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Time-dependence of biological activity induced by covalent insulin-receptor complexes in rat adipocytes.

ABSTRACT: Lipogenesis in isolated adipocyte preparations is stimulated when photosensitive insulin derivatives are attached covalently to specific receptors. This response was compared quantitatively with that to reversibly associated insulin, and it was shown that both covalent and reversible insulin-receptor complexes behave very similarly. The extent of stimulation of lipogenesis was studied as a function of time. Cells were incubated in buffer for various times before addition to vials containing 0 (basal) or 10 ng of monocomponent insulin/ml (maximal) and [U-3H]glucose. After 60 min, the toluene-soluble [3H]lipids were measured. The maximal stimulation induced by reversibly bound insulin was virtually constant over a period of 4 h. In contrast, adipocytes to which N alpha B2-(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin had been covalently attached at the start of the experiment showed a loss of stimulation with time when incubated at 37 degrees C. This loss was decreased in the presence of lysosomotropic agents such as chloroquine at concentrations (approx. 200 microM) that had very little or no effect on the basal and maximal lipogenesis rates. A simple method was used to transform the measured rate of loss of stimulation into a rate of loss of effective units. A half-time of 80 min was calculated for the effective covalent insulin-receptor units in adipocytes at 37 degrees C at pH 7.4. This is very close to values reported by others for the internalization of covalent complexes in these cells, suggesting that this may be the causative event for the deactivation of the insulin-receptor unit. The inhibitory effect of chloroquine on the deactivation may indicate that the insulin-receptor complex can function even after internalization.

SUBMITTER: Schuttler A

PROVIDER: S-EPMC1152837 | biostudies-other | 1985 Nov

REPOSITORIES: biostudies-other

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Project description:Catecholamine treatment of isolated rat adipocytes decreases insulin binding and inhibits insulin stimulation of the glucose-transport system. There is increasing evidence that the insulin signal is transmitted after insulin is bound to the receptor via a tyrosine kinase, which is an intrinsic part of the receptor. To find whether the receptor kinase is modified by catecholamines, we solubilized and partially purified the insulin receptor of isoprenaline-treated adipocytes and studied the effect of insulin on its kinase activity. (1) Insulin increased the tyrosine autophosphorylation of the insulin receptor kinase from catecholamine-treated cells only 4-fold, compared with a 12-fold stimulation in control cells. (2) The rate of insulin-stimulated 32P incorporation into the receptor of isoprenaline-treated cells at non-saturating [32P]ATP concentrations (5 muM) was decreased to 5-8% of the values for receptor from control cells. (3) 125I-insulin binding to the partially purified receptor from catecholamine-treated cells was also markedly decreased. The insulin receptor from catecholamine treated cells bound 25-50% of the amount of insulin bound by the receptor from control cells at insulin concentrations of 10 pM-0.1 muM. Part of the impaired insulin-responsiveness of the receptor kinase of catecholamine-treated cells is therefore explained by impaired binding properties; however, an additional inhibition of the kinase activity of the insulin receptor from catecholamine-treated cells is evident. (4) This inhibition of kinase activity decreased when the concentration of [gamma-32P]ATP in the phosphorylation assay was increased. A Lineweaver-Burk analysis revealed that the Km for ATP of the receptor kinase from isoprenaline-treated cells was increased to approx. 100 muM, compared with approx. 25 muM for receptor of control cells. (5) We conclude from the data that catecholamine treatment of rat adipocytes modulates the kinase activity of the insulin receptor by increasing its Km for ATP and that this is part of the mechanism leading to insulin-resistance in these cells.

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Time-dependence of biological activity induced by covalent insulin-receptor complexes in rat adipocytes.

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