Mechanism of activation of guanine nucleotide exchange factor by insulin
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Immunoprecipitation
Insulin receptor substrate
To determine whether the impaired insulin-stimulated glucose uptake in obese individuals is associated with altered insulin receptor signaling, we measured both glucose uptake and early steps in the insulin action pathway in intact strips of human skeletal muscle. Biopsies of rectus abdominus muscle were taken from eight obese and eight control subjects undergoing elective surgery (body mass index 52.9 +/- 3.6 vs 25.7 +/- 0.9). Insulin-stimulated 2-deoxyglucose uptake was 53% lower in muscle strips from obese subjects. Additional muscle strips were incubated in the basal state or with 10(-7) M insulin for 2, 15, or 30 min. In the lean subjects, tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), measured by immunoblotting with anti-phosphotyrosine antibodies, was significantly increased by insulin at all time points. In the skeletal muscle from the obese subjects, insulin was less effective in stimulating tyrosine phosphorylation (maximum receptor and IRS-1 phosphorylation decreased by 35 and 38%, respectively). Insulin stimulation of IRS-1 immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity also was markedly lower in obese subjects compared with controls (10- vs 35-fold above basal, respectively). In addition, the obese subjects had a lower abundance of the insulin receptor, IRS-1, and the p85 subunit of PI 3-kinase (55, 54, and 64% of nonobese, respectively). We conclude that impaired insulin-stimulated glucose uptake in skeletal muscle from severely obese subjects is accompanied by a deficiency in insulin receptor signaling, which may contribute to decreased insulin action.
Insulin receptor substrate
IRS1
IRS2
Basal (medicine)
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We have previously shown that in the insulin-resistant obese hyperglycemic mouse (ob/ob) there is a deficiency in the number of insulin receptor sites on hepatocytes, adipocytes, and thymic lymphocytes. We now find that concentration of insulin receptors on liver plasma membranes is decreased in the db/db mouse, another form of inherited obesity, and in normal mice that became obese after treatment with gold thioglucose, while thin mice, heterozygous for the ob mutation (ob/+), have normal insulin binding. With acute and chronic food restriction of the ob/ob and gold thioglucose obese mice, there is reduction in hyperinsulinemia and an associated increase in the insulin receptor concentration toward normal. In contrast, when fasting ob/ob mice were given exogenous insulin to maintain the hyperinsulinemia, insulin receptors failed to increase. Thus, in all cases, there was a consistent relationship between the degree of hyperinsulinemia and of insulin receptor loss. These findings suggest that decreased insulin binding is a characteristic feature of the insulin resistance of obesity, and that sustained hyperinsulinemia is a major factor in the control of the concentration of insulin receptors on target cells.
Hyperinsulinemia
Insulin receptor substrate
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Syp is a protein tyrosine phosphatase implicated in insulin and growth factor signaling. To evaluate the role of syp in insulin's regulation of plasma glucose, we generated knockout mice. Homozygous knockout mice die prior to day 10.5 of embryonic development. Hemizygous mice express half the levels of syp protein compared with their wild type littermates but do not display any gross morphological changes. Total body weight (age 2-10 weeks) and plasma insulin and glucose levels both in fasting and glucose-challenged states were comparable in the wild type and the hemizygous mice. No differences were observed in insulin-induced glucose uptake in soleus muscle and epididymal fat; insulin inhibition of lipolysis was also similar. We injected insulin into the portal vein of the mice to examine upstream events of the insulin signaling cascade. Tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) from hemizygous tissue was similar to that of wild type tissue. Association of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 increased an average of 2-fold in both groups. We did not observe an increase of IRS-1/syp association after insulin administration, but we did note a significant basal association in both wild type and hemizygous tissue. Our results do not support a major role for syp in the acute in vivo metabolic actions of insulin. Syp is a protein tyrosine phosphatase implicated in insulin and growth factor signaling. To evaluate the role of syp in insulin's regulation of plasma glucose, we generated knockout mice. Homozygous knockout mice die prior to day 10.5 of embryonic development. Hemizygous mice express half the levels of syp protein compared with their wild type littermates but do not display any gross morphological changes. Total body weight (age 2-10 weeks) and plasma insulin and glucose levels both in fasting and glucose-challenged states were comparable in the wild type and the hemizygous mice. No differences were observed in insulin-induced glucose uptake in soleus muscle and epididymal fat; insulin inhibition of lipolysis was also similar. We injected insulin into the portal vein of the mice to examine upstream events of the insulin signaling cascade. Tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) from hemizygous tissue was similar to that of wild type tissue. Association of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 increased an average of 2-fold in both groups. We did not observe an increase of IRS-1/syp association after insulin administration, but we did note a significant basal association in both wild type and hemizygous tissue. Our results do not support a major role for syp in the acute in vivo metabolic actions of insulin.
