Satellite cells derived from obese humans with type 2 diabetes and differentiated into myocytes in vitro exhibit abnormal response to IL-6

Obesity and type 2 diabetes are associated with chronically elevated systemic levels of IL-6, a pro-inflammatory cytokine with a role in skeletal muscle metabolism that signals through the IL-6 receptor (IL-6Rα). We hypothesized that skeletal muscle in obesity-associated type 2 diabetes develops a resistance to IL-6. By utilizing western blot analysis, we demonstrate that IL-6Rα protein was down regulated in skeletal muscle biopsies from obese persons with and without type 2 diabetes. To further investigate the status of IL-6 signaling in skeletal muscle in obesity-associated type 2 diabetes, we isolated satellite cells from skeletal muscle of people that were healthy (He), obese (Ob) or were obese and had type 2 diabetes (DM), and differentiated them in vitro into myocytes. Down-regulation of IL-6Rα was conserved in Ob myocytes. In addition, acute IL-6 administration for 30, 60 and 120 minutes, resulted in a down-regulation of IL-6Rα protein in Ob myocytes compared to both He myocytes (P<0.05) and DM myocytes (P<0.05). Interestingly, there was a strong time-dependent regulation of IL-6Rα protein in response to IL-6 (P<0.001) in He myocytes, not present in the other groups. Assessing downstream signaling, DM, but not Ob myocytes demonstrated a trend towards an increased protein phosphorylation of STAT3 in DM myocytes (P = 0.067) accompanied by a reduced SOCS3 protein induction (P<0.05), in response to IL-6 administration. Despite this loss of negative control, IL-6 failed to increase AMPKα2 activity and IL-6 mRNA expression in DM myocytes. There was no difference in fusion capacity of myocytes between cell groups. Our data suggest that negative control of IL-6 signaling is increased in myocytes in obesity, whereas a dysfunctional IL-6 signaling is established further downstream of IL-6Rα in DM myocytes, possibly representing a novel mechanism by which skeletal muscle function is compromised in type 2 diabetes.     

Resveratrol inhibition of inducible nitric oxide synthase in skeletal muscle involves AMPK but not SIRT1

The plant-derived polyphenol resveratrol (RSV) modulates life span and metabolism, and it is thought that these effects are largely mediated by activating the deacetylase enzyme SIRT1. However, RSV also activates the cell energy sensor AMP-activated protein kinase (AMPK). We have previously reported that AMPK activators inhibit inducible nitric oxide synthase (iNOS), a key proinflammatory mediator of insulin resistance in endotoxemia and obesity. The aim of this study was to evaluate whether RSV inhibits iNOS induction in insulin target tissues and to determine the role of SIRT1 and AMPK activation in this effect. We found that RSV (40 mg/kg ip) treatment decreased iNOS induction and NO production in skeletal muscle and white adipose tissue, but not in liver, of endotoxin (LPS)-challenged mice. This effect of the polyphenol was recapitulated in vitro, where RSV (10-80 μM) robustly inhibited iNOS protein induction and NO production in cytokine/LPS-treated L6 myocytes and 3T3-L1 adipocytes. However, no effect of RSV was observed on iNOS induction in FAO hepatocytes. Further studies using inhibitors of SIRT1 revealed that the deacetylase enzyme is not involved in RSV action on iNOS. In marked contrast, RSV activates AMPK in L6 myocytes, and blunting its activation using Compound C or RNA interference partly blocked the inhibitory effect of RSV on NO production. These results show that RSV specifically inhibits iNOS induction in muscle through a mechanism involving AMPK but not SIRT1 activation. This anti-inflammatory action of RSV likely contributes to the therapeutic effect of this plant polyphenol.     

SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase

Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser(428), and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKKbeta, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases.     

