Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of obese rats: possible involvement in obesity-linked insulin resistance

The mammalian target of rapamycin (mTOR) pathway integrates insulin and nutrient signaling in numerous cell types. Recent studies also suggest that this pathway negatively modulates insulin signaling to phosphatidylinositol 3-kinase/Akt in adipose and muscle cells. However, it is still unclear whether activation of the mTOR pathway is increased in obesity and if it could be involved in the promotion of insulin resistance. In this paper we show that basal (fasting state) activation of mTOR and its downstream target S6K1 is markedly elevated in liver and skeletal muscle of obese rats fed a high fat diet compared with chow-fed, lean controls. Time-course studies also revealed that mTOR and S6K1 activation by insulin was accelerated in tissues of obese rats, in association with increased inhibitory phosphorylation of insulin receptor substrate-1 (IRS-1) on Ser636/Ser639 and impaired Akt activation. The relationship between mTOR/S6K1 overactivation and impaired insulin signaling to Akt was also examined in hepatic cells in vitro. Insulin caused a time-dependent activation of mTOR and S6K1 in HepG2 cells. This was associated with increased IRS-1 phosphorylation on Ser636/Ser639. Inhibition of mTOR/S6K1 by rapamycin blunted insulin-induced Ser636/Ser639 phosphorylation of IRS-1, leading to a rapid (approximately 5 min) and persistent increase in IRS-1-associated phosphatidylinositol 3-kinase activity and Akt phosphorylation. These results show that activation of the mTOR pathway is increased in liver and muscle of high fat-fed obese rats. In vitro studies with rapamycin suggest that mTOR/S6K1 overactivation contributes to elevated serine phosphorylation of IRS-1, leading to impaired insulin signaling to Akt in liver and muscle of this dietary model of obesity.     

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppressor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.     

Rescuing 3T3-L1 adipocytes from insulin resistance induced by stimulation of Akt-mammalian target of rapamycin/p70 S6 kinase (S6K1) pathway and serine phosphorylation of insulin receptor substrate-1: effect of reduced expression of p85alpha subunit of phosphatidylinositol 3-kinase and S6K1 kinase

Phosphorylation of insulin receptor substrate-1 (IRS-1) on serine residues has been recognized as a mechanism responsible for a diminution of insulin action and insulin resistance. Potential approaches to improve insulin sensitivity may include interference with and/or reduction in expression of certain signaling intermediates that participate in the pathogenesis of insulin resistance. In this study, we transduced fully differentiated 3T3-L1 adipocytes with a constitutively active myristoylated Akt that led to hyperactivation of mammalian target of rapamycin and p70 S6 kinase (S6K1), increased serine phosphorylation of IRS-1, and reduction in insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and glucose transport. We then reduced expression of the PI 3-kinase regulatory subunit, p85alpha, or expression of S6K1 kinase using small interfering RNA transfections, which led to a reduction in p85alpha expression of 70% at 48 h (P < 0.05) and S6K1 of 49% (P < 0.05). Reduction in expression of either p85alpha or S6K1 achieved with small interfering RNA in the presence of myristoylated Akt rescued 3T3-L1 adipocytes from the insulin resistance induced by serine phosphorylation of IRS-1 and completely restored insulin-stimulated activation of PI 3-kinase and glucose uptake. We conclude that reduction in expression of p85alpha or S6K1 could represent therapeutic targets to mitigate insulin resistance.     

Phosphorylation of human insulin receptor substrate-1 at Serine 629 plays a positive role in insulin signaling

