Caffeine modulates phosphorylation of insulin receptor substrate-1 and impairs insulin signal transduction in rat skeletal muscle

Caffeine decreases insulin sensitivity and insulin-stimulated glucose transport in skeletal muscle; however, the precise mechanism responsible for this deleterious effect is not understood fully. We investigated the effects of incubation with caffeine on insulin signaling in rat epitrochlearis muscle. Caffeine (≥1 mM, ≥15 min) suppressed insulin-stimulated insulin receptor substrate (IRS)-1 Tyr(612) phosphorylation in a dose- and time-dependent manner. These responses were associated with inhibition of the insulin-stimulated phosphorylation of phosphatidylinositol 3-kinase (PI3K) Tyr(458), Akt Ser(473), and glycogen synthase kinase-3β Ser(9) and with inhibition of insulin-stimulated 3-O-methyl-d-glucose (3MG) transport but not with inhibition of the phosphorylation of insulin receptor-β Tyr(1158/62/63). Furthermore, caffeine enhanced phosphorylation of IRS-1 Ser(307) and an IRS-1 Ser(307) kinase, inhibitor-κB kinase (IKK)-α/β Ser(176/180). Blockade of IKK/IRS-1 Ser(307) by caffeic acid ameliorated the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation and 3MG transport. Caffeine also increased the phosphorylation of IRS-1 Ser(789) and an IRS-1 Ser(789) kinase, 5'-AMP-activated protein kinase (AMPK). However, inhibition of IRS-1 Ser(789) and AMPK phosphorylation by dantrolene did not rescue the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation or 3MG transport. In addition, caffeine suppressed the phosphorylation of insulin-stimulated IRS-1 Ser(636/639) and upstream kinases, including the mammalian target of rapamycin and p70S6 kinase. Intravenous injection of caffeine at a physiological dose (5 mg/kg) in rats inhibited the phosphorylation of insulin-stimulated IRS-1 Tyr(612) and Akt Ser(473) in epitrochlearis muscle. Our results indicate that caffeine inhibits insulin signaling partly through the IKK/IRS-1 Ser(307) pathway, via a Ca(2+)- and AMPK-independent mechanism in skeletal muscle.     

Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling

Ser/Thr phosphorylation of insulin receptor substrate IRS-1 regulates insulin signaling, but the relevant phosphorylated residues and their potential functions during insulin-stimulated signal transduction are difficult to resolve. We used a sequence-specific polyclonal antibody directed against phosphorylated Ser(302) to study IRS-1-mediated signaling during insulin and insulin-like growth factor IGF-I stimulation. Insulin or IGF-I stimulated phosphorylation of Ser(302) in various cell backgrounds and in murine muscle. Wortmannin or rapamycin inhibited Ser(302) phosphorylation, and amino acids or glucose stimulated Ser(302) phosphorylation, suggesting a role for the mTOR cascade. The Ser(302) kinase associates with IRS-1 during immunoprecipitation, but its identity is unknown. The NH(2)-terminal c-Jun kinase did not phosphorylate Ser(302). Replacing Ser(302) with alanine significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and p85 binding and reduced insulin-stimulated phosphorylation of p70(S6K), ribosomal S6 protein, and 4E-BP1; however, this mutation had no effect on insulin-stimulated Akt or glycogen synthase kinase 3beta phosphorylation. Replacing Ser(302) with alanine reduced insulin/IGF-I-stimulated DNA synthesis. We conclude that Ser(302) phosphorylation integrates nutrient availability with insulin/IGF-I signaling to promote mitogenesis and cell growth.     