Berberine suppresses neuroinflammatory responses through AMP‐activated protein kinase activation in BV‐2 microglia

The AMPK cascade is a sensor of cellular energy change, which monitors the AMP/ATP ratio to regulate cellular metabolism by restoring ATP levels, but its regulation of neuroinflammation mechanism remains unclear. Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been shown to improve several metabolic disorders, such as obesity and type II diabetes. However, the effect of berberine on neuroinflammatory responses in microglia are poorly understood. This study shows that berberine represses proinflammatory responses through AMP‐activated protein kinase (AMPK) activation in BV‐2 microglia. Our findings also demonstrate that berberine significantly down‐regulates LPS‐ or interferon (IFN)‐γ‐induced nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2) expression in BV‐2 microglia cells. Berberine also inhibited LPS‐ or IFN‐γ‐induced nitric oxide production. In addition, berberine effectively inhibited proinflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6 expression. On the other hand, upon various inflammatory stimulus including LPS and IFN‐γ, berberine suppressed the phosphorylated of ERK but not p38 and JNK in BV‐2 microglia. AMPK activation is catalyzed by upstream kinases such as LKB1 and Ca2+/calmodulin‐dependent protein kinase kinase‐II (CaMKK II). Moreover, berberine induced LKB1 (Ser428), CaMKII (Thr286), and AMPK (Thr172) phosphorylation, but not AMPK (Ser485). Furthermore, the inhibitory effect of berberine on iNOS and COX‐2 expression was abolished by AMPK inhibition via Compound C, an AMPK inhibitor. Berberine‐suppressed ERK phosphorylation was also reversed by Compound C treatment. Our data demonstrate that berberine significantly induces AMPK signaling pathways activation, which is involved in anti‐neuroinflammation. J. Cell. Biochem. 110: 697–705, 2010. © 2010 Wiley‐Liss, Inc.     

Metformin inhibits inflammatory response via AMPK-PTEN pathway in vascular smooth muscle cells

Atherosclerosis is a chronic inflammation of the coronary arteries. Vascular smooth muscle cells (VSMCs) stimulated by cytokines and chemokines accelerate the inflammatory response and migrate to the injured endothelium during the progression of atherosclerosis. Activation of AMP activated protein kinase (AMPK), a key sensor maintaining metabolic homeostasis, suppresses the inflammatory response. However, how AMPK regulates the inflammatory response is poorly understood. To identify the mechanism of this response, we focused on phosphatase and tensin homolog (PTEN), which is a negative regulator of inflammation. We investigated that activation of AMPK-induced PTEN expression and suppression of the inflammatory response through the AMPK-PTEN pathway in VSMCs. We treated with the well-known AMPK activator metformin to induce PTEN expression. PTEN was induced by metformin (2mM) and inhibited by compound C (10μM) and AMPK siRNA. Tumor necrosis factor-alpha (TNF-α) was used to induce inflammation. The inflammatory response was confirmed by cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) expression, and activation of nuclear factor (NF)-κB. Metformin suppressed COX-2 and iNOS mRNA and protein expression dose dependently. Treatment with compound C and bpv (pic) in the presence of metformin, iNOS and COX-2 protein expression increased. NF-κB activation decreased in response to metformin and was restored by inhibiting AMPK and PTEN. Inhibiting AMPK and PTEN restored ROS levels stimulated with TNF-α. Taken together, PTEN could be a possible downstream regulator of AMPK, and the AMPK-PTEN pathway might be important in the regulation of the inflammatory response in VSMCs.     

Adiponectin-mediated modulation of hypertrophic signals in the heart

Patients with diabetes and other obesity-linked conditions have increased susceptibility to cardiovascular disorders. The adipocytokine adiponectin is decreased in patients with obesity-linked diseases. Here, we found that pressure overload in adiponectin-deficient mice resulted in enhanced concentric cardiac hypertrophy and increased mortality that was associated with increased extracellular signal-regulated kinase (ERK) and diminished AMP-activated protein kinase (AMPK) signaling in the myocardium. Adenovirus-mediated supplemention of adiponectin attenuated cardiac hypertrophy in response to pressure overload in adiponectin-deficient, wild-type and diabetic db/db mice. In cultures of cardiac myocytes, adiponectin activated AMPK and inhibited agonist-stimulated hypertrophy and ERK activation. Transduction with a dominant-negative form of AMPK reversed these effects, suggesting that adiponectin inhibits hypertrophic signaling in the myocardium through activation of AMPK signaling. Adiponectin may have utility for the treatment of hypertrophic cardiomyopathy associated with diabetes and other obesity-related diseases.     

