ATP-sensitive K+ channel-mediated glucose uptake is independent of IRS-1/phosphatidylinositol 3-kinase signaling

We previously found that disruption of Kir6.2-containing ATP-sensitive K+ (KATP) channels increases glucose uptake in skeletal muscle, but the mechanism is not clear. In the present study, we generated knockout mice lacking both Kir6.2 and insulin receptor substrate-1 (IRS-1). Because IRS-1 is the major substrate of insulin receptor kinase, we expected disruption of the IRS-1 gene to reduce glucose uptake in Kir6.2 knockout mice. However, the double-knockout mice do not develop insulin resistance or glucose intolerance. An insulin tolerance test reveals the glucose-lowering effect of exogenous insulin in double-knockout mice and in Kir6.2 knockout mice to be similarly enhanced compared with wild-type mice. The basal 2-deoxyglucose uptake rate in skeletal muscle of double-knockout mice is increased similarly to the rate in Kir6.2 knockout mice. Accordingly, disruption of the IRS-1 gene affects neither systemic insulin sensitivity nor glucose uptake in skeletal muscles of Kir6.2-deficient mice. In addition, no significant changes were observed in phosphatidylinositol 3-kinase (PI3K) activity and its downstream signal in skeletal muscle due to lack of the Kir6.2 gene. Disruption of Kir6.2-containing Katp channels clearly protects against IRS-1-associated insulin resistance by increasing glucose uptake in skeletal muscles by a mechanism separate from the IRS-1/PI3K pathway.     

Roles of phosphatase and tensin homolog in skeletal muscle

The phosphatase and tensin homolog (PTEN), originally identified as a tumor suppressor, is an important regulator of the PI3K–Akt pathway. PTEN plays crucial roles in various cellular processes, including cell survival, cell growth, cell proliferation, cell differentiation, and cell metabolism. In metabolic tissues, PTEN expression affects insulin sensitivity and glucose homeostasis. In skeletal muscle, the deletion of PTEN regulates muscle development and protects the mutant mice from insulin resistance and diabetes. Notably, the regulatory role of PTEN in skeletal muscle stem cells has been recently reported. In this review, we mainly discuss the role of PTEN in regulating the development, glucose metabolism, stem cell fate decision, and regeneration of skeletal muscle.     

Shikonin stimulates glucose uptake in 3T3-L1 adipocytes via an insulin-independent tyrosine kinase pathway

Type 2 diabetes is due to defects in both insulin action and secretion. In an attempt to discover small molecules that stimulate glucose uptake, similar to insulin, a cell-based glucose uptake screening assay was performed using 3T3-L1 adipocytes. Shikonin, a substance originally isolated from the root of the Chinese plant that has been used as an ointment for wound healing, was thus identified. Shikonin stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. Stimulation of glucose uptake was also observed in rat primary adipocytes and cardiomyocytes. Like insulin, shikonin-stimulated glucose uptake was inhibited by genistein, a tyrosine kinase inhibitor, and enhanced by vanadate, a tyrosine phosphatase inhibitor. However, in contrast to insulin, shikonin-stimulated glucose uptake was not strongly inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). In vitro phosphorylation analyses revealed that shikonin did not induce tyrosine phosphorylation of the insulin receptor, but significantly induced both Thr-308 and Ser-473 phosphorylation of Akt. Our results suggest that in 3T3-L1 adipocytes, shikonin action is not mediated primarily via the insulin receptor/PI3K pathway, but rather via another distinct tyrosine kinase-dependent pathway leading to glucose uptake involving Akt phosphorylation.     