Mammalian Target of Rapamycin Pathway Regulates Insulin Signaling via Subcellular Redistribution of Insulin Receptor Substrate 1 and Integrates Nutritional Signals and Metabolic Signals of Insulin

A pathway sensitive to rapamycin, a selective inhibitor of mammalian target of rapamycin (mTOR), down-regulates effects of insulin such as activation of Akt (protein kinase B) via proteasomal degradation of insulin receptor substrate 1 (IRS-1). We report here that the pathway also plays an important role in insulin-induced subcellular redistribution of IRS-1 from the low-density microsomes (LDM) to the cytosol. After prolonged insulin stimulation, inhibition of the redistribution of IRS-1 by rapamycin resulted in increased levels of IRS-1 and the associated phosphatidylinositol (PI) 3-kinase in both the LDM and cytosol, whereas the proteasome inhibitor lactacystin increased the levels only in the cytosol. Since rapamycin but not lactacystin enhances insulin-stimulated 2-deoxyglucose (2-DOG) uptake, IRS-1-associated PI 3-kinase localized at the LDM was suggested to be important in the regulation of glucose transport. The amino acid deprivation attenuated and the amino acid excess enhanced insulin-induced Ser/Thr phosphorylation and subcellular redistribution and degradation of IRS-1 in parallel with the effects on phosphorylation of p70 S6 kinase and 4E-BP1. Accordingly, the amino acid deprivation increased and the amino acid excess decreased insulin-stimulated activation of Akt and 2-DOG uptake. Furthermore, 2-DOG uptake was affected by amino acid availability even when the degradation of IRS-1 was inhibited by lactacystin. We propose that subcellular redistribution of IRS-1, regulated by the mTOR-dependent pathway, facilitates proteasomal degradation of IRS-1, thereby down-regulating Akt, and that the pathway also negatively regulates insulin-stimulated glucose transport, probably through the redistribution of IRS-1. This work identifies a novel function of mTOR that integrates nutritional signals and metabolic signals of insulin.     

Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation

Nutritional excess and/or obesity represent well-known predisposition factors for the development of non-insulin-dependent diabetes mellitus (NIDDM). However, molecular links between obesity and NIDDM are only beginning to emerge. Here, we demonstrate that nutrients suppress phosphatidylinositol 3 (PI3)-kinase/Akt signaling via Raptor-dependent mTOR (mammalian target of rapamycin)-mediated phosphorylation of insulin receptor substrate 1 (IRS-1). Raptor directly binds to and serves as a scaffold for mTOR-mediated phosphorylation of IRS-1 on Ser636/639. These serines lie close to the Y(632)MPM motif that is implicated in the binding of p85alpha/p110alpha PI3-kinase to IRS-1 upon insulin stimulation. Phosphomimicking mutations of these serines block insulin-stimulated activation of IRS-1-associated PI3-kinase. Knockdown of Raptor as well as activators of the LKB1/AMPK pathway, such as the widely used antidiabetic compound metformin, suppress IRS-1 Ser636/639 phosphorylation and reverse mTOR-mediated inhibition on PI3-kinase/Akt signaling. Thus, diabetes-related hyperglycemia hyperactivates the mTOR pathway and may lead to insulin resistance due to suppression of IRS-1-dependent PI3-kinase/Akt signaling.     

Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function.

Incubation of cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins, accompanied by elevation in their Ser(P)/Thr(P) content and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, selectively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissociation from IR, and the decrease in its Tyr(P) content following 60 min of insulin treatment. Four conserved phosphorylation sites within the phosphotyrosine binding/SAIN domains of IRS-1 and IRS-2 served as in vitro substrates for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylinositol 3-kinase. Furthermore, PKB and IRS-1 formed stable complexes in vivo, and overexpression of PKB enhanced Ser phosphorylation of IRS-1. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-1; however, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced the rate of Tyr dephosphorylation of IRS-1, while inclusion of vanadate reversed this effect. These results implicate a wortmannin-sensitive Ser/Thr kinase, different from PKB, as the kinase that phosphorylates IRS-1 and acts as the feedback control regulator that turns off insulin signals by inducting the dissociation of IRS proteins from IR. In contrast, insulin-stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine binding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein-tyrosine phosphatases and enables it to maintain its Tyr-phosphorylated active conformation. These findings implicate PKB as a positive regulator of IRS-1 functions.     

