatty acids, and the stimulation of insulin secretion to both fuel and nonfuel stimuli., DA - 20040329IS - 0012-1797LA - engPT - Journal ArticleSB - AIMSB - IM

Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and- stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys22-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys22 with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-kappaB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-alpha at Thr172 and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state.

Purpose of reviewThe prevalence of cardiovascular disease is markedly increased in patients with type 2 diabetes and to a somewhat lesser extent impaired glucose tolerance (IGT). This has generally been attributed to the concurrent presence of cardiovascular disease risk factors, including dyslipide

An increasing body of evidence has revealed that activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK)-activated protein kinase increases fatty acid oxidation by lowering the concentration of malonyl coenzyme A (CoA), an inhibitor of carnitine palmitoyl transferase 1. Studies carried out primarily in skeletal muscle suggest that AMPK modulates the concentration of malonyl CoA by concurrently phosphorylating and inhibiting acetyl CoA carboxylase (ACC), the rate limiting enzyme in malonyl CoA synthesis, and phosphorylating and activating malonyl CoA decarboxylase (MCD), an enzyme involved in its degradation. We have recently observed that AMPK and MCD activities are increased and ACC activity diminished in skeletal muscle, liver and, surprisingly, in adipose tissue 30 min following exercise (treadmill run) in normal rats. In liver and adipose tissue these changes were associated with a decrease in the activity of glycerol-3-phosphate acyltransferase (GPAT), which catalyses the first committed reaction in glycerolipid synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. Similar changes in ACC, MCD and GPAT were observed following the administration of 5-aminoimidazole 4-carboxamide-riboside (AICAR), further indicating that the exercise-induced alterations in these enzymes were AMPK-mediated. Conclusions: (1) AMPK plays a major role in regulating lipid metabolism in multiple tissues following exercise. (2) The net effect of its activation is to increase fatty acid oxidation and diminish glycerolipid synthesis. (3) The relevance of these findings to the regulation of muscle glycogen repletion in the post-exercise state and to the demonstrated ability of AMPK activation to decrease adiposity and increase insulin sensitivity in rodents remains to be determined.

Preexposure to a low concentration of glucose upregulates glucose transport into skeletal muscle, whereas exposure to a high concentration of glucose has the opposite effect. This autoregulatory process occurs independently of insulin, and the mechanism by which it operates is incompletely understood. Activation of the energy-sensing enzyme AMP-activated protein kinase (AMPK) has been shown to increase insulin-independent glucose transport into skeletal muscle in response to such stimuli as exercise and hypoxia. In the present study, we examined whether AMPK could also mediate glucose autoregulation. The activity of the α2 isoform of AMPK and 2-deoxyglucose uptake were assessed in incubated rat extensor digitorum longus muscle after preincubation for 4 h in media containing 0, 3, 6, or 25 mmol/l glucose. The principal findings were as follows. First, AMPK activity was highest in muscles incubated with no added glucose, and it decreased as the concentration of glucose was increased. In keeping with these findings, the concentration of malonyl CoA was increased, and acetyl CoA carboxylase phosphorylation at serine 79 was decreased as the medium glucose concentration was raised. Second, decreases in AMPK activity at the higher glucose concentrations correlated closely with decreases in glucose transport (2-deoxyglucose uptake), measured during a subsequent 20-min incubation at 6 mmol/l glucose (r2 = 0.93, P < 0.001). Third, the decrease in AMPK activity at the higher glucose concentrations was not associated with changes in whole-tissue concentrations of creatine phosphate or adenine nucleotides; however, it did correlate with increases in the rate of glycolysis, as estimated by lactate release. The results suggest that glucose autoregulates its own transport into skeletal muscle by a mechanism involving AMPK. They also suggest that this autoregulatory mechanism is not paralleled by changes in whole-tissue concentrations of creatine phosphate ATP, or AMP, but they leave open the possibility that alterations in a cytosolic pool of these compounds play a regulatory role.

Based on available evidence, we would propose the following. (i) Excesses of glucose and free fatty acids cause insulin resistance in skeletal muscle and damage to the endothelial cell by a similar mechanism. (ii) Key pathogenetic events in this mechanism very likely include increased fatty acid esterification, protein kinase C activation, an increase in oxidative stress (demonstrated to date in endothelium) and alterations in the inhibitor κB kinase/nuclear factor κB system. (iii) Activation of AMP-activated protein kinase (AMPK) inhibits all of these events and enhances insulin signalling in the endothelial cell. It also enhances insulin action in muscle; however, the mechanism by which it does so has not been well studied. (iv) The reported beneficial effects of exercise and metformin on cardiovascular disease and insulin resistance in humans could be related to the fact that they activate AMPK. (v) The comparative roles of AMPK in regulating metabolism, signalling and gene expression in muscle and endothelial cells warrant further study.

Changes in the concentration of malonyl-CoA in many tissues have been related to alterations in the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in its formation. In contrast, little is known about the physiological role of malonyl-CoA decarboxylase (MCD), an enzyme responsible for malonyl-CoA catabolism. In this study, we examined the effects of voluntary exercise on MCD activity in rat liver, skeletal muscle, and adipose tissue. In addition, the activity ofsn-glycerol-3-phosphate acyltransferase (GPAT), which like MCD and ACC can be regulated by AMP-activated protein kinase (AMPK), was assayed. Thirty min after the completion of a treadmill run, MCD activity was increased ∼2-fold, malonyl-CoA levels were reduced, and ACC and GPAT activities were diminished by 50% in muscle and liver. These events appeared to be mediated via activation of AMPK since: 1) AMPK activity was concurrently increased by exercise in both tissues; 2) similar findings were observed after the injection of 5-amino 4 imidazole carboxamide, an AMPK activator; 3) changes in the activity of GPAT and ACC paralleled that of MCD; and 4) the increase in MCD activity in muscle was reversed in vitro by incubating immunoprecipitated enzyme from the exercised muscle with protein phosphatase 2A, and it was reproduced by incubating immunopurified MCD from resting muscle with purified AMPK. An unexpected finding was that exercise caused similar changes in the activities of ACC, MCD, GPAT, and AMPK and the concentration of malonyl-CoA in adipose tissue. In conclusion: MCD, GPAT, and ACC are coordinately regulated by AMPK in liver and adipose tissue in response to exercise, and except for GPAT, also in muscle. The results suggest that AMPK activation plays a major role in regulating lipid metabolism in many cells following exercise. They also suggest that in each of them, it acts to increase fatty acid oxidation and decrease its esterification.