Mgat5 and Pten interact to regulate cell growth and polarity

Phosphatase and tensin homolog (Pten) phosphatase opposes intracellular phosphoinositide 3-kinase (PI3K)/Akt signaling and is a potent tumor suppressor, while Golgi beta1,6 N-acetylglucosaminyltransferase V (Mgat5) is positively associated with cancer progression and metastasis. beta1,6GlcNAc-branched N-glycans on receptor glycoproteins promote their surface residency and sensitizes cells to growth factor signaling. Here we demonstrate that the Pten heterozygosity in mouse embryonic fibroblasts enhances cell adhesion-dependent PI3K/Akt signaling, cell spreading, and proliferation, while Pten/Mgat5 double mutant cells are normalized. However, planar asymmetry typical of fibroblasts and invasive carcinomas is not fully rescued, suggesting that Mgat5 and Pten function together to regulate the membrane dynamics of PI3K/Akt signaling typical of motile cells. Pten heterozygosity was associated with increased surface beta1,6GlcNAc-branched N-glycans, suggesting positive feedback from PI3K signaling to N-glycan branching. In vivo, Mgat5(-/-) Pten(+/-) and Mgat5(+/-)Pten(+/-)mutant mice showed a small but significant increase in longevity compared with Pten(+/-) mice. Taken together, our results reveal that Mgat5 and Pten interact in an opposing manner to regulate cellular sensitivities to extracelluar growth cues.     

Knockdown of NYGGF4 increases glucose transport in C2C12 mice skeletal myocytes by activation IRS-1/PI3K/AKT insulin pathway

NYGGF4, an obesity-related gene, is proposed to be involved in the development of insulin resistance. Skeletal muscle is a primary target organ for insulin and NYGGF4 showed a relatively high expression level in skeletal muscle. Therefore, this study aimed to explore the effect of NYGGF4 on insulin sensitivity of skeletal muscle cells. RNA interference (RNAi) was adopted to silence NYGGF4 expression in mice C2C12 skeletal myocytes. A remarkably increased insulin-stimulated glucose uptake and GLUT4 translocation was observed in NYGGF4 silencing C2C12 cells. Importantly, the enhanced glucose uptake induced by NYGGF4 silencing could be abrogated by the PI3K inhibitor LY294002. In addition, the crucial molecules involved in PI3K insulin signaling pathway were detected by western blotting. The results showed that NYGGF4 knockdown dramatically activate the insulin-stimulated phosphorylation of IRS-1 and AKT. Taken together, these data demonstrate that NYGGF4 knockdown increases glucose transport in myocytes by activation of the IRS-1/PI3K/AKT insulin pathway.     

Apelin stimulates glucose uptake through the PI3K/Akt pathway and improves insulin resistance in 3T3-L1 adipocytes

Apelin, a cytokine mainly secreted by adipocytes, is closely related with insulin resistance. The underlying molecular mechanisms of how apelin affects insulin resistance, however, are poorly understood. This study aimed to investigate the effect of apelin on glucose metabolism and insulin resistance in 3T3-L1 adipocytes. After 10 ng/ml TNF-α treatment for 24 h, insulin-stimulated glucose uptake was reduced by 47% in 3T3-L1 adipocytes. Apelin treatment improved glucose uptake in a time- and dose-dependent manner. Treatment of 1,000 nM apelin for 60 min maximally augmented glucose uptake in insulin-resistant 3T3-L1 adipocytes. Furthermore, apelin pre-incubation also increased adipocytes' insulin-stimulated glucose uptake, and PI3K/Akt pathway were involved in these effects. In addition, immunocytochemistry staining and western blotting analysis indicated that apelin could increase glucose transporter 4 translocation from the cytoplasm to the plasma membrane. Apelin also increased the anti-inflammatory adipokine adiponectin mRNA expression while reducing that of pro-inflammatory adipokine interleukin-6 in insulin-resistant 3T3-L1 adipocytes. These results suggest that apelin stimulates glucose uptake through the PI3K/Akt pathway, promotes GLUT4 translocation from the cytoplasm to the plasma membrane, and modulates inflammatory responses in insulin-resistant 3T3-L1 adipocytes.     

