. Resistin inhibits glucose uptake in L6 cells independently of changes in insulin signaling and GLUT4 translocation. Am J Physiol Endocrinol Metab 285: E106-E115, 2003. First published March 4, 2003 10.1152/ajpendo.00457.2002-Elevated levels of resistin have been proposed to cause insulin resistance and therefore may serve as a link between obesity and type 2 diabetes. However, its role in skeletal muscle metabolism is unknown. In this study, we examined the effect of resistin on insulinstimulated glucose uptake and the upstream insulin-signaling components in L6 rat skeletal muscle cells that were either incubated with recombinant resistin or stably transfected with a vector containing the myc-tagged mouse resistin gene. Transfected clones expressed intracellular resistin, which was released in the medium. Incubation with recombinant resistin resulted in a dose-dependent inhibition of insulin-stimulated 2-deoxyglucose (2-DG) uptake. The inhibitory effect of resistin on insulin-stimulated 2-DG uptake was not the result of impaired GLUT4 translocation to the plasma membrane. Furthermore, resistin did not alter the insulin receptor (IR) content and its phosphorylation, nor did it affect insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation, its association with the p85 subunit of phosphatidylinositol (PI) 3-kinase, or IRS-1-associated PI 3-kinase enzymatic activity. Insulin-stimulated phosphorylation of Akt/protein kinase B-␣, one of the downstream targets of PI 3-kinase and p38 MAPK phosphorylation, was also not affected by resistin. Expression of resistin also inhibited insulin-stimulated 2-DG uptake when compared with cells expressing the empty vector (L6Neo) without affecting GLUT4 translocation, GLUT1 content, and IRS-1/PI 3-kinase signaling. We conclude that resistin does not alter IR signaling but does affect insulin-stimulated glucose uptake, presumably by decreasing the intrinsic activity of cell surface glucose transporters. skeletal muscle; glucose transporter-4

Small GTPases of the Rho family are well known intracellular signaling proteins that act as molecular switches to control actin cytoskeleton organization in many cell types including smooth muscle (1-4). Recent studies indicate that RhoA-dependent signaling pathway controls vascular smooth muscle cell functions such as contraction, migration, and proliferation (5-6). In VSMCs, 1 the contractile effect of RhoA results from the activation of Rho-dependent kinase (ROK-␣), which phosphorylates the regulatory subunit of myosin light chain phosphatase (MBS) and thereby inhibits the phosphatase activity (7-8), thus allowing an increase in the level of phosphorylated myosin light chain and contraction at a constant intracellular calcium level [Ca 2ϩ ] i (9). This phenomenon is defined as Ca 2ϩ sensitization (10). ROK-␣ and another isoform Rho kinase, ROCK1, are serine/ threonine protein kinases that contain an amino-terminal catalytic kinase domain, a central coiled-coil domain in which Rho/GTP binds, and a carboxyl-terminal pleckstrin homology (PH) domain that is split by a cysteine-rich region (11-12). Insulin receptor substrate proteins (IRS) also contain an amino-terminal PH domain and phosphotyrosine binding domain domain. The PH domain is required for efficient phosphorylation of IRS-1 by the insulin receptor (13-15). In addition, IRS-1 also interacts with 14-3-3 proteins, a process apparently dependent on serine phosphorylation of IRS-1 (16).Recent studies from our laboratory (17) have shown that insulin rapidly stimulates myosin-associated phosphatase (MBP) activity by causing a site-specific decrease in MBS T695 phosphorylation by inactivating thrombin-stimulated Rho and one of its downstream effectors, Rho kinase. Furthermore, inhibition of PI3-kinase, nitric-oxide synthase (NOS), and cGMP signaling pathways abolished insulin-stimulated MBP activation suggesting the involvement of these signaling pathways in MBP activation (18). Thus, insulin stimulates MBP in VSMCs by activating the NO/cGMP signaling pathway that also inactivates Rho/Rho kinase (17). The effects of insulin on MBP activation and vasorelaxation were severely impaired in VSMCs isolated from diabetic Goto-Kakizaki rats and spontaneous hypertensive rats (SHR) due to defective IRS-1/PI3-kinase signaling as well as up-regulation of Rho kinase activity (18,19). These observations prompted us to explore in detail potential interactions between Rho signaling and insulin signaling pathways.In the present study, VSMCs were infected with an activated RhoA V14 , dominant negative RhoA N19 , and cGK I␣. We examined the effects of insulin and thrombin on ROK-␣/IRS-1 association, IRS-1 tyrosine phosphorylation, IRS-1/p85 PI3-kinase association, and PI3-kinase activation and its impact on MBS T695 site-specific phosphorylation and MBP activation. * This work was supported by a American Heart Association Established Investigator grant, medical education funds from Winthrop University Hospital, and by Deutsche Forschungsgemeinschaft Grant SFB355. The costs of ...

