We evaluated the role of the G alpha-q (G␣q) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous G␣q function by single cell microinjection of anti-G␣q/11 antibody or RGS2 protein (a GAP protein for G␣q), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. G␣q/11 antibody and RGS2 inhibited insulin-induced GLUT4 translocation by 60 or 75%, respectively, indicating that activated G␣q is important for insulin-induced glucose transport. We then assessed the effect of overexpressing wild-type G␣q (WT-G␣q) or a constitutively active G␣q mutant (Q209L-G␣q) by using an adenovirus expression vector. In the basal state, Q209L-G␣q expression stimulated 2-deoxy-D-glucose uptake and GLUT4 translocation to 70% of the maximal insulin effect. This effect of Q209L-G␣q was inhibited by wortmannin, suggesting that it is phosphatidylinositol 3-kinase (PI3-kinase) dependent. We further show that Q209L-G␣q stimulates PI3-kinase activity in p110␣ and p110␥ immunoprecipitates by 3-and 8-fold, respectively, whereas insulin stimulates this activity mostly in p110␣ by 10-fold. Nevertheless, only microinjection of anti-p110␣ (and not p110␥) antibody inhibited both insulin-and Q209L-G␣q-induced GLUT4 translocation, suggesting that the metabolic effects induced by Q209L-G␣q are dependent on the p110␣ subunit of PI3-kinase. In summary, (i) G␣q appears to play a necessary role in insulin-stimulated glucose transport, (ii) G␣q action in the insulin signaling pathway is upstream of and dependent upon PI3-kinase, and (iii) G␣q can transmit signals from the insulin receptor to the p110␣ subunit of PI3-kinase, which leads to GLUT4 translocation.

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3␤, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C (PKC ) activity was also augmented in small-t-antigenexpressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC expression or PKC siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.Protein phosphorylation plays a key role in many cellular processes, including insulin signal transduction (24), and the phosphorylation state of a target protein is regulated by opposing kinase and phosphatase activities (24). Thus, the balance of enzyme activity between kinases and phosphatases is critical for the mediation of insulin's effects and, in turn, for the pathogenesis of insulin-resistant states.Tyrosine phosphorylation is essential for insulin action, and several lines of evidence have demonstrated that protein tyrosine phosphatases can play a role in insulin-resistant states (3, 4). For example, protein tyrosine phosphatase 1B (PTP1B) directly interacts with the activated insulin receptor and exhibits high specific activity for IRS-1 (22, 49). It has been reported previously that hyperglycemia can impair insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1, at least in part because of the increased expression and activity of PTP1B (37,41), and that overexpression of PTP1B inhibits insulin-stimulated glucose metabolism in 3T3-L1 adipocytes and L6 myocytes (12,18,51).Serine/thre...

OBJECTIVE—To document an association between arterial wall stiffness and reduced flow volume in the lower-extremity arteries of diabetic patients. RESEARCH DESIGN AND METHODS—We recruited 60 consecutive type 2 diabetic patients who had no history or symptoms of peripheral arterial disease (PAD) in the lower extremities and normal ankle/brachial systolic blood pressure index at the time of the study (non-PAD group) and 20 age-matched nondiabetic subjects (control group). We used an automatic device to measure pulse wave velocity (PWV) in the lower extremities as an index of arterial wall stiffness. At the popliteal artery, we evaluated flow volume and the resistive index as an index of arterial resistance to blood flow using gated two-dimensional cine-mode phase-contrast magnetic resonance imaging. RESULTS—Consistent with previous reports, we confirmed that the non-PAD group had an abnormally higher PWV compared with that of the control group (P < 0.001). To further demonstrate decreased flow volume and abnormal flow pattern at the popliteal artery in patients with a higher degree of arterial wall stiffness, we assigned the 60 non-PAD patients to tertiles based on their levels of PWV. In the highest group, magnetic resonance angiograms of the calf and foot arteries showed decreased intravascular signal intensity, indicating the decreased arterial inflow in those arteries. The highest group was also characterized by the lowest late diastolic and total flow volumes as well as the highest resistive index among the groups. From stepwise multiple regression analysis, PWV and autonomic function were identified as independent determinants for late diastolic flow volume (r2 = 0.300; P < 0.001). CONCLUSIONS—Arterial wall stiffness was associated with reduced arterial flow volume in the lower extremities of diabetic patients.

Phosphatidylinositol 3-kinase (PI 3-K) is implicated in cellular events including glucose transport, glycogen synthesis, and protein synthesis. It is activated in insulin-stimulated cells by binding of the Src homology 2 (SH2) domains in its 85-kDa regulatory subunit to insulin receptor substrate-1 (IRS-1), and, others. We have previously shown that IRS-1-associated PI 3-kinase activity is not essential for insulin-stimulated glucose transport in 3T3-L1 adipocytes, and that alternate pathways exist in these cells. We now show that adenovirusmediated overexpression of the p85N-SH2 domain in these cells behaves in a dominant-negative manner, interfering with complex formation between endogenous PI 3-K and its SH2 binding targets. This not only inhibited insulin-stimulated IRS-1-associated PI 3-kinase activity, but also completely blocked anti-phosphotyrosine-associated PI 3-kinase activity, which would include the non-IRS-1-associated activity. This resulted in inhibition of insulin-stimulated glucose transport, glycogen synthase activity and DNA synthesis. Further, Ser/Thr phosphorylation of downstream molecules Akt and p70 S6 kinase was inhibited. However, co-expression of a membrane-targeted p110 CAAX with the p85N-SH2 protein rescued glucose transport, supporting our argument that the p85N-SH2 protein specifically blocks insulin-mediated PI 3-kinase activity, and, that the signaling pathways downstream of PI 3-kinase are intact. Unexpectedly, GTP-bound Ras was elevated in the basal state. Since p85 is known to interact with GTPase-activating protein in 3T3-L1 adipocytes, the overexpressed p85N-SH2 peptide could titrate out cellular GTPase-activating protein by direct association, such that it is unavailable to hydrolyze GTP-bound Ras. However, insulin-induced mitogen-activated protein kinase phosphorylation was inhibited. Thus, PI 3-kinase may be required for this action at a step independent of and downstream of Ras. We conclude that, in 3T3-L1 adipocytes, non-IRS-1-associated PI 3-kinase activity is crucial for insulin's metabolic signaling, and that overexpressed p85N-SH2 protein inhibits a variety of insulin's ultimate biological effects.Insulin binding to its cell surface receptors initiates diverse metabolic and mitogenic signals by activation of a complex signaling cascade of protein tyrosine and serine/threonine kinases, as well as lipid kinases (1, 2). Phosphatidylinositol (PI) 1 3-kinase (PI 3-kinase), a dual protein and lipid kinase is a heterodimeric enzyme composed of a 110-kDa catalytic subunit (p110) associated with an 85-kDa regulatory subunit (p85). Two isoforms of the catalytic subunit (p110␣ and p110␤) and several isoforms of the regulatory subunit (p55␣, p55PIK, p85␣, and p85␤) have been cloned so far. The regulatory subunit contains several well known functional domains: one Src homology 3 (SH3) domain, homology to the breakpoint cluster region (bcr) gene, two proline-rich motifs, and two Src homology region 2 (SH2) domains (3). The p85␣ isoform is insulinresponsive and is predominantly ex...

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.