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.

These effects are inhibited by wortmannin in the p110*-expressing cells, indicating that the phosphatidylinositol 3-kinase activity of the protein is required. Overexpression of an identical protein containing a point mutation in the kinase domain, p110*⌬kin, was incapable of mediating either action, confirming that neither the microinjection process nor a nonspecific effect of the protein was responsible for the observed effects. These data suggest that although insulin is capable of inducing numerous signaling pathways, the isolated activation of phosphatidylinositol 3-kinase can initiate the signaling cascade leading to both actin rearrangement and GLUT4 translocation in the absence of insulin stimulation.Insulin regulates plasma glucose levels primarily through stimulation of glucose uptake into target tissues and suppression of hepatic glucose production (1). Glucose transport into adipose tissue and skeletal muscle is predominantly induced by translocation of the GLUT4 glucose transporter from an intracellular vesicular pool to the plasma membrane (2, 3). Despite the importance of this biological effect, the signaling mechanisms leading to insulin-stimulated GLUT4 translocation remain poorly defined. Recent work has demonstrated that this event is dependent on insulin-stimulated phosphatidylinositol 3-kinase (PI3K) 1 but is independent of activation of the Ras/ mitogen-activated protein kinase pathway (4). Insulin can cause rapid rearrangement of the actin cytoskeleton, and this is also PI3K-dependent and Ras-independent (5, 6). Furthermore, disassembly of the actin network with cytochalasin D has been shown to inhibit insulin-stimulated GLUT4 translocation in L6 muscle cells (7).PI3K is a heterodimer of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110) (8). The p110 subunit binds to the p85 subunit at a region between the two Src homology 2 (SH2) modules of p85 termed the inter-SH2 domain (iSH2) (12, 13). Binding of the SH2 domains of the p85 subunit to phosphotyrosines in the insulin receptor substrate proteins (9, 10) or directly to the receptor itself (11) serves to recruit and activate the p110 catalytic subunit, leading to phosphorylation of the D-3 position of phosphatidylinositol (8). Insulin-stimulated activation of PI3K is required for many cellular responses, ranging from gene expression to mitogenesis (14).Recent reports demonstrate that a constitutively active mutant of PI3K (p110*), which is composed of an amino-terminal fusion of the p110 catalytic subunit with the iSH2 activation domain of p85, is capable of stimulating PI3K-dependent gene expression in the absence of added growth factors (15). Using single cell microinjection of an expression vector for p110* and fluorescence microscopy, we have studied the direct role of PI3K activation on actin rearrangement and GLUT4 translocation in 3T3-L1 adipocytes. EXPERIMENTAL PROCEDURES MaterialsPorcine insulin was kindly provided by Lilly. Rabbit polyclonal GLUT4 antibody (F349) and construction of the CMV expression vecto...

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5-phosphatase-defective SHIP2 (⌬IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor ␤ subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or ⌬IP-SHIP2. Because WT-SHIP2 possesses the 5-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of ⌬IP-SHIP2, indicating that ⌬IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C, whereas these activations were increased by expression of ⌬IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of ⌬IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3␤ and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of ⌬IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.Insulin binding to the extracellular ␣ subunit of the insulin receptor activates the intrinsic tyrosine kinase activity of the intracellular ␤ subunit. The activated insulin receptor phosphorylates the insulin receptor substrate (IRS) family of proteins on the tyrosine residues. IRS proteins propagate insulin signals to the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, which activates the p110 catalytic subunit. Insulin-induced PI 3-kinase activation is shown to be extremely important for the subsequent performance of a variety of insulin...

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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.