Denervation has been shown to impair the ability of insulin to stimulate glycogen synthesis and, to a lesser extent, glucose transport in rat skeletal muscle. Insulin binding to its receptor, activation of the receptor tyrosine kinase and phosphatidylinositol 3'-kinase do not appear to be involved. On the other hand, it has been shown that denervation causes an increase in the total diacylglycerol (DAG) content and membrane-associated protein kinase C (PKC) activity. In this study, we further characterize these changes in PKC and assess other possible signaling abnormalities that might be related to the decrease of glycogen synthesis. The results reveal that PKC-epsilon and -theta;, but not -alpha or -zeta, are increased in the membrane fraction 24 h after denervation and that the timing of these changes parallels the impaired ability of insulin to stimulate glycogen synthesis. At 24 h, these changes were associated with a 65% decrease in glycogen synthase (GS) activity ratio and decreased electrophoretic mobility, indicative of phosphorylation in GS in muscles incubated in the absence of insulin. Incubation of the denervated soleus with insulin for 30 min minimally increased glucose incorporation into glycogen; however, it increased GS activity threefold, to a value still less than that of control muscle, and it eliminated the gel shift. In addition, insulin increased the apparent abundance of GS kinase (GSK)-3 and protein phosphatase (PP)1 alpha in the supernatant fraction of muscle homogenate to control values, and it caused the same increases in GSK-3 and Akt/protein kinase B (PKB) phosphorylation and Akt/PKB activity that it did in nondenervated muscle. No alterations in hexokinase I or II activity were observed after denervation; however, in agreement with a previous report, glucose 6-phosphate levels were diminished in 24-h-denervated soleus, and they did not increase after insulin stimulation. These results indicate that alterations in the distribution of PKC-epsilon and -theta; accompany the impairment of glycogen synthesis in the 24-h-denervated soleus. They also indicate that the basal rate of glycogen synthesis and its stimulation by insulin in these muscles are diminished despite a normal activation of Akt/PKB and phosphorylation of GSK-3. The significance of the observed alterations to GSK-3 and PP1 alpha distribution remain to be determined.

Nitric oxide (NO) plays an important role in the control of numerous vascular functions including basal Na+-K+-ATPase activity in arterial tissue. Hyperglycemia inhibits Na+-K+-ATPase activity in rabbit aorta, in part, through diminished bioactivity of NO. The precise mechanism(s) for such observations, however, are not yet clear. The purpose of this study was to examine the role of superoxide in modulating NO-mediated control of Na+-K+-ATPase in response to hyperglycemia. Rabbit aorta incubated with hyperglycemic glucose concentrations (44 mM) demonstrated a 50% reduction in Na+-K+-ATPase activity that was abrogated by superoxide dismutase. Hyperglycemia also produced a 50% increase in steady-state vascular superoxide measured by lucigenin-enhanced chemiluminescence that was closely associated with reduced Na+-K+-ATPase activity. Specifically, the hyperglycemia-induced increase in vascular superoxide was endothelium dependent, inhibited by l-arginine, and stimulated by N ω-nitro-l-arginine. Aldose reductase inhibition with zopolrestat also inhibited the hyperglycemia-induced increase in vascular superoxide. In each manipulation of vascular superoxide, a reciprocal change in Na+-K+-ATPase activity was observed. Finally, a commercially available preparation of Na+-K+-ATPase was inhibited by pyrogallol, a superoxide generator. These data suggest that hyperglycemia induces an increase in endothelial superoxide that inhibits the stimulatory effect of NO on vascular Na+-K+-ATPase activity.

Apoptosis has been observed in vascular cells, nerve, and myocardium of diabetic humans and experimental animals, although whether it contributes to or is a marker of complications in these tissues is unclear. Previous studies have shown that incubation of human umbilical vein endothelial cells (HUVECs) with 30 vs. 5 mmol/l glucose for 72 h causes a significant increase in apoptosis, possibly related to an increase in oxidative stress. We report here that this increase in apoptosis (assessed morphologically by TdT-mediated dUTP nick- end labeling staining) is preceded (24 h of incubation) by inhibition of fatty acid oxidation, by increases in diacylglycerol synthesis, the concentration of malonyl CoA, and caspase-3 activity, and by decreases in mitochondrial membrane potential and cellular ATP content. In addition, the phosphorylation of Akt in the presence of 150 μU/ml insulin was impaired. No increases in ceramide content or its de novo synthesis were observed. AMP-activated protein kinase (AMPK) activity was not diminished; however, incubation with the AMPK activator 5-aminoimidazole-4-carboxamide-riboside increased AMPK activity twofold and completely prevented all of these changes. Likewise, expression of a constitutively active AMPK in HUVEC prevented the increase in caspase-3 activity. The results indicate that alterations in fatty-acid metabolism, impaired Akt activation by insulin, and increased caspase-3 activity precede visible evidence of apoptosis in HUVEC incubated in a hyperglycemic medium. They also suggest that AMPK could play an important role in protecting the endothelial cell against the adverse effects of sustained hyperglycemia.

Background Malonyl-CoA, an allosteric inhibitor of carnitine palmitoyl transferase, controls the oxidation of fatty acids in muscle and other tissues by regulating their entrance into mitochondria. The level of malonyl-CoA in muscle is influenced by the uptake of energy substrates such as glucose, the precursor of its synthesis. The desert gerbil, Psammomys obesus, develops a severe insulin resistance with hyperinsulinemia and hyperglycemia when transferred from its native nutrition to a relative high-energy (HE) rodent chow. In keeping with this it shows a low rate of glucose transport and a failure of insulin to suppress hepatic glucose production during a hyperinsulinemic-euglycemic clamp. Methods The concentration of malonyl-CoA has been determined by radio-enzymatic assay together with the levels of citrate and malate in the gastrocnemius muscle of diabetes-prone (DP) and diabetes-resistant (DR) P. obesus during the administration of exogenous insulin, during an hyperinsulinemic-euglycemic clamp and following a 48-h fast. Results Muscle GLUT4 protein was reduced by 44% in DP Psammomys on a HE diet, compared with normoglycemic-normoinsulinemic animals on a low-energy (LE) diet. Muscle levels of malonyl-CoA, its precursor citrate and the citrate counter-ion malate were not elevated in DP Psammomys on the HE diet despite the hyperinsulinemia. Likewise, the administration of external insulin in subcutaneous (sc) implants to DP Psammomys did not evoke hypoglycemia, decrease glucose production or increase the concentration of malonyl-CoA and citrate in muscle, as it did in both albino rats and a selected line of DR Psammomys. In contrast, fasting significantly reduced muscle malonyl-CoA and citrate levels in the DP and DR Psammomys and promoted the fat oxidative pathway. Conclusion Since non-diabetic Psammomys on a LE diet already show insulin resistance in the fed state, the sustained low malonyl-CoA levels in these animals imply a readiness for the oxidation of fatty acids. In a desert gerbil, adjusted to a food-scarce environment, such a continuing utilization of fatty acids as energy substrate by muscle would preserve the available glucose for glucose-dependent tissues and would diminish the need for gluconeogenesis. Copyright © 2002 John Wiley & Sons, Ltd.

The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H2O2)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H2O2-induced senescence, resveratrol also prevented the H2O2-induced decrease in proliferation (as indicated by 3H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol’s effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by the AMPK-FOXO3 pathway and in some situations, but not all, by SIRT1.