Lipid-induced Muscle Insulin Resistance Is Mediated by GGPPS via Modulation of the RhoA/Rho Kinase Signaling Pathway

Elevated circulating free fatty acid levels are important contributors to insulin resistance in the muscle and liver, but the underlying mechanisms require further elucidation. Here, we show that geranylgeranyl diphosphate synthase 1 (GGPPS), which is a branch point enzyme in the mevalonic acid pathway, promotes lipid-induced muscle insulin resistance through activation of the RhoA/Rho kinase signaling pathway. We have found that metabolic perturbation would increase GGPPS expression in the skeletal muscles of db/db mice and high fat diet-fed mice. To address the metabolic effects of GGPPS activity in skeletal muscle, we generated mice with specific GGPPS deletions in their skeletal muscle tissue. Heterozygous knock-out of GGPPS in the skeletal muscle improved systemic insulin sensitivity and glucose homeostasis in mice fed both normal chow and high fat diets. These metabolic alterations were accompanied by activated PI3K/Akt signaling and enhanced glucose uptake in the skeletal muscle. Further investigation showed that the free fatty acid-stimulated GGPPS expression in the skeletal muscle was able to enhance the geranylgeranylation of RhoA, which further induced the inhibitory phosphorylation of IRS-1 (Ser-307) by increasing Rho kinase activity. These results implicate a crucial role of the GGPPS/RhoA/Rho kinase/IRS-1 pathway in skeletal muscle, in which it mediates lipid-induced systemic insulin resistance in obese mice. Therefore, skeletal muscle GGPPS may represent a potential pharmacological target for the prevention and treatment of obesity-related type 2 diabetes.     

Role of tumor suppressor PTEN in tumor necrosis factor alpha-induced inhibition of insulin signaling in murine skeletal muscle C2C12 cells.

In an attempt to clarify the role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in muscle insulin resistance, we investigated the effect of PTEN on phosphoinositide 3 (PI3)-kinase/Akt related insulin signaling pathway in skeletal muscle-like C2C12 cells damaged by tumor necrosis factor-alpha (TNFalpha). C2C12 cells cultured with TNFalpha (10 ng/ml) for 1 h displayed a marked decrease of insulin-stimulated 2-[14C]-deoxy-D-glucose (2-DG) uptake in parallel with an elevation of PTEN mRNA and protein levels. However, pretreatment of PTEN antisense oligonucleotide (AS) (1 micromol/l for 3 days) for specific inhibition of PTEN expression in C2C12 cells abolished the TNFalpha-induced changes in 2-DG uptake. Similar pretreatment with PTEN AS, but not with sense oligonucleotide (1 micromol/l for 3 days), eliminated the ability of TNFalpha to impair insulin-stimulated signals including p85 regulatory subunit of PI3-kinase expression and the degree of Akt serine phosphorylation as well as protein expression in glucose transporter subtype 4. Data taken from cultured C2C12 cells emphasize the negative regulatory of muscle PI3-kinase/Akt signaling pathways as the major substrate of PTEN but also support the concept that PTEN contributes to the development of insulin resistance in skeletal muscle.     

Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice

Bioactive components from bitter melon (BM) have been reported to improve glucose metabolism in vivo, but definitive studies on efficacy and mechanism of action are lacking. We sought to investigate the effects of BM bioactives on body weight, muscle lipid content and insulin signaling in mice fed a high-fat diet and on insulin signaling in L6 myotubes. Male C57BL/6J mice were randomly divided into low-fat diet control (LFD), high-fat diet (HFD) and HFD plus BM (BM) groups. Body weight, body composition, plasma glucose, leptin, insulin and muscle lipid profile were determined over 12 weeks. Insulin signaling was determined in the mouse muscle taken at end of study and in L6 myotubes exposed to the extract. Body weight, plasma glucose, insulin, leptin levels and HOMA-IR values were significantly lower in the BM-fed HFD group when compared to the HFD group. BM supplementation significantly increased IRS-2, IR β, PI 3K and GLUT4 protein abundance in skeletal muscle, as well as phosphorylation of IRS-1, Akt1 and Akt2 when compared with HFD (P<.05 and P<.01). BM also significantly reduced muscle lipid content in the HFD mice. BM extract greatly increased glucose uptake and enhanced insulin signaling in L6 myotubes. This study shows that BM bioactives reduced body weight, improved glucose metabolism and enhanced skeletal muscle insulin signaling. A contributing mechanism to the enhanced insulin signaling may be associated with the reduction in skeletal muscle lipid content. Nutritional supplementation with this extract, if validated for human studies, may offer an adjunctive therapy for diabetes.     

Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle

Inducible nitric oxide synthase (iNOS) is induced by inflammatory cytokines in skeletal muscle and fat. It has been proposed that chronic iNOS induction may cause muscle insulin resistance. Here we show that iNOS expression is increased in muscle and fat of genetic and dietary models of obesity. Moreover, mice in which the gene encoding iNOS was disrupted (Nos2-/- mice) are protected from high-fat-induced insulin resistance. Whereas both wild-type and Nos2-/- mice developed obesity on the high-fat diet, obese Nos2-/- mice exhibited improved glucose tolerance, normal insulin sensitivity in vivo and normal insulin-stimulated glucose uptake in muscles. iNOS induction in obese wild-type mice was associated with impairments in phosphatidylinositol 3-kinase and Akt activation by insulin in muscle. These defects were fully prevented in obese Nos2-/- mice. These findings provide genetic evidence that iNOS is involved in the development of muscle insulin resistance in diet-induced obesity.     

Palmitic acid acutely stimulates glucose uptake via activation of Akt and ERK1/2 in skeletal muscle cells

Chronic exposure to saturated fatty acids can cause insulin resistance. However, the acute effects of fatty acids are not clear and need to be elucidated because plasma fatty acid concentrations fluctuate postprandially. Here, we present the acute effects of palmitate (PA) on skeletal muscle cells and their underlying molecular mechanisms. Immuno-fluorescence results showed that PA rapidly induced GLUT4 translocation and stimulated glucose uptake in rat skeletal muscle cell line L6. Phosphorylation of AMP-activated protein kinase (AMPK), Akt, and extracellular signal-related kinase1/2 (ERK1/2) was enhanced by PA in a time-dependent manner. Cell surface-bound PA was sufficient to stimulate Akt phosphorylation. The inhibitors of PI3 kinase (PI3K), AMPK, Akt, and ERK1/2 could decrease PA-induced glucose uptake, and PI3K inhibitor decreased AMPK, Akt, and ERK1/2 phosphorylation. Weakening AMPK activity reduced phosphorylation of Akt but not ERK1/2, and Akt inhibitor could not affect ERK1/2 activation either. Meanwhile, ERK1/2 inhibitors had no effect on Akt phosphorylation. Taken together, our data suggest that PA-mediated glucose uptake in skeletal muscle cells may be stimulated by the binding of PA to cell surface and followed by PI3K/AMPK/Akt and PI3K/ERK1/2 pathways independently.     

Hormone-sensitive lipase knockout mice have increased hepatic insulin sensitivity and are protected from short-term diet-induced insulin resistance in skeletal muscle and heart

Insulin resistance in skeletal muscle and heart plays a major role in the development of type 2 diabetes and diabetic heart failure and may be causally associated with altered lipid metabolism. Hormone-sensitive lipase (HSL) is a rate-determining enzyme in the hydrolysis of triglyceride in adipocytes, and HSL-deficient mice have reduced circulating fatty acids and are resistant to diet-induced obesity. To determine the metabolic role of HSL, we examined the changes in tissue-specific insulin action and glucose metabolism in vivo during hyperinsulinemic euglycemic clamps after 3 wk of high-fat or normal chow diet in awake, HSL-deficient (HSL-KO) mice. On normal diet, HSL-KO mice showed a twofold increase in hepatic insulin action but a 40% decrease in insulin-stimulated cardiac glucose uptake compared with wild-type littermates. High-fat feeding caused a similar increase in whole body fat mass in both groups of mice. Insulin-stimulated glucose uptake was reduced by 50-80% in skeletal muscle and heart of wild-type mice after high-fat feeding. In contrast, HSL-KO mice were protected from diet-induced insulin resistance in skeletal muscle and heart, and these effects were associated with reduced intramuscular triglyceride and fatty acyl-CoA levels in the fat-fed HSL-KO mice. Overall, these findings demonstrate the important role of HSL on skeletal muscle, heart, and liver glucose metabolism.     

