SUMMARY Insulin resistance and elevated glucagon levels result in non-suppressible hepatic glucose production and hyperglycemia in patients with type 2 diabetes. The CREB co-activator complex controls transcription of hepatic gluconeogenic enzyme genes. Here we show that both the antidiabetic agent metformin and insulin phosphorylate the transcriptional co-activator CBP at serine 436 via PKCι/λ. This event triggers the dissociation of the CREB-CBP-TORC2 transcription complex and reduces gluconeogenic enzyme gene expression. Mice carrying a germline mutation of this CBP phosphorylation site (S436A) demonstrate resistance to the hypoglycemic effect of both insulin and metformin. Obese, hyperglycemic mice display hepatic insulin resistance, but metformin is still effective in treating the hyperglycemia of these mice since it stimulates CBP phosphorylation by bypassing the block in insulin signaling.

Angiotensin II (AII), acting via its G-protein linked receptor, is an important regulator ofcardiac, vascular, and renal function. Following injection of All into rats, we find that there is also a rapid tyrosine phosphorylation of the major insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in the heart. This phenomenon appears to involve JAK2 tyrosine kinase, which associates with the ATI receptor and IRS-1/IRS-2 after AII stimulation. AII-induced phosphorylation leads to binding of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and IRS-2; however, in contrast to other ligands, AII injection results in an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity. The latter occurs without any reduction in insulin receptor or IRS phosphorylation or in the interaction of the p85 and pllO subunits of PI 3-kinase with each other or with IRS-1/IRS-2. These effects ofAII are inhibited by ATI receptor antagonists. Thus, there is direct cross-talk between insulin and AII signaling pathways at the level of both tyrosine phosphorylation and PI 3-kinase activation. These interactions may play an important role in the association of insulin resistance, hypertension, and cardiovascular disease.Insulin resistance occurs in a wide variety of pathological states and is a central component of non-insulin dependent diabetes mellitus (1). The frequent clustering of insulin resistance, hypertension, central obesity, hypertriglyceridemia, and accelerated atherosclerosis has lead to the definition of a common metabolic condition often referred to as syndrome X (2, 3). Over the past decade, many of the proteins involved in insulin action have been defined at a molecular level (4). The insulin receptor is a protein tyrosine kinase which, when activated by insulin binding, undergoes rapid autophosphorylation and phosphorylates intracellular protein substrates, including Shc, one or more 50-60 kDa proteins, and two related high molecular weight insulin receptor substrates, IRS-1 and IRS-2 (4, 5). Following tyrosine phosphorylation, IRS-1 and IRS-2 act as docking proteins for several Src homology 2 domaincontaining molecules, including phosphatidylinositol 3-kinase (PI 3-kinase), Grb2, SHPTP2, NCK, and Fyn (4, 6, 7). The interaction between the IRS proteins and PI 3-kinase occurs through the p85 regulatory subunit of the enzyme and results in an increase in catalytic activity of the pllO subunit (6, 8). PI 3-kinase is essential for many insulin-sensitive metabolic processes including stimulation of glucose transport, activation of the p70 S6 and Akt serine kinases, and stimulation of glycogen and protein synthesis (9-13).Angiotensin II (AII) plays an important role in cardiovascular and neuroendocrine physiology and fluid volume homeostasis, and may also act as a growth factor for heart and vascular smooth muscle (14). Angiotensin-converting enzyme inhibitors are a cornerstone in the therapy of human hypertension and cardiac failure (15). Most of the known actions of AII are exerted through the AT1 rece...