Type 2 diabetes (T2D) is a metabolic disease characterized by insulin resistance, β-cell dysfunction, and elevated hepatic glucose output. Over 350 million people worldwide have T2D, and the International Diabetes Federation projects that this number will increase to nearly 600 million by 2035. There is a great need for more effective treatments for maintaining glucose homeostasis and improving insulin sensitivity. AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase whose activation elicits insulin-sensitizing effects, making it an ideal therapeutic target for T2D. AMPK is an energy-sensing enzyme that is activated when cellular energy levels are low, and it signals to stimulate glucose uptake in skeletal muscles, fatty acid oxidation in adipose (and other) tissues, and reduces hepatic glucose production. There is substantial evidence suggesting that AMPK is dysregulated in animals and humans with metabolic syndrome or T2D, and that AMPK activation (physiological or pharmacological) can improve insulin sensitivity and metabolic health. Numerous pharmacological agents, natural compounds, and hormones are known to activate AMPK, either directly or indirectly - some of which (for example, metformin and thiazolidinediones) are currently used to treat T2D. This paper will review the regulation of the AMPK pathway and its role in T2D, some of the known AMPK activators and their mechanisms of action, and the potential for future improvements in targeting AMPK for the treatment of T2D.

Recent studies have highlighted the importance of an inhibitory phosphorylation site, Ser(485/491), on the α-subunit of AMP-activated protein kinase (AMPK); however, little is known about the regulation of this site in liver and skeletal muscle. We examined whether the inhibitory effects of insulin on AMPK activity may be mediated through the phosphorylation of this inhibitory Ser(485/491) site in hepatocytes, myotubes and incubated skeletal muscle. HepG2 and C2C12 cells were stimulated with or without insulin for 15-min. Similarly, rat extensor digitorum longus (EDL) muscles were treated +/- insulin for 10-min. Insulin significantly increased Ser(485/491) p-AMPK under all conditions, resulting in a subsequent reduction in AMPK activity, ranging from 40% to 70%, despite no change in p-AMPK Thr(172). Akt inhibition both attenuated the increase in Ser(485/491) p-AMPK caused by insulin, and prevented the decrease in AMPK activity. Similarly, the growth factor IGF-1 stimulated Ser(485/491) AMPK phosphorylation, and this too was blunted by inhibition of Akt. Inhibition of the mTOR pathway with rapamycin, however, had no effect on insulin-stimulated Ser(485/491) p-AMPK. These data suggest that insulin and IGF-1 diminish AMPK activity in hepatocytes and muscle, most likely through Akt activation and the inhibitory phosphorylation of Ser(485/491) on its α-subunit.

Although a correlation exists between obesity and insulin resistance, roughly 25 % of obese individuals are insulin sensitive. AMP-activated protein kinase (AMPK) is a cellular energy sensor that among its many actions, integrates diverse physiological signals to restore energy balance. In addition, in many situations it also increases insulin sensitivity. In this context, AMPK activity is decreased in very obese individuals undergoing bariatric surgery who are insulin resistant compared to equally obese patients who are insulin sensitive. In this review, we will both explore what distinguishes these individuals, and evaluate the evidence that diminished AMPK is associated with insulin resistance and metabolic syndrome-associated disorders in other circumstances.

Liraglutide is a glucagon-like peptide-1 (GLP-1) mimetic used for the treatment of Type 2 diabetes. Similar to the actions of endogenous GLP-1, liraglutide potentiates the post-prandial release of insulin, inhibits glucagon release and increases satiety. Recent epidemiological studies and clinical trials have suggested that treatment with GLP-1 mimetics may also diminish the risk of cardiovascular disease in diabetic patients. The mechanism responsible for this effect has yet to be determined; however, one possibility is that they might do so by a direct effect on vascular endothelium. Since low grade inflammation of the endothelium is an early event in the pathogenesis of atherosclerotic cardiovascular disease (ASCVD), we determined the effects of liraglutide on inflammation in cultured human aortic endothelial cells (HAECs). Liraglutide reduced the inflammatory responses to TNFα and LPS stimulation, as evidenced by both reduced protein expression of the adhesion molecules VCAM-1 and E-Selectin, and THP-1 monocyte adhesion. This was found to result from increased cell Ca2+ and several molecules sensitive to Ca2+ with known anti inflammatory actions in endothelial cells, including CaMKKβ, CaMKI, AMPK, eNOS and CREB. Treatment of the cells with STO-609, a CaMKK inhibitor, diminished both the activation of AMPK, CaMKI and the inhibition of TNFα and LPS-induced monocyte adhesion by liraglutide. Likewise, expression of an shRNA against AMPK nullified the anti-inflammatory effects of liraglutide. The results indicate that liraglutide exerts a strong anti-inflammatory effect on HAECs. They also demonstrate that this is due to its ability to increase intracellular Ca2+ and activate CAMKKβ, which in turn activates AMPK.

Numerous studies have shown a correlation between changes in protein kinase C (PKC) distribution and/or activity and insulin resistance in skeletal muscle. To investigate which PKC isoforms might be involved and how they affect insulin action and signaling, studies were carried out in rat soleus muscle incubated with phorbol esters. Muscles preincubated for 1 h with 1 μM phorbol 12,13-dibutyrate (PDBu) showed an impaired ability of insulin to stimulate glucose incorporation into glycogen and a translocation of PKC-α, -βI, -θ, and -ε, and probably -βII, from the cytosol to membranes. Preincubation with 1 μM PDBu decreased activation of the insulin receptor tyrosine kinase by insulin and to an even greater extent the phosphorylation of Akt/protein kinase B and glycogen synthase kinase-3. However, it failed to diminish the activation of phosphatidylinositol 3′-kinase by insulin. Despite these changes in signaling, the stimulation by insulin of glucose transport (2-deoxyglucose uptake) and glucose incorporation into lipid and oxidation to CO2was unaffected. The results indicate that preincubation of skeletal muscle with phorbol ester leads to a translocation of multiple conventional and novel PKC isoforms and to an impairment of several, but not all, events in the insulin-signaling cascade. They also demonstrate that these changes are associated with an inhibition of insulin-stimulated glycogen synthesis but that, at the concentration of PDBu used here, glucose transport, its incorporation into lipid, and its oxidation to CO2are unaffected.

Abstract —It is generally accepted that endothelial cells generate most of their ATP by anaerobic glycolysis and that very little ATP is derived from the oxidation of fatty acids or glucose. Previously, we have reported that, in cultured human umbilical vein endothelial cells (HUVECs), activation of AMP-activated protein kinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximide riboside (AICAR) is associated with an increase in the oxidation of 3 H-palmitate. In the present study, experiments carried out with cultured HUVECs revealed the following: (1) AICAR-induced increases in palmitate oxidation during a 2-hour incubation are associated with a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitor of carnitine palmitoyl transferase 1), which temporally parallels the increase in AMPK activity and a decrease in the activity of acetyl CoA carboxylase (ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitine is omitted from the medium or oxidation of the medium-chain fatty acid octanoate. (3) When intracellular lipid pools are prelabeled with 3 H-palmitate, the measured rate of palmitate oxidation is 3-fold higher, and in the presence of AICAR, it accounts for nearly 40% of calculated ATP generation. (4) Incubation of HUVECs in a glucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonyl CoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied, the contribution of glucose oxidation to ATP production is minimal. The results indicate that the AMPK–ACC–malonyl CoA–carnitine palmitoyl transferase 1 mechanism plays a key role in the physiological regulation of fatty acid oxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids from both intracellular and extracellular sources, and that when this is taken into account, fatty acids can be a major substrate for ATP generation. Finally, they suggest that AMPK is likely to be a major factor in modulating the response of the endothelium to stresses that alter its energy state.

