Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells.

Hypertension is associated with insulin-resistant states such as diabetes and obesity. Nitric oxide (NO) contributes to regulation of blood pressure. To gain insight into potential mechanisms linking hypertension with insulin resistance we directly measured and characterized NO production from human umbilical vein endothelial cells (HUVEC) in response to insulin using an amperometric NO-selective electrode. Insulin stimulation of HUVEC resulted in rapid, dose-dependent production of NO with a maximal response of approximately 100 nM NO (200,000 cells in 2 ml media; ED50 approximately 500 nM insulin). Although HUVEC have many more IGF-1 receptors than insulin receptors (approximately 400,000, and approximately 40,000 per cell respectively), a maximally stimulating dose of IGF-1 generated a smaller response than insulin (40 nM NO; ED50 approximately 100 nM IGF-1). Stimulation of HUVEC with PDGF did not result in measurable NO production. The effects of insulin and IGF-1 were completely blocked by inhibitors of either tyrosine kinase (genestein) or nitric oxide synthase (L-NAME). Wortmannin (an inhibitor of phosphatidylinositol 3-kinase [PI 3-kinase]) inhibited insulin-stimulated production of NO by approximately 50%. Since PI 3-kinase activity is required for insulin-stimulated glucose transport, our data suggest that NO is a novel effector of insulin signaling pathways that are also involved with glucose metabolism.

Paternal versus maternal transmission of a stimulatory G-protein alpha subunit knockout produces opposite effects on energy metabolism.

Heterozygous disruption of Gnas, the gene encoding the stimulatory G-protein alpha subunit (G(s)alpha), leads to distinct phenotypes depending on whether the maternal (m-/+) or paternal (+/p-) allele is disrupted. G(s)alpha is imprinted, with the maternal allele preferentially expressed in adipose tissue. Hence, expression is decreased in m-/+ mice but normal in +/p- mice. M-/+ mice become obese, with increased lipid per cell in white and brown adipose tissue, whereas +/p- mice are thin, with decreased lipid in adipose tissue. These effects are not due to abnormalities in thyroid hormone status, food intake, or leptin secretion. +/p- mice are hypermetabolic at both ambient temperature (21 degrees C) and thermoneutrality (30 degrees C). In contrast, m-/+ mice are hypometabolic at ambient temperature and eumetabolic at thermoneutrality M-/+ and wild-type mice have similar dose-response curves for metabolic response to a beta(3)-adrenergic agonist, CL316243, indicating normal sensitivity of adipose tissue to sympathetic stimulation. Measurement of urinary catecholamines suggests that +/p- and m-/+ mice have increased and decreased activation of the sympathetic nervous system, respectively. This is to our knowledge the first animal model in which a single genetic defect leads to opposite effects on energy metabolism depending on parental inheritance. This probably results from deficiency of maternal- and paternal-specific Gnas gene products, respectively.

Phosphorylation of critical serine residues in Gem separates cytoskeletal reorganization from down-regulation of calcium channel activity.

Gem is a small GTP-binding protein that has a ras-like core and extended chains at each terminus. The primary structure of Gem and other RGK family members (Rad, Rem, and Rem2) predicts a GTPase deficiency, leading to the question of how Gem functional activity is regulated. Two functions for Gem have been demonstrated, including inhibition of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. These functions for Gem have been ascribed to its interaction with the calcium channel beta subunit and Rho kinase beta, respectively. We show here that these functions are separable and regulated by distinct structural modifications to Gem. Phosphorylation of serines 261 and 289, located in the C-terminal extension, is required for Gem-mediated cytoskeletal reorganization, while GTP and possibly calmodulin binding are required for calcium channel inhibition. In addition to regulating cytoskeletal reorganization, phosphorylation of serine 289 in conjunction with serine 23 results in bidentate 14-3-3 binding, leading to increased Gem protein half-life. Evidence presented shows that phosphorylation of serine 261 is mediated via a cdc42/protein kinase Czeta-dependent pathway. These data demonstrate that phosphorylation of serines 261 and 289, outside the GTP-binding region of Gem, controls its inhibition of Rho kinase beta and associated changes in the cytoskeleton.

Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.

Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells.

