Insulin-stimulated IRS-1 Tyrosine Phosphorylation Research Articles

Methylglyoxal induces multiple serine phosphorylation in insulin receptor substrate 1 via the TAK1-p38-mTORC1 signaling axis in adipocytes.

Certain metabolic intermediates produced during metabolism are known to regulate a wide range of cellular processes. Methylglyoxal (MG), a natural metabolite derived from glycolysis, has been shown to negatively influence systemic metabolism by inducing glucose intolerance, insulin resistance, and diabetic complications. MG plays a functional role as a signaling molecule that initiates signal transduction. However, the specific relationship between MG-induced activation of signal transduction and its negative effects on metabolism remains unclear. Here, we found that MG activated mammalian target of rapamycin complex 1 (mTORC1) signaling via p38 mitogen-activated protein kinase in adipocytes, and that the transforming growth factor-β-activated kinase 1 (TAK1) is needed to activate p38-mTORC1 signaling following treatment with MG. We also found that MG increased the phosphorylation levels of serine residues in insulin receptor substrate (IRS)-1, which is involved in its negative regulation, thereby attenuating insulin-stimulated tyrosine phosphorylation in IRS-1. The negative effect of MG on insulin-stimulated IRS-1 tyrosine phosphorylation was exerted due to the MG-induced activation of the TAK1-p38-mTORC1 signaling axis. The involvement of the TAK1-p38-mTORC1 signaling axis in the induction of IRS-1 multiple serine phosphorylation was not unique to MG, as the proinflammatory cytokine, tumor necrosis factor-α, also activated the same signaling axis. Therefore, our findings suggest that MG-induced activation of the TAK1-p38-mTORC1 signaling axis caused multiple serine phosphorylation on IRS-1, potentially contributing to insulin resistance.

1879-P: Targeted Disruption of Muscle-Specific ROCK2 Results in Insulin Resistance In Vivo

Insulin signaling is essential for the maintenance of glucose homeostasis. ROCK2 has been shown to participate in insulin signaling and glucose metabolism in cultured cell lines. However, the in vivo function of ROCK2 in muscle remains to be elucidated. To explore the physiological role of muscle ROCK2 in the regulation of whole-body glucose homeostasis and insulin sensitivity in vivo, mice lacking ROCK2 in skeletal muscle selectively (myogenic-Cre; ROCK2lox/lox) were studied. Here we show that muscle-specific ROCK1-deficient mice displayed insulin resistance, as revealed by the failure of blood glucose levels to decrease after insulin injection. However, glucose tolerance was normal in the absence of muscle-specific ROCK2. These effects were independent of changes in adiposity. To determine the mechanism(s) by which muscle-specific deletion of ROCK2 causes insulin resistance, we measured the ability of insulin to activate downstream distal pathways in skeletal muscle. Insulin-stimulated IRS-1 tyrosine phosphorylation in skeletal muscle was markedly reduced by ∼80% in myogenic-Cre; ROCK2lox/lox mice compared with control (ROCK2lox/lox) mice. Concurrently, impaired insulin-induced phosphorylation of Akt, AS160, GSK, and S6K was found when ROCK2 was specifically deleted in muscle. Of pathological significance, ROCK2 activity in skeletal muscle was markedly decreased in obese type 2 diabetic mice in response to insulin, along with an impairment of IRS-1 phosphorylation. These data suggest that ROCK2 deficiency results in systemic insulin resistance by impairing insulin signaling in skeletal muscle, at the level of IRS-1. Thus, our results identify ROCK2 as a novel regulator of glucose homeostasis and insulin sensitivity in vivo, which could lead to new treatment approaches for obesity and type 2 diabetes. Disclosure H. Kim: None. R.M. Pereira: None. A.G. Uner: None. H. Lee: None. Y. Kim: None.