The function of insulin receptor substrate-1 (IRS-1) is regulated by both tyrosine and serine/threonine phosphorylation. Phosphorylation of some serine/threonine residues in IRS-1 dampens insulin signaling, whereas phosphorylation of other serine/threonine residues enhances insulin signaling. Phosphorylation of human IRS-1 at Ser(629) was increased by insulin in Chinese hamster ovary cells expressing the insulin receptor (1.26 +/- 0.09-fold; P < 0.05) and L6 cells (1.35 +/- 0.29-fold; P < 0.05) expressing human IRS-1. Sequence analysis surrounding Ser(629) revealed conformity to the consensus phosphorylation sequence recognized by Akt. Phosphorylation of IRS-1 at Ser(629) in cells was decreased upon treatment with either an Akt inhibitor or by coexpression with kinase dead Akt, whereas Ser(629) phosphorylation was increased by coexpression with constitutively active Akt. In addition, Ser(629) of IRS-1 is directly phosphorylated by Akt in vitro. In cells, preventing phosphorylation of Ser(629) by a Ser(629)Ala mutation resulted in increased phosphorylation of Ser(636), a known negative regulator of IRS-1, without affecting phosphorylation of Tyr(632) or Ser(616). Cells expressing the Ser(629)Ala mutation, along with increased Ser(636) phosphorylation, had decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol 3'-kinase with IRS-1 and decreased phosphorylation of Akt at Ser(473). Finally, in vitro phosphorylation of a Ser(629)-containing IRS-1 fragment with Akt reduces the subsequent ability of ERK to phosphorylate Ser(636/639). These results suggest that a feed-forward mechanism may exist whereby insulin activation of Akt leads to phosphorylation of IRS-1 at Ser(629), resulting in decreased phosphorylation of IRS-1 at Ser(636) and enhanced downstream signaling. Understanding the complex phosphorylation patterns of IRS-1 is crucial to elucidating the factors contributing to insulin resistance and, ultimately, the pathogenesis of type 2 diabetes.     

Inhibition of insulin signaling and adipogenesis by rapamycin: effect on phosphorylation of p70 S6 kinase vs eIF4E-BP1

OBJECTIVE: Insulin-responsive adipogenic signaling molecules include insulin receptor substrates (IRS)-1 and -2, phosphoinositide 3-kinase (PI3K), and protein kinase B (PKB; also known as Akt). Mammalian target of rapamycin (mTOR) is a PKB substrate, and regulates p70 S6 kinase (p70 S6K). Since p70 S6K is an insulin-responsive kinase downstream of PI3K and PKB, its potential role in adipogenic insulin signaling was investigated. DESIGN: We measured the effect of rapamycin, a specific inhibitor of mTOR, on insulin-induced 3T3-L1 adipogenesis and on insulin-stimulated p70 S6K activation. RESULTS: Rapamycin partially reduced differentiation, measured by Oil Red O staining, triacylglycerol accumulation (by up to 46%), and peroxisome proliferator-activated receptor gamma protein expression (by 50%). In contrast, rapamycin completely inhibited insulin-stimulated p70 S6K activation, assessed by phosphorylation of p70 S6K and its substrate, S6. Expression of a constitutively activated form of p70 S6K did not promote 3T3-L1 adipogenesis. The considerable residual differentiation in the presence of rapamycin, despite the complete blockade of p70 S6K activation, prompted us to measure the phosphorylation of another rapamycin-sensitive protein, eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). Insulin-stimulated 4E-BP1 phosphorylation in 3T3-L1 preadipocytes was only partially affected by rapamycin, consistent with the differentiation data. Phosphorylation of eIF4E itself, an expected consequence of 4E-BP1 phosphorylation, was also only partially inhibited. CONCLUSION: Our data suggest that adipogenic mTOR signaling occurs via the 4E-BP1/eIF4E pathway, rather than through p70 S6K.     

游离脂肪酸诱导3T3-L1脂肪细胞胰岛素抵抗的分子机制

目的研究游离脂肪酸（FFA）对3T3-L1脂肪细胞IKKβ及胰岛素信号转导蛋白的影响,探讨FFA诱导胰岛素抵抗（IR）的分子机制。方法诱导成熟的3T3-L1脂肪细胞与0.3-1.0mmol/L的软脂酸（PA）培养6-24h,以2-脱氧-〔^3H〕-D-葡萄糖摄入法观察葡萄糖的转运率,用Western blot检测IKKβ蛋白、IKKβ Ser181磷酸化、IRS-1蛋白、IRS-1 Ser307磷酸化、PI3Kp85蛋白及GluT4蛋白的表达。结果0.3-1.0mmol/LPA作用6-24h后,3T3-L1脂肪细胞的葡萄糖消耗明显减少,同时,Western blot显示,PA对IKKβ及GluT4蛋白的表达无明显影响,却能明显增加IKKβ Ser181及IRS-1 Ser307磷酸化,同时减少IRS-1蛋白和PI3Kp85蛋白的表达。结论FFA可以诱导IR,其分子机制可能与FFA激活IKKβ,使IRS-1丝氨酸残基磷酸化增加而酪氨酸残基磷酸化减少,进而使其下游的PI-3Kp85蛋白表达减少抑制葡萄糖转运有关。     

