Differential contribution of insulin receptor substrates 1 versus 2 to insulin signaling and glucose uptake in l6 myotubes

Insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) are pivotal in relaying insulin signaling in insulin-responsive tissues such as muscle. However, the precise contribution of IRS-1 vis-a-vis IRS-2 in insulin-mediated metabolic and mitogenic responses has not been compared directly in differentiated muscle cells. This study aimed to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using small interfering RNA (siRNA)-mediated specific gene silencing. In L6 myotubes, transfection of siRNA targeted specifically against IRS-1 (siIRS-1) or IRS-2 (siIRS-2) reduced the cognate protein expression by 70-75%. Insulin-induced ERK phosphorylation was much more sensitive to IRS-2 than IRS-1 ablation, whereas p38MAPK phosphorylation was reduced by 43 or 62% in myotubes treated with siIRS-1 or siIRS-2, respectively. Insulin-induced Akt1 and Akt2 phosphorylation was reduced in myotubes treated with siIRS-1, but only Akt2 phosphorylation was reduced in myotubes treated with siIRS-2. In contrast, siIRS-1 treatment caused a marked reduction in insulin-induced actin remodeling, glucose uptake, and GLUT4 translocation, and siIRS-2 was without effect on these responses. Notably, combined siIRS-1 and siIRS-2, although reducing each IRS by around 75%, caused no further drop in glucose uptake than that achieved with siIRS-1 alone, but abolished p38MAPK phosphorylation. We conclude that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake are regulated mainly by IRS-1, whereas IRS-2 contributes selectively to ERK signaling, and Akt2 and p38MAPK lie downstream of both IRS in muscle cells.     

Inhibition of mitochondrial respiratory chain in the brain of rats after hepatic failure induced by acetaminophen

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.     

Zinc stimulates glucose consumption by modulating the insulin signaling pathway in L6 myotubes: essential roles of Akt–GLUT4, GSK3β and mTOR–S6K1

The present study was performed to evaluate the insulin-like effects of zinc in normal L6 myotubes as well as its ability to alleviate insulin resistance. Glucose consumption was measured in both normal and insulin-resistant L6 myotubes. Western blotting and immunofluorescence revealed that zinc exhibited insulin-like glucose transporting effects by activating key markers that are involved in the insulin signaling cascade (including Akt, GLUT4 and GSK3β), and downregulating members of the insulin signaling feedback cascade such as mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K1). In normal L6 myotubes, zinc enhanced glucose consumption via a mechanism that might involve the activation of Akt phosphorylation, glucose transporter 4 (GLUT4) translocation and GSK3β phosphorylation. In contrast, zinc exerted insulin-mimetic effects in insulin-resistant L6 myotubes by upregulating Akt phosphorylation, GLUT4 translocation and GSK3β phosphorylation, and downregulating the expression of mTOR and S6K1. In conclusion, zinc might enhance glucose consumption by modulating insulin signaling pathways including Akt–GLUT4, GSK3β, mTOR and S6K1.     

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.     

Tumor necrosis factor alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner

Insulin stimulation produced a reliable 3-fold increase in glucose uptake in primary neonatal rat myotubes, which was accompanied by a similar effect on GLUT4 translocation to plasma membrane. Tumor necrosis factor (TNF)-alpha caused insulin resistance on glucose uptake and GLUT4 translocation by impairing insulin stimulation of insulin receptor (IR) and IR substrate (IRS)-1 and IRS-2 tyrosine phosphorylation, IRS-associated phosphatidylinositol 3-kinase activation, and Akt phosphorylation. Because this cytokine produced sustained activation of stress and proinflammatory kinases, we have explored the hypothesis that insulin resistance by TNF-alpha could be mediated by these pathways. In this study we demonstrate that pretreatment with PD169316 or SB203580, inhibitors of p38 MAPK, restored insulin signaling and normalized insulin-induced glucose uptake in the presence of TNF-alpha. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-alpha was still produced. Moreover, TNF-alpha produced inhibitor kappaB kinase (IKK)-beta activation and inhibitor kappaB-beta and -alpha degradation in a p38 MAPK-dependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-alpha produced serine phosphorylation of IR and IRS-1 (total and on Ser(307) residue), and these effects were completely precluded by pretreatment with either PD169316 or salicylate. Consequently, TNF-alpha, through activation of p38 MAPK and IKK, produces serine phosphorylation of IR and IRS-1, impairing its tyrosine phosphorylation by insulin and the corresponding activation of phosphatidylinositol 3-kinase and Akt, leading to insulin resistance on glucose uptake and GLUT4 translocation.     

Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis

Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin.     

Depletion of mitochondrial DNA causes impaired glucose utilization and insulin resistance in L6 GLUT4myc myocytes

Mitochondrial dysfunction contributes to a number of human diseases, such as hyperlipidemia, obesity, and diabetes. The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of diabetes. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long term treatment with ethidium bromide. L6 GLUT4myc cells cultured with 0.2 mug/ml ethidium bromide (termed depleted cells) revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The mRNA level and expression of insulin receptor substrate (IRS)-1 associated with insulin signaling were decreased by 76 and 90% in the depleted cells, respectively. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Moreover, insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reductions in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.     

Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression

Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs) normally active or middle-aged (56.6 yrs) individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane. On the other hand, middle-aged active cells retained both insulin-stimulated increases in glucose uptake and GLUT4 translocation to the plasma membrane. Middle-aged active cells also had significantly higher mRNA expression of GLUT1 and GLUT4 compared to middle-aged sedentary cells, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact on the metabolism of human myotubes during aging and may contribute to aging-associated insulin resistance through impaired GLUT4 localization.     

MAP4K4 gene silencing in human skeletal muscle prevents tumor necrosis factor-alpha-induced insulin resistance

Tumor necrosis factor-alpha (TNF-alpha) induces skeletal muscle insulin resistance by impairing insulin signaling events involved in GLUT4 translocation. We tested whether mitogenic-activated protein kinase kinase kinase kinase isoform 4 (MAP4K4) causes the TNF-alpha-induced negative regulation of extracellular signal-regulated kinase-1/2 (ERK-1/2), c-Jun NH2-terminal kinase (JNK), and the insulin receptor substrate-1 (IRS-1) on the insulin signaling pathway governing glucose metabolism. Using small interfering RNA (siRNA) to suppress the expression of MAP4K4 protein 85% in primary human skeletal muscle cells, we provide evidence that TNF-alpha-induced insulin resistance on glucose uptake was completely prevented. MAP4K4 silencing inhibited TNF-alpha-induced negative signaling inputs by preventing excessive JNK and ERK-1/2 phosphorylation, as well as IRS-1 serine phosphorylation. These results highlight the MAPK4K4/JNK/ERK/IRS module in the negative regulation of insulin signaling to glucose transport in response to TNF-alpha. Depletion of MAP4K4 also prevented TNF-alpha-induced insulin resistance on Akt and the Akt substrate 160 (AS160), providing evidence that appropriate insulin signaling inputs for glucose metabolism were rescued. Silencing of MAP2K1 and MAP2K4, signaling proteins downstream of MAP4K4, recapitulated the effect of MAP4K4 siRNA in TNF-alpha-treated cells. Thus, strategies to inhibit MAP4K4 may be efficacious in the prevention of TNF-alpha-induced inhibitory signals that cause skeletal muscle insulin resistance on glucose metabolism in humans. Moreover, in myotubes from insulin-resistant type II diabetic patients, siRNA against MAP4K4, MAP2K4, or MAP2K1 restored insulin action on glucose uptake to levels observed in healthy subjects. Collectively, our results demonstrate that MAP4K4 silencing prevents insulin resistance in human skeletal muscle and restores appropriate signaling inputs to enhance glucose uptake.     

Cross-talk mechanisms in the development of insulin resistance of skeletal muscle cells palmitate rather than tumour necrosis factor inhibits insulin-dependent protein kinase B (PKB)/Akt stimulation and glucose uptake.