Patients with hepatitis C virus (HCV) infection have a greater risk of developing type 2 diabetes mellitus. However, the mechanism of this association is unclear. In this study, we examined the potential defects in upstream insulin signaling pathways in liver specimens obtained from nonobese/nondiabetic subjects with HCV infection. Fasting liver biopsy specimens were obtained from 42 HCV-infected subjects and 10 non-HCV-infected subjects matched for age and body mass index. Liver tissues were exposed to insulin and examined for the contents and phosphorylation/activation status of the upstream insulin signaling molecules by immunoprecipitation and Western blot analysis. HCV infection resulted in a trend toward a 2-fold to 3-fold increase in insulin receptor (IR) and insulin receptor substrate (IRS)-1 contents when compared with non-HCV. In contrast, insulin-stimulated IRS-1 tyrosine phosphorylation was decreased by 2-fold in HCV-infected subjects compared with non-HCV-infected subjects (P < .05). The observed reductions in IRS-1 tyrosine phosphorylation were accompanied by a 3.4-fold decrease in IRS-1/p85 phosphatidylinositol 3-kinase (PI3-kinase) association and a 2.5-fold decrease in IRS-1-associated PI3-kinase enzymatic activity (P < .05 vs. non-HCV). This was accompanied by a marked reduction in insulinstimulated Akt phosphorylation without any alterations in mitogen-activated protein kinase (MAPK) phosphorylation. Cellular contents of the hepatic p85 subunit of PI3-kinase were comparable between HCV-infected and non-HCV-infected subjects. In conclusion, we found that (1) HCV infection leads to a postreceptor defect in IRS-1 association with the IR and (2) insulin signaling defects in hepatic IRS-1 tyrosine phosphorylation and PI3-kinase association/activation may contribute to insulin resistance, which leads to the development of type 2 diabetes mellitus in patients with HCV infection. (HEPATOLOGY 2003;38:1384-1392

Hyperinsulinemia (HI) and insulin resistance (IR) are frequently associated with hypertension and atherosclerosis. However, the exact roles of HI and IR in the development of hypertension are unclear. Mitogen-activated protein kinases (MAPK) are well-characterized intracellular mediators of cell proliferation. In this study, we examined the contribution of MAPK pathway in insulin-stimulated mitogenesis using primary vascular smooth muscle cells (VSMCs) isolated from aortas of normotensive Wistar-Kyoto rats (WKY) and spontaneous hypertensive rats (SHR). VSMCs were grown to confluence in culture, serum starved, and examined for DNA synthesis {using [3H]thymidine (TDR), immunoprecipitated MAPK activity, and MAPK phosphatase (MKP-1) induction}. Basal rate of TDR incorporation into DNA was twofold higher in SHR compared with WKY ( P < 0.005). Insulin caused a dose-dependent increase in TDR incorporation (150% over basal levels with 100 nM in 12 h). Stimulation was sustained for 24 h with a decline toward basal in 36 h. Pretreatment with insulin-like growth factor I (IGF-I) receptor antibody did not abolish mitogenesis mediated by 10–100 nM insulin, suggesting that insulin effect is mediated via its own receptors. Insulin had a small mitogenic effect in WKY (33% over basal). Insulin-stimulated mitogenesis was accompanied by a dose-dependent increase in MAPK activity in SHR, with a peak activation (>2-fold over basal) between 5 and 10 min with 100 nM insulin. Insulin had very small effects on MAPK activity in WKY. In contrast, serum-stimulated MAPK activation was comparable in WKY and SHR. Pretreatment with MEK inhibitor, PD-98059, completely blocked insulin’s effect on MAPK activation and mitogenesis. Inhibition of phosphatidylinositol 3-kinase with wortmannin also prevented insulin’s effects on MAPK activation and mitogenesis. In WKY, insulin and IGF-I treatment resulted in a rapid induction of MKP-1, the dual-specificity MAPK phosphatase. In contrast, VSMCs from SHR were resistant to insulin with respect to MPK-1 expression. We conclude that insulin is mitogenic in SHR, and the effect appears to be mediated by sustained MAPK activation due to impaired insulin-mediated MKP-1 mRNA expression, which may act as an inhibitory feedback loop in attenuating MAPK signaling.