A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

Insulin receptor substrate-2-dependent interleukin-4 signaling in macrophages is impaired in two models of type 2 diabetes mellitus

We have shown previously that hyperinsulinemia inhibits interferon-alpha-dependent activation of phosphatidylinositol 3-kinase (PI3-kinase) through mammalian target of rapamycin (mTOR)-induced serine phosphorylation of insulin receptor substrate (IRS)-1. Here we report that chronic insulin and high glucose synergistically inhibit interleukin (IL)-4-dependent activation of PI3-kinase in macrophages via the mTOR pathway. Resident peritoneal macrophages (PerMPhis) from diabetic (db/db) mice showed a 44% reduction in IRS-2-associated PI3-kinase activity stimulated by IL-4 compared with PerMPhis from heterozygote (db/+) control mice. IRS-2 from db/db mouse PerMPhis also showed a 78% increase in Ser/Thr-Pro motif phosphorylation without a difference in IRS-2 mass. To investigate the mechanism of this PI3-kinase inhibition, 12-O-tetradecanoylphorbol-13-acetate-matured U937 cells were treated chronically with insulin (1 nm, 18 h) and high glucose (4.5 g/liter, 48 h). In these cells, IL-4-stimulated IRS-2-associated PI3-kinase activity was reduced by 37.5%. Importantly, chronic insulin or high glucose alone did not impact IL-4-activated IRS-2-associated PI3-kinase. Chronic insulin + high glucose did reduce IL-4-dependent IRS-2 tyrosine phosphorylation and p85 association by 54 and 37%, respectively, but did not effect IL-4-activated JAK/STAT signaling. When IRS-2 Ser/Thr-Pro motif phosphorylation was examined, chronic insulin + high glucose resulted in a 92% increase in IRS-2 Ser/Thr-Pro motif phosphorylation without a change in IRS-2 mass. Pretreatment of matured U937 cells with rapamycin blocked chronic insulin + high glucose-dependent IRS-2 Ser/Thr-Pro motif phosphorylation and restored IL-4-dependent IRS-2-associated PI3-kinase activity. Taken together these results indicate that IRS-2-dependent IL-4 signaling in macrophages is impaired in models of type 2 diabetes mellitus through a mechanism that relies on insulin/glucose-dependent Ser/Thr-Pro motif serine phosphorylation mediated by the mTOR pathway.     

Hepatitis C virus core protein upregulates serine phosphorylation of insulin receptor substrate-1 and impairs the downstream akt/protein kinase B signaling pathway for insulin resistance

Chronic hepatitis C virus (HCV) infection has a significantly increased prevalence of type 2 diabetes mellitus (T2DM). Insulin resistance is a critical component of T2DM pathogenesis. Several mechanisms are likely to be involved in the pathogenesis of HCV-related insulin resistance. Since we and others have previously observed that HCV core protein activates c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase, we examined the contribution of these pathways to insulin resistance in hepatocytes. Our experimental findings suggest that HCV core protein alone or in the presence of other viral proteins increases Ser(312) phosphorylation of the insulin receptor substrate-1 (IRS-1). Hepatocytes infected with cell culture-grown HCV genotype 1a or 2a displayed a significant increase in the Ser(473) phosphorylation status of the Ser/Thr kinase protein kinase B (Akt/PKB), while Thr(308) phosphorylation was not significantly altered. HCV core protein-mediated Ser(312) phosphorylation of IRS-1 was inhibited by JNK (SP600125) and phosphatidylinositol-3 kinase (LY294002) inhibitors. A functional assay also suggested that hepatocytes expressing HCV core protein alone or infected with cell culture-grown HCV exhibited a suppression of 2-deoxy-d-[(3)H]glucose uptake. Inhibition of the JNK signaling pathway significantly restored glucose uptake despite HCV core expression in hepatocytes. Taken together, our results demonstrated that HCV core protein increases IRS-1 phosphorylation at Ser(312) which may contribute in part to the mechanism of insulin resistance.     

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.     

Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3-L1 adipocytes

In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1-associated phosphoinositide 3 (PI 3)-kinase activity in response to physiological insulin concentrations. Insulin-induced membrane ruffling, which is dependent on PI 3-kinase activation, was also markedly reduced. These inhibitory effects were associated with an increase in IRS-1 Ser307 phosphorylation. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser307. The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation. In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces the phosphorylation of IRS-1 on Ser307 by an mTOR-dependent pathway. This, in turn, leads to a decrease in early proximal signaling events induced by physiological insulin concentrations. On the other hand, prolonged osmotic stress alters IRS-1 function by inducing its degradation, which could contribute to the down-regulation of insulin action.     