Insulin resistance in skeletal muscle is one of the earliest symptoms associated with non-insulin-dependent diabetes mellitus (NIDDM). Tumour necrosis factor (TNF) and nonesterified fatty acids have been proposed to be crucial factors in the development of the insulin-resistant state. We here show that, although TNF downregulated insulin-induced insulin receptor (IR) and IR substrate (IRS)-1 phosphorylation as well as phosphoinositide 3-kinase (PI3-kinase) activity in pmi28 myotubes, this was, unlike in adipocytes, not sufficient to affect insulin-induced glucose transport. Rather, TNF increased membrane expression of GLUT1 and glucose transport in these muscle cells. In contrast, the nonesterified fatty acid palmitate inhibited insulin-induced signalling cascades not only at the level of IR and IRS-1 phosphorylation, but also at the level protein kinase B (PKB/Akt), which is thought to be directly involved in the insulin-induced translocation of GLUT4, and inhibited insulin-induced glucose uptake. Palmitate also abrogated TNF-dependent enhancement of basal glucose uptake, suggesting that palmitate has the capacity to render muscle cells resistant not only to insulin but also to TNF with respect to glucose transport by GLUT4 and GLUT1, respectively. Our data illustrate the complexity of the mechanisms governing insulin resistance of skeletal muscle, questioning the role of TNF as a direct inhibitor of glucose homoeostasis in this tissue and shedding new light on an as yet unrecognized multifunctional role for the predominant nonesterified fatty acid palmitate in this process.     

Resistin inhibits glucose uptake in L6 cells independently of changes in insulin signaling and GLUT4 translocation

Elevated levels of resistin have been proposed to cause insulin resistance and therefore may serve as a link between obesity and type 2 diabetes. However, its role in skeletal muscle metabolism is unknown. In this study, we examined the effect of resistin on insulin-stimulated glucose uptake and the upstream insulin-signaling components in L6 rat skeletal muscle cells that were either incubated with recombinant resistin or stably transfected with a vector containing the myc-tagged mouse resistin gene. Transfected clones expressed intracellular resistin, which was released in the medium. Incubation with recombinant resistin resulted in a dose-dependent inhibition of insulin-stimulated 2-deoxyglucose (2-DG) uptake. The inhibitory effect of resistin on insulin-stimulated 2-DG uptake was not the result of impaired GLUT4 translocation to the plasma membrane. Furthermore, resistin did not alter the insulin receptor (IR) content and its phosphorylation, nor did it affect insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation, its association with the p85 subunit of phosphatidylinositol (PI) 3-kinase, or IRS-1-associated PI 3-kinase enzymatic activity. Insulin-stimulated phosphorylation of Akt/protein kinase B-alpha, one of the downstream targets of PI 3-kinase and p38 MAPK phosphorylation, was also not affected by resistin. Expression of resistin also inhibited insulin-stimulated 2-DG uptake when compared with cells expressing the empty vector (L6Neo) without affecting GLUT4 translocation, GLUT1 content, and IRS-1/PI 3-kinase signaling. We conclude that resistin does not alter IR signaling but does affect insulin-stimulated glucose uptake, presumably by decreasing the intrinsic activity of cell surface glucose transporters.     

Hepatitis C virus activates the mTOR/S6K1 signaling pathway in inhibiting IRS-1 function for insulin resistance

