Insulin-stimulated Receptor Tyrosine Kinase Activity Research Articles

Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats.

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.

Phorbol Esters Induce Insulin Receptor Phosphorylation in Transfected Fibroblasts without Affecting Tyrosine Kinase Activity

The effects of phorbol ester induced activation of protein kinase C on insulin receptor phosphorylation and tyrosine kinase activity have been investigated in transfected fibroblasts expressing high levels of the human insulin receptor. Receptor phosphorylation was stimulated more than two-fold over basal levels upon treating CHO.T cells with PMA. This phosphorylation was additive with, rather than antagonistic to, that induced by insulin. Furthermore, PMA treatment was completely without effect on insulin-stimulated receptor tyrosine kinase activity. Similar results were obtained in NIH3T3 HIR3.5 and Rat 1 HIRc-B cells. It is concluded that the previously reported inhibitory effect of PMA on receptor kinase activity is not of general regulatory significance in all cell types.

Cyclic AMP impairs the rapid effect of insulin to enhance cell-surface insulin-binding capacity in rat adipocytes.

The aim of this study was to characterize further the interaction between cyclic AMP (cAMP) and insulin binding and action. Rat adipocytes were preincubated at 37 degrees C for 20 min, and after energy depletion with KCN, cell-surface 125I-insulin binding was measured. As recently reported [Eriksson, Lönnroth & Smith (1992) Diabetes 41, 707-714], preincubation with insulin rapidly increased the number of cell-