Alterations in the concentration of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, have been linked to the regulation of fatty acid oxidation in skeletal muscle. During contraction decreases in muscle malonyl-CoA concentration have been related to activation of AMP-activated protein kinase (AMPK), which phosphorylates and inhibits acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in malonyl-CoA formation. We report here that the activity of malonyl-CoA decarboxylase (MCD) is increased in contracting muscle. Using either immunopurified enzyme or enzyme partially purified by (NH(4))(2)SO(4) precipitation, 2-3-fold increases in the V(max) of MCD and a 40% decrease in its K(m) for malonyl-CoA (190 versus 119 micrometer) were observed in rat gastrocnemius muscle after 5 min of contraction, induced by electrical stimulation of the sciatic nerve. The increase in MCD activity was markedly diminished when immunopurified enzyme was treated with protein phosphatase 2A or when phosphatase inhibitors were omitted from the homogenizing solution and assay mixture. Incubation of extensor digitorum longus muscle for 1 h with 2 mm 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, a cell-permeable activator of AMPK, increased MCD activity 2-fold. Here, too, addition of protein phosphatase 2A to the immunopellets reversed the increase of MCD activity. The results strongly suggest that activation of AMPK during muscle contraction leads to phosphorylation of MCD and an increase in its activity. They also suggest a dual control of malonyl-CoA concentration by ACC and MCD, via AMPK, during exercise.

Questions concerning whether malonyl-CoA is regulated in human muscle and whether malonyl-CoA modulates fatty acid oxidation are still unanswered. To address these questions, whole-body fatty acid oxidation and the concentration of malonyl-CoA, citrate, and malate were determined in the vastus lateralis muscle of 16 healthy nonobese Swedish men during a sequential euglycemic-hyperinsulinemic clamp. Insulin was infused at rates of 0.25 and 1.0 mU x kg(-1) x min(-1), and glucose was infused at rates of 2.0 +/- 0.2 and 8.1 +/- 0.7 mg x kg(-1) x min(-1), respectively. During the low-dose insulin infusion, whole-body fatty acid oxidation, as determined by indirect calorimetry, decreased by 22% from a basal rate of 0.94 +/- 0.06 to 0.74 +/- 0.07 mg x kg(-1) x min(-1) (P = 0.005), but no increase in malonyl-CoA was observed. In contrast, during the high-dose insulin infusion, malonyl-CoA increased from 0.20 +/- 0.01 to 0.24 +/- 0.01 nmol/g (P < 0.001), and whole-body fatty acid oxidation decreased by an additional 41% to 0.44 +/- 0.06 mg x kg(-1) x min(-1) (P < 0.001). The increase in malonyl-CoA was associated with 30-50% increases in the concentrations of citrate (102 +/- 6 vs. 137 +/- 7 nmol/g, P < 0.001), an allosteric activator of the rate-limiting enzyme in the malonyl-CoA formation, acetyl-CoA carboxylase, and malate (80 +/- 6 vs. 126 +/- 9 nmol/g, P = 0.002), an antiporter for citrate efflux from the mitochondria. Significant correlations were observed between the concentration of malonyl-CoA and both glucose utilization (r = 0.53, P = 0.002) and the sum of the concentrations of citrate and malate (r = 0.52, P < 0.001), a proposed index of the cytosolic concentration of citrate. In addition, an inverse correlation between malonyl-CoA concentration and fatty acid oxidation was observed (r = -0.32, P = 0.03). The results indicate that an infusion of insulin and glucose at a high rate leads to increases in the concentration of malonyl-CoA in skeletal muscle and to decreases in whole-body and, presumably, muscle fatty acid oxidation. Furthermore, they suggest that the increase in malonyl-CoA in this situation is due, at least in part, to an increase in the cytosolic concentration of citrate. Because cytosolic citrate is also an inhibitor of phosphofructokinase, an attractive hypothesis is that changes in its concentration are part of an autoregulatory mechanism by which glucose modulates its own use and the use of fatty acids as fuels for skeletal muscle.

In liver, insulin and glucose acutely increase the concentration of malonyl-CoA by dephosphorylating and activating acetyl-CoA carboxylase (ACC). In contrast, in incubated rat skeletal muscle, they appear to act by increasing the cytosolic concentration of citrate, an allosteric activator of ACC, as reflected by increases in the whole cell concentrations of citrate and malate [Saha, A. K., D. Vavvas, T. G. Kurowski, A. Apazidis, L. A. Witters, E. Shafrir, and N. B. Ruderman. Am. J. Physiol. 272 ( Endocrinol. Metab. 35): E641–E648, 1997]. We report here that sustained increases in plasma insulin and glucose may also increase the concentration of malonyl-CoA in rat skeletal muscle in vivo by this mechanism. Thus 70 and 125% increases in malonyl-CoA induced in skeletal muscle by infusions of glucose for 1 and 4 days, respectively, and a twofold increase in its concentration during a 90-min euglycemic-hyperinsulinemic clamp were all associated with significant increases in the sum of whole cell concentrations of citrate and/or malate. Similar correlations were observed in muscle of the hyperinsulinemic fa/fa rat, in denervated muscle, and in muscle of rats infused with insulin for 5 h. In muscle of 48-h-starved rats 3 and 24 h after refeeding, increases in malonyl-CoA were not accompanied by consistent increases in the concentrations of malate or citrate. However, they were associated with a decrease in the whole cell concentration of long-chain fatty acyl-CoA (LCFA-CoA), an allosteric inhibitor of ACC. The results suggest that increases in the concentration of malonyl-CoA, caused in rat muscle in vivo by sustained increases in plasma insulin and glucose or denervation, may be due to increases in the cytosolic concentration of citrate. In contrast, during refeeding after starvation, the increase in malonyl-CoA in muscle is probably due to another mechanism.