Activation of the hexosamine pathway leads to phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 and impairs the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin insulin biosynthetic pathway in RIN pancreatic beta-cells

Many adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP). There is evidence for an autocrine role of the insulin signaling in beta-cell function. We tested the hypothesis that activation of the HBP induces defects in insulin biosynthesis by affecting the insulin-mediated protein translation signaling. Exposure of human pancreatic islets and RIN beta-cells to glucosamine resulted in reduction in glucose- and insulin-stimulated insulin biosynthesis, which in RIN beta-cells was associated with impairment in insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation at Tyr(608) and Tyr(628), which are essential for engaging phosphatidylinositol 3-kinase (PI 3-kinase). These changes were accompanied by impaired activation of PI 3-kinase, and activation of Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway. RIN beta-cells exposed to high glucose exhibited increased c-Jun N-terminal kinase (JNK) and ERK1/2 activity, which was associated with increased IRS-1 phosphorylation at serine (Ser)(307) and Ser(612), respectively, that inhibits coupling of IRS-1 to the insulin receptor and is upstream of the inhibition of IRS-1 tyrosine phosphorylation. Azaserine reverted the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Glucosamine mimicked the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser(307) and Ser(612). Inhibition of JNK and MAPK kinase-1 activity reverted the negative effects of glucosamine on insulin-mediated protein synthesis. These results suggest that activation of the HBP accounts, in part, for glucose-induced phosphorylation at Ser(307) and Ser(612) of IRS-1 mediated by JNK and ERK1/2, respectively. These changes result in impaired coupling of IRS-1 and PI 3-kinase, and activation of the Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway.     

Regulation of insulin signalling by hyperinsulinaemia: role of IRS-1/2 serine phosphorylation and the mTOR/p70 S6K pathway

Aim/hypothesis Several epidemiological studies have suggested an association between chronic hyperinsulinaemia and insulin resistance. However, the causality of this relationship remains uncertain.Methods We performed chronic hyperinsulinaemic–euglycaemic clamps and delineated, by western blotting, an IR/IRSs/phosphatidylinositol 3-kinase(PI[3]K)/Akt pathway in insulin-responsive tissues of hyperinsulinaemic rats. IRS-1/2 serine phosphorylation, IR/protein tyrosine phosphatase 1B (PTP1B) association, and mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70 S6K) activity were also evaluated in the liver, skeletal muscle and white adipose tissue of hyperinsulinaemic animals.Results We found that chronic hyperinsulinaemic rats have insulin resistance and reduced levels of glycogen content in liver and muscle. In addition, we demonstrated an impairment of the insulin-induced IR/IRSs/PI(3)K/Akt pathway in liver and muscle of chronic hyperinsulinaemic rats that parallels increases in IRS1/2 serine phosphorylation, IR/PTP1B association and mTOR activity. Despite a higher association of IR/PTP1B, there was an increase in white adipose tissue of chronic hyperinsulinaemic rats in IRS-1/2 protein levels, tyrosine phosphorylation and IRSs/PI(3)K association, which led to an increase in basal Akt serine phosphorylation. No increases in IRS-1/2 serine phosphorylation and mTOR activity were observed in white adipose tissue. Rapamycin reversed the insulin resistance and the changes induced by hyperinsulinaemia in the three tissues studied.Conclusions/interpretation Our data provide evidence that chronic hyperinsulinaemia itself, imposed on normal rats, appears to have a dual effect, stimulating insulin signalling in white adipose tissue, whilst decreasing it in liver and muscle. The underlying mechanism of these differential effects may be related to the ability of hyperinsulinaemia to increase mTOR/p70 S6K pathway activity and IRS-1/2 serine phosphorylation in a tissue-specific fashion. In addition, we demonstrated that inhibition of the mTOR pathway with rapamycin can prevent insulin resistance caused by chronic hyperinsulinaemia in liver and muscle. These findings support the hypothesis that defective and tissue-selective insulin action contributes to the insulin resistance observed in hyperinsulinaemic states.     