The cystine/glutamate exchanger (xCT) provides intracellular cyst(e)ine for production of glutathione, a major cellular antioxidant. Using xCT overexpression and underexpression, we present evidence that xCT-dependent glutathione production modulates both neuroprotection from oxidative stress and cell proliferation. In embryonic and adult rat brain, xCT protein was enriched at the CSF-brain barrier (i.e., meninges) and also expressed in the cortex, hippocampus, striatum, and cerebellum. To examine the neuroprotective role of xCT, various non-neuronal cell types (astrocytes, meningeal cells, and peripheral fibroblasts) were cocultured with immature cortical neurons and exposed to oxidative glutamate toxicity, a model involving glutathione depletion. Cultured meningeal cells, which naturally maintain high xCT expression, were more neuroprotective than astrocytes. Selective xCT overexpression in astrocytes was sufficient to enhance glutathione synthesis/release and confer potent glutathione-dependent neuroprotection from oxidative stress. Moreover, normally nonprotective fibroblasts could be re-engineered to be neuroprotective with ectopic xCT overexpression indicating that xCT is a key step in the pathway to glutathione synthesis. Conversely, astrocytes and meningeal cells derived from sut/sut mice (xCT loss-of-function mutants) showed greatly reduced proliferation in culture attributable to increased oxidative stress and thiol deficiency, because growth could be rescued by the thiol-donor ␤-mercaptoethanol. Strikingly, sut/sut mice developed brain atrophy by early adulthood, exhibiting ventricular enlargement, thinning of the cortex, and shrinkage of the striatum. Our results indicate that xCT can provide neuroprotection by enhancing glutathione export from non-neuronal cells such as astrocytes and meningeal cells. Furthermore, xCT is critical for cell proliferation during development in vitro and possibly in vivo.

Obesity and stress inhibit insulin action by activating protein kinases that enhance serine phosphorylation of IRS1 and have been thus associated to insulin resistance and the development of type II diabetes. The protein kinase C (PKC) is activated by free-fatty acids, and its activity is higher in muscle from obese diabetic patients. However, a molecular link between PKC and insulin resistance has not been defined yet. Here we show that PKC phosphorylates IRS1 at serine 1101 blocking IRS1 tyrosine phosphorylation and downstream activation of the Akt pathway. Mutation of Ser 1101 to alanine makes IRS1 insensitive to the effect of PKC and restores insulin signaling in culture cells. These results provide a novel mechanism linking the activation of PKC to the inhibition of insulin signaling.

(1995) Nature 377, 173-177) purified and cloned 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, which has significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice, we performed immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays using specific antibodies to 4PS. Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle, binds to the p85 subunit of PI 3-kinase, and activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1-deficient mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression. Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine-associated PI 3-kinase activity in IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal transduction to these two major pathways of insulin signaling.Stimulation of the insulin and IGF-1 1 receptor tyrosine kinases results in rapid autophosphorylation and subsequent phosphorylation of cytoplasmic substrates. A major substrate of the insulin receptor is IRS-1, a cytoplasmic protein of 160 -185 kDa on SDS-PAGE (1-3). Following insulin/IGF-1 stimulation, IRS-1 is rapidly phosphorylated on multiple tyrosines (4). This results in docking of several SH2 domain proteins, including: the p85 subunit of PI 3-kinase (5-8), an upstream element in insulin-stimulated glucose transport and activation of p70 S6 kinase (9, 10); Grb 2, an adapter molecule linking IRS-1 to activation of Ras and mitogen-activated protein kinase (11-13); and the tyrosine phosphatase SHPTP2 (14, 15). Insulin and IGF-1 receptors can also phosphorylate other cytoplasmic proteins. These include Shc, a cytoplasmic protein which binds to Grb 2 (16), a p62 protein which associates with Ras-GAP (17), and a 55-60-kDa protein which associates with PI 3-kinase (18,19).Abundant evidence from Xenopus oocytes (20), cell culture systems (21-23), and animal models (24,25) has demonstrated the central role of IRS-1 in mediating downstream effects of insulin and IGF-1. Recently, we (26) and others (27) have shown that mice made IRS-1-deficient by targeted gene knockout exhibit hyperinsulinemia, glucose intolerance, and marked growth retardation. However, IRS-1-deficient mice continue to exhibit some insulin-stimulated glucose disposal and phosphotyrosine-associated PI 3-kinase activation, suggesting the presence of an IRS-1-independent pathway of signaling. Immunoblots from both liver and muscle tissue of IRS-1 (Ϫ/Ϫ) animals reveal a ϳ180-kDa protein (tentatively designated IRS-2) which is tyrosine-phosphorylated within 1 min of insulin stimulation and binds to PI 3-kinase...