BACKGROUND: Previously, we demonstrated that insulin stimulates production of nitric oxide (NO) in endothelial cells. However, specific insulin-signaling pathways mediating production of NO have not been elucidated. METHODS AND RESULTS: We developed methods for transfection of human umbilical vein endothelial cells (HUVECs) and direct measurement of NO to begin defining insulin-signaling pathways related to NO production. HUVECs were cotransfected with enhanced Green Fluorescent Protein (eGFP) and another gene of interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an approximately 3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase-deficient mutant insulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-stimulated production of NO. Overexpression of an inhibitory mutant of Ras had a much smaller effect. CONCLUSIONS: Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly to this process, whereas the contribution of Ras is small.

Insulin stimulates both endothelin and nitric oxide activity in the human forearm.

BACKGROUND: The mechanism of the hemodynamic effect of insulin in the skeletal muscle circulation has not been fully elucidated. The purpose of this study was to assess whether the hemodynamic response to insulin involves the concurrent release of endothelin (ET-1) and nitric oxide (NO), 2 substances with opposing vasoactive properties. METHODS AND RESULTS: Bioactivity of ET-1 and NO was assessed without insulin and during insulin infusion in the forearm circulation of healthy subjects by use of blockers of ET-1 receptors and by NO synthesis inhibition. In the absence of hyperinsulinemia, ET-1 receptor blockade did not result in any significant change in forearm blood flow from baseline (P=0.29). Intra-arterial insulin administration did not significantly modify forearm blood flow (P=0. 88). However, in the presence of hyperinsulinemia, ET-1 receptor antagonism was associated with a significant vasodilator response (P<0.001). In the presence of ET-1 receptor blockade, the vasoconstrictor response to NO inhibition by N(G)-monomethyl-L-arginine was significantly higher after insulin infusion than in the absence of hyperinsulinemia (P=0.006). CONCLUSIONS: These findings suggest that in the skeletal muscle circulation, insulin stimulates both ET-1 and NO activity. An imbalance between the release of these 2 substances may be involved in the pathophysiology of hypertension and atherosclerosis in insulin-resistant states associated with endothelial dysfunction.

Non-insulin-mediated glucose disappearance in subjects with IDDM. Discordance between experimental results and minimal model analysis.

Both insulin and glucose contribute to the regulation of glucose metabolism in vivo. We directly measured the ability of glucose per se to promote glucose disposal in subjects with insulin-dependent diabetes mellitus (IDDM). We compared our results with predictions of the minimal model of glucose metabolism. To identify minimal model parameters, a frequently sampled intravenous glucose tolerance test (FSIVGTT) was administered to each subject while they were connected to a Biostator (a device that monitors blood glucose and gives insulin to mimic normal insulin secretion). Data from this test reflected normal glucose tolerance and were in excellent agreement with minimal model predictions. The FSIVGTT was then repeated without the Biostator in the same diabetic subjects in order to directly measure the effect of glucose per se to promote glucose disposal in the absence of an incremental insulin effect (a basal insulin drip was maintained). To compare these results with minimal model predictions, the equations describing glucose disappearance in the absence of an incremental insulin effect were solved using parameters identified from the Biostator experiment. The glucose disappearance measured in the absence of an incremental insulin response was much slower than the minimal model predictions. Thus, the minimal model appears to overestimate the effect of glucose per se on glucose uptake and underestimate the contribution of incremental insulin.

Insulin signalling: metabolic pathways and mechanisms for specificity.

Biological actions of insulin are mediated by the insulin receptor, a member of a large family of receptor tyrosine kinases (RTK). Signal transduction by the insulin receptor follows a paradigm for RTK signalling. Many intracellular signalling molecules contain multiple modular domains that mediate protein-protein interactions and participate in the formation of signalling complexes. Phosphorylation cascades are also a prominent feature of RTK signalling. Distal pathways are difficult to dissect because branching paths emerge from downstream effectors and several upstream inputs converge upon single branch points. Thus, insulin action is determined by complicated signalling networks rather than simple linear pathways. Interestingly, many signalling molecules downstream from the insulin receptor are also activated by a plethora of RTKs. Therefore, mechanisms that generate specificity are required. In this review we discuss recent advances in the elucidation of specific metabolic insulin signalling pathways related to glucose transport, one of the most distinctive biological actions of insulin. We also present examples of potential mechanisms underlying specificity in insulin signalling including interactions between multiple branching pathways, subcellular compartmentalization, tissue-specific expression of key effectors and modulation of signal frequency and amplitude.