Requirement for PIKfyve enzymatic activity in acute and long-term insulin cellular effects

PIKfyve is a phosphoinositide 5-kinase that can also act as a protein kinase. PIKfyve's role in acute insulin action has been suggested on the basis of its association with the insulin stimulatable phosphatidylinositol-3-kinase and the ability of acute insulin to recruit and phosphorylate PIKfyve on intracellular membranes of 3T3-L1 adipocytes. Here we have examined several classical insulin-regulated long- and short-term responses in insulin-sensitive cells expressing high levels of either active PIKfyve or kinase-dead mutants with a dominant-negative effect. Up-regulation of PIKfyve protein expression was documented in the early stages of differentiation of cultured 3T3-L1 fibroblasts into adipocytes and a kinase-dead mutant, PIKfyveDeltaK, introduced into the preadipocyte stage profoundly delayed the hormone-induced adipogenesis. Next, insulin-induced mitogenesis was markedly inhibited in HEK293 stable cell lines, inducibly expressing the dominant-negative kinase-dead PIKfyve(K1831E) mutant but not in cells expressing PIKfyve(WT). Similarly, expression of the dominant negative mutants PIKfyve(K1831E) or PIKfyveDeltaK strongly inhibited insulin-stimulated translocation of GLUT4 in 3T3-L1 adipocytes, or GLUT1-mediated glucose uptake in Chinese hamster ovary T cells expressing the human insulin receptor. Expression of PIKfyveDeltaK and PIKfyve(WT) in Chinese hamster ovary T cells decreased or increased, respectively, insulin-stimulated Akt phosphorylation at Ser473 but not at Thr308. Furthermore, a powerful inhibition of PIKfyve was documented at a very low concentration (ID(50) = 6 micro M) of the cell-permeable kinase inhibitor curcumin. When introduced into 3T3-L1 adipocytes, curcumin markedly inhibited insulin-induced GLUT4 translocation and glucose transport. Together these data indicate that PIKfyve enzymatic activity functions as a positive regulatory intermediate in insulin acute and long-term biological responses and identify Ser473 in Akt as one potential PIKfyve downstream target.     

Angiotensin II impairs the insulin signaling pathway promoting production of nitric oxide by inducing phosphorylation of insulin receptor substrate-1 on Ser312 and Ser616 in human umbilical vein endothelial cells

It has been suggested that serine (Ser) phosphorylation of insulin receptor substrate-1 (IRS-1) decreases the ability of IRS-1 to be phosphorylated on tyrosine, thereby attenuating insulin signaling. There is evidence that angiotensin II (AII) may impair insulin signaling to the IRS-1/phosphatydilinositol 3-kinase (PI 3-kinase) pathway by enhancing Ser phosphorylation. Insulin stimulates NO production by a pathway involving IRS-1/PI3-kinase/Akt/endothelial NO synthase (eNOS). We addressed the question of whether AII affects insulin signaling involved in NO production in human umbilical vein endothelial cells and tested the hypothesis that the inhibitory effect of AII on insulin signaling was caused by increased site-specific Ser phosphorylation in IRS-1. Exposure of human umbilical vein endothelial cells to AII resulted in inhibition of insulin-stimulated production of NO. This event was associated with impaired IRS-1 phosphorylation at Tyr612 and Tyr632, two sites essential for engaging the p85 subunit of PI3-kinase, resulting in defective activation of PI 3-kinase, Akt, and eNOS. This inhibitory effect of AII was reversed by the type 1 receptor antagonist losartan. AII increased c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 activity, which was associated with a concomitant increase in IRS-1 phosphorylation at Ser312 and Ser616, respectively. Inhibition of JNK and ERK1/2 activity reversed the negative effects of AII on insulin-stimulated NO production. Our data suggest that AII, acting via the type 1 receptor, increases IRS-1 phosphorylation at Ser312 and Ser616 via JNK and ERK1/2, respectively, thus impairing the vasodilator effects of insulin mediated by the IRS-1/PI 3-kinase/Akt/eNOS pathway.     