Skeletal muscle reprogramming by activation of calcineurin improves insulin action on metabolic pathways

The protein phosphatase calcineurin is a signaling intermediate that induces the transformation of fast-twitch skeletal muscle fibers to a slow-twitch phenotype. This reprogramming of the skeletal muscle gene expression profile may have therapeutic applications for metabolic disease. Insulin-stimulated glucose uptake in skeletal muscle is both impaired in individuals with type II diabetes mellitus and positively correlated with the percentage of slow- versus fast-twitch muscle fibers. Using transgenic mice expressing activated calcineurin in skeletal muscle, we report that skeletal muscle reprogramming by calcineurin activation leads to improved insulin-stimulated 2-deoxyglucose uptake in extensor digitorum longus (EDL) muscles compared with wild-type mice, concomitant with increased protein expression of the insulin receptor, Akt, glucose transporter 4, and peroxisome proliferator-activated receptor-gamma co-activator 1. Transgenic mice exhibited elevated glycogen deposition, enhanced amino acid uptake, and increased fatty acid oxidation in EDL muscle. When fed a high-fat diet, transgenic mice maintained superior rates of insulin-stimulated glucose uptake in EDL muscle and were protected against diet-induced glucose intolerance. These results validate calcineurin as a target for enhancing insulin action in skeletal muscle.     

The interrelation between aPKC and glucose uptake in the skeletal muscle during contraction and insulin stimulation

Contraction and insulin increase glucose uptake in skeletal muscle. While the insulin pathway, better characterized, requires activation of phosphoinositide 3‐kinase (PI3K) and atypical protein kinase (aPKC), muscle contraction seems to share insulin‐activated components to increase glucose uptake. This study aimed to investigate the interrelation between the pathway involved in glucose uptake evoked by insulin and muscle contraction. Isolated muscle of rats was treated with solvent (control), insulin, wortmannin (PI3K inhibitor) and the combination of insulin plus wortmannin. After treatment, muscles were electrically stimulated (contracted) or remained at rest. Glucose transporter 4 (GLUT4) localization, glucose uptake and phospho‐aPKC (aPKC activated form) were assessed. Muscle contraction and insulin increased glucose uptake in all conditions when compared with controls not stimulating an effect that was accompanied by an increase in GLUT4 and of phospho‐aPKC at the muscle membrane. Contracted muscles treated with insulin did not show additive effects on glucose uptake or aPKC activity compared with the response when these stimuli were applied alone. Inhibition of PI3K blocked insulin effect on glucose uptake and aPKC but not in the contractile response. Thus, muscle contraction seems to stimulate aPKC and glucose uptake independently of PI3K. Therefore, aPKC may be a convergence point and a rate limit step in the pathway by which, insulin and contraction, increase glucose uptake in skeletal muscle. Copyright © 2014 John Wiley & Sons, Ltd.     

A high-fructose diet impairs Akt and PKCzeta phosphorylation and GLUT4 translocation in rat skeletal muscle.

The molecular mechanism of insulin resistance induced by high-fructose feeding is not fully understood. The present study investigated the role of downstream signaling molecules of phosphatidylinositol 3-kinase (PI3K) in the insulin-stimulated skeletal muscle of high-fructose-fed rats. Rats were divided into chow-fed and fructose-fed groups. The results of the euglycemic clamp study (insulin infusion rates: 6 mU/kg BW/min) showed a significant decrease in the glucose infusion rate (GIR) and the metabolic clearance rate of glucose (MCR) in fructose-fed rats compared with chow-fed rats. In skeletal muscle removed immediately after the clamp procedure, high-fructose feeding did not alter protein levels of protein kinase B (PKB/Akt), protein kinase C zeta (PKCzeta), or glucose transporter 4 (GLUT4). However, insulin-stimulated phosphorylation of Akt and PKCzeta and GLUT4 translocation to the plasma membrane were reduced. Our findings suggest that insulin resistance in fructose-fed rats is associated with impaired Akt and PKCzeta activation and GLUT4 translocation in skeletal muscle.     

