Phosphorylation of insulin receptor substrate-1 serine 307 correlates with JNK activity in atrophic skeletal muscle

c-Jun NH(2)-terminal kinase (JNK) has been shown to negatively regulate insulin signaling through serine phosphorylation of residue 307 within the insulin receptor substrate-1 (IRS-1) in adipose and liver tissue. Using a rat hindlimb suspension model for muscle disuse atrophy, we found that JNK activity was significantly elevated in atrophic soleus muscle and that IRS-1 was phosphorylated on Ser(307) prior to the degradation of the IRS-1 protein. Moreover, we observed a corresponding reduction in Akt activity, providing biochemical evidence for the development of insulin resistance in atrophic skeletal muscle.     

Vascular smooth muscle insulin resistance, but not hypertrophic signaling, is independent of angiotensin II-induced IRS-1 phosphorylation by JNK

Angiotensin II (ANG II) has been implicated in the pathogenesis of diabetic micro- and macrovascular disease. In vascular smooth muscle cells (VSMCs), ANG II phosphorylates and degrades insulin receptor substrate-1 (IRS-1). While the pathway responsible for IRS-1 degradation in this system is unknown, c-Jun NH(2)-terminal kinase (JNK) has been linked with serine phosphorylation of IRS-1 and insulin resistance. We investigated the role of JNK in ANG II-induced IRS-1 phosphorylation, degradation, Akt activation, glucose uptake, and hypertrophic signaling, focusing on three IRS-1 phosphorylation sites: Ser302, Ser307, and Ser632. Maximal IRS-1 phosphorylation on Ser632 occurred at 5 min, on Ser307 at 30 min, and on Ser302 at 60 min. The JNK inhibitor SP600125 reduced ANG II-induced IRS-1 Ser307 phosphorylation (by 80%), IRS-1 Ser302 phosphorylation (by 70%), and IRS-1 Ser632 phosphorylation (by 50%). However, JNK inhibition had no effect on ANG II-mediated IRS-1 degradation, nor did it reverse the ANG II-induced decrease in Akt phosphorylation or glucose uptake. Transfection of VSMCs with mutants S307A, S302A, or S632A of IRS-1 did not block ANG II-mediated IRS-1 degradation. In contrast, JNK inhibition attenuated insulin-induced upregulation of collagen and smooth muscle α-actin in ANG II-pretreated cells. We conclude that phosphorylation of Ser307, Ser302, and Ser632 of IRS-1 is not involved in ANG II-mediated IRS-1 degradation, and that JNK alone does not mediate ANG II-stimulated IRS-1 degradation, but rather is responsible for the hypertrophic effects of insulin on smooth muscle.     

Free fatty acid-induced muscle insulin resistance and glucose uptake dysfunction: Evidence for PKC activation and oxidative stress-activated signaling pathways

In the present study, we examined the effects of free fatty acids (FFAs) on insulin sensitivity and signaling cascades in the C2C12 skeletal muscle cell culture system. Our data clearly manifested that the inhibitory effects of PKC on insulin signaling may at least in part be explained by the serine/threonine phosphorylation of IRS-1. Both oleate and palmitate treatment were able to increase the Serine³⁰⁷ phosphorylation of IRS-1. IRS-1 Serine³⁰⁷ phosphorylation is inducible which causes the inhibition of IRS-1 tyrosine phosphorylation by either IκB-kinase (IKK) or c-jun N-terminal kinase (JNK) as seen in our proteomic kinases screen. Furthermore, our proteomic data have also manifested that the two FFAs activate the IKKα/β, the stress kinases S6 kinase p70 (p70SK), stress-activated protein kinase (SAPK), JNK, as well as p38 MAP kinase (p38MAPK). On the other hand, the antioxidant, Taurine at 10 mM concentrations was capable of reversing the oleate-induced insulin resistance in myocytes as manifested from the glucose uptake data. Our current data point out the importance of FFA-induced insulin resistance via multiple signaling mechanisms.     

Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in tumor necrosis factor-treated cells through targeting multiple serine kinases

The hypoglycemic effects of high dose salicylates in the treatment of diabetes were documented before the advent of insulin. However, the molecular mechanisms by which salicylates exert these anti-diabetic effects are not well understood. In this study, we analyzed the effects of aspirin (acetylsalicylic acid) on serine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells treated with tumor necrosis factor (TNF)-alpha. Phosphorylation of IRS-1 at Ser307, Ser267, and Ser612 was monitored by immunoblotting with phospho-specific IRS-1 antibodies. In 3T3-L1 and Hep G2 cells, phosphorylation of IRS-1 at Ser307 in response to TNF-alpha treatment correlated with phosphorylation of JNK, c-Jun, and degradation of IkappaBalpha. Moreover, phosphorylation of IRS-1 at Ser307 in embryo fibroblasts derived from either JNK or IKK knockout mice was reduced when compared with that in the wild-type controls. Taken together, these data suggest that serine phosphorylation of IRS-1 in response to TNF-alpha is mediated, in part, by JNK and IKK. Interestingly, aspirin treatment inhibited the phosphorylation of IRS-1 at Ser307 as well as the phosphorylation of JNK, c-Jun, and degradation of IkappaBalpha. Furthermore, other serine kinases including Akt, extracellular regulated kinase, mammalian target of rapamycin, and PKCzeta were also activated by TNF-alpha (as assessed by phospho-specific antibodies). Phosphorylation of IRS-1 at Ser267 and Ser612 correlated with the activation of these kinases. Phosphorylation of Akt and the mammalian target of rapamycin (but not extracellular regulated kinase or PKCzeta) in response to TNF-alpha was inhibited by aspirin treatment. Finally, aspirin rescued insulin-induced glucose uptake in 3T3-L1 adipocytes pretreated with TNF-alpha. We conclude that aspirin may enhance insulin sensitivity by protecting IRS proteins from serine phosphorylation catalyzed by multiple kinases.     

The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307)

Tumor necrosis factor alpha (TNFalpha) inhibits insulin action, in part, through serine phosphorylation of IRS proteins; however, the phosphorylation sites that mediate the inhibition are unknown. TNFalpha promotes multipotential signal transduction cascades, including the activation of the Jun NH(2)-terminal kinase (JNK). Endogenous JNK associates with IRS-1 in Chinese hamster ovary cells. Anisomycin, a strong activator of JNK in these cells, stimulates the activity of JNK bound to IRS-1 and inhibits the insulin-stimulated tyrosine phosphorylation of IRS-1. Serine 307 is a major site of JNK phosphorylation in IRS-1. Mutation of serine 307 to alanine eliminates phosphorylation of IRS-1 by JNK and abrogates the inhibitory effect of TNFalpha on insulin-stimulated tyrosine phosphorylation of IRS-1. These results suggest that phosphorylation of serine 307 might mediate, at least partially, the inhibitory effect of proinflammatory cytokines like TNFalpha on IRS-1 function.     

C-reactive protein suppresses insulin signaling in endothelial cells: role of spleen tyrosine kinase

Although few epidemiological studies have demonstrated that C-reactive protein (CRP) is related to insulin resistance, no study to date has examined the molecular mechanism. Here, we show that recombinant CRP attenuates insulin signaling through the regulation of spleen tyrosine kinase (Syk) on small G-protein RhoA, jun N-terminal kinase (JNK) MAPK, insulin receptor substrate-1 (IRS-1), and endothelial nitric oxide synthase in vascular endothelial cells. Recombinant CRP suppressed insulin-induced NO production, inhibited the phosphorylation of Akt and endothelial nitric oxide synthase, and stimulated the phosphorylation of IRS-1 at the Ser307 site in a dose-dependent manner. These events were blocked by treatment with an inhibitor of RhoA-dependent kinase Y27632, or an inhibitor of JNK SP600125, or the transfection of dominant negative RhoA cDNA. Next, anti-CD64 Fcgamma phagocytic receptor I (FcgammaRI), but not anti-CD16 (FcgammaRIIIa) or anti-CD32 (FcgammaRII) antibody, partially blocked the recombinant CRP-induced phosphorylation of JNK and IRS-1 and restored, to a certain extent, the insulin-stimulated phosphorylation of Akt. Furthermore, we identified that recombinant CRP modulates the phosphorylation of Syk tyrosine kinase in endothelial cells. Piceatannol, an inhibitor of Syk tyrosine kinase, or infection of Syk small interference RNA blocked the recombinant CRP-induced RhoA activity and the phosphorylation of JNK and IRS-1. In addition, piceatannol also restrained CRP-induced endothelin-1 production. We conclude that recombinant CRP induces endothelial insulin resistance and dysfunction, and propose a new mechanism by which recombinant CRP induces the phosphorylation of JNK and IRS-1 at the Ser307 site through a Syk tyrosine kinase and RhoA-activation signaling pathway.     

ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹⁶. Ang II-induced Ser³⁰⁷ and Ser⁶¹⁶ phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.     

