A Nonradioactive Assay for the Insulin Receptor Tyrosine Kinase: Use in Monitoring Receptor Kinase Activity after Activation of Overexpressed Protein Kinase Cα and High Glucose Treatment

Boge, Annegret; Roth, Richard A.

doi:10.1006/abio.1995.9992

Cited by 18 publications

(15 citation statements)

References 4 publications

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“…The material was then centrifuged at 100,000g for 60 min at 4°C and the supernatant collected and stored at Ϫ70°C. Muscle IR tyrosine phosphorylation was determined using an ELISA (12,20). Ninety-six-well microtiter plates were coated with 0.2 g anti-IR antibody MA-20, 100 l solubilized muscle extract (600 pg IR/ml) was added to each well, and receptors were allowed to bind overnight at 4°C.…”

Section: Subjectsmentioning

confidence: 99%

Plasma Adiponectin Concentration Is Associated With Skeletal Muscle Insulin Receptor Tyrosine Phosphorylation, and Low Plasma Concentration Precedes a Decrease in Whole-Body Insulin Sensitivity in Humans

et al. 2002

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Adiponectin, the most abundant adipose-specific protein, has been found to be negatively associated with degree of adiposity and positively associated with insulin sensitivity in Pima Indians and other populations. Moreover, adiponectin administration to rodents has been shown to increase insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and also increase whole-body insulin sensitivity. To further characterize the relationship between plasma adiponectin concentration and insulin sensitivity in humans, we examined 1) the cross-sectional association between plasma adiponectin concentration and skeletal muscle IR tyrosine phosphorylation and 2) the prospective effect of plasma adiponectin concentration at baseline on change in insulin sensitivity. Fasting plasma adiponectin concentration, body composition (hydrodensitometry or dual energy X-ray absorptiometry), insulin sensitivity (insulinstimulated glucose disposal, hyperinsulinemic clamp), and glucose tolerance (75-g oral glucose tolerance test) were measured in 55 Pima Indians (47 men and 8 women, aged 31 ؎ 8 years, body fat 29 ؎ 8% [mean ؎ SD]; 50 with normal glucose tolerance, 3 with impaired glucose tolerance, and 2 with diabetes). Group 1 (19 subjects) underwent skeletal muscle biopsies for the measurement of basal and insulin-stimulated tyrosine phosphorylation of the IR (stimulated by 100 nmol/l insulin). The fold increase after insulin stimulation was calculated as the ratio between maximal and basal phosphorylation.Group 2 (38 subjects) had follow-up measurements of insulin-stimulated glucose disposal. Cross-sectionally, plasma adiponectin concentration was positively associated with insulin-stimulated glucose disposal (r ‫؍‬ 0.58, P < 0.0001) and negatively associated with percent body fat (r ‫؍‬ ؊0.62, P < 0.0001) in the whole group. In group 1 plasma adiponectin was negatively associated with the basal (r ‫؍‬ ؊0.65, P ‫؍‬ 0.003) and positively associated with the fold increase in IR tyrosine phosphorylation (r ‫؍‬ 0.69, P ‫؍‬ 0.001) before and after the adjustment for percent body fat (r ‫؍‬ ؊0.58, P ‫؍‬ 0.01 and r ‫؍‬ 0.54, P ‫؍‬ 0.02, respectively). Longitudinally, after adjustment for age, sex, and percent body fat, low plasma adiponectin concentration at baseline was associated with a decrease in insulin sensitivity (P ‫؍‬ 0.04). In conclusion, our cross-sectional data suggest a role of physiological concentration of fasting plasma adiponectin in the regulation of skeletal muscle IR tyrosine phosphorylation. Prospectively, low plasma adiponectin concentration at baseline precedes a decrease in insulin sensitivity. Our data indicate that adiponectin plays an important role in regulation of insulin sensitivity in humans. Diabetes 50:1884 -1888, 2002 A dipose tissue serves not only as an energy storage organ, but also secretes hormones and metabolites that are thought to regulate insulin sensitivity and energy metabolism (1,2). Adiponectin, the most abundant adipose-specific protein, is exclusively expressed in and secreted from a...

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Section: Subjectsmentioning

confidence: 99%

Plasma Adiponectin Concentration Is Associated With Skeletal Muscle Insulin Receptor Tyrosine Phosphorylation, and Low Plasma Concentration Precedes a Decrease in Whole-Body Insulin Sensitivity in Humans

et al. 2002

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“…Cell lysates were cleared by centrifugation (12,000 g at 4°C for 15 min) and stored at Ϫ80°C until assayed. IR autophosphorylation was detected by a plate-capture enzyme-linked immunosorbent assay (ELISA) [Boge and Roth, 1995]. Ninety-six-well microtiter plates (Nunc Immunoplate I Maxisorp) were coated with 100 µl of a specific monoclonal anti-insulin receptor antibody (2 µg/ml in 50 mM bicarbonate buffer, pH 9.0) for 16 h at 4°C.…”