Altered subcellular distribution of estrogen receptor alpha is implicated in estradiol-induced dual regulation of insulin signaling in 3T3-L1 adipocytes

We investigated the mechanisms by which estrogen alters insulin signaling in 3T3-L1 adipocytes. Treatment with 17beta-estradiol (E2) did not affect insulin-induced tyrosine phosphorylation of insulin receptor. E2 enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1/p85 association, phosphorylation of Akt, and 2-deoxyglucose uptake at 10(-8) m, but inhibited these effects at 10(-5) m. A concentration of 10(-5) m E2 enhanced insulin-induced phosphorylation of IRS-1 at Ser(307), which was abolished by treatment with a c-Jun NH(2)-terminal kinase inhibitor. In addition, the effect of E2 was abrogated by pretreatment with a specific estrogen receptor antagonist, ICI182,780. Membrane-impermeable E2, E2-BSA, did not affect the insulin-induced phosphorylation of Akt at 10(-8) m, but inhibited it at 10(-5) m. Furthermore, E2 decreased the amount of estrogen receptor alpha at the plasma membrane at 10(-8) m, but increased it at 10(-5) m. In contrast, the subcellular distribution of estrogen receptor beta was not altered by the treatment. These results indicate that E2 affects the metabolic action of insulin in a concentration-specific manner, that high concentrations of E2 inhibit insulin signaling by modulating phosphorylation of IRS-1 at Ser(307) via a c-Jun NH(2)-terminal kinase-dependent pathway, and that the subcellular redistribution of estrogen receptor alpha in response to E2 may explain the dual effect of E2.     

Mechanism for differential effect of protein-tyrosine phosphatase 1B on Akt versus mitogen-activated protein kinase in 3T3-L1 adipocytes

We investigated the effect of overexpression of protein-tyrosine phosphatase 1B (PTP1B) on insulin signaling in 3T3-L1 adipocytes. Overexpression of a wild-type PTP1B in L1 adipocytes as well as in L6 myocytes, led to a profound decrease in insulin-stimulated phosphorylation of MAPK. Even though the decrease in insulin receptor substrate protein-1 (IRS-1) phosphorylation was identical with that seen in L6 myocytes, overexpression of wild-type PTP1B in L1 adipocytes was associated with modest impairment of insulin-stimulated Akt phosphorylation in addition to a small, but significant, attenuation in insulin-stimulated glucose uptake, when compared with a phosphatase-negative mutant. Regarding the relatively small effect on Akt phosphorylation, we obtained identical results in rat 1 fibroblasts overexpressing human insulin receptor, suggesting that the higher expression levels of insulin receptor and IRS-1 might be responsible. With regard to the large effect on MAPK phosphorylation, we found that PTP1B overexpression led to the impaired phosphorylation of both IRS-1 and Shc, resulting in a decrease in their association with Grb2. Furthermore, phosphorylation of Shc stimulated by platelet-derived growth factor was also attenuated, without any change in its receptors, suggesting that PTP1B directly regulates Shc phosphorylation. These data demonstrate that PTP1B negatively regulates insulin signaling in the MAPK cascade to a much greater extent than the Akt pathway in some cell lines, especially in L1 adipocytes.     

MAP kinases and mTOR mediate insulin-induced phosphorylation of Insulin Receptor Substrate-1 on serine residues 307, 612 and 632