Hepatitis C virus (HCV) infection significantly increases the prevalence of type 2 diabetes mellitus (T2DM). Insulin receptor substrate 1 (IRS-1) plays a key role in insulin signaling, thus enabling metabolic regulation in mammalian cells. We have previously shown that HCV infection modulates phosphorylation of Akt, a downstream target of IRS-1. In this study, we further examined the status of total IRS-1 and the downstream regulation of the Akt pathway in understanding mTOR/S6K1 signaling using HCV genotype 2a (clone JFH1)-infected hepatocytes. Inhibition of IRS-1 expression was observed in HCV-infected hepatocytes compared to that in a mock-infected control. The status of the tuberous sclerosis complex (TSC-1/TSC-2) was significantly decreased after HCV infection of human hepatocytes, showing a modulation of the downstream Akt pathway. Subsequent study indicated an increased level of Rheb and mTOR expression in HCV-infected hepatocytes. Interestingly, the phosphoS6K1 level was higher in HCV-infected hepatocytes, suggesting a novel mechanism for IRS-1 inhibition. Ectopic expression of TSC-1/TSC-2 significantly recovered the IRS-1 protein expression level in HCV-infected hepatocytes. Further analyses indicated that HCV core protein plays a significant role in modulating the mTOR/S6K1 signaling pathway. Proteasome inhibitor MG 132 recovered IRS-1 and TSC1/2 expression, suggesting that degradation occurred via the ubiquitin proteasome pathway. A functional consequence of IRS-1 inhibition was reflected in a decrease in GLUT4 protein expression and upregulation of the gluconeogenic enzyme PCK2 in HCV-infected hepatocytes. Together, these observations suggested that HCV infection activates the mTOR/S6K1 pathway in inhibiting IRS-1 function and perturbs glucose metabolism via downregulation of GLUT4 and upregulation of PCK2 for insulin resistance.     

Serotonin (5-Hydroxytryptamine), a novel regulator of glucose transport in rat skeletal muscle

In this study we show that serotonin (5-hydroxytryptamine (5-HT)) causes a rapid stimulation in glucose uptake by approximately 50% in both L6 myotubes and isolated rat skeletal muscle. This activation is mediated via the 5-HT2A receptor, which is expressed in L6, rat, and human skeletal muscle. In L6 cells, expression of the 5-HT2A receptor is developmentally regulated based on the finding that receptor abundance increases by over 3-fold during differentiation from myoblasts to myotubes. Stimulation of the 5-HT2A receptor using methylserotonin (m-HT), a selective 5-HT2A agonist, increased muscle glucose uptake in a manner similar to that seen in response to 5-HT. The agonist-mediated stimulation in glucose uptake was attributable to an increase in the plasma membrane content of GLUT1, GLUT3, and GLUT4. The stimulatory effects of 5-HT and m-HT were suppressed in the presence of submicromolar concentrations of ketanserin (a selective 5-HT2A antagonist) providing further evidence that the increase in glucose uptake was specifically mediated via the 5-HT2A receptor. Treatment of L6 cells with insulin resulted in tyrosine phosphorylation of IRS1, increased cellular production of phosphatidylinositol 3,4,5-phosphate and a 41-fold activation in protein kinase B (PKB/Akt) activity. In contrast, m-HT did not modulate IRS1, phosphoinositide 3-kinase, or PKB activity. The present results indicate that rat and human skeletal muscle both express the 5-HT2A receptor and that 5-HT and specific 5-HT2A agonists can rapidly stimulate glucose uptake in skeletal muscle by a mechanism which does not depend upon components that participate in the insulin signaling pathway.     

Protein arginine methylation regulates insulin signaling in L6 skeletal muscle cells

Protein N-arginine methyltransferase (PRMT)1 catalyzes arginine methylation in a variety of substrates, although the potential role of PRMT1 in insulin action has not been defined. We therefore investigated the effect of PRMT1-mediated methylation on insulin signaling and glucose uptake in skeletal L6 myotubes. Exposure of L6 myotubes to insulin rapidly induced translocation of PRMT1 and increased its catalytic activity in membrane fraction. Several proteins in the membrane fraction were arginine-methylated after insulin treatment, which were inhibited by pretreatment with an inhibitor of methyltransferase, 5′-deoxy-5′-(methylthio)adenosine (MTA), or a small interfering RNA against PRMT1 (PRMT1-siRNA). Inhibition of arginine methylation with MTA or PRMT1-siRNA diminished later phase of insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) β and IRS-1, association of IRS-1 with p85α subunit of PI3-K, and glucose uptake. Our results suggest that PRMT1-mediated methylation serves as a positive modulator of IR/IRS-1/PI3-K pathway and subsequent glucose uptake in skeletal muscle cells.     

Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice.

Decreased GLUT4 expression, impaired insulin receptor (IR), IRS-1, and pp60/IRS-3 tyrosine phosphorylation are characteristics of adipocytes from insulin-resistant animal models and obese NIDDM humans. However, the sequence of events leading to the development of insulin signaling defects and the significance of decreased GLUT4 expression in causing adipocyte insulin resistance are unknown. The present study used male heterozygous GLUT4 knockout mice (GLUT4(+/-)) as a novel model of diabetes to study the development of insulin signaling defects in adipocytes with the progression of whole body insulin resistance and diabetes. Male GLUT4(+/-) mice with normal fed glycemia and insulinemia (N/N), normal fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with hyperinsulinemia (H/H) exist at all ages. The expression of GLUT4 protein and the maximal insulin-stimulated glucose transport was 50% decreased in adipocytes from all three groups. Insulin signaling was normal in N/N adipose cells. From 35 to 70% reductions in insulin-stimulated tyrosine phosphorylation of IR, IRS-1, and pp60/IRS-3 were noted with no changes in the cellular content of IR, IRS-1, and p85 in N/H adipocytes. Insulin-stimulated protein tyrosine phosphorylation was further decreased to 12-23% in H/H adipose cells accompanied by 42% decreased IR and 80% increased p85 expression. Insulin-stimulated, IRS-1-associated PI3 kinase activity was decreased by 20% in N/H and 68% reduced in H/H GLUT4(+/-) adipocytes. However, total insulin-stimulated PI3 kinase activity was normal in H/H GLUT4(+/-) adipocytes. Taken together, these results strongly suggest that hyperinsulinemia triggers a reduction of IR tyrosine kinase activity that is further exacerbated by the appearance of hyperglycemia. However, the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake. Furthermore, the data indicate that the cellular content of GLUT4 is the rate-limiting factor in mediating maximal insulin-stimulated glucose uptake in GLUT4(+/-) adipocytes.     

Monoclonal antibody to six transmembrane epithelial antigen of prostate-4 influences insulin sensitivity by attenuating phosphorylation of P13K (P85) and Akt: Possible mitochondrial mechanism

We examined the effects of anti-six-transmembrane epithelial antigen of the prostate-4 (STEAP4) antibodies on glucose transport in mature adipocytes and determined the mechanism of insulin resistance in obesity. Western blotting was performed to determine STEAP4 expression, to assess translocation of insulin-sensitive glucose transporter 4 (GLUT4), and to measure phosphorylation and total protein content of insulin-signaling proteins. Confocal laser microscopy and flow cytometry were used to detect intracellular reactive oxygen species (ROS) and fluctuations in mitochondrial membrane potential (ΔΨ). ATP production was measured by using a luciferase-based luminescence assay kit. After the application of anti-STEAP4 antibodies at 0.002?mg/mL, adipocytes exhibited reduced insulin-stimulated glucose transport by attenuating the phosphorylation of IRS-1, PI3K (p85), and Akt. The antibodies also potentially increase the level of ROS and decrease cellular ATP production and ΔΨ. In conclusion, (i) STEAP4 regulates the function of IRS-1, PI3K, and Akt and decreases insulin-induced GLUT4 translocation and glucose uptake; (ii) ROS-related mitochondrial dysfunction may be related to a reduced IRS-1 correlation with the PI3K signaling pathway, leading to insulin resistance. These observations highlight the potential role of STEAP4 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity and may provide new insights into the mechanisms of insulin resistance in obesity.     