Malonyl-CoA is an allosteric inhibitor of carnitine palmitoyltransferase (CPT) I, the enzyme that controls the transfer of long-chain fatty acyl (LCFA)-CoAs into the mitochondria where they are oxidized. In rat skeletal muscle, the formation of malonyl-CoA is regulated acutely (in minutes) by changes in the activity of the β-isoform of acetyl-CoA carboxylase (ACCβ). This can occur by at least two mechanisms: one involving cytosolic citrate, an allosteric activator of ACCβand a precursor of its substrate cytosolic acetyl-CoA, and the other involving changes in ACCβphosphorylation. Increases in cytosolic citrate leading to an increase in the concentration of malonyl-CoA occur when muscle is presented with insulin and glucose, or when it is made inactive by denervation, in keeping with a diminished need for fatty acid oxidation in these situations. Conversely, during exercise, when the need of the muscle cell for fatty acid oxidation is increased, decreases in the ATP/AMP and/or creatine phosphate-to-creatine ratios activate an isoform of an AMP-activated protein kinase (AMPK), which phosphorylates ACCβand inhibits both its basal activity and activation by citrate. The central role of cytosolic citrate links this malonyl-CoA regulatory mechanism to the glucose-fatty acid cycle concept of Randle et al. (P. J. Randle, P. B. Garland. C. N. Hales, and E. A. Newsholme. Lancet 1: 785–789, 1963) and to a mechanism by which glucose might autoregulate its own use. A similar citrate-mediated malonyl-CoA regulatory mechanism appears to exist in other tissues, including the pancreatic β-cell, the heart, and probably the central nervous system. It is our hypothesis that by altering the cytosolic concentrations of LCFA-CoA and diacylglycerol, and secondarily the activity of one or more protein kinase C isoforms, changes in malonyl-CoA provide a link between fuel metabolism and signal transduction in these cells. It is also our hypothesis that dysregulation of the malonyl-CoA regulatory mechanism, if it leads to sustained increases in the concentrations of malonyl-CoA and cytosolic LCFA-CoA, could play a key role in the pathogenesis of insulin resistance in muscle. That it may contribute to abnormalities associated with the insulin resistance syndrome in other tissues and the development of obesity has also been suggested. Studies are clearly needed to test these hypotheses and to explore the notion that exercise and some pharmacological agents that increase insulin sensitivity act via effects on malonyl-CoA and/or cytosolic LCFA-CoA.

We thank Taegtmeyer et al. (1) and Zaccardi et al. (2) for their insightful comments on our recent article (3). Both are very supportive of our view that insulin resistance (IR) has a protective role against insulin-mediated metabolic stress in overweight and obese patients with type 2 diabetes (T2D). Our article is similar to several recent articles (4–7). With this consolidation of views, clinicians should consider the implications of this concept for their patients with obesity-associated T2D, as overriding IR could cause harm for critical tissues such as the heart. We entirely agree with Taegtmeyer et al. (1) that “in the management of diabetes of patients with heart failure the …

Nearly 20 years ago, it was suggested that individuals exist who are not obese on the basis of height and weight, but who, like people with overt obesity, are hyperinsulinemic, insulin-resistant, and predisposed to type 2 diabetes, hypertriglyceridemia, and premature coronary heart disease. Since then it has become increasingly clear that such metabolically obese, normal-weight (MONW) individuals are very common in the general population and that they probably represent one end of the spectrum of people with the insulin resistance syndrome. Available evidence also suggests that MONW individuals could account for the higher prevalence of type 2 diabetes, cardiovascular disease, and other disorders in people with a BMI in the 20-27 kg/m2 range who have gained modest amounts of weight (2-10 kg of adipose mass) in adult life. Specific factors that appear to predispose MONW, as well as more obese individuals, to insulin resistance include central fat distribution, inactivity, and a low VO2max. Because these factors are potentially reversible and because insulin resistance may contribute to the pathogenesis of many diseases, it is our premise that a compelling argument can be made for identifying MONW individuals and treating them with diet, exercise, and possibly pharmacological agents before these diseases become overt, or at least early after their onset. One reason for doing so is that disorders such as type 2 diabetes may be accompanied by irreversible consequences, e.g., ischemic heart disease and nephropathy, at the time of diagnosis or shortly thereafter. Another is that MONW individuals in general should be younger and more amenable and responsive to diet and exercise therapy than are obese patients with established disease. That long-term diet and exercise can work is suggested by two large studies in which, over 5-6 years, the incidence of diabetes was diminished in nonobese and minimally obese patients with impaired glucose tolerance. Based on these considerations and the emerging worldwide epidemic of type 2 diabetes, we believe that studies to assess whether therapies aimed at young MONW individuals can prevent the development of type 2 diabetes and other diseases, including perhaps obesity itself, are urgently needed.

Malonyl CoA is an inhibitor of carnitine palmitoyl transferase 1 (CPT1), the enzyme that regulates the transfer of long chain fatty acyl CoA into mitochondria. By virtue of this effect, it is thought to play a key role in regulating fatty acid oxidation. Thus, when the supply of glucose to muscle is increased, malonyl CoA levels increase in keeping with a decreased need for fatty acid oxidation, and fatty acids are preferentially esterified to form diaglycerol and triglycerides. In contrast, during exercise, when the need for fatty acid oxidation is increased, malonyl CoA levels fall. Changes in glucose supply regulate malonyl CoA by modulating the concentration of cytosolic citrate, an allosteric activator of acetyl CoA carboxylase (ACC), the rate-limiting enzyme for malonyl CoA formation and a precursor of its substrate cytosolic acetyl CoA. Conversely, exercise lowers the concentration of malonyl CoA, by activating an AMP-activated protein kinase, which phosphorylates and inhibits ACC. A number of reports have linked sustained increases in the concentration of malonyl CoA in muscle to insulin resistance. In this paper, we review these reports, as well as the notion that changes in malonyl CoA contribute to the increases in long chain fatty acyl CoA, (LCFA CoA), diacylglycerol and triglyceride content and changes in protein kinase C activity and distribution observed in insulin-resistant muscle. We also review the implications of the malonyl CoA/LCFA CoA hypothesis to two other proposed mechanisms for insulin resistance, the glucose-fatty acid cycle and the hexosamine theory.

The concentration of malonyl-CoA, a negative regulator of fatty acid oxidation, diminishes acutely in contracting skeletal muscle. To determine how this occurs, the activity and properties of acetyl-CoA carboxylase beta (ACC-beta), the skeletal muscle isozyme that catalyzes malonyl-CoA formation, were examined in rat gastrocnemius-soleus muscles at rest and during contractions induced by electrical stimulation of the sciatic nerve. To avoid the problem of contamination of the muscle extract by mitochondrial carboxylases, an assay was developed in which ACC-beta was first purified by immunoprecipitation with a monoclonal antibody. ACC-beta was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a Km for acetyl-CoA (120 microM) similar to that reported for ACC-beta purified by other means. After 5 min of contraction, ACC-beta activity was decreased by 90% despite an apparent increase in the cytosolic concentration of citrate, a positive regulator of ACC. SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these muscles showed a decrease in the electrophoretic mobility of ACC, suggesting that phosphorylation could account for the decrease in ACC activity. In keeping with this notion, citrate activation of ACC purified from contracting muscle was markedly depressed. In addition, homogenization of the muscles in a buffer free of phosphatase inhibitors and containing the phosphatase activators glutamate and MgCl2 or treatment of immunoprecipitated ACC-beta with purified protein phosphatase 2A abolished the decreases in both ACC-beta activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-beta activity after the onset of contractions (50% by 20 s) and its slow restoration to initial values during recovery (60-90 min) were paralleled temporally by reciprocal changes in the activity of the alpha2 but not the alpha1 isoform of 5'-AMP-activated protein kinase (AMPK). In conclusion, the results suggest that the decrease in ACC activity during muscle contraction is caused by an increase in its phosphorylation, most probably due, at least in part, to activation of the alpha2 isoform of AMPK. They also suggest a dual mechanism for ACC regulation in muscle in which inhibition by phosphorylation takes precedence over activation by citrate. These alterations in ACC and AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase in fatty acid oxidation observed in skeletal muscle during exercise.

