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Excessive secretion of glucagon is a major contributor to the development of diabetic hyperglycemia. Secretion of glucagon is regulated by various nutrients, with glucose being a primary determinant of the rate of alpha cell glucagon secretion. The intra-islet action of insulin is essential to exert the effect of glucose on the alpha cells since, in the absence of insulin, glucose is not able to suppress glucagon release in vivo. However, the precise mechanism by which insulin suppresses glucagon secretion from alpha cells is unknown. In this study, we show that insulin induces activation of GABAA receptors in the alpha cells by receptor translocation via an Akt kinase-dependent pathway. This leads to membrane hyperpolarization in the alpha cells and, ultimately, suppression of glucagon secretion. We propose that defects in this pathway(s) contribute to diabetic hyperglycemia.

Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.

We report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2-deoxyglucose (10 W WM) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation. We further show that wortmannin and the cytoskeleton disruptors cytochalasin D and latrunculin B completely blocked these insulin effects. The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti-diabetic drugs.z 1998 Federation of European Biochemical Societies.

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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...

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulinstimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.

To test whether the tyrosine kinase activity of the insulin receptor is crucial for insulin action, we have constructed mutations of the human insulin receptor at Lys-1030, which is in the presumed ATP-binding region. By using oligonucleotide-directed mutagenesis, this lysine residue was replaced with either methionine, arginine, or alanine. Chinese hamster ovary cells were transfected by mutant cDNAs and the expressed insulin receptors were characterized. We show here that none of these mutants exhibited insulin-activated autophosphorylation and kinase activity in vitro. They also do not mediate insulin-and antibody-stimulated uptake of 2-deoxyglucose. The tyrosine kinase activity is thus required for a key physiological response of insulin.Insulin initiates its diverse biological effects by binding to its receptor, an integral membrane glycoprotein composed of two a (Mr = 135,000) and two ,B (Mr = 95,000) subunits linked by disulfide bonds (1, 2). An immediate consequence of insulin binding to the a subunit is the activation of an intrinsic tyrosine kinase activity located in the ,3 subunit (3, 4). Since tyrosine kinase activity is also found associated with receptors of several other hormones (5), and many of the effects of insulin are caused by changes in the phosphorylation state of various proteins (6), it has been suggested that some or all of the effects of insulin are mediated by the receptor's kinase activity. A variety of experiments supports this hypothesis (7)(8)(9)(10)(11)(12), including the finding that a long-term effect of insulin is blocked by a monoclonal antibody that inhibits the insulin receptor (IR) kinase (13). However, other studies utilizing polyclonal and monoclonal antireceptor antibodies show a stimulation of glucose uptake in adipocytes without stimulating the kinase activity of the receptor (14-17). Thus, the role the kinase activity plays in the responses to insulin is still uncertain.The isolation and sequencing of the human placental IR cDNA has now provided primary structural information about the receptor protein (18,19). The a subunit (735 amino acids) contains a cysteine-rich crosslinking domain and presumably the insulin-binding site; the /3 subunit (620 amino acids) contains the single transmembrane domain of the receptor and the tyrosine kinase domain with the presumed ATP-binding region and potential tyrosine phosphorylation sites (18, 19). Furthermore, the functional human IR has been expressed in Chinese hamster ovary (CHO) cells that are stably transformed with the human IR cDNA (20). This system permits the further analysis of the structure-function relationship of the receptor molecule and the role of the receptor kinase in eliciting the various physiological responses to insulin. In a previous study, Ellis et al. (21) have shown that removal of the C-terminal region of the IR renders the cytoplasmic domain unstable, and it is removed, presumably by processing. The resulting truncated receptor is capable of binding insulin normally but exhibits no kinase act...