Melatonin alleviates insulin resistance through the PI3K/AKT signaling pathway in ovary granulosa cells of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine gynecological disorder. Insulin resistance (IR) is a major cause of PCOS. Melatonin, a critical endogenous hormone, has beneficial effects on the female reproductive system. This study aims to investigate the molecular effect of melatonin on IR in human ovarian granulosa cells (GCs). Hormone levels of the subjects were determined through clinical examination. The expression levels of insulin receptor substrate (IRS)-1 and glucose transporter (GLUT4) in GCs from PCOS patients and a human granulosa cell line (SVOG) were examined using qRT-PCR and western blot. The IR cell model was established by inducing SVOG cells with palmitic acid (PA). IR was detected in GCs of PCOS patients and SVOG by measuring glucose content and glucose uptake. Cell viability and apoptosis levels were detected by CCK-8 assay and flow cytometry. PI3K/Akt pathway expression in SVOG was assessed by western blot. PCOS patients had higher levels of luteinizing hormone (LH), testosterone, and LH/follicle-stimulating hormone. PA decreased cell viability, promoted apoptosis, and reduced glucose uptake in SVOG cells. IRS-1 and GLUT4 mRNA and protein expression was downregulated, and glucose uptake capacity was reduced in PCOS GCs and SVOG cells. Melatonin significantly upregulated IRS-1 and GLUT4 expression, downregulated p-IRS-1 (Ser307), and improved glucose uptake in PCOS patients' GCs and SVOG cells. PA decreased PI3K and Akt phosphorylation, whereas melatonin increased p-PI3K and p-Akt levels. Melatonin can reduce IR in GCs and PA-induced SVOG cells via the PI3K/Akt signaling pathway, providing more evidence for treating polycystic ovary syndrome.     

Direct cross-talk of interleukin-6 and insulin signal transduction via insulin receptor substrate-1 in skeletal muscle cells

The exercise-induced interleukin (IL)-6 production and secretion within skeletal muscle fibers has raised the question of a putative tissue-specific function of IL-6 in the energy metabolism of the muscle during and after the exercise. In the present study, we followed the hypothesis that IL-6 signaling may directly interact with insulin receptor substrate (IRS)-1, a keystone in the insulin signaling cascade. We showed that IL-6 induces a rapid recruitment of IRS-1 to the IL-6 receptor complex in cultured skeletal muscle cells. Moreover, IL-6 induced a rapid and transient phosphorylation of Ser-318 of IRS-1 in muscle cells and in muscle tissue, but not in the liver of IL-6-treated mice, probably via the IL-6-induced co-recruitment of protein kinase C-delta. This Ser-318 phosphorylation improved insulin-stimulated Akt phosphorylation and glucose uptake in myotubes since transfection with an IRS-1/Glu-318 mutant simulating a permanent phospho-Ser-318 modification increased Akt phosphorylation and glucose uptake. Noteworthily, two inhibitory mechanisms of IL-6 on insulin action, phosphorylation of the inhibitory Ser-307 residue of IRS-1 and induction of SOCS-3 expression, were only found in liver but not in muscle of IL-6-treated mice. Thus, the data provided evidence for a possible molecular mechanism of the physiological metabolic effects of IL-6 in skeletal muscle, thereby exerting short term beneficial effects on insulin action.     

Epigallocatechin-3-O-gallate (EGCG) protects the insulin sensitivity in rat L6 muscle cells exposed to dexamethasone condition

The tea polyphenol epigallocatechin-3-O-gallate (EGCG) displays some antidiabetic effects; however the mechanisms are incompletely understood. In the present study, the investigation of the effects of EGCG on insulin resistance was performed in rat L6 cells treated with dexamethasone. We found that dexamethasone increased Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and reduced phosphorylation of AMPK and Akt. Furthermore, glucose uptake and glucose transporter (GLUT4) translocation were inhibited by dexamethasone. However, the treatment of EGCG improved insulin-stimulated glucose uptake by increasing GLUT4 translocation to plasma membrane. Furthermore, we also demonstrated these EGCG effects essentially depended on the AMPK and Akt activation. Together, our data suggested that EGCG inhibited dexamethasone-induced insulin resistance through AMPK and PI3K/Akt pathway.