Activation of mTOR/p70S6 kinase by ANG II inhibits insulin-stimulated endothelial nitric oxide synthase and vasodilation

Elevated tissue levels of angiotensin II (ANG II) are associated with impairment of insulin actions in metabolic and cardiovascular tissues. ANG II-stimulated activation of mammalian target of rapamycin (mTOR)/p70 S6 kinase (p70S6K) in cardiovascular tissues is implicated in cardiac hypertrophy and vascular remodeling. However, the role of ANG II-stimulated mTOR/p70S6K in vascular endothelium is poorly understood. In the present study, we observed that ANG II stimulated p70S6K in bovine aortic endothelial cells. ANG II increased phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser(636/639) and inhibited the insulin-stimulated phosphorylation of endothelial nitric oxide synthase (eNOS). An inhibitor of mTOR, rapamycin, attenuated the ANG II-stimulated phosphorylation of p70S6K and phosphorylation of IRS-1 (Ser(636/639)) and blocked the ability of ANG II to impair insulin-stimulated phosphorylation of eNOS, nitric oxide production, and mesenteric-arteriole vasodilation. Moreover, point mutations of IRS-1 at Ser(636/639) to Ala prevented the ANG II-mediated inhibition of insulin signaling. From these results, we conclude that activation of mTOR/p70S6K by ANG II in vascular endothelium may contribute to impairment of insulin-stimulated vasodilation through phosphorylation of IRS-1 at Ser(636/639). This ANG II-mediated impairment of vascular actions of insulin may help explain the role of ANG II as a link between insulin resistance and hypertension.     

Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.     

Mechanisms of high-glucose/insulin-mediated desensitization of acute insulin-stimulated glucose transport and Akt activation

High-glucose/low-dose insulin-mediated insulin resistance of glucose transport was studied in 3T3-L1 adipocytes. In this model, proximal insulin signaling, including insulin receptor substrate (IRS)-1-bound phosphatidylinositol 3-kinase (PI 3-kinase) activation, is preserved, but insulin-stimulated protein kinase B (Akt) activation is markedly impaired. To assess a difference in acute insulin-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], cells were labeled with [32P]orthophosphate, and glycerophosphoinositides were quantified by HPLC. Although basal PtdIns(3,4,5)P3 was similar, insulin stimulated its production 33.6% more in controls (P < 0.03) than in insulin-resistant cells. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein, a lipid phosphatase that dephosphorylates PtdIns(3,4,5)P3 in the 3-position, was significantly and specifically increased in insulin-resistant cells. Treatment with rapamycin [a specific inhibitor of mammalian target of rapamycin complex 1 (mTORC1)] inhibited the increased PTEN expression and partially restored insulin-stimulated glucose transport and Akt activation to insulin-resistant cells. Acute insulin markedly stimulated Ser(636/639) phosphorylation of IRS-1; this was rapamycin inhibited but was significantly decreased in cells that had been preexposed to insulin, whereas total IRS-1 was unaffected. These findings were essentially paralleled by changes in the activation of p70 S6 kinase and S6-ribosomal protein. Overexpression of uncoupling protein-1 or manganese superoxide dismutase did not prevent the development of insulin-resistant glucose transport and impaired Akt activation in high-glucose/low-insulin-pretreated cells. The insulin resistance associated with glucotoxicity in our model reflects in part decreased availability of PtdIns(3,4,5)P3, which correlates with increased PTEN protein expression. Chronic activation of mTORC1 plays a role in stimulating PTEN expression and possibly in activation or induction of a phosphoprotein phosphatase. No evidence was found for a role for increased mitochondrial superoxide production in this model.     