Insulin signal transduction pathways.

Insulin initiates its pleiotropic effects by activating the insulin receptor tyrosine kinase to phosphorylate several intracellular proteins. Recent studies have demonstrated that phosphotyrosine residues bind specifically to proteins that contain src homology 2 (SH2) domains, and that this interaction mediates the regulation of multiple intracellular signaling pathways. This article reviews recent progress in elucidating the detailed pathways that lead from the insulin receptor to the ultimate biologic actions of insulin.

Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor.

PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B contains this motif (RYRDVS(50)), and wild-type PTP1B (but not mutants with substitutions for Ser(50)) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3(IR) cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with [(32)P]-orthophosphate. Insulin stimulation caused a significant increase in phosphorylation of wild-type PTP1B that could be blocked by pretreatment of cells with wortmannin or cotransfection of a dominant inhibitory Akt mutant. Similar results were observed with endogenous PTP1B in untransfected HepG2 cells. Cotransfection of constitutively active Akt caused robust phosphorylation of wild-type PTP1B both in the absence and presence of insulin. By contrast, PTP1B-S50A did not undergo phosphorylation in response to insulin. We tested the functional significance of phosphorylation at Ser(50) by evaluating insulin receptor autophosphorylation in transfected Cos-7 cells. Insulin treatment caused robust receptor autophosphorylation that could be substantially reduced by coexpression of wild-type PTP1B. Similar results were obtained with coexpression of PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an impaired ability to dephosphorylate the insulin receptor. Moreover, cotransfection of constitutively active Akt significantly inhibited the ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the insulin receptor. We conclude that PTP1B is a novel substrate for Akt and that phosphorylation of PTP1B by Akt at Ser(50) may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.

Caveolin-1 interacts with the insulin receptor and can differentially modulate insulin signaling in transfected Cos-7 cells and rat adipose cells.

Caveolae may function as microdomains for signaling that help to determine specific biological actions mediated by the insulin receptor (IR). Caveolin-1, a major component of caveolae, contains a scaffolding domain (SD) that binds to a caveolin-1 binding motif in the kinase domain of the IR in vitro. To investigate the potential role of caveolin-1 in insulin signaling we overexpressed wild-type (Cav-WT) or mutant (Cav-Mut; F92A/V94A in SD) caveolin-1 in either Cos-7 cells cotransfected with IR or rat adipose cells (low and high levels of endogenous caveolin-1, respectively). Cav-WT coimmunoprecipitated with the IR to a much greater extent than Cav-Mut, suggesting that the SD is important for interactions between caveolin-1 and the IR in intact cells. We also constructed several IR mutants with a disrupted caveolin-1 binding motif and found that these mutants were poorly expressed and did not undergo autophosphorylation. Interestingly, overexpression of Cav-WT in Cos-7 cells significantly enhanced insulin-stimulated phosphorylation of Elk-1 (a mitogen-activated protein kinase-dependent pathway) while overexpression of Cav-Mut was without effect. In contrast, in adipose cells, overexpression of either Cav-WT or Cav-Mut did not affect insulin-stimulated phosphorylation of a cotransfected ERK2 (but did significantly inhibit basal phosphorylation of ERK2). Furthermore, we also observed a small inhibition of insulin-stimulated translocation of GLUT4 when either Cav-WT or Cav-Mut was overexpressed in adipose cells. Thus, interaction of caveolin-1 with IRs may differentially modulate insulin signaling to enhance insulin action in Cos-7 cells but inhibit insulin's effects in adipose cells.

Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 in the activation loop of protein kinase C-zeta.