Insulin receptor substrate
IRS1
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Insulin receptor substrate
IRS2
IRS1
Insulin oscillation
Pancreatic Islets
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Immunoprecipitation
Insulin receptor substrate
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Diabetes induced by streptozotocin (STZ) in laboratory rats leads to impaired glucose metabolism in the heart and changes in myocardial contractile protein isoform expression and cardiac function. The purpose of this study was to investigate in vivo insulin signaling responses in the myocardium of STZ-diabetic rats. Insulin rapidly stimulated tyrosine phosphorylation of the insulin receptor, insulin receptor substrate-1 (IRS-1) and, to a lesser extent, IRS-2 in normal and diabetic myocardium. In diabetic rats, there was 2-fold higher insulin receptor content and insulin-stimulated receptor tyrosine phosphorylation in comparison with control rats. IRS-1 tyrosine phosphorylation also increased in STZ diabetes in spite of a decrease in IRS-1 content, resulting in a 4-fold higher ratio of phosphorylated to total IRS-1. This was associated with 2-fold higher IRS-1 precipitable phosphatidylinositide 3-kinase activity in diabetic animals. Insulin stimulation of glycogen synthase activity was significantly diminished in STZ diabetes, consistent with resistance to insulin in a step downstream from phosphatidylinositide 3-kinase activation. Under the same experimental conditions, there was a marked increase in insulin stimulation of myocardial c-fos messenger RNA content in diabetic animals in comparison with controls. These data demonstrate altered early steps in insulin signaling in STZ-diabetic rat myocardium. Consequent oppositely directed disturbances in growth regulatory and glucose regulatory responses to insulin may contribute to the development of myocardial functional abnormalities in this model of diabetes.
Insulin receptor substrate
IRS1
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Insulin resistance is a predominant feature in women with polycystic ovarian syndrome (PCO). The cellular mechanisms for this insulin resistance have not been defined. In this study, major steps in the insulin action cascade, receptor binding, kinase activity, and glucose transport activity were evaluated in isolated adipocytes prepared from PCO subjects (n = 8) without acanthosis nigricans and in a group of age and weight-matched controls [normal cycling (NC) n = 8]. The PCO group was hyperinsulinemic and displayed elevated insulin responses to an iv glucose load. The binding of 125I-insulin to adipocytes was similar in cells from PCO and NC subjects. In PCO, autophosphorylation of the insulin receptor-subunit in the absence of insulin was normal but a significant decrease (30% below control) in maximal insulin stimulated autophosphorylation was observed. However, receptor kinase activity measured against the exogenous substrate poly glu:tyr (4:1) was normal. Cells from PCO subjects transported glucose at the same rate, in both the absence and presence of a maximal insulin concentration, as those from NC subjects. Strikingly, there was a large rightward shift in the insulin dose-response curve for transport stimulation in PCO cells (EC50 = 87 +/- 14 pmol in NC vs. 757 +/- 138 in PCO, P less than 0.0005); 8-fold greater insulin concentrations were required to attain comparable glucose transport rates in cells from PCO against NC. In conclusion, our results suggest that insulin resistance in PCO, as assessed in the adipocyte, is accompanied by normal function of insulin receptors, but involves a novel postreceptor defect in the insulin signal transduction chain between the receptor kinase and glucose transport.