Malonyl-CoA is an inhibitor of carnitine palmitoyltransferase I, the enzyme that controls the oxidation of fatty acids by regulating their transfer into the mitochondria. Despite this, knowledge of how malonyl-CoA levels are regulated in skeletal muscle, the major site of fatty acid oxidation, is limited. Two- to fivefold increases in malonyl-CoA occur in rat soleus muscles incubated with glucose or glucose plus insulin for 20 min [Saha, A. K., T. G. Kurowski, and N. B. Ruderman. Am. J. Physiol. 269 (Endocrinol. Metab. 32): E283-E289, 1995]. In addition, as reported here, acetoacetate in the presence of glucose increases malonyl-CoA levels in the incubated soleus. The increases in malonyl-CoA in all of these situations correlated closely with increases in the concentration of citrate (r2 = 0.64) and to an even greater extent the sum of citrate plus malate (r2 = 0.90), an antiporter for citrate efflux from the mitochondria. Where measured, no increase in the activity of acetyl-CoA carboxylase (ACC) was found. Inhibition of ATP citrate lyase with hydroxycitrate markedly diminished the increases in malonyl-CoA in these muscles, indicating that citrate was the major substrate for the malonyl-CoA precursor, cytosolic acetyl-CoA. Studies with enzyme purified by immunoprecipitation indicated that the observed increases in citrate could have also allosterically activated ACC. The results suggest that in the presence of glucose, insulin and acetoacetate acutely increase malonyl-CoA levels in the incubated soleus by increasing the cytosolic concentration of citrate. This novel mechanism could complement the glucose-fatty acid cycle in determining how muscle chooses its fuels. It could also provide a means by which glucose acutely modulates signal transduction in muscle and other cells (e.g., the pancreatic beta-cell) in which its metabolism is determined by substrate availability.

Individuals with type 2 diabetes are insulin resistant and as a group have a lower exercise capacity ( V o2max) than age- and weight-matched people without diabetes (1,2). In this issue, Regensteiner et al. (3) report that rosiglitazone (RSG), a thiazolidinedione (TZD) commonly used to treat insulin resistance, also improves exercise capacity in patients with type 2 diabetes. Following 4 months of treatment with 4 mg/day RSG, the authors observed expected improvements in insulin sensitivity as determined by homeostasis model assessment and a hyperinsulinemic-euglycemic clamp in 10 middle-aged men and women with type 2 diabetes. A novel finding was that RSG caused a modest but significant increase in V o2max (1.4 ml · kg−1 · min−1 or ∼7.1%). These observations raise three fundamental questions: 1 ) Why is type 2 diabetes associated with a decrease in exercise capacity? 2 ) How might TZDs, such as RSG, counteract this? and 3 ) Is the effect of RSG on exercise capacity likely to be clinically relevant? Recent studies suggest several explanations for the decrease in V o2max in patients with type 2 diabetes. One of these is the presence of mitochondrial dysfunction. As reported by Kelley et al. and Ritov et al., (4–6) skeletal muscle of sedentary middle-aged individuals with established type 2 diabetes (HbA1c 8.0 ± 0.2%) exhibits reduced mitochondrial oxidative enzyme (succinate dehydrogenase) activity and electron transport chain capacity (rotenone-sensitive NADH:O2 oxido-reductase activity) (∼26 and 59%, respectively), smaller mitochondria, and higher intramyocellular triglyceride content than muscle of normal control subjects. Their data also suggested that subsarcolemmal mitochondria were especially affected. In addition to these …

Despite a strong correlation between obesity and insulin resistance, 25% of severely obese (BMI40) individuals are insulin sensitive. In this review, we will examine the factors in adipose tissue that distinguish the two groups, as well as reasons for believing the insulin-sensitive group will be less disease prone.Obesity has been linked to the metabolic syndrome with an increase in visceral (intra-abdominal) compared to subcutaneous fat. Recent studies in which adipose tissue of insulin-sensitive and insulin-resistant patients with severe obesity were compared indicate that the insulin-resistant group is also distinguished by increases in oxidative stress and decreases in AMP-activated protein kinase (AMPK) activity. In contrast, changes in the expression of genes for SIRT1, inflammatory cytokines, mitochondrial biogenesis and function, and the two α-isoforms of AMPK showed more depot variation. Studies of how these and other changes in adipose tissue respond to bariatric surgery are still in their infancy.Available data suggest that increases in oxidative stress, decreases in AMPK activity and SIRT1 gene expression, depot-specific changes in inflammatory, mitochondrial and other genes distinguish adipose tissue of insulin resistant from insulin-sensitive individuals with severe obesity.

Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a novel regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a novel SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.

The effect of insulin on Na+ pump activity, measured as ouabain-sensitive (OS) 86Rb uptake, was studied in the rabbit aorta. In the absence of insulin, incubation of endothelium-intact rings for 3 h in a medium containing a high concentration of glucose (44 mM) decreased OS 86Rb uptake by 42% compared with that observed at 5.5 mM glucose. Addition of insulin (0.1-10 microU/ml) increased OS86 86Rb uptake at both glycose concentrations and eliminated the differences between the groups. Insulin also increased OS 86Rb uptake in endothelium-intact and -denuded (ED) rings in the presence of the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine. Removal of the endothelium before the incubations did not diminish the insulin-induced increase in OS 86Rb uptake, which was concentration dependent. The NO donor sodium nitroprusside increased OS 86Rb uptake in ED rings, and its effect and that of insulin were additive. Phorbol 12,13-dibutyrate, a direct activator of protein kinase C (PKC), also increased OS 86Rb uptake in ED rings; however, its effect and that of insulin were not additive. The PKC inhibitor bisindolylmaleimide totally inhibited insulin-induced, but not sodium nitroprusside-induced, increases in OS 86Rb uptake. The results suggest that insulin activates the Na+ pump in the aorta and reverses the inhibition of the pump caused by hyperglycemia. This effect of insulin can occur at physiological concentrations, is independent of endothelium-derived NO, and is presumably mediated by an increase in PKC activity, In contrast, activation of the Na+ pump by NO appears to be independent of PKC.

Background— Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling, through the activity of protein kinase C-β (PKCβ) and nuclear factor κB, reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results— We measured protein expression and insulin response in freshly isolated endothelial cells from patients with type 2 diabetes mellitus (n=40) and nondiabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes mellitus ( P =0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in nondiabetic subjects but not in diabetic patients ( P =0.003), consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients, indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P =0.02). Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes mellitus. Endothelial nuclear factor κB activation was higher in diabetes mellitus and was reduced with PKCβ inhibition. Conclusions— We provide evidence for the presence of altered eNOS activation, reduced insulin action, and inflammatory activation in the endothelium of patients with diabetes mellitus. Our findings implicate PKCβ activity in endothelial insulin resistance.