Previous studies have suggested that 1) atypical protein kinase C (PKC) isoforms are required for insulin stimulation of glucose transport, and 2) 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is required for activation of atypical PKCs. Presently, we evaluated the role of PDK-1, both in the activation of PKC-zeta, and the translocation of epitope-tagged glucose transporter 4 (GLUT4) to the plasma membrane, during insulin action in transiently transfected rat adipocytes. Overexpression of wild-type PDK-1 provoked increases in the activity of cotransfected hemagglutinin (HA)-tagged PKC-zeta and concomitantly enhanced HA-tagged GLUT4 translocation. Expression of both kinase-inactive PDK-1 and an activation-resistant form of PKC-zeta that is mutated at Thr-410, the immediate target of PDK-1 in the activation loop of PKC-zeta, inhibited insulin-induced increases in both HA-PKC-zeta activity and HA-GLUT4 translocation to the same extent as kinase-inactive PKC-zeta. Moreover, the inhibitory effects of kinase-inactive PDK-1 were fully reversed by cotransfection of wild-type PDK-1 and partly reversed by wild-type PKC-zeta, but not by wild-type PKB. In contrast to the T410A PKC-zeta mutant, an analogous double mutant of PKB (T308A/S473A) that is resistant to PDK-1 activation had only a small effect on insulin-stimulated HA-GLUT4 translocation and did not inhibit HA-GLUT4 translocation induced by overexpression of wild-type PDK-1. Our findings suggest that both PDK-1 and its downstream target, Thr-410 in the activation loop of PKC-zeta, are required for insulin-stimulated glucose transport.

Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells.

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Previously, we have demonstrated that insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicited a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, we also studied a mutant IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did not promote translocation of GLUT4-HA, but actually inhibited the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4.

Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells.

Stimulation of glucose transport is among the most important metabolic actions of insulin. Studies in adipose cells have demonstrated that insulin stimulates its receptor to phosphorylate tyrosine residues in IRS-1, leading to activation of phosphatidylinositol 3-kinase, which plays a necessary role in mediating the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface. Akt is a serine-threonine kinase recently identified as a direct downstream target of phosphatidylinositol 3-kinase. A previous study in 3T3-L1 cells showed that overexpression of a constitutively active mutant of Akt is sufficient to recruit GLUT4 to the cell surface. Since effects of overexpression of signaling molecules in tissue culture models do not always reflect physiological function, we have overexpressed a dominant inhibitory mutant of Akt in rat adipose cells to investigate the effects of inhibiting endogenous Akt in a physiologically relevant insulin target cell. Cells were transfected with either wild type (Akt-WT), constitutively active (Akt-myr), or dominant inhibitory (Akt-K179A) forms of Akt, and effects of overexpression of these constructs on insulin-stimulated translocation of a cotransfected epitope-tagged GLUT4 were studied. Overexpression of Akt-WT resulted in significant translocation of GLUT4 to the cell surface even in the absence of insulin. Interestingly, overexpression of Akt-myr resulted in an even larger effect that was independent of insulin. More importantly, overexpression of Akt-K179A (kinase-inactive mutant) significantly inhibited insulin-stimulated translocation of GLUT4. Taken together, our data suggest that Akt is not only capable of stimulating the translocation of GLUT4 but that endogenous Akt is likely to play a significant physiological role in insulin-stimulated glucose uptake in insulin targets such as muscle and adipose tissue.

Tyr(612) and Tyr(632) in human insulin receptor substrate-1 are important for full activation of insulin-stimulated phosphatidylinositol 3-kinase activity and translocation of GLUT4 in adipose cells.

To examine contributions of specific YXXM motifs in human insulin receptor substrate-1 (IRS-1) to mediating the metabolic actions of insulin, we studied IRS-1 mutants containing various substitutions of Phe for Tyr. In transfected NIH-3T3(IR) cells, insulin stimulation caused a 5-fold increase in phosphatidylinositol 3-kinase (PI3K) activity coimmunoprecipitated with wild-type IRS-1. No PI3K activity was associated with IRS1-F6 (Phe substituted for Tyr at positions 465, 612, 632, 662, 941, and 989). Adding back both Tyr(612) and Tyr(632) fully restored IRS-1-associated PI3K activity, whereas adding back either Tyr(612) or Tyr(632) alone was associated with intermediate PI3K activity. In rat adipose cells transfected with epitope-tagged GLUT4, insulin stimulation caused a 2-fold increase in cell surface GLUT4-HA. Cotransfection of cells with GLUT4-HA and either wild-type IRS-1 or IRS1-Y612/Y632 increased basal cell surface GLUT4-HA (in the absence of insulin) to approximately 80% of the levels seen in insulin-stimulated control cells, whereas overexpression of IRS1-F6 had no effect on the insulin dose-response curve. Overexpression of IRS1-Y612 or IRS1-Y632 caused intermediate effects. Thus, both Tyr(612) and Tyr(632) are important for IRS-1 to fully activate PI3K and mediate translocation of GLUT4 in response to insulin.