The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitate-induced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1

Dietary administration of the histone deacetylase (HDAC) inhibitor butyric acid – a short chain fatty acid present in milk products and also bacterially produced in the intestine – has been shown to increase energy expenditure and favour insulin sensitivity in mice through induction of PGC1α (peroxisome proliferator-activated receptor gamma co-activator 1α) and AMPK (AMP-activated protein kinase) in skeletal muscle, and a consequential increase of mitochondrial fatty acid oxidation. Here, we investigate whether such physiological improvements are associated to epigenetic effects dependent on increased histone acetylation and whether butyrate exerts a direct action on skeletal muscle insulin signalling. We show that sodium butyrate (NaBut) ameliorates the insulin-resistant phenotype, induced in L6 myotubes by prolonged exposure to palmitate, by i) increasing the insulin-induced phosphorylation of both PKB (protein kinase B) and MAPK (mitogen activated protein kinase), the two branches of insulin signalling and ii) increasing histone H3 acetylation – even in the presence of palmitate - on chromatin in proximity of the Irs1 (insulin receptor substrate 1) transcriptional start site. Consequently, NaBut induced Irs1 mRNA and protein overexpression, which in turn relayed higher insulin-stimulated IRS1 tyrosine phosphorylation and PI 3-kinase (phosphoinositide 3-kinase) association, suggesting that the increased IRS1 expression may mediate the insulin-sensitizing effects of NaBut. Furthermore, downstream of PKB, NaBut induced GSK3β gene upregulation. Our observations indicate that NaBut – through its action as HDAC inhibitor – can promote insulin responsiveness in L6 myotubes under conditions of lipid-induced insulin resistance.

Synergistic induction of insulin resistance by endothelin-1 and cAMP in 3T3-L1 adipocytes

Both endothelin-1 (ET-1) and cAMP are implicated for inducing insulin resistance. Since we have shown previously that there is a crosstalk between ET-1 and cAMP signaling pathways in regulating glucose uptake in 3T3-L1 adipocytes, we extended our investigation in this study on whether they may have a synergistic effect on inducing insulin resistance. Our results showed that it was indeed the case. Insulin-stimulated glucose uptake, phosphorylation of PKB, IRS-1-associated PI3K, and IRS-1 tyrosine phosphorylation were all inhibited by ET-1 and 8-bromo cAMP in a synergistic manner. IRS-1 protein levels were similarly decreased by ET-1 and 8-bromo cAMP, attributable to suppressed mRNA expression. In addition, after correction for the loss in IRS-1 protein, the inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation or IRS-1-associated PI3K was mainly caused by cAMP. Moreover, whereas IRS-2 protein levels were increased by cAMP or ET-1 plus cAMP, insulin-stimulated IRS-2-associated PI3K activities were abolished by both treatments. Furthermore, ET-1 and β-adrenergic agonists had similar synergistic inhibition on insulin-stimulated glucose uptake. In conclusion, we have shown that ET-1 and cAMP may synergistically induce insulin resistance in adipocytes via inhibiting IRS-1 expression as well as insulin-stimulated IRS-1/IRS-2 activities.

Modified Si-Miao-San inhibits inflammation and promotes glucose disposal in adipocytes through regulation of AMP-kinase

Modified Si-Miao-San (mSMS) is composed of Rhizoma Coptidis, Cortex Phellodendri, Rhizoma Coptidis Semen Coicis and Atractylodes Rhizome. The prescription is used for the management of diabetes and insulin resistance in the clinic. This study aims to investigate its regulation of glucose disposal in adipocytes. Differentiated 3T3-L1 adipocytes were stimulated with conditioned medium derived from activated macrophages to induce insulin resistance and observed the effects of Mac-CM on insulin-mediated glucose uptake along the insulin receptor substrate-1/PI3K/Akt signaling pathway. Moreover, its regulation of AMPK phosphorylation was also investigated. mSMS enhanced AMPK phosphorylation and promoted basal glucose uptake in adipocytes; mSMS inhibited NF-κB activation by reducing P65 phosphorylation and improved insulin-stimulated IRS-1 tyrosine and Akt phosphorylation, leading to the restoration of insulin-mediated glucose uptake when cells were exposed to inflammatory stimulation. These beneficial effects were diminished in the presence of the AMPK inhibitor compound C. mSMS positively regulated AMPK activity, and this action contributed to improving insulin PI3K signaling by the beneficial regulation of IRS-1 function through inhibition of inflammation in adipocytes.