Section: Insulin Receptor Radioimmunoassay and Insulin Receptor Autopmentioning

confidence: 99%

Insulin-stimulated cell growth in insulin receptor substrate-1–deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase–independent pathway

et al. 1998

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In many human breast cancers and cultured cell lines, insulin receptor expression is elevated, and insulin, via its own insulin receptor, can stimulate cell growth. It has recently been demonstrated that the enzyme phosphatidylinositol-3-kinase (PI3-K) mediates various aspects of insulin receptor signaling including cell growth. In order to understand the mechanisms for insulin-stimulated cell growth in human breast cancer, we measured insulin-stimulable PI3-K activity in a non-transformed breast epithelial cell line, MCF-10A, and in two malignantly transformed cell lines, ZR-75-1 and MDA-MB157. All three cell lines express comparable amounts of insulin receptors whose tyrosine autophosphorylation is increased by insulin, and in these cell lines insulin stimulates growth. In MDA-MB157 and MCF-10A cells, insulin stimulated PI3-K activity three- to fourfold. In ZR-75-1 cells, however, insulin did not stimulate PI3-K activity. In ZR-75-1 cells PI3-K protein was present, and its activity was stimulated by epidermal growth factor, suggesting that there might be a defect in insulin receptor signaling upstream of PI3-K and downstream of the insulin receptor. Next, we studied insulin receptor substrate-1 (IRS-1), a major endogenous substrate for the insulin receptor which, when tyrosine is phosphorylated by the insulin receptor, interacts with and activates PI3-K. In ZR-75-1 cells, there were reduced levels of protein for IRS-1. In these cells, both Shc tyrosine phosphorylation and mitogen-activated protein kinase (MAP-K) activity were increased by the insulin receptor (indicating that the p21ras pathway may account for insulin-stimulated cell growth in ZR-75-1 cells). The PI3-K inhibitor LY294002 (50 microM) reduced insulin-stimulated growth in MCF-10A and MDA-MB157 cell lines, whereas it did not modify insulin effect on ZR-75-1 cell growth. The MAP-K/Erk (MEK) inhibitor PD98059 (50 microM) consistently reduced insulin-dependent growth in all three cell lines. Taken together, these data suggest that in breast cancer cells insulin may stimulate cell growth via PI3-K-dependent or-independent pathways.

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“…Biotinylation of secondary antibodies used in both assays was performed as previously described [37].…”

Section: Insulin Receptor and Pc-1 Elisamentioning

confidence: 99%

Increased adipose tissue PC-1 protein content, but not tumour necrosis factor-a gene expression, is associated with a reduction of both whole body insulin sensitivity and insulin receptor tyrosine-kinase activity

et al. 1997

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The pathophysiology of non-insulin-dependent diabetes mellitus (NIDDM) is complex, consisting of both abnormal insulin secretory patterns, and resistance to the actions of insulin [1][2][3][4][5][6][7][8]. While insulin resistance is a characteristic feature of many NIDDM patients, it is also observed in non-diabetic subjects [1]. In fact, it has been estimated that up to 25 % of normoglycaemic non-obese individuals are as insulin resistant as NIDDM patients [1], and it has been suggested that a significant proportion of these individuals will eventually develop diabetes if either insulin secretion declines and/or insulin resistance increases [1]. However, in most individuals, the cellular mechanisms causing human insulin resistance are not understood [3][4][5][6][7][8].Insulin regulates cellular functions by binding to the insulin receptor, a tetrameric plasma membrane glycoprotein of the receptor tyrosine-kinase family [6][7][8]. After binding, insulin activates receptor tyrosine-kinase activity, leading to the phosphorylation of key intracellular substrates, and insulin action ensues [6][7][8]. Many studies have been performed on insulin receptor tyrosine-kinase activity in NIDDM Diabetologia (1997) 40: 282-289 Increased adipose tissue PC-1 protein content, but not tumour necrosis factor-a gene expression, is associated with a reduction of both whole body insulin sensitivity and insulin receptor tyrosine-kinase activity Summary In the present study we measured PC-1 content, tumour necrosis factor (TNF)-a gene expression, and insulin stimulation of insulin receptor tyrosine-kinase activity in adipose tissue from nonobese, non-diabetic subjects. These parameters were correlated with in vivo insulin action as measured by the intravenous insulin tolerance test (K itt values). PC-1 content was negatively correlated with K itt values (r = -0.5, p = 0.04) and positively with plasma insulin levels both fasting (r = 0.58, p = 0.009) and after 120 min during oral glucose tolerance test (OGTT) (r = 0.67, p = 0.002). Moreover, adipose tissue PC-1 content was higher in relatively insulin-resistant subjects (K itt values lower than 6) than in relatively insulin-sensitive subjects (K itt values higher than 6) (525 ± 49 ng/mg protein vs 336 ± 45, respectively, p = 0.012). Adipose tissue insulin receptor tyrosinekinase activity in response to insulin was significantly lower at all insulin concentrations tested (p = 0.017, by two-way analysis of variance test) in insulin-resistant than in insulin-sensitive subjects (K itt values lower or higher than 6, respectively). In contrast to PC-1, no significant correlation was observed between adipose tissue TNF-a mRNA content and K itt values, and plasma insulin levels, both fasting and at after 120 min during OGTT. Also, no difference was observed in TNF-a mRNA content between subjects with K itt values higher or lower than 6. These studies in adipose tissue, together with our previous studies in skeletal muscle raise the possibility that PC-1, by regulating insulin receptor funct...

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A Nonradioactive Assay for the Insulin Receptor Tyrosine Kinase: Use in Monitoring Receptor Kinase Activity after Activation of Overexpressed Protein Kinase Cα and High Glucose Treatment

Cited by 18 publications

References 4 publications

Plasma Adiponectin Concentration Is Associated With Skeletal Muscle Insulin Receptor Tyrosine Phosphorylation, and Low Plasma Concentration Precedes a Decrease in Whole-Body Insulin Sensitivity in Humans

Plasma Adiponectin Concentration Is Associated With Skeletal Muscle Insulin Receptor Tyrosine Phosphorylation, and Low Plasma Concentration Precedes a Decrease in Whole-Body Insulin Sensitivity in Humans

Insulin-stimulated cell growth in insulin receptor substrate-1–deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase–independent pathway

Increased adipose tissue PC-1 protein content, but not tumour necrosis factor-a gene expression, is associated with a reduction of both whole body insulin sensitivity and insulin receptor tyrosine-kinase activity

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