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Receptor-associated protein kinase activity has been shown in all primary target tissues of insulin action in the rat and a function of insulin receptor phosphorylation in signal transmission was proposed. Insulin receptor phosphorylation so far has not been demonstrated in human target tissues of insulin. We describe here insulin receptor kinase activity in human skeletal muscle. Insulin (10 −8moll) stimulates the phosphorylation of a 95-kDa protein from skeletal muscle 2-fold. The phosphoprotein is quantitatively immunoprecipitated with insulin receptor antibody identifying it as the β-subunit of the insulin receptor. The insulin stimulation of phosphorylation is detectable also at physiological insulin concentrations (10 −9moll) showing that receptor phosphorylation could be involved in insulin action in human skeletal muscle as well.

The insulin receptor purified from skeletal muscle of patients with non-insulin-dependent diabetes mellitus (NIDDM) displayed a 25–55% reduction in insulin-stimulated autophosphorylation and tyrosyl-specific phosphotransferase activity relative to controls. This decrease was not explained by alterations of muscle fiber composition, insulin binding affinity or capacity, or the Kmvalues for ATP; the lower kinase activity was entirely attributed to a decrease in the Vmaxof the enzyme. Phosphorylation sites in the β-subunit of the control and diabetic receptor were identified by tryptic digestion and reverse-phase high performance liquid chromatography. Autophosphorylation occurred primarily in two regions of the β-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C terminus containing Tyr-1316 and 1322. Autophosphorylation of the regulatory region at all three tyrosyl residues (tris-phosphorylation) appears to be necessary to activate the receptor kinase (White, M. F., Shoelson, S. E., Stepman, E. W., Keutmann, H. & Kahn, C. R. (1988) J. Biol. Chem. 263, 2969–2980). The receptor from NIDDM patients showed a decreased level of tris-phosphorylation of the regulatory region which was closely associated (r2= 0.97) with the decreased kinase activity. In contrast, weak associations were found between kinase activity and the bis-phosphorylated forms of the regulatory region (r2= 0.51) and the C terminus (r2= 0.35). Therefore, the reduced formation of the tris-phosphorylated regulatory region in the diabetic receptors suggests that a defective autophosphorylation cascade leading to tris-phosphorylation of the regulatory region may cause, in part, the reduced insulin-stimulated kinase activity of the insulin receptor in muscle of NIDDM patients.

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.

Phorbol ester treatment of isolated rat adipocytes inhibits insulin stimulation of the glucose transport system. We studied whether this effect is related to a modification of the insulin receptor kinase. Insulin receptor of tetradecanoyl-beta-phorbol acetate (TPA)-treated adipocytes was solubilized and partially purified, and its kinase activity was studied in vitro. We found that insulin (10(-7) M) increased the tyrosine autophosphorylation of the insulin receptor kinase from TPA-treated cells only 3-fold in contrast to a 12-fold stimulation in control cells. The rate of insulin-stimulated 32P incorporation into the receptor of TPA-treated cells at insulin concentrations between 10(-10) M and 10(-7) M and at nonsaturating [32P]ATP levels (5 microM) was reduced to only 5-8% of the values found with receptor from control cells. 125I-insulin binding to the solubilized receptor of TPA-treated cells was reduced as well, apparently due to a decreased affinity of the binding site. Decreased binding was however, not sufficient to explain the difference of kinase activity. The inhibition of kinase activity of the receptor from TPA-treated cells decreased when the concentration of [gamma-32P]ATP in the phosphorylation assay was increased. A Lineweaver-Burk analysis of receptor phosphorylation revealed that the Km for ATP of the receptor kinase from TPA-treated cells was increased to greater than 100 microM compared to 25 microM for the receptor of control cells. An analogous change of the Km for ATP was found when we studied the phosphorylation of a synthetic polymer of tyrosine and glutamic acid as a substrate of the receptor kinase. We conclude from the data that phorbol treatment of rat adipocytes modulates the kinase activity of the insulin receptor by increasing its Km for ATP and that this is part of a mechanism leading to insulin resistance in these cells.

The insulin effect on glucose uptake is not sufficiently explained by a simple glucose-carrier translocation model. Recent studies rather suggest a two-step model of carrier translocation and carrier activation. We used several pharmacological tools to characterize the proposed model further. We found that inositol phosphate (IP)-oligosaccharides isolated from the drug Actovegin, as well as the alkaloid vinblastine, show a partial insulin-like effect on glucose-transport activity of fat-cells (3-O-methylglucose uptake, expressed as % of equilibrium value per 4 s: basal 5.8%, insulin 59%, IP-oligosaccharides 30%, vinblastine 29%) without inducing carrier translocation. On the other hand, two newly developed anti-diabetic compounds (alpha-activated carbonic acids, BM 130795 and BM 13907) induced carrier translocation to the same extent as insulin and phorbol esters [cytochalasin-B-binding sites in plasma membranes: basal 5 pmol/mg of protein, insulin 13 pmol/mg of protein, TPA (12-O-tetradecanoylphorbol 13-acetate) 11.8 pmol/mg of protein, BM 130795 10.8 pmol/mg of protein], but produce also only 40-50% of the insulin effect on glucose-transport activity (basal 5.8%, insulin 59%, TPA 23%, BM 130795 35%). Almost the full insulin effect was mimicked by a combination of phorbol esters and IP-oligosaccharides (basal 7%, insulin 50%, IP-oligosaccharides 30%, TPA 23%, IP-oligosaccharides + TPA 45%). None of these substances stimulated insulin-receptor kinase in vitro or in vivo, suggesting a post-kinase site of action. The data confirm the following aspects of the proposed model: (1) carrier translocation and carrier activation are two independently regulated processes; (2) the full insulin effect is mimicked only by a simultaneous stimulation of carrier translocation and intrinsic carrier activity, suggesting that insulin acts through a synergism of both mechanisms; (3) IP-oligosaccharides might be involved in the transmission of a stimulatory signal on carrier activity.