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Our laboratory has demonstrated that insulin rapidly stimulates myosin-bound phosphatase (MBP) activity in vascular smooth muscle cells (VSMCs). In this study, we examined whether diabetes is accompanied by alterations in MBP activation and elucidated the components of the signaling pathway that mediate the effects of diabetes. VSMCs isolated from Goto-Kakizaki (GK) diabetic rats (a model for type 2 diabetes) exhibited marked impairment in MBP activation by insulin that was accompanied by failure of insulin to decrease the phosphorylation of a regulatory myosin-bound subunit (

Sustained elevations in cytosolic calcium concentrations ([Ca2+]i) have been shown to render insulin target cells resistant to insulin action. In this study we examined the mechanisms of the detrimental effect of high levels of [Ca2+]i on insulin-induced 2-deoxyglucose (2-DOG) uptake. To elevate [Ca2+]i, we incubated rat adipocytes with either 40 mM potassium (K+) or 20 ng/ml PTH for 1 h for in vitro experiments and injected rats with PTH (injections of 50 micrograms, ip, every hour for 3 h) for in vivo studies. Adipocytes with elevated [Ca2+]i demonstrated a 30% decrease in insulin-stimulated 2-DOG uptake. A calcium channel blocker (nitrendipine) and a cAMP antagonist (RpcAMP) each partially restored insulin-stimulated glucose transport, but together they completely restored 2-DOG uptake. Concomitantly, we found a significant increase in phosphorylation of GLUT-4 in adipocytes with elevated [Ca2+]i. This change in GLUT-4 phosphorylation was also attenuated by nitrendipine and RpcAMP. These observations confirm that elevated [Ca2+]i diminishes insulin-stimulated glucose transport and suggest that increased phosphorylation of GLUT-4 in adipocytes with high [Ca2+]i may alter its intrinsic activity.

Our laboratory has recently demonstrated that insulin induces relaxation of vascular smooth muscle cells (VSMCs) by activating myosin-bound phosphatase (MBP) and by inhibiting Rho kinase (Begum N, Duddy N, Sandu OA, Reinzie J, and Ragolia L. Mol Endocrinol 14: 1365-1376, 2000). In this study, we tested the hypothesis that insulin via the nitric oxide (NO)/cGMP pathway may inactivate Rho, resulting in a decrease in phosphorylation of the myosin-bound subunit (MBS(Thr695)) of MBP and in its activation. Treatment of confluent serum-starved VSMCs with insulin prevented thrombin-induced increases in membrane-associated RhoA, Rho kinase activation, and site-specific phosphorylation of MBS(Thr695) of MBP and caused MBP activation. Preexposure to N(G)-monomethyl-L-arginine, a NO synthase inhibitor, and R-p-8-(4-chlorophenylthio)cGMP, a cGMP antagonist, attenuated insulin's inhibitory effect on Rho translocation and restored thrombin-mediated Rho kinase activation and site-specific MBS(Thr695) phosphorylation, resulting in MBP inactivation. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked insulin's inhibitory effects by abolishing thrombin-mediated Rho signaling and promoted dephosphorylation of MBS(Thr695). Furthermore, expression of a dominant-negative RhoA decreased basal as well as thrombin-induced MBS(Thr695) phosphorylation and caused insulin activation of MBP. Collectively, these results indicate that insulin inhibits Rho signaling by decreasing RhoA translocation via the NO/cGMP signaling pathway to cause MBP activation via site-specific dephosphorylation of its regulatory subunit MBS.