Insulin-Receptor Autophosphorylation and Endogenous Substrate Phosphorylation in Human Adipocytes From Control, Obese, and NIDDM Subjects

Abstract

We identified a possible endogenous substrate (pp185) of the insulin-receptor kinase in human adipocytes by treating intact cells with insulin and immunoblotting the cellular extracts with polyclonal antiphosphotyrosine antibody. This 185,000-Mr protein was phosphorylated on tyrosine residues in response to insulin in both rat and human adipocytes. The time course of pp185 phosphorylation at 37 degrees C was rapid and corresponded closely to insulin-receptor autophosphorylation but preceded insulin-stimulated glucose transport. Unlike many growth factor receptors, including the insulin receptor, pp185 was not adsorbed to wheat-germ agglutinin. We found that pp185 phosphorylation occurred at 12 degrees C and that the phosphoprotein was associated with both cytoplasmic and membrane fractions at this temperature. Furthermore, pp185 phosphorylation was induced to the same extent as insulin by vanadate and hydrogen peroxide, compounds previously shown to mimic the biologic effects of insulin. In addition, dose-response analysis of insulin-stimulated glucose transport, receptor autophosphorylation, and pp185 phosphorylation resulted in ED50 values of 0.3, 12, and 12 ng/ml, respectively. These results demonstrate the magnitude of "spare" autophosphorylation and pp185 phosphorylation with respect to glucose transport stimulation in human adipocytes. To determine whether the insulin resistance characteristic of non-insulin-dependent diabetes mellitus (NIDDM) and obesity is associated with a defect in receptor autophosphorylation and/or endogenous substrate phosphorylation, we estimated the extent of beta-subunit and pp185 phosphorylation in adipocytes from NIDDM, obese, and healthy subjects. Although the efficiency of coupling between receptor activation and pp185 phosphorylation was normal in obesity and NIDDM, the capacity for insulin-receptor autophosphorylation was approximately 50% lower in NIDDM subjects compared with nondiabetic obese or lean subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of PTP-1 and Insulin Receptor Kinase by Fractions from Cinnamon: Implications for Cinnamon Regulation of Insulin Signalling

“…One possible interpretation is that, in intact adipocytes, cinnamon compounds act directly on the insulin receptor to simulate of its kinase activity and/or inhibit PTPases which may attenuate the receptor kinase signal. Bioactivity and inhibition of tyrosine phosphatase by cinnamon would be consistent with the proposed idea that insulin resistance in adult-onset diabetes may be due to elevated tyrosine phosphatase activity [7, 8, 14]. …”

“…This insulin resistance could be the result of a number of defects in the insulin signalling pathway [2, 3, 4, 5, 6]. Much focus has been placed on evidence that insulin receptor kinase autophosphorylation and subsequent phosphorylation of its principal substrate, IRS-1, are markedly decreased in insulin-responsive tissues of subjects with severe obesity or NIDDM [7, 8, 9]. Dephosphorylation of the receptor β-subunit is associated with the deactivation of its kinase activity and therefore is associated with insulin signal down-regulation [10].…”

Circulating and cellular adiponectin in polycystic ovary syndrome: relationship to glucose tolerance and insulin action

“…Cells were concentrated by centrifugation (50 x g), then rapidly washed twice in 17 C, BSA-free HWS buffer, as described previously (30). A 2x-concentrated solubilization buffer was then added-final concentrations: 20 mM TrisHCl, 145 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 0.5% NP-40, 200 mM sodium orthovanadate, 200 mM PMSF, 1 mM leupeptin, 10 mg/mL aprotinin, pH 7.5.…”

Long-term treatment with an angiotensin II receptor blocker decreases adipocyte size and improves insulin signaling in obese Zucker rats

“…A number of studies [8,17,18] have suggested that insulin-stimulated activation of PI3K pathway is impaired in adipose tissue of insulin-resistant rodent and humans. Insulin signaling in this tissue is not critical for the maintenance of euglycemia, but it is necessary for the maintenance of normal triglyceride stores, inhibition of lipolysis and insulin-stimulated glucose uptake [19].…”