Free fatty acid induced impairment of insulin signaling is prevented by the diastereomeric mixture of calophyllic acid and isocalophyllic acid in skeletal muscle cells

Elevated fatty acid levels play a pathogenic role in the development of insulin resistance, associated with type 2 diabetes. Interventions with ability to ameliorate fatty acid-induced insulin resistance might be useful for the management of diabetes. Here, we explored the effect of the diastereomeric mixture of calophyllic acid and isocalophyllic acid (F015) on palmitate-induced insulin resistance in skeletal muscle cells. An incubation of L6 myotubes with palmitate inhibited insulin-stimulated glucose uptake and translocation of GLUT4 to cell surface. Addition of F015 strongly prevented these inhibitions. Furthermore, F015 effectively inhibited the ability of palmitate to reduce insulin-stimulated phosphorylation of IRS-1, AKT and GSK-3β in L6 myotubes. F015 presented a strong inhibition on palmitate-induced production of reactive oxygen species and associated inflammation, as the activation JNK, ERK1/2 and p38 MAPK were greatly reduced. F015 also inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-stimulated IRS-1 tyrosine phosphorylation in presence of palmitate, resulted in enhanced insulin sensitivity. Results suggest that F015 inhibits palmitate-induced, reactive oxygen species-associated MAPK kinase activation and restored insulin sensitivity through regulating IRS-1 function. All these indicate F015 to be a potentially therapeutic candidate for insulin resistance and type 2 diabetes.

Insulin and Metabolic Stress Stimulate Multisite Serine/Threonine Phosphorylation of Insulin Receptor Substrate 1 and Inhibit Tyrosine Phosphorylation

IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (αpS/TmAb(Irs1)). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K → Akt → mechanistic target of rapamycin (mTOR) → S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)(Irs1)) was enhanced by inhibition of the PI3K → Akt → mTOR pathway and correlated with decreased Ser(P)-302(Irs1), Ser(P)-307(Irs1), Ser(P)-318(Irs1), Ser(P)-325(Irs1), and Ser(P)-346(Irs1). Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302(Irs1), Ser(P)-307(Irs1), and four others) correlated significantly with impaired insulin-stimulated Tyr(P)(Irs1). Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)(Irs1) in CHO(IR)/IRS1 cells.

Activation of Hepatic Inflammatory Pathways by Catecholamines Is Associated With Hepatic Insulin Resistance in Male Ischemic Stroke Rats

Patients who experience acute ischemic stroke may develop hyperglycemia, even in the absence of diabetes. In the current study we determined the effects of acute stroke on hepatic insulin signaling, TNF-α expression, endoplasmic reticulum (ER) stress, the activities of c-Jun N-terminal kinase (JNK), inhibitor κB kinase β (IKK-β), and nuclear factor-κB (NF-κB) pathways. Rats with cerebral ischemia developed higher blood glucose, and insulin levels, and insulin resistance index, as well as hepatic gluconeogenic enzyme expression compared with the sham-treated group. The hepatic TNF-α mRNA and protein levels were elevated in stroke rats in association with increased ER stress, phosphorylation of JNK1/2 and IKK-β proteins, IκB/NF-κB signaling, and phosphorylation of insulin receptor-1 (IRS-1) at serine residue. The basal and insulin-stimulated tyrosine phosphorylation of IRS-1 and AKT proteins was reduced. In addition, acute stroke increased circulating catecholamines in association with hepatic adrenergic signaling activation. After administration of a nonselective β-adrenergic receptor blocker (propranolol) before induction of cerebral ischemic injury, hepatic adrenergic transduction, TNF-α expression, ER stress, and the activation of the JNK1/2, IKK-β, and NF-κB pathways, and serine phosphorylation of IRS-1 were all attenuated. In contrast, the phosphorylated IRS-1 at tyrosine site and AKT levels were partially restored with improved poststroke hyperglycemia and insulin resistance index. These results suggest that acute ischemic stroke can activate proinflammatory pathways in the liver by the catecholamines and is associated with the development of hepatic insulin resistance.