Regulation of leptin promoter function by Sp1, C/EBP, and a novel factor.

Leptin is a hormone produced in adipose cells that regulates energy expenditure, food intake, and adiposity. To understand leptin's transcriptional regulation, we are studying its promoter. Four conserved and functional regions were identified. Mutations in the C/EBP and TATA motifs each caused an approximately 10-fold decrease in promoter activity. The C/EBP motif bound recombinant C/EBP alpha and mediated trans-activation by C/EBP alpha, -beta, and -delta. Mutation of a consensus Sp1 site reduced promoter activity 2.5-fold and abolished binding of Sp1. Mutation of a fourth factor-binding site, denoted LP1, abolished protein binding and reduced promoter activity 2-fold. Factor binding to the LP1 motif was observed with adipocyte, but not with nonadipocyte extracts. Adipocytes from fa/fa Zucker rats transcribed the reporter plasmids more efficiently than did control adipocytes. No effect on the transient expression of leptin was noted upon treatment with a thiazolidinedione, BRL49653, or upon cotransfection with peroxisome proliferator-activated receptor-gamma/retinoid X receptor-alpha or sterol response element-binding protein-1. Mutations of the Sp1, LP1, and C/EBP sites in pairwise combinations diminished promoter activity to the extent predicted assuming these motifs contribute independently to leptin promoter function. Our identification of motifs regulating leptin transcription is an important step in the elucidation of the mechanisms underlying hormonal and metabolic regulation of this gene.

Alpha2-Heremans Schmid glycoprotein inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in rat adipose cells.

Alpha2-Heremans Schmid glycoprotein (alpha2-HSG) is a member of the fetuin family of serum proteins whose biological functions are not completely understood. There is a consensus that alpha2-HSG plays a role in the regulation of tissue mineralization. However, one aspect of alpha2-HSG function that remains controversial is its ability to inhibit the insulin receptor tyrosine kinase and the biological actions of insulin. Interestingly, some studies suggest that alpha2-HSG differentially inhibits mitogenic, but not metabolic, actions of insulin. However, these previous studies were not carried out in bona fide insulin target cells. Therefore, in the present study we investigate the effects of alpha2-HSG in the physiologically relevant rat adipose cell. We studied insulin-stimulated translocation of the insulin-responsive glucose transporter GLUT4 in transfected rat adipose cells overexpressing human alpha2-HSG. In addition, we measured insulin-stimulated glucose transport in adipose cells cultured with conditioned medium from the transfected cells as well as in freshly isolated adipose cells treated with purified human alpha2-HSG. Compared with control cells, we were unable to demonstrate any significant effect of alpha2-HSG on insulin-stimulated translocation of GLUT4 or glucose transport. In contrast, we did demonstrate that overexpression of alpha2-HSG in adipose cells inhibits both basal and insulin-stimulated phosphorylation of Elk-1 (a transcription factor phosphorylated and activated by mitogen-activated protein kinase and other related upstream kinases). Interestingly, we did not observe any major effects of alpha2-HSG to inhibit insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate-1, -2, or -3, in either transfected or freshly isolated adipose cells. We conclude that alpha2-HSG inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in adipose cells by a mechanism that may involve effector molecules downstream of insulin receptor substrate proteins.

Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans.

Insulin resistance plays an important role in the pathophysiology of diabetes and is associated with obesity and other cardiovascular risk factors. The "gold standard" glucose clamp and minimal model analysis are two established methods for determining insulin sensitivity in vivo, but neither is easily implemented in large studies. Thus, it is of interest to develop a simple, accurate method for assessing insulin sensitivity that is useful for clinical investigations. We performed both hyperinsulinemic isoglycemic glucose clamp and insulin-modified frequently sampled iv glucose tolerance tests on 28 nonobese, 13 obese, and 15 type 2 diabetic subjects. We obtained correlations between indexes of insulin sensitivity from glucose clamp studies (SI(Clamp)) and minimal model analysis (SI(MM)) that were comparable to previous reports (r = 0.57). We performed a sensitivity analysis on our data and discovered that physiological steady state values [i.e. fasting insulin (I(0)) and glucose (G(0))] contain critical information about insulin sensitivity. We defined a quantitative insulin sensitivity check index (QUICKI = 1/[log(I(0)) + log(G(0))]) that has substantially better correlation with SI(Clamp) (r = 0.78) than the correlation we observed between SI(MM) and SI(Clamp). Moreover, we observed a comparable overall correlation between QUICKI and SI(Clamp) in a totally independent group of 21 obese and 14 nonobese subjects from another institution. We conclude that QUICKI is an index of insulin sensitivity obtained from a fasting blood sample that may be useful for clinical research.