Aim/hypothesis Insulin-induced IRS-1 serine phosphorylation could be physiologically important to regulate insulin action. In a hyperinsulinaemic state such as obesity or Type 2 diabetes, this phosphorylation could be modified and exacerbate insulin resistance. We aimed at identifying serine residues in IRS-1 phosphorylated in response to insulin stimulation and at determining the involved kinases.Methods 3T3-L1 adipocytes, muscle and adipose tissue of mice were subjected to Western Blot analysis with phosphospecific antibodies to identify phosphorylation sites in IRS-1 following insulin treatment. Pharmacological inhibitors were used to determine the serine kinases involved in this phosphorylation.Results In 3T3-L1 adipocytes, insulin promoted the phosphorylation of serine 307, 612 and 632 with Serine^612/632 more rapidly phosphorylated than Serine³⁰⁷. Insulin-induced phosphorylation of Serine³⁰⁷ was dependent on the activation of a PI 3-kinase/mTOR pathway. The phosphorylation of Serine^612/632 required the activation of the MAP kinase pathway following short-term insulin stimulation and activation of the PI 3-kinase/mTOR pathway following prolonged insulin stimulation. Phosphorylation of Serine³⁰⁷ and Serine⁶³² occurred in vivo in skeletal muscle and white adipose tissue of mice injected with insulin and was dependent on the activation of mTOR. Moreover, inhibition of mTOR led to a persistent PI 3-kinase activation by insulin.Conclusion/Interpretation Insulin-induced IRS-1 serine phosphorylation is a complex process involving different sites and kinases. This complexity could be physiologically important to accurately regulate insulin signalling. Abnormal phosphorylation of these serine residues in hyperinsulinaemic state could participate in the down-regulation of insulin signalling.Abbreviations PI 3-kinase phosphatidylinositol 3-kinase - mTOR mammalian target of rapamycin - APS adaptor with a PH and SH2 domains - Shc Src Homology Collagen - SH2 Src Homology 2 - PTB phosphotyrosine binding - MAP mitogen-activated protein - MEK mitogen-activated protein kinase kinase - PKB protein kinase B - PDGF platelet derived growth factor - JNK c-Jun NH2 terminal kinase - PMA phorbol myristate acetate - PIP3 phosphatidylinositol 3,4,5 triphosphates     

Regulation of glucose transport by ROCK1 differs from that of ROCK2 and is controlled by actin polymerization

A role of Rho-associated coiled-coil-containing protein kinase (ROCK)1 in regulating whole-body glucose homeostasis has been reported. However, cell-autonomous effects of ROCK1 on insulin-dependent glucose transport in adipocytes and muscle cells have not been elucidated. To determine the specific role of ROCK1 in glucose transport directly, ROCK1 expression in 3T3-L1 adipocytes and L6 myoblasts was biologically modulated. Here, we show that small interfering RNA-mediated ROCK1 depletion decreased insulin-induced glucose transport in adipocytes and myoblasts, whereas adenovirus-mediated ROCK1 expression increased this in a dose-dependent manner, indicating that ROCK1 is permissive for glucose transport. Inhibition of ROCK1 also impaired glucose transporter 4 translocation in 3T3-L1 adipocytes. Importantly, the ED?? of insulin for adipocyte glucose transport was reduced when ROCK1 was expressed, leading to hypersensitivity to insulin. These effects are dependent on actin cytoskeleton remodeling, because inhibitors of actin polymerization significantly decreased ROCK1's effect to promote insulin-stimulated glucose transport. Unlike ROCK2, ROCK1 binding to insulin receptor substrate (IRS)-1 was not detected by immunoprecipitation, although cell fractionation demonstrated both ROCK isoforms localize with IRS-1 in low-density microsomes. Moreover, insulin's ability to increase IRS-1 tyrosine 612 and serine 632/635 phosphorylation was attenuated by ROCK1 suppression. Replacing IRS-1 serine 632/635 with alanine reduced insulin-stimulated phosphatidylinositol 3-kinase activation and glucose transport in 3T3-L1 adipocytes, indicating that phosphorylation of these serine residues of IRS-1, which are substrates of the ROCK2 isoform in vitro, are crucial for maximal stimulation of glucose transport by insulin. Our studies identify ROCK1 as an important positive regulator of insulin action on glucose transport in adipocytes and muscle cells.     