Inositol polyphosphate multikinase: an emerging player for the central action of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an essential enzyme indispensable for energy sensing and metabolic homeostasis at both the cellular and whole-body levels. Phosphorylation of AMPK, a key step for its activation, is known to be regulated by upstream kinases such as liver kinase B1 (LKB1) and calmodulin-dependent protein kinase kinase-beta (CaMKKβ). Recent evidence shows that inositol polyphosphate multikinase (IPMK), which possesses both inositol phosphate kinase and lipid inositol kinase activities, can physiologically regulate AMPK signaling in cultured cells and in the arcuate nucleus. IPMK-mediated regulation of AMPK occurs through the dynamic protein interactions of IPMK with AMPK in response to glucose availability. Here we review and discuss a novel role for the hypothalamic IPMK signaling in the control of AMPK and central energy homeostasis.     

Cellular energy sensing and signaling by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy sensing/signaling protein that, when activated, increases ATP production by stimulating glucose uptake and fatty acid oxidation while at the same time inhibiting ATP=consuming processes such as protein synthesis. Chronic activation of AMPK inhibits expression of lipogenic enzymes in the liver and enhances expression of mitochondrial oxidative enzymes in skeletal muscle. Deficiency of muscle LKB1, the upstream kinase of AMPK, results in greater fluctuation in energy charge during muscle contraction and decreased capacity for exercise at higher work rates. Because AMPK enhances both glucose uptake and fatty acid oxidation in skeletal muscle, it has become a target for prevention and treatment of type 2 diabetes and obesity.     

A liver-derived secretory protein, selenoprotein P, causes insulin resistance

The liver may regulate glucose homeostasis by modulating the sensitivity/resistance of peripheral tissues to insulin, by way of the production of secretory proteins, termed hepatokines. Here, we demonstrate that selenoprotein P (SeP), a liver-derived secretory protein, causes insulin resistance. Using serial analysis of gene expression (SAGE) and DNA chip methods, we found that hepatic SeP mRNA levels correlated with insulin resistance in humans. Administration of purified SeP impaired insulin signaling and dysregulated glucose metabolism in both hepatocytes and myocytes. Conversely, both genetic deletion and RNA interference-mediated knockdown of SeP improved systemic insulin sensitivity and glucose tolerance in mice. The metabolic actions of SeP were mediated, at least partly, by inactivation of adenosine monophosphate-activated protein kinase (AMPK). In summary, these results demonstrate a role of SeP in the regulation of glucose metabolism and insulin sensitivity and suggest that SeP may be a therapeutic target for type 2 diabetes.     

Metformin sensitizes insulin signaling through AMPK-mediated PTEN down-regulation in preadipocyte 3T3-L1 cells

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.     

Targeting AMPK signaling pathway by natural products for treatment of diabetes mellitus and its complications

Diabetes affects a large population of the world. Lifestyle, obesity, dietary habits, and genetic factors contribute to this metabolic disease. A target pathway to control diabetes is the 5′-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. AMPK is a heterotrimeric protein with α, β, and γ subunits. In several studies, AMPK activation enhanced glucose uptake into cells and inhibited intracellular glucose production. Impairment of AMPK activity is present in diabetes, according to some studies. Drugs used in the treatment of diabetes, such as metformin, are also known to act through regulation of AMPK. Thus, drugs that activate and regulate AMPK are potential candidates for the treatment of diabetes. In addition, many patients encounter important adverse effects, like hypoglycemia, while using allopathic drugs. As a result, the investigation of plant-derived natural drugs that lack adverse side effects and treat diabetes is necessary. Natural products like berberine, quercetin, resveratrol, and so forth have shown significant potential in regulating and activating the AMPK pathway which can lead to manage diabetes mellitus and its complications.     

Reciprocal Regulation of AMP-activated Protein Kinase and Phospholipase D

AMP-activated protein kinase (AMPK), a critical sensor of energy sufficiency, acts as central metabolic switch in cell metabolism. Once activated by low energy status, AMPK phosphorylates key regulatory substrates and turns off anabolic biosynthetic pathways. In contrast, the mammalian/mechanistic target of rapamycin (mTOR) is active when there are sufficient nutrients for anabolic reactions. A critical factor regulating mTOR is phosphatidic acid (PA), a central metabolite of membrane lipid biosynthesis and the product of the phospholipase D (PLD)-catalyzed hydrolysis of phosphatidylcholine. PLD is a downstream target of the GTPase Rheb, which is turned off in response to AMPK via the tuberous sclerosis complex. Although many studies have linked AMPK with mTOR, very little is known about the connection between AMPK and PLD. In this report, we provide evidence for reciprocal regulation of PLD by AMPK and regulation of AMPK by PLD and PA. Suppression of AMPK activity led to an increase in PLD activity, and conversely, activation of AMPK suppressed PLD activity. Suppression of PLD activity resulted in elevated AMPK activity. Exogenously supplied PA abolished the inhibitory effects of elevated AMPK activity on mTOR signaling. In contrast, exogenously supplied PA could not overcome the effect AMPK activation if either mTOR or Raptor was suppressed, indicating that the inhibitory effects of PLD and PA on AMPK activity are mediated by mTOR. These data suggest a reciprocal feedback mechanism involving AMPK and the PLD/mTOR signaling node in cancer cells with therapeutic implications.     

Identification of protein kinase D as a novel contraction-activated kinase linked to GLUT4-mediated glucose uptake, independent of AMPK

Contraction-induced glucose uptake is only partly mediated by AMPK activation. We examined whether the diacylglycerol-sensitive protein kinase D (PKD; also known as novel PKC isoform mu) is also involved in the regulation of glucose uptake in the contracting heart. As an experimental model, we used suspensions of cardiac myocytes, which were electrically stimulated to contract or treated with the contraction-mimicking agent oligomycin. Induction of contraction at 4 Hz in cardiac myocytes or treatment with 1 microM oligomycin enhanced (i) autophosphorylation of PKD at Ser916 by 5.1- and 3.8-fold, respectively, (ii) phosphorylation of PKD's downstream target cardiac-troponin-I (cTnI) by 2.9- and 2.1-fold, respectively, and (iii) enzymatic activity of immunoprecipitated PKD towards the substrate peptide syntide-2 each by 1.5-fold. Although AMPK was also activated under these same conditions, in vitro phosphorylation assays and studies with cardiac myocytes from AMPKalpha2(-/-) mice indicated that activation of PKD occurs independent of AMPK activation. CaMKKbeta, and the cardiac-specific PKC isoforms alpha, delta, and epsilon were excluded as upstream kinases for PKD in contraction signaling because none of these kinases were activated by oligomycin. Stimulation of glucose uptake and induction of GLUT4 translocation in cardiac myocytes by contraction and oligomycin each were sensitive to inhibition by the PKC/PKD inhibitors staurosporin and calphostin-C. Together, these data elude to a role of PKD in contraction-induced GLUT4 translocation. Finally, the combined actions of PKD on cTnI phosphorylation and on GLUT4 translocation would efficiently link accelerated contraction mechanics to increased energy production when the heart is forced to increase its contractile activity.     

Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase

Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.     

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.     

Macrophage α1 AMP-activated Protein Kinase (α1AMPK) Antagonizes Fatty Acid-induced Inflammation through SIRT1

In this study, we aim to determine cellular mechanisms linking nutrient metabolism to the regulation of inflammation and insulin resistance. The nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 show striking similarities in nutrient sensing and regulation of metabolic pathways. We find that the expression, activity, and signaling of the major isoform α1AMPK in adipose tissue and macrophages are substantially down-regulated by inflammatory stimuli and in nutrient-rich conditions, such as exposure to lipopolysaccharide (LPS), free fatty acids (FFAs), and diet-induced obesity. Activating AMPK signaling in macrophages by 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside or constitutively active α1AMPK (CA-α1) significantly inhibits; although inhibiting α1AMPK by short hairpin RNA knock-down or dominant-negative α1AMPK (DN-α1) increases LPS- and FFA-induced tumor necrosis factor α expression. Chromatin immunoprecipitation and luciferase reporter assays show that activation of AMPK by CA-α1 in macrophages significantly inhibits LPS- or FFA-induced NF-κB signaling. More importantly, in a macrophage-adipocyte co-culture system, we find that inactivation of macrophage AMPK signaling inhibits adipocyte insulin signaling and glucose uptake. Activation of AMPK by CA-α1 increases the SIRT1 activator NAD⁺ content and SIRT1 expression in macrophages. Furthermore, α1AMPK activation mimics the effect of SIRT1 on deacetylating NF-κB, and the full capacity of AMPK to deacetylate NF-κB and inhibit its signaling requires SIRT1. In conclusion, AMPK negatively regulates lipid-induced inflammation, which acts through SIRT1, thereby contributing to the protection against obesity, inflammation, and insulin resistance. Our study defines a novel role for AMPK in bridging the signaling between nutrient metabolism and inflammation.