A sustained increase in plasma NEFA upregulates the Toll-like receptor network in human muscle.

Insulin-sensitive tissues (muscle, liver) of individuals with obesity and type 2 diabetes mellitus are in a state of low-grade inflammation, characterised by increased Toll-like receptor (TLR) expression and TLR-driven signalling. However, the cause of this mild inflammatory state is unclear. We tested the hypothesis that a prolonged mild increase in plasma NEFA will increase TLR expression and TLR-driven signalling (nuclear factor κB [NFκB] and mitogen-activated kinase [MAPK]) and impair insulin action in muscle of lean healthy individuals. Twelve lean, normal-glucose-tolerant participants were randomised to receive a 48 h infusion (30 ml/h) of saline or Intralipid followed by a euglycaemic-hyperinsulinaemic clamp. Vastus lateralis muscle biopsies were performed before and during the clamp. Lipid infusion impaired insulin-stimulated IRS-1 tyrosine phosphorylation and reduced peripheral insulin sensitivity (p < 0.01). The elevation in circulating NEFA increased expression of TLR3, TLR4 and TLR5, and several MAPK (MAPK8, MAP4K4, MAP2K3) and inhibitor of κB kinase-NFκB (CHUK [IKKA], c-REL [REL] and p65 [RELA, NFKB3, p65]) signalling genes (p < 0.05). The lipid infusion also increased extracellular signal-regulated kinase (ERK) phosphorylation (p < 0.05) and tended to reduce the content of inhibitor of kappa Bα (p = 0.09). The muscle content of most diacylglycerol, ceramide and acylcarnitine species was unaffected. In summary, insulin resistance induced by prolonged low-dose lipid infusion occurs together with increased TLR-driven inflammatory signalling and impaired insulin-stimulated IRS-1 tyrosine phosphorylation. A sustained, mild elevation in plasma NEFA is sufficient to increase TLR expression and TLR-driven signalling (NFκB and MAPK) in lean individuals. The activation of this pathway by NEFA may be involved in the pathogenesis of insulin resistance in humans. ClinicalTrials.gov NCT01740817.

Pinusolide improves high glucose-induced insulin resistance via activation of AMP-activated protein kinase

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a crucial role in the maintenance of cellular energy homeostasis, and several natural compounds that activate AMPK possibly enhance glucose uptake by muscle cells. In this study, we found that pinusolide stimulated AMPK phosphorylation and glucose uptake and these effects were significantly reduced by siRNA LKB1 or compound C, suggesting that enhanced glucose uptake by pinusolide is predominantly accomplished via an LKB1-mediated AMPK activation pathway. An insulin resistance state was induced by exposing cells to 30mM glucose, as indicated by reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and glucose uptake. Under these conditions, the phosphorylation of AMPK and ACC were decreased. Surprisingly, disrupted insulin signaling and decreased AMPK activity by high glucose concentrations were prevented by pinusolide. Moreover, this treatment increased insulin-stimulated glucose uptake via AMPK activation. Taken together, our findings suggest a link between high glucose and insulin resistance in muscle cells, and provide further evidence that pinusolide attenuates blockade of insulin signaling by enhancing IRS-1 tyrosine phosphorylation by the activating the AMPK pathway. In addition, this study indicates the targeting of AMPK represents a new therapeutic strategy for hyperglycemia-induced insulin resistance and type 2 diabetes.