Mammalian target of rapamycin regulates IRS-1 serine 307 phosphorylation

Insulin signaling can be negatively regulated by phosphorylation of serine 307 of the insulin receptor substrate (IRS)-1. Rapamycin, an inhibitor of the kinase mTOR, can prevent serine 307 phosphorylation and the development of insulin resistance. We further investigated the role of mTOR in regulating serine 307 phosphorylation, demonstrating that serine 307 phosphorylation in response to insulin, anisomycin, or tumor necrosis factor was quantitatively and temporally associated with activation of mTOR and could be inhibited by rapamycin. Amino acid stimulation activated mTOR and resulted in IRS-1 serine 307 phosphorylation without activating PKB or JNK. Okadaic acid, an inhibitor of the phosphatase PP2A, activated mTOR and stimulated the phosphorylation of serine 307 in a rapamycin-sensitive manner, indicating serine 307 phosphorylation requires mTOR activity but not PP2A, suggesting that mTOR itself may be responsible for phosphorylating serine 307. Finally, we demonstrated that serine 307 phosphorylated IRS-1 is detected primarily in the cytosolic fraction.     

GSK-3beta activity is increased in skeletal muscle after burn injury in rats

Previous reports suggest that burn-induced muscle proteolysis can be inhibited by treatment with GSK-3beta inhibitors, suggesting that burn injury may be associated with increased GSK-3beta activity. The influence of burn injury on muscle GSK-3beta activity, however, is not known. We determined the effect of a 30% total body surface full-thickness burn injury in rats on muscle GSK-3beta activity by measuring GSK-3beta activity and tissue levels of serine 9 phosphorylated GSK-3beta, p(Ser9)-GSK-3beta, by Western blot analysis and immunohistochemistry. Because burn-induced muscle wasting is, at least in part, mediated by glucocorticoids, we used dexamethasone-treated cultured muscle cells in which GSK-3beta expression was reduced with small interfering RNA (siRNA) to further assess the role of GSK-3beta in muscle atrophy. Burn injury resulted in a seven-fold increase in GSK-3beta activity in skeletal muscle. This effect of burn was accompanied by reduced tissue levels of p(Ser9)-GSK-3beta, suggesting that burn injury stimulates GSK-3beta in skeletal muscle secondary to inhibited phosphorylation of the enzyme. In addition, burn injury resulted in inhibited phosphorylation and activation of Akt, an upstream regulatory mechanism of GSK-3beta activity. Reducing the expression of GSK-3beta in cultured muscle cells with siRNA inhibited dexamethasone-induced protein degradation by approximately 50%. The results suggest that burn injury stimulates GSK-3beta activity in skeletal muscle and that GSK-3beta may, at least in part, regulate glucocorticoid-mediated muscle wasting.     

Interleukin-1alpha inhibits insulin signaling with phosphorylating insulin receptor substrate-1 on serine residues in 3T3-L1 adipocytes

Proinflammatory cytokines are recently reported to inhibit insulin signaling causing insulin resistance. IL-1alpha is also one of the proinflammatory cytokines; however, it has not been clarified whether IL-1alpha may also cause insulin resistance. Here, we investigated the effects of IL-1alpha treatment on insulin signaling in 3T3-L1 adipocytes. IL-1alpha treatment up to 4 h did not alter insulin-stimulated insulin receptor tyrosine phosphorylation, whereas tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the association with phosphatidylinositol 3-kinase were partially inhibited with the maximal inhibition in around 15 min. IRS-1 was transiently phosphorylated on some serine residues around 15 min after IL-1alpha stimulation, when several serine kinases, IkappaB kinase, c-Jun-N-terminal kinase, ERK, and p70S6K were activated. Chemical inhibitors for these kinases inhibited IL-1alpha-induced serine phosphorylation of IRS-1. Tyrosine phosphorylation of IRS-1 was recovered only by the IKK inhibitor or JNK inhibitor, suggesting specific involvement of these two kinases. Insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake were not inhibited only by IL-1alpha. Interestingly, Akt phosphorylation was synergistically inhibited by IL-1alpha in the presence of IL-6. Taken together, short-term IL-1alpha treatment transiently causes insulin resistance at IRS-1 level with its serine phosphorylation. IL-1alpha may suppress insulin signaling downstream of IRS-1 in the presence of other cytokines, such as IL-6.     