PDGF-induced vascular smooth muscle cell proliferation is associated with dysregulation of insulin receptor substrates

In vascular smooth muscle cells (VSMCs), platelet-derived growth factor (PDGF) plays a major role in inducing phenotypic switching from contractile to proliferative state. Importantly, VSMC phenotypic switching is also determined by the phosphorylation state/expression levels of insulin receptor substrate (IRS), an intermediary signaling component that is shared by insulin and IGF-I. To date, the roles of PDGF-induced key proliferative signaling components including Akt, p70S6kinase, and ERK1/2 on the serine phosphorylation/expression of IRS-1 and IRS-2 isoforms remain unclear in VSMCs. We hypothesize that PDGF-induced VSMC proliferation is associated with dysregulation of insulin receptor substrates. Using human aortic VSMCs, we demonstrate that prolonged PDGF treatment led to sustained increases in the phosphorylation of protein kinases such as Akt, p70S6kinase, and ERK1/2, which mediate VSMC proliferation. In addition, PDGF enhanced IRS-1/IRS-2 serine phosphorylation and downregulated IRS-2 expression in a time- and concentration-dependent manner. Notably, phosphoinositide 3-kinase (PI 3-kinase) inhibitor (PI-103) and mammalian target of rapamycin inhibitor (rapamycin), which abolished PDGF-induced Akt and p70S6kinase phosphorylation, respectively, blocked PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. In contrast, MEK1/ERK inhibitor (U0126) failed to block PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. PDGF-induced IRS-2 downregulation was prevented by lactacystin, an inhibitor of proteasomal degradation. Functionally, PDGF-mediated IRS-1/IRS-2 dysregulation resulted in the attenuation of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Pharmacological inhibition of PDGF receptor tyrosine kinase with imatinib prevented IRS-1/IRS-2 dysregulation and restored insulin receptor signaling. In conclusion, strategies to inhibit PDGF receptors would not only inhibit neointimal growth but may provide new therapeutic options to prevent dysregulated insulin receptor signaling in VSMCs in nondiabetic and diabetic states.     

Insulin resistance in the skeletal muscle of women with PCOS involves intrinsic and acquired defects in insulin signaling

Insulin resistance in polycystic ovary syndrome (PCOS) is due to a postbinding defect in signaling that persists in cultured skin fibroblasts and is associated with constitutive serine phosphorylation of the insulin receptor (IR). Cultured skeletal muscle from obese women with PCOS and age- and body mass index-matched control women (n = 10/group) was studied to determine whether signaling defects observed in this tissue in vivo were intrinsic or acquired. Basal and insulin-stimulated glucose transport and GLUT1 abundance were significantly increased in cultured myotubes from women with PCOS. Neither IR beta-subunit abundance and tyrosine autophosphorylation nor insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity differed in the two groups. However, IRS-1 protein abundance was significantly increased in PCOS, resulting in significantly decreased PI 3-kinase activity when normalized for IRS-1. Phosphorylation of IRS-1 on Ser312, a key regulatory site, was significantly increased in PCOS, which may have contributed to this signaling defect. Insulin signaling via IRS-2 was also decreased in myotubes from women with PCOS. In summary, decreased insulin-stimulated glucose uptake in PCOS skeletal muscle in vivo is an acquired defect. Nevertheless, there are intrinsic abnormalities in glucose transport and insulin signaling in myotubes from affected women, including increased phosphorylation of IRS-1 Ser312, that may confer increased susceptibility to insulin resistance-inducing factors in the in vivo environment. These abnormalities differ from those reported in other insulin resistant states consistent with the hypothesis that PCOS is a genetically unique disorder conferring an increased risk for type 2 diabetes.     