Endothelial Metabolic Inflammation: A Link between High Fat Feeding,Insulin Resistance, and Impaired Trans-Endothelial Insulin Transport

Copyright: © 2012 Wang H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Obesity and its associated metabolic syndrome and type 2 diabetes are becoming epidemics in the United States. The most recent data show that nationwide incidence of obesity (BMI > 30 kg/ m) and type 2 diabetes has reached to 27.8% and 8.7%, respectively (CDC Behavioral Risk Factor Surveillance System 2012). Endothelial dysfunction, characterized by a deficiency of bio-available nitric oxide (NO), has been found to precede the development of type 2 diabetes and is significantly correlated with insulin resistance [1]. Early studies have shown that feeding rodent animals with a high fat diet (HFD) (~60% of calories) produces not only obesity [2] but also a state of insulin resistance [3]. These HFD-fed rodents develop striking hyperinsulinemia with significantly reduced whole body insulin sensitivity and glucose disposal rates, severe impairments in both muscle and adipose tissue insulin signaling and glucose uptake and an impairment of insulin-mediated suppression of hepatic glucose output [4-6]. Moreover, obesity has been shown to be a state of low-grade chronic systemic inflammation known as the metabolic inflammation characterized by elevated levels of pro-inflammatory cytokines (such as TNFα, IL-6, IL-1β, CCL2 etc.), accumulation of leukocytes within adipose tissue and other organs, activation of macrophages in both liver and fat and activation of pro-inflammatory signaling pathways in multiple organs or tissues [7,8]. The mechanisms causing the metabolic inflammation have been related to excess nutrient intake (metabolic stress) including HFD feeding [7,8]. Dietary fat intake not only significantly increases circulating free fatty acids (FFAs) concentration but also affects the composition of circulating FFAs [9]. Four-week HFD feeding has been shown to cause metabolic endotoxemia leading to the metabolic inflammation in mice [10]. The lipopolysaccharide (LPS) -induced inflammatory responses in macrophages have been shown to be mediated by Toll-Like Receptor-4 (TLR4) (pattern recognition receptors that sense lipopeptides and lipopolysaccharides of bacterial walls) [11]. Interestingly, saturated fatty acids (SFAs), but not unsaturated fatty acids, can induce an inflammatory response like LPS through activation of TLR4 [12,13]. It has also been proposed that nutrients per se are naturally inflammatory [7]. While the flood of nutrients in a short period of time may induce a brief episode of stress signaling in the target cells, long-chain SFAs, particularly palmitate, have been shown to directly activate TLR4 that may require CD36 (a class B scavenger receptor) [14-16], leading to IKKβ/NFκB and c-jun N-terminal kinase (JNK) pathway activation, increased production of pro-inflammatory cytokines TNFα, IL-1β and IL-6 [13,17-19] and significant insulin resistance as reflected by impairments in insulinstimulated tyrosine phosphorylation of IRS-1, serine phosphorylation of Akt and eNOS, and NO production. Interestingly, recent studies have shown evidence that vascular endothelium that line up the inner wall of vasculature appear to be the first responder to the environmental insult, high fat feeding, leading to the vascular endothelial metabolic inflammation and insulin resistance.

Acute effects of different diet compositions on skeletal muscle insulin signalling in obese individuals during caloric restriction