Reversal of denervation-induced insulin resistance by SHIP2 protein synthesis blockade

Short-term muscle denervation is a reproducible model of tissue-specific insulin resistance. To investigate the molecular basis of insulin resistance in denervated muscle, the downstream signaling molecules of the insulin-signaling pathway were examined in intact and denervated soleus muscle of rats. Short-term denervation induced a significant fall in glucose clearance rates (62% of control, P < 0.05) as detected by euglycemic hyperinsulinemic clamp and was associated with a significant decrease in insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR; 73% of control, P < 0.05), IR substrate 1 (IRS1; 69% of control, P < 0.05), and IRS2 (82% of control, P < 0.05) and serine phosphorylation of Akt (39% of control, P < 0.05). Moreover, denervation reduced insulin-induced association between IRS1/IRS2 and p85/phosphatidylinositol (PI) 3-kinase. Nevertheless, denervation caused an increase in PI 3-kinase activity associated with IRS1 (275%, P < 0.05) and IRS2 (180%, P < 0.05), but the contents of phosphorylated PI detected by HPLC were significantly reduced in lipid fractions. In the face of the apparent discrepancy, we evaluated the expression and activity of the 5-inositol, lipid phosphatase SH2 domain-containing inositol phosphatase (SHIP2), and the serine phosphorylation of p85/PI 3-kinase. No major differences in SHIP2 expression were detected between intact and denervated muscle. However, serine phosphorylation of p85/PI 3-kinase was reduced in denervated muscle, whereas the blockade of SHIP2 expression by antisense oligonucleotide treatment led to partial restoration of phosphorylated PI contents and to improved glucose uptake. Thus modulation of the functional status of SHIP2 may be a major mechanism of insulin resistance induced by denervation.     

Overproduction of angiotensinogen from adipose tissue induces adipose inflammation, glucose intolerance, and insulin resistance

Although obesity is associated with overactivation of the white adipose tissue (WAT) renin-angiotensin system (RAS), a causal link between the latter and systemic insulin resistance is not established. We tested the hypothesis that overexpression of angiotensinogen (Agt) from WAT causes systemic insulin resistance via modulation of adipose inflammation. Glucose tolerance, systemic insulin sensitivity, and WAT inflammatory markers were analyzed in mice overexpressing Agt in the WAT (aP2-Agt mice). Proteomic studies and in vitro studies using 3T3-L1 adipocytes were performed to build a mechanistic framework. Male aP2-Agt mice exhibited glucose intolerance, insulin resistance, and lower insulin-stimulated glucose uptake by the skeletal muscle. The difference in glucose tolerance between genotypes was normalized by high-fat (HF) feeding, and was significantly improved by treatment with angiotensin-converting enzyme (ACE) inhibitor captopril. aP2-Agt mice also had higher monocyte chemotactic protein-1 (MCP-1) and lower interleukin-10 (IL-10) in the WAT, indicating adipose inflammation. Proteomic studies in WAT showed that they also had higher monoglyceride lipase (MGL) and glycerol-3-phosphate dehydrogenase levels. Treatment with angiotensin II (Ang II) increased MCP-1 and resistin secretion from adipocytes, which was prevented by cotreating with inhibitors of the nuclear factor-κB (NF-κB) pathway or nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In conclusion, we show for the first time that adipose RAS overactivation causes glucose intolerance and systemic insulin resistance. The mechanisms appear to be via reduced skeletal muscle glucose uptake, at least in part due to Ang II-induced, NADPH oxidase and NFκB-dependent increases in WAT inflammation.     

Selective modulation of the CAP/Cbl pathway in the adipose tissue of high fat diet treated rats

A high-fat diet (HFD) is associated with reduced glucose uptake in muscle, but not in adipose tissue. In the present study, we investigated whether a HFD can modulate glucose uptake in adipose tissue by increasing signal transduction through the CAP/Cbl pathway, independently of the PI3-K/Akt pathway. Our results suggest that, in HFD, the differential regulation of insulin-induced glucose uptake between skeletal muscle and adipose tissue may, in part, be a consequence of the CAP/Cbl/C3G pathway, since the expression of CAP and Cbl, and also the activation of this pathway were increased in adipose tissue but not in muscle.