Insulin stimulates IGFBP-2 expression in 3T3-L1 adipocytes through the PI3K/mTOR pathway

Insulin-like growth factor binding protein 2 (IGFBP-2) has been implicated in the etiology of several diseases, including the metabolic syndrome. Although IGFBP-2 derives mostly from the liver, recent evidence in mice and humans indicate that aging and obesity are associated with altered IGFBP-2 levels in white adipocytes. The present study was aimed at determining the mechanisms that control IGFBP-2 expression in mature adipocytes. IGFBP-2 mRNA and protein expression in serum-deprived 3T3-L1 adipocytes were twofold increased by acute insulin treatment. Co-treatments with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or the mammalian target of rapamycin (mTOR) inhibitor rapamycin blunted the effects of insulin. Coherently, IGFBP-2 mRNA levels were robustly increased in adipocytes lacking either TSC2 or 4E-BP1. Insulin triggered the recruitment of CAAT/enhancer binding protein α (C/EBPα) to the IGFBP-2 proximal promoter. These findings suggest that insulin upregulates IGFBP-2 expression through a PI3K/mTOR/C/EBPα pathway in white adipocytes.     

Overexpression of the dual-specificity phosphatase MKP-4/DUSP-9 protects against stress-induced insulin resistance

Insulin resistance, a hallmark of type 2 diabetes and obesity, is associated with increased activity of MAP and stress-activated protein (SAP) kinases, which results in decreased insulin signaling. Our goal was to investigate the role of MAP kinase phosphatase-4 (MKP-4) in modulating this process. We found that MKP-4 expression is up-regulated during adipocyte and myocyte differentiation in vitro and up-regulated during fasting in white adipose tissue in vivo. Overexpression of MKP-4 in 3T3-L1 cells inhibited ERK and JNK phosphorylation and, to a lesser extent, p38MAPK phosphorylation. As a result, the phosphorylation of IRS-1 serine 307 induced by anisomycin was abolished, leading to a sensitization of insulin signaling with recovery of insulin-stimulated IRS-1 tyrosine phosphorylation, IRS-1 docking with phosphatidylinositol 3-kinase, and Akt phosphorylation. MKP-4 also reversed the effect of TNF-alpha to inhibit insulin signaling; alter IL-6, Glut1 and Glut4 expression; and inhibit insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Overexpression of MKP-4 in the liver of ob/ob mice decreased ERK and JNK phosphorylation, leading to a reduction in fed and fasted glycemia, improved glucose intolerance, decreased expression of gluconeogenic and lipogenic genes, and reduced hepatic steatosis. Thus, MKP-4 has a protective effect against the development of insulin resistance through its ability to dephosphorylate and inactivate crucial mediators of stress-induced insulin resistance, such as ERK and JNK, and increasing MKP-4 activity might provide a therapy for insulin-resistant disorders.     

Nitric oxide stimulates glucose transport through insulin-independent GLUT4 translocation in 3T3-L1 adipocytes

OBJECTIVE: It is well known that nitric oxide synthase (NOS) is expressed and that it modulates glucose transport in skeletal muscles. Recent studies have shown that adipose tIssues also express inducible and endothelial nitric oxide synthase (eNOS). In the present study, we investigated whether nitric oxide (NO) induces glucose uptake in adipocytes, and the signaling pathway involved in the NO-stimulated glucose uptake in 3T3-L1 adipocytes. METHODS: First, we determined the expression of eNOS in 3T3-L1 adipocytes, and then these cells were treated with the NO donor sodium nitroprusside (SNP) and/or insulin, and glucose uptake and phosphorylation of insulin receptor substrate (IRS)-1 and Akt were evaluated. Moreover, we examined the effects of a NO scavenger, a guanylate cyclase inhibitor or dexamethasone on SNP-stimulated glucose uptake and GLUT4 translocation. RESULTS: SNP at a concentration of 50 mmol/l increased 2-deoxyglucose uptake (1.8-fold) without phosphorylation of IRS-1 and Akt. Treatment with the NO scavenger or guanylate cyclase inhibitor decreased SNP-stimulated glucose uptake to the basal level. Dexamethasone reduced both insulin- and SNP-stimulated glucose uptake with impairment of GLUT4 translocation. CONCLUSION: NO is capable of stimulating glucose transport through GLUT4 translocation in 3T3-L1 adipocytes, via a mechanism different from the insulin signaling pathway.