ObjectiveThe cellular effects of restricting fat versus carbohydrate during a low-calorie diet are unclear. The aim of this study was to examine acute effects of energy and macronutrient restriction on skeletal muscle insulin signalling in obesity. Materials/MethodsEighteen obese individuals without diabetes underwent euglycemic–hyperinsulinemic clamp and skeletal muscle biopsy after: (a) 5days of eucaloric diet (30% fat, 50% carbohydrate), and (b) 5days of a 30% calorie-restricted diet, either low fat/high carbohydrate (LF/HC: 20% fat, 60% carbohydrate) or high-fat/low carbohydrate (HF/LC: 50% fat, 30% carbohydrate). ResultsWeight, body composition, and insulin sensitivity were similar between groups after eucaloric diet. Weight loss was similar between groups after hypocaloric diet, 1.3±1.3kg (p<0.0001 compared with eucaloric). Whole-body insulin sensitivity was unchanged after calorie restriction and similar between groups. However, ex vivo skeletal muscle insulin signalling differed depending on macronutrient composition of calorie-restricted diet. Skeletal muscle of the LF/HC group had increased insulin-stimulated tyrosine phosphorylation of IRS-1, decreased insulin-stimulated Ser307 phosphorylation of IRS-1, and increased IRS-1-associated phosphatidylinositol (PI)3-kinase activity. Conversely, insulin stimulation of tyrosine phosphorylated IRS-1 was absent and serine 307 phosphorylation of IRS-1 was increased on HF/LC, with blunting of IRS-1-associated PI3-kinase activity. ConclusionAcute caloric restriction with an LF/HC diet alters skeletal muscle insulin signalling in a way that improves insulin sensitivity, while acute caloric restriction with an HF/LC diet induces changes compatible with insulin resistance. In both cases, ex vivo changes in skeletal muscle insulin signalling appear prior to changes in whole body insulin sensitivity.

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.

Cyclin-dependent Kinase-5 Is a Key Molecule in Tumor Necrosis Factor-α-induced Insulin Resistance

The mechanism of TNF-α-induced insulin resistance has remained unresolved with evidence for down-regulation of insulin effector targets effects or blockade of proximal as well as distal insulin signaling events depending upon the dose, time, and cell type examined. To address this issue we examined the acute actions of TNF-α in differentiated 3T3L1 adipocytes. Acute (5-15 min) treatment with 20 ng/ml (~0.8 nm) TNF-α had no significant effect on IRS1-associated phosphatidylinositol 3-kinase. In contrast, TNF-α increased insulin-stimulated cyclin-dependent kinase-5 (CDK5) phosphorylation on tyrosine residue 15 through an Erk-dependent pathway and up-regulated the expression of the CDK5 regulator protein p35. In parallel, TNF-α stimulation also resulted in the phosphorylation and GTP loading of the Rho family GTP-binding protein, TC10α. TNF-α enhanced the depolymerization of cortical F-actin and inhibited insulin-stimulated glucose transporter-4 (GLUT4) translocation. Treatment with the MEK inhibitor, PD98059, blocked the TNF-α-induced increase in CDK5 phosphorylation and the depolymerization of cortical F-actin. Conversely, siRNA-mediated knockdown of CDK5 or treatment with the MEK inhibitor restored the impaired insulin-stimulated GLUT4 translocation induced by TNF-α. Furthermore, siRNA-mediated knockdown of p44/42 Erk also rescued the TNF-α inhibition of insulin-stimulated GLUT4 translocation. Together, these data demonstrate that TNF-α-mediated insulin resistance of glucose uptake can occur through a MEK/Erk-dependent activation of CDK5.

Betel nut extract and arecoline block insulin signaling and lipid storage in 3T3-L1 adipocytes

According to several population-based studies, betel nut chewing is associated with metabolic syndrome and diabetes in British South Asians and Taiwanese. However, the underlying molecular mechanism is not yet clear. Arecoline is an alkaloid-type natural product found in betel nuts. Our aim was to clarify the influence of betel nut extract and arecoline on lipid accumulation and insulin signaling in adipocytes. We found that betel nut extract and arecoline blocked lipid storage in 3T3-L1 adipocytes. The possible mechanism may function by inhibiting the expression of the insulin receptor, glucose transporter-4, fatty acid synthase, and the lipid droplet proteins perilipin and adipophilin. In addition, betel nut extract and arecoline increased the basal level of IRS-1 serine(307) phosphorylation and decreased insulin-stimulated IRS-1 tyrosine, Akt, and PI3 kinase phosphorylation. In conclusion, betel nut extract and arecoline have diabetogenic potential on adipocytes that may result in insulin resistance and diabetes at least in part via the obstruction of insulin signaling and the blockage of lipid storage.