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Hyperinsulinemia
IRS1
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Postprandial cellular glucose uptake is dependent on an insulin-signaling cascade in muscle and adipose tissue, resulting in the translocation of the insulin-dependent glucose transporter 4 (Glut4) into the plasma membrane. Additionally, extended food deprivation is characterized by suppressed insulin signaling and decreased Glut4 expression. Northern elephant seals are adapted to prolonged fasts characterized by high levels of plasma glucose. To address the hypothesis that the fasting-induced decrease in insulin is associated with reduced insulin signaling in prolonged fasted seals, we compared the adipose protein levels of the cellular insulin-signaling pathway, Glut4 and plasma glucose, insulin, cortisol, and adiponectin concentrations between Early ( n = 9; 2–3 wks postweaning) and Late ( n = 8; 6–8 wks postweaning) fasted seals. Plasma adiponectin (230 ± 13 vs. 177 ± 11 ng/ml), insulin (2.7 ± 0.4 vs. 1.0 ± 0.1 μU/ml), and glucose (9.8 ± 0.5 vs. 8.0 ± 0.3 mM) decreased, while cortisol (124 ± 6 vs. 257 ± 30 nM) doubled with fasting. Glut4 increased (31%) with fasting despite the significant decreases in the cellular content of phosphatidylinositol 3-kinase as well as phosphorylated insulin receptor, insulin receptor substrate-1, and Akt2. Increased Glut4 may have contributed to the decrease in plasma glucose, but the decrease in insulin and insulin signaling suggests that Glut4 is not insulin-dependent in adipose tissue during prolonged fasting in elephant seals. The reduction of plasma glucose independent of insulin may make these animals an ideal model for the study of insulin resistance.
GLUT4
Elephant seal
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Magnesium deficiency (plants)
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Muscle insulin resistance develops when plasma free fatty acids (FFAs) are acutely increased to supraphysiological levels (approximately 1,500-4,000 micromol/l). However, plasma FFA levels >1,000 micromol/l are rarely observed in humans under usual living conditions, and it is unknown whether insulin action may be impaired during a sustained but physiological FFA increase to levels seen in obesity and type 2 diabetes mellitus (T2DM) (approximately 600-800 micromol/l). It is also unclear whether normal glucose-tolerant subjects with a strong family history of T2DM (FH+) would respond to a low-dose lipid infusion as individuals without any family history of T2DM (CON). To examine these questions, we studied 7 FH+ and 10 CON subjects in whom we infused saline (SAL) or low-dose Liposyn (LIP) for 4 days. On day 4, a euglycemic insulin clamp with [3-3H]glucose and indirect calorimetry was performed to assess glucose turnover, combined with vastus lateralis muscle biopsies to examine insulin signaling. LIP increased plasma FFA approximately 1.5-fold, to levels seen in T2DM. Compared with CON, FH+ were markedly insulin resistant and had severely impaired insulin signaling in response to insulin stimulation. LIP in CON reduced insulin-stimulated glucose disposal (Rd) by 25%, insulin-stimulated insulin receptor tyrosine phosphorylation by 17%, phosphatidylinositol 3-kinase activity associated with insulin receptor substrate-1 by 20%, and insulin-stimulated glycogen synthase fractional velocity over baseline (44 vs. 15%; all P < 0.05). In contrast to CON, a physiological elevation in plasma FFA in FH+ led to no further deterioration in Rd or to any additional impairment of insulin signaling. In conclusion, a 4-day physiological increase in plasma FFA to levels seen in obesity and T2DM impairs insulin action/insulin signaling in CON but does not worsen insulin resistance in FH+. Whether this lack of additional deterioration in insulin signaling in FH+ is due to already well-established lipotoxicity, or to other molecular mechanisms related to insulin resistance that are nearly maximally expressed early in life, remains to be determined.
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