Calorie restriction prevents the development of insulin resistance and impaired insulin signaling in skeletal muscle of ovariectomized rats

Insulin resistance of skeletal muscle glucose transport due to prolonged loss of ovarian function in ovariectomized (OVX) rats is accompanied by other features of the metabolic syndrome and may be confounded by increased calorie consumption. In this study, we investigated the role of calorie consumption in the development of insulin resistance in OVX rats. In addition, we examined the cellular mechanisms underlying skeletal muscle insulin resistance in OVX rats. Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (SHAM). OVX rats either had free access to food, pair feeding (PF) with SHAM or received a 35% reduction in food intake (calorie restriction; CR) for 12weeks. Compared with SHAM, ovariectomy induced skeletal muscle insulin resistance, which was associated with decreases (32-70%) in tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), IRS-1 associated p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), and Akt Ser(473) phosphorylation whereas insulin-stimulated phosphorylation of IRS-1 Ser(307), SAPK/JNK Thr(183)/Tyr(185), and p38 mitogen-activated protein kinase (MAPK) Thr(180)/Tyr(182) was increased (24-62%). PF improved the serum lipid profile but did not restore insulin-stimulated glucose transport, indicating that insulin resistance in OVX rats is a consequence of ovarian hormone deprivation. In contrast, impaired insulin sensitivity and defective insulin signaling were not observed in the skeletal muscle of OVX+CR rats. Therefore, we provide evidence for the first time that CR effectively prevents the development of insulin resistance and impaired insulin signaling in the skeletal muscle of OVX rats.     

Aspirin inhibits serine phosphorylation of IRS-1 in muscle and adipose tissue of septic rats

Whole body insulin resistance has been demonstrated in septic patients and in infected animals. In this study, we demonstrate that sepsis induces insulin resistance and that pretreatment with aspirin inhibits sepsis-induced insulin resistance. Sepsis was observed to lead to serine phosphorylation of IRS-1, a phenomenon which was reversed by aspirin in muscle and WAT, in parallel with a reduction in JNK activity. In addition, our data show an impairment of insulin activation of IR and IRS-1 tyrosine phosphorylation in septic rats and, consistent with the reduction of IRS-1 serine phosphorylation observed in septic animals pretreated with aspirin, there was an increase in IRS-1 protein levels and tyrosine phosphorylation in muscle and WAT. Overall, these results provide important new insights into the mechanism of sepsis-induced insulin resistance.     

Targeted disruption of iNOS prevents LPS-induced S-nitrosation of IRbeta/IRS-1 and Akt and insulin resistance in muscle of mice

We have previously demonstrated that the insulin resistance associated with inducible nitric oxide synthase (iNOS) induction in two different models of obesity, diet-induced obesity and the ob/ob mice, is mediated by S-nitrosation of proteins involved in insulin signal transduction: insulin receptor beta-subunit (IRbeta), insulin receptor substrate 1(IRS-1), and Akt. S-nitrosation of IRbeta and Akt impairs their kinase activities, and S-nitrosation of IRS-1 reduces its tissue expression. In this study, we observed that LPS-induced insulin resistance in the muscle of wild-type mice, as demonstrated by reduced insulin-induced tyrosine phosphorylation of IRbeta and IRS-1, reduced IRS-1 expression and reduced insulin-induced serine phosphorylation of Akt. This resistance occurred in parallel with enhanced iNOS expression, which was accompanied by S-nitrosation of IRbeta/IRS-1 and Akt. In the muscle of iNOS(-/-) mice, we did not observe enhanced iNOS expression or any S-nitrosation of IRbeta/IRS-1 and Akt after LPS treatment. Moreover, insulin resistance was not present. The preservation of insulin-induced tyrosine phosphorylation of IRbeta and IRS-1, of IRS-1 protein expression, and of insulin-induced serine phosphorylation of Akt observed in LPS-treated iNOS(-/-) mice strongly suggests that the insulin resistance induced by LPS is iNOS mediated, probably through S-nitrosation of proteins of early steps of insulin signaling.     

Inhibition of insulin sensitivity by free fatty acids requires activation of multiple serine kinases in 3T3-L1 adipocytes