Background— The liver-secreted protein fetuin-A induces peripheral insulin resistance in vitro. In a pilot study, we observed that higher fetuin-A levels were associated with diabetes mellitus in older persons. However, this finding has not been confirmed in large cohorts. We sought to confirm the association of fetuin-A with incident diabetes mellitus in older persons and to determine whether the association differs by age, sex, and race and among persons with cardiovascular disease (CVD). Methods and Results— Among 3710 community-living individuals ≥65 years of age without diabetes mellitus at baseline, fetuin-A was measured in serum collected in 1992 to 1993. Participants were followed up for 10.6 years (median) for incident diabetes mellitus. Cox regression models evaluated the association of fetuin-A with incident diabetes mellitus. Interaction terms evaluated heterogeneity by age, sex, race, and CVD. Mean age was 75 years; 60 were female; 15 were black; and 16 had CVD. Mean fetuin-A concentrations were 0.47±0.10 g/L. During follow-up, 305 incident diabetes cases occurred. Each 0.10-g/L (SD)-greater fetuin-A was associated with 19 higher risk of diabetes mellitus (hazard ratio, 1.19; 95 confidence interval, 1.06–1.33) after adjustment for demographics, lifestyle factors, albumin, kidney function, and CVD. Further adjustment for potential mediators (body mass index, waist circumference, hypertension, lipids, and C-reactive protein) moderately attenuated the association (hazard ratio, 1.13; 95 confidence interval, 1.00–1.28). Results were similar by sex, race, and CVD status but were stronger in persons P for interaction=0.01). Conclusions— Higher fetuin-A is associated with incident diabetes mellitus in older persons regardless of sex, race, or prevalent CVD status. The association may be attenuated in those ≥75 years of age.

AMP-activated protein kinase (AMPK) and the NAD+-dependent histone/protein deacetylase sirtuin 1 (SIRT1) are metabolic sensors that can increase each other's activity. They are also both activated by the antidiabetic drug metformin and downregulated in the liver under conditions of nutrient excess (e.g., hyperglycemia, high-fat diet, obesity). In these situations, the abundance of the tumor suppressor p53 is increased; however, the relevance of this to the changes in AMPK and SIRT1 is not known. In the present study we investigated this question in HepG2 cells under high glucose conditions. Metformin induced activation of AMPK and SIRT1 and decreased p53 protein abundance. It also decreased triglyceride accumulation and cytosolic oxidative stress (a trigger for p53 accumulation) and increased the deacetylation of p53 at a SIRT1-targeted site. The decrease in p53 abundance caused by metformin was abolished by inhibition of murine double minute 2 (MDM2), a ubiquitin ligase that mediates p53 degradation, as well as by overexpression of a dominant-negative AMPK or a shRNA-mediated knockdown of SIRT1. In addition, overexpression of p53 decreased SIRT1 gene expression and protein abundance, as well as AMPK activity in metformin-treated cells. It also diminished the triglyceride-lowering action of metformin, an effect that was rescued by incubation with the SIRT1 activator SRT2183. Collectively, these findings suggest the existence of a novel reciprocal interaction between AMPK/SIRT1 and p53 that may have implications for the pathogenesis and treatment of metabolic diseases.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a heterodimer composed of an 85-kDa subunit that binds tyrosyl-phosphorylated proteins via its SH2 domains and a 110-kDa catalytic subunit. Expression and mutagenesis experiments have shown that the 110-kDa subunit is a dual specificity kinase that possesses both lipid and serine kinase activities. Except for the 85- and 110-kDa subunits of PI 3-kinase, however, no endogenous substrates for the serine kinase have been identified. The results of the present study show that another target of this kinase is the insulin receptor substrate, IRS-1. Serine phosphorylation of IRS-1 as well as the 85-kDa subunit of PI 3-kinase was demonstrated in immunoprecipitates of PI 3-kinase and IRS-1 isolated from rat adipocytes incubated with insulin. In adipocytes incubated in the absence of insulin, only the serine phosphorylation of p85 was observed in immunoprecipitates of PI 3-kinase. Both the serine and lipid kinase activities of PI 3-kinase were abolished by the fungal metabolite Wortmannin. Wortmannin also partially inhibited the ability of insulin to stimulate glucose transport and inhibit lipolysis in fat cells. These data raise the possibility that the serine kinase activity of PI 3-kinase is involved in insulin signaling. They also suggest that inhibition of the lipid or serine kinase activities of PI 3-kinase could explain the effect of Wortmannin to diminish insulin action.

Insulin resistance is present in liver and muscle of subjects with type 2 diabetes and obesity. Recent studies suggest that such insulin resistance could be related to abnormalities in lipid-mediated signal transduction; however, the nature of these abnormalities is unclear. To examine this question further, tissue levels of diacylglycerol (DAG), malonyl-CoA, and triglyceride (TG) were determined in liver and soleus muscle of obese insulin-resistant KKAy mice and lean C57 BL control mice. In addition, the effects of treatment with pioglitazone, an antidiabetic agent that acts by increasing insulin sensitivity in muscle, liver, and other tissues, were assessed. The KKAy mice were hyperglycemic (407 vs. 138 mg/dl), hypertriglyceridemic (337 vs. 109 mg/dl), hyperinsulinemic (631 vs. 15 mU/ml), and weighed more (42 vs. 35 g) than the control mice. They also had 1.5- to 2.0-fold higher levels of malonyl-CoA in both liver and muscle, higher DAG (twofold) and TG (1.3-fold) levels in muscle, and higher TG (threefold), but not DAG, levels. Treatment of the KKAy mice with pioglitazone for 4 days decreased plasma glucose, TGs, and insulin by approximately 50% and restored hepatic and muscle malonyl-CoA levels to control values. In contrast, pioglitazone increased hepatic and muscle DAG levels two- or threefold. It has no effect on muscle or hepatic TG content, and it slightly increased hepatic TGs in the control group. The results indicate that abnormalities in tissue lipids occur in both liver and muscle of the KKAy mouse and that they are differentially altered when insulin sensitivity is enhanced by treatment with pioglitazone.(ABSTRACT TRUNCATED AT 250 WORDS)