Alpha-synuclein overexpression negatively regulates insulin receptor substrate 1 by activating mTORC1/S6K1 signaling

Alpha-synuclein (α-Syn) is a major component of Lewy bodies, a pathological feature of Parkinson's and other neurodegenerative diseases collectively known as synucleinopathies. Among the possible mechanisms of α-Syn-mediated neurotoxicity is interference with cytoprotective pathways such as insulin signaling. Insulin receptor substrate (IRS)-1 is a docking protein linking IRs to downstream signaling pathways such as phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K)1; the latter exerts negative feedback control on insulin signaling, which is impaired in Alzheimer's disease. Our previous study found that α-Syn overexpression can inhibit protein phosphatase (PP)2A activity, which is involved in the protective mechanism of insulin signaling. In this study, we found an increase in IRS-1 phosphorylation at Ser636 and decrease in tyrosine phosphorylation, which accelerated IRS-1 turnover and reduced insulin-Akt signaling in α-Syn-overexpressing SK-N-SH cells and transgenic mice. The mTOR complex (C)1/S6K1 blocker rapamycin inhibited the phosphorylation of IRS-1 at Ser636 in cells overexpressing α-Syn, suggesting that mTORC1/S6K1 activation by α-Syn causes feedback inhibition of insulin signaling via suppression of IRS-1 function. α-Syn overexpression also inhibited PP2A activity, while the PP2A agonist C2 ceramide suppressed both S6K1 activation and IRS-1 Ser636 phosphorylation upon α-Syn overexpression. Thus, α-Syn overexpression negatively regulated IRS-1 via mTORC1/S6K1 signaling while activation of PP2A reverses this process. These results provide evidence for a link between α-Syn and IRS-1 that may represent a novel mechanism for α-Syn-associated pathogenesis.     

Modulation of insulin receptor substrate-1 tyrosine phosphorylation by an Akt/phosphatidylinositol 3-kinase pathway

Serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) has been implicated as a negative regulator of insulin signaling. Prior studies have indicated that this negative regulation by protein kinase C involves the mitogen-activated protein kinase and phosphorylation of serine 612 in IRS-1. In the present studies, the negative regulation by platelet-derived growth factor (PDGF) was compared with that induced by endothelin-1, an activator of protein kinase C. In contrast to endothelin-1, the inhibitory effects of PDGF did not require mitogen-activated protein kinase or the phosphorylation of serine 612. Instead, three other serines in the phosphorylation domain of IRS-1 (serines 632, 662, and 731) were required for the negative regulation by PDGF. In addition, the PDGF-activated serine/threonine kinase called Akt was found to inhibit insulin signaling. Moreover, this inhibition required the same IRS-1 serine residues as the inhibition by PDGF. Finally, the negative regulatory effects of PDGF and Akt were inhibited by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), one of the downstream targets of Akt. These studies implicate the phosphatidylinositol 3-kinase/Akt kinase cascade as an additional negative regulatory pathway for the insulin signaling cascade.     

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.     

Role of mTOR in the degradation of IRS-1: regulation of PP2A activity

We have investigated the role of PI 3-kinase and mTOR in the degradation of IRS-1 induced by insulin. Inhibition of mTOR with rapamycin resulted in approximately 50% inhibition of the insulin-induced degradation of IRS-1. In contrast, inhibition of PI-3 kinase, an upstream activator of mTOR, leads to a complete block of the insulin-induced degradation. Inhibition of either PI-3 kinase or mTOR prevented the mobility shift in IRS-1 in response to insulin, a shift that is caused by Ser/Thr phosphorylation. These results indicate that insulin stimulates PI 3-kinase-mediated degradation of IRS-1 via both mTOR-dependent and -independent pathways. Platelet-derived growth factor (PDGF) stimulation leads to a lower level of degradation, but significant phosphorylation of IRS-1. Both the degradation and phosphorylation of IRS-1 in response to PDGF are completely inhibited by rapamycin, suggesting that PDGF stimulates IRS-1 degradation principally via the mTOR-dependent pathway. Inhibition of the serine/threonine phosphatase PP2A with okadaic acid also induced the phosphorylation and degradation of IRS-1. IRS-1 phosphorylation and degradation in response to okadaic acid were not inhibited by rapamycin, suggesting that the action of mTOR in the degradation of IRS-1 results from inhibition of PP2A. Consistent with this, treatment of cells with rapamycin stimulated PP2A activity. While the role of mTOR in the phosphorylation of IRS-1 appears to proceed primarily through the regulation of PP2A, we also provide evidence that the regulation of p70S6 kinase phosphorylation requires the direct activity of mTOR.