Insulin receptor substrate (IRS) has been suggested as a molecular target of free fatty acids (FFAs) for insulin resistance. However, the signaling pathways by which FFAs lead to the inhibition of IRS function remain to be established. In this study, we explored the FFA-signaling pathway that contributes to serine phosphorylation and degradation of IRS-1 in adipocytes and in dietary obese mice. Linoleic acid, an FFA used in this study, resulted in a reduction in insulin-induced glucose uptake in 3T3-L1 adipocytes. This mimics insulin resistance induced by high-fat diet in C57BL/6J mice. The reduction in glucose uptake is associated with a decrease in IRS-1, but not IRS-2 or GLUT4 protein abundance. Decrease in IRS-1 protein was proceeded by IRS-1 (serine 307) phosphorylation that was catalyzed by serine kinases inhibitor kappaB kinase (IKK) and c-JUN NH2-terminal kinase (JNK). IKK and JNK were activated by linoleic acid and inhibition of the two kinases led to prevention of IRS-1 reduction. We demonstrate that protein kinase C (PKC) theta is expressed in adipocytes. In 3T3-L1 adipocytes and fat tissue, PKCtheta was activated by fatty acids as indicated by its phosphorylation status, and by its protein level, respectively. Activation of PKCtheta contributes to IKK and JNK activation as inhibition of PKCtheta by calphostin C blocked activation of the latter kinases. Inhibition of either PKCtheta or IKK plus JNK by chemical inhibitors resulted in protection of IRS-1 function and insulin sensitivity in 3T3-L1 adipocytes. These data suggest that: 1) activation of PKCtheta contributes to IKK and JNK activation by FFAs; 2) IKK and JNK mediate PKCtheta signals for IRS-1 serine phosphorylation and degradation; and 3) this molecular mechanism may be responsible for insulin resistance associated with hyperlipidemia.     

Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302

Inhibitory serine phosphorylation is a potential molecular mechanism for insulin resistance. We have developed a new variant of the yeast two-hybrid method, referred to as disruptive yeast tri-hybrid (Y3H), to identify inhibitory kinases and sites of phosphorylation in insulin receptors (IR) and IR substrates, IRS-1. Using IR and IRS-1 as bait and prey, respectively, and c-Jun NH(2)-terminal kinase (JNK1) as the disruptor, we now show that phosphorylation of IRS-1 Ser-307, a previously identified site, is necessary but not sufficient for JNK1-mediated disruption of IR/IRS-1 binding. We further identify a new phosphorylation site, Ser-302, and show that this too is necessary for JNK1-mediated disruption. Seven additional kinases potentially linked to insulin resistance similarly block IR/IRS-1 binding in the disruptive Y3H, but through distinct Ser-302- and Ser-307-independent mechanisms. Phosphospecific antibodies that recognize sequences surrounding Ser(P)-302 or Ser(P)-307 were used to determine whether the sites were phosphorylated under relevant conditions. Phosphorylation was promoted at both sites in Fao hepatoma cells by reagents known to promote Ser/Thr phosphorylation, including the phorbol ester phorbol 12-myristate 13-acetate, anisomycin, calyculin A, and insulin. The antibodies further showed that Ser(P)-302 and Ser(P)-307 are increased in animal models of obesity and insulin resistance, including genetically obese ob/ob mice, diet-induced obesity, and upon induction of hyperinsulinemia. These findings demonstrate that phosphorylation at both Ser-302 and Ser-307 is necessary for JNK1-mediated inhibition of the IR/IRS-1 interaction and that Ser-302 and Ser-307 are phosphorylated in parallel in cultured cells and in vivo under conditions that lead to insulin resistance.     

Role of Protein Farnesylation in Burn-Induced Metabolic Derangements and Insulin Resistance in Mouse Skeletal Muscle

Objective

Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle.

Methods

A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated.

Results

Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277.

Conclusions

Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients.     

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.     

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.     

Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) inhibits insulin signal transduction in a variety of cell backgrounds, which might contribute to peripheral insulin resistance. However, because of the large number of potential phosphorylation sites, the mechanism of inhibition has been difficult to determine. One serine residue located near the phosphotyrosine-binding (PTB) domain in IRS-1 (Ser(307) in rat IRS-1 or Ser(312) in human IRS-1) is phosphorylated via several mechanisms, including insulin-stimulated kinases or stress-activated kinases like JNK1. During a yeast tri-hybrid assay, phosphorylation of Ser(307) by JNK1 disrupted the interaction between the catalytic domain of the insulin receptor and the PTB domain of IRS-1. In 32D myeloid progenitor cells, phosphorylation of Ser(307) inhibited insulin stimulation of the phosphatidylinositol 3-kinase and MAPK cascades. These results suggest that inhibition of PTB domain function in IRS-1 by phosphorylation of Ser(307) (Ser(312) in human IRS-1) might be a general mechanism to regulate insulin signaling.     

Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in growth hormone treated animals

In this study, we demonstrate that pretreatment with aspirin inhibits GH-induced insulin resistance. GH was observed to lead to serine phosphorylation of IRS-1, a phenomenon which was reversed by aspirin in liver, muscle and WAT in parallel with a reduction in JNK activity. In addition, our data show an impairment of insulin activation in the IR/IRS/PI(3)kinase pathway and a reduction in IRS-1 protein levels in rats treated with GH, which was also reversed in the animals pretreated with aspirin. Overall, these results provide new insights into the mechanism of GH-induced insulin resistance.