An endothelium-derived factor with the properties of nitric oxide (NO) has recently been implicated in the regulation of basal Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity in vascular smooth muscle. To determine whether this factor also plays a role in the stimulation of ouabain-sensitive (OS) 86Rb uptake by specific agonists, studies were carried out using rabbit aortic rings. In endothelium-intact rings incubated for 3 h with Krebs-Henseleit solution containing 5.5 mM glucose, endothelin (ET) caused a concentration-dependent increase in OS 86Rb uptake (maximal increase = 205%, with 100 nM ET). Incubation with phenylephrine (Phe; 0.1 and 1 microM) or phorbol 12,13-dibutyrate (PDBu; 0.1 microM), under the same conditions, increased OS 86Rb uptake by 128, 144, and 140%, respectively. Removal of endothelium before incubation decreased the ability of ET to stimulate OS 86Rb uptake by 38–45%, but it did not diminish the stimulation of OS 86Rb uptake by Phe or PDBu. An increase in the concentration of glucose from 5.5 to 44 mM diminished ET-stimulated OS 86Rb uptake by 50% in endothelium-intact rings but had no effect on Phe- or PDBu-induced increases in OS 86Rb uptake. Addition of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 0.3 mM) to the medium decreased ET-stimulated OS 86Rb uptake by 40%. Guanosine 3',5'-cyclic monophophate (cGMP) formation in endothelium-intact rings was also increased (65%) by ET but not by Phe or PDBu. The increase in cGMP by ET was totally inhibited by L-NMMA or endothelium denudation.(ABSTRACT TRUNCATED AT 250 WORDS)

It has long been known that excesses of glucose and branched chain amino acids, such as leucine, lead to insulin resistance in skeletal muscle. A recent study in incubated rat muscle suggests that both molecules may do so by virtue of their ability to downregulate the fuel sensing and signaling enzyme AMP-activated protein kinase (AMPK) and activate mTOR/p70S6 kinase (p70S6K) signaling. The results also demonstrated that inhibition of mTOR/p70S6K with rapamycin prevented the development of insulin resistance but had no effect on AMPK activity (Thr172 phosphorylation of its catalytic subunit). In contrast, activation of AMPK by both AICAR and α-lipoic acid led to the phosphorylation of specific molecules that diminished both mTOR/p70S6K signaling and insulin resistance. These findings suggest that downregulation of AMPK precedes mTOR/p70S6K activation in mediating glucose and leucine-induced insulin resistance, although the mechanism by which it does so remains to be determined. Also requiring study is how an excess of the two nutrients leads to AMPK downregulation.

We investigated the effects of AMPK on H(2)O(2)-induced premature senescence in primary human keratinocytes. Incubation with 50 µM H(2)O(2) for 2 h resulted in premature senescence with characteristic increases in senescence-associated ß-galactosidase (SA-gal) staining 3 days later and no changes in AMPK or p38 MAPK activity. The increase in SA-gal staining was preceded by increases in both p53 phosphorylation (S15) (1 h) and transactivation (6 h) and the abundance of the cyclin inhibitor p21(CIP1) (16 h). Incubation with AICAR or resveratrol, both of which activated AMPK, prevented the H(2)O(2)-induced increases in both SA-Gal staining and p21 abundance. In addition, AICAR diminished the increase in p53 transactivation. The decreases in SA-Gal expression induced by resveratrol and AICAR were prevented by the pharmacological AMPK inhibitor Compound C, expression of a DN-AMPK or AMPK knock-down with shRNA. Likewise, both knockdown of AMPK and expression of DN-AMPK were sufficient to induce senescence, even in the absence of exogenous H(2)O(2). As reported by others, we found that AMPK activation by itself increased p53 phosphorylation at S15 in embryonic fibroblasts (MEF), whereas under the same conditions it decreased p53 phosphorylation in the keratinocytes, human aortic endothelial cells, and human HT1080 fibrosarcoma cells. In conclusion, the results indicate that H(2)O(2) at low concentrations causes premature senescence in human keratinocytes by activating p53-p21(CIP1) signaling and that these effects can be prevented by acute AMPK activation and enhanced by AMPK downregulation. They also suggest that this action of AMPK may be cell or context-specific.

We studied the extraction and analysis of integral membrane proteins possessing hydrophobic and hydrophilic domains and found that a nonionic detergent called MEGA-10, used in lysis buffers, had a superior extraction effect compared to most conventional detergents. A sodium dodecyl sulfate (SDS) concentration of >0.4% (w/v) in the sample buffer was crucial for those proteins to be clearly analyzed by electrophoresis and Western blotting. Furthermore, MEGA-10 had the tendency to maximally extract proteins around its critical micelle concentration (CMC) of 0.24% (w/v). These solutions can greatly assist functional investigations of membrane proteins in the proteomics era.

Diabetic retinopathy (DR) is a leading cause of blindness in adults worldwide.1 During its course retinal cells of both vascular and neural origin undergo apoptosis that leads to interruptions in nutritive blood flow, neural dysfunction, and, ultimately, impaired vision. One of the earliest cells to undergo apoptosis in this setting is the microvascular pericyte (PC).2 Various theories have attributed PC apoptosis to hyperglycemia-induced increases in sorbitol, hexosamines, advanced glycation-end products, and protein kinase C (PKC) activity, all of which have also been linked to the death of endothelial and neural cells in the retina.3–7 In addition to hyperglycemia, dyslipidemia has recently been implicated in the pathogenesis of DR in humans. Thus, results from the Action to Control Cardiovascular Risk in Diabetes (ACCORD) eye study8 and especially the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study9 demonstrated that the lipid-lowering agent fenofibrate reduced progression to retinopathy, necessitating laser treatment by 31% in patients with type 2 diabetes and good glycemic control (HbA1C, ∼7.0%). Like hyperglycemia, dyslipidemia, including elevated serum free fatty acid (FFA) levels, is a common feature in patients with poorly controlled types 1 and 2 diabetes.10,11 We have demonstrated that elevated levels of the FFA palmitate increase apoptosis in bovine retinal PCs by an effect dependent on increases in oxidative stress, ceramide synthesis, and NF-κB activation.12 Evidence that elevated FFAs, and specifically saturated fatty acids such as palmitate, can cause dysfunction is strongly suggested by studies with other cultured cells. Thus, the incubation of pancreatic β cells, cardiomyocytes, skeletal muscle myotubes, and vascular endothelium with elevated levels of FFAs has all been shown to cause apoptosis, insulin resistance, or both.13–17 In addition to lowering plasma lipids, fenofibrate, the main drug tested in the FIELD study, is an activator of AMP-activated protein kinase (AMPK).18 AMPK is a fuel and stress-sensing enzyme that is activated by such antidiabetic and lipid-lowering therapies as metformin, the thiazolidinediones and statins, and cellular energy deficits caused by hypoxia and exercise. Once activated, AMPK phosphorylates key metabolic enzymes resulting in an increase in processes that generate adenosine triphosphate (ATP), such as fatty acid oxidation (FAox), and a decrease in others that consume ATP but are not acutely necessary for survival, such as fatty acid and triglyceride synthesis.17,19,20 In this regard, AMPK has the potential to protect cells against the adverse effects of high glucose and FFAs by preventing the accumulation of damaging or toxic secondary metabolites such as diacylglycerol (DAG) or ceramides and by effects on multiple transcriptional activators and coactivators.21,22 In the present study, we tested whether AMPK activation protects PCs against palmitate-induced cytotoxicity. We also compared the effects on the PC of palmitate with that of oleate, a fatty acid that, at similar concentrations, is not toxic to PC.12,23