Vasodilator response to systemic but not to local hyperinsulinemia in the human forearm.

Insulin-mediated vasodilation has been proposed as an important determinant of whole-body insulin-stimulated glucose disposal. However, it is not clear whether the vasodilator effect of insulin results from a direct action of the hormone or whether alternative mechanisms are involved. To better characterize the mechanism of insulin-mediated vasorelaxation, we compared forearm blood flow (FBF) responses to local (intra-arterial) and systemic (intravenous, euglycemic clamp) hyperinsulinemia in 10 healthy lean subjects using venous occlusion plethysmography. In addition, we assessed the effect of nitric oxide (NO) synthase inhibition by NG-monomethyl-L-arginine (L-NMMA) on the vasodilator and metabolic responses to hyperinsulinemia. Similar forearm concentrations of insulin were achieved during local and systemic infusion (231+/-39 versus 265+/-22 microU/mL; P=0.54). Of note, FBF did not change significantly in response to local hyperinsulinemia (from 2.6+/-0.3 to 2.4+/-0.3 mL . min-1 . dL-1; P=0.50). In contrast, systemic hyperinsulinemia caused a 52% increase in FBF (from 2.5+/-0.2 to 3. 8+/-0.5 mL . min-1 . dL-1; P<0.004), which was reversed by L-NMMA (FBF decreased from 3.8+/-0.5 to 2.3+/-0.2 mL . min-1 . dL-1; P=0. 004). We conclude that systemic, but not local, hyperinsulinemia induces vasodilation in the forearm. Our findings suggest that insulin-mediated vasodilation is not due solely to a direct stimulatory effect of insulin but involves additional mechanisms activated only during systemic hyperinsulinemia.

Repeatability characteristics of simple indices of insulin resistance: implications for research applications.

The objectives of this study were to evaluate test characteristics, such as normality of distribution, variation, and repeatability, of simple fasting measures of insulin sensitivity and to use the results to choose among these measures. Duplicate fasting samples of insulin and glucose were collected before 4 h of euglycemic hyperinsulinemic clamping using insulin infusion rates ranging from 40-600 mU/m2 x min. Currently recommended estimates of insulin sensitivity, including the fasting insulin, 40/insulin, the homeostasis model assessment, the logarithmic transformation of the homeostasis model assessment, and the Quantitative Insulin Sensitivity Check Index, were evaluated. The normality of distribution and the variability of the tests (coefficient of variation and discriminant ratio) were compared between the measures and against the "gold standard" hyperinsulinemic clamp. Data from 253 clamp studies in 152 subjects were examined, including 79 repeated studies for repeatability analysis. In subjects ranging from lean to diabetic, the log transformed fasting measures combining insulin and glucose had normal distributions and test characteristics superior to the other simple indices (logarithmic transformation of the homeostasis model assessment coefficient of variation, 0.55; discriminant ratio, 13; Quantitative Insulin Sensitivity Check Index coefficient of variation, 0.05; discriminant ratio, 10) and statistically comparable to euglycemic hyperinsulinemic clamps (coefficient of variation, 0.10; discriminant ratio, 6.4). These favorable characteristics helped explain the superior correlations of these measures with the hyperinsulinemic clamps among insulin-resistant subjects. Furthermore, therapeutic changes in insulin sensitivity were as readily demonstrated with these simple measures as with the hyperinsulinemic clamp. The test characteristics of the logarithmic transformation of the homeostasis model assessment and the Quantitative Insulin Sensitivity Check Index are superior to other simple indices of insulin sensitivity. This helps explain their excellent correlations with formal measures both at baseline and with changes in insulin sensitivity and supports their broader application in clinical research.