Effect of NF-κB decoy on insulin resistance of adipocytes from patients with type 2 diabetes mellitus

Aim This study aimed to investigate whether NF-κB contributes to insulin resistance in type 2 diabetes (T2DM). Methods Subcutaneous abdominal adipose tissue was obtained from T2DM patients and non-diabetic control subjects. Pre-adipocytes were cultured and differentiated into adipocytes in vitro. Upon insulin stimulation, IRS-1 tyrosine and AKT (Ser473) phosphorylation were examined by immunoprecipitation and immunoblotting, while levels of inflammatory mediators IL-6 and MCP-1, and the DNA-binding activity of NF-κB, were examined by ELISA and electrophoretic mobility shift assay (EMSA), respectively. NF-κB decoy molecules were introduced into T2DM adipocytes, and their effects on all these molecular events evaluated. Results Compared with cells from non-diabetic subjects, adipocytes from T2DM patients showed signs of insulin resistance, with significantly reduced IRS-1 tyrosine and AKT (Ser 473) phosphorylation levels in response to insulin stimulation. At the same time, T2DM cells displayed elevated levels of IL-6 and MCP-1, and NF-κB activity. Introduction of NF-κB decoy molecules significantly inhibited both IL-6 secretion and NF-κB activity, while enhancing insulin-stimulated IRS-1 tyrosine and AKT (Ser473) phosphorylation in T2DM adipocytes. Conclusion Abdominal subcutaneous fat cells from T2DM patients display signs of insulin resistance and microinflammatory status. NF-κB decoy molecules inhibited NF-κB overactivation and also partly reversed insulin resistance. These results provide evidence of a link between inflammation and insulin resistance in T2DM cells, suggesting a potential contribution of inflammation to the mechanism of insulin resistance.

P38 Mitogen-Activated Protein Kinase-Dependent Transactivation of ErbB Receptor Family

OBJECTIVEStress stimuli such as tumor necrosis factor (TNF) have been shown to induce insulin receptor substrate (IRS)-1 serine phosphorylation and insulin resistance by transactivation of ErbB receptors. We aimed at elucidating the potential role of p38 mitogen-activated protein kinase (p38MAPK) in mediating stress-induced ErbB receptors activation.RESEARCH DESIGN AND METHODSp38MAPK effect on ErbBs transactivation and insulin signaling was assessed in Fao or HepG2 cells, exposed to stress stimuli, and on metabolic parameters in ob/ob and C57/BL6 mice.RESULTSHigh-fat diet–fed mice and ob/ob mice exhibited elevated hepatic p38MAPK activation associated with glucose intolerance and hyperinsulinemia. Liver expression of dominant-negative (DN)-p38MAPKα in ob/ob mice reduced fasting insulin levels and improved glucose tolerance, whereas C57/BL6 mice overexpressing wild-type p38MAPKα exhibited enhanced IRS-1 serine phosphorylation and reduced insulin-stimulated IRS-1 tyrosine phosphorylation. Fao or HepG2 cells exposed to TNF, anisomycin, or sphingomyelinase demonstrated rapid transactivation of ErbB receptors leading to PI3-kinase/Akt activation and IRS-1 serine phosphorylation. p38MAPK inhibition either by SB203580, by small interfering RNA, or by DN-p38MAPKα decreased ErbB receptors transactivation and IRS-1 serine phosphorylation and partially restored insulin-stimulated IRS-1 tyrosine phosphorylation. When cells were incubated with specific ErbB receptors antagonists or in cells lacking ErbB receptors, anisomycin- and TNF-induced IRS-1 serine phosphorylation was attenuated, despite intact p38MAPK activation. The stress-induced p38MAPK activation leading to ErbB receptors transactivation was associated with intracellular reactive oxygen species generation and was attenuated by treatment with antioxidants.CONCLUSIONSHepatic p38MAPK is activated following various stress stimuli. This event is upstream to ErbB receptors transactivation and plays an important role in stress-induced IRS-1 serine phosphorylation and insulin resistance.

In vivo activation of ROCK1 by insulin is impaired in skeletal muscle of humans with type 2 diabetes

To determine whether serine/threonine ROCK1 is activated by insulin in vivo in humans and whether impaired activation of ROCK1 could play a role in the pathogenesis of insulin resistance, we measured the activity of ROCK1 and the protein content of the Rho family in vastus lateralis muscle of lean, obese nondiabetic, and obese type 2 diabetic subjects. Biopsies were taken after an overnight fast and after a 3-h hyperinsulinemic euglycemic clamp. Insulin-stimulated GDR was reduced 38% in obese nondiabetic subjects compared with lean, 62% in obese diabetic subjects compared with lean, and 39% in obese diabetic compared with obese nondiabetic subjects (all comparisons P < 0.001). Insulin-stimulated IRS-1 tyrosine phosphorylation is impaired 41-48% in diabetic subjects compared with lean or obese subjects. Basal activity of ROCK1 was similar in all groups. Insulin increased ROCK1 activity 2.1-fold in lean and 1.7-fold in obese nondiabetic subjects in muscle. However, ROCK1 activity did not increase in response to insulin in muscle of obese type 2 diabetic subjects without change in ROCK1 protein levels. Importantly, insulin-stimulated ROCK1 activity was positively correlated with insulin-mediated GDR in lean subjects (P < 0.01) but not in obese or type 2 diabetic subjects. Moreover, RhoE GTPase that inhibits the catalytic activity of ROCK1 by binding to the kinase domain of the enzyme is notably increased in obese type 2 diabetic subjects, accounting for defective ROCK1 activity. Thus, these data suggest that ROCK1 may play an important role in the pathogenesis of resistance to insulin action on glucose disposal in muscle of obese type 2 diabetic subjects.

Over-expression of LYRM1 inhibits glucose transport in rat skeletal muscles via attenuated phosphorylation of PI3K (p85) and Akt

To explore the effect of LYRM1 over-expression on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells, and to understand the underlying mechanisms, Rat myoblasts (L6) transfected with either an empty expression vector (pcDNA3.1Myc/His B) or a LYRM1 expression vector were differentiated into myotubes. Glucose uptake was determined by measuring 2-deoxy-D-[(3)H] glucose uptake into L6 myotubes. Western blotting was performed to assess the translocation of insulin-sensitive glucose transporter 4 (GLUT4). It was also used to measure the phosphorylation and total protein contents of insulin-signaling proteins, such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase (PI3K) p85, Akt, ERK1/2, P38, and JNK. LYRM1 over-expression in L6 myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1, PI3K (p85), and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, P38, and JNK. LYRM1 regulates the function of IRS-1, PI3K, and Akt, and decreases GLUT4 translocation and glucose uptake in response to insulin. These observations highlight the potential role of LYRM1 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity.

SOCS3 inhibits insulin signaling in porcine primary adipocytes.

Insulin resistance is a major player in the pathogenesis of type II diabetes, the metabolic syndrome, and obesity. SOCS3 plays an important role in the development of insulin resistance. To investigate the role of SOCS3 in porcine adipocyte insulin signaling, we first detected the effect of insulin on SOCS3 mRNA and protein expression in porcine primary adipocytes by real-time RT-PCR and Western blotting. Then, we constructed a recombinant adenovirus encoding SOCS3 gene (Ad-SOCS3) which was used to infect differentiated porcine primary adipocytes for 3 days. The expression and phosphorylation of main insulin signaling components were detected by Western blotting. The results showed that 100nM insulin could induce SOCS3 mRNA expression but not protein expression, and overexpression of SOCS3 decreased IRS1 protein level, insulin-stimulated IRS1 tyrosine phosphorylation, PI3K activation, and Akt phosphorylation, but increased IRS1 serine phosphorylation in porcine primary adipocytes. These results indicate that SOCS3 is an important negative regulator of insulin signaling in porcine adipocytes. Thus, SOCS3 may be a novel therapeutic target for the prevention or treatment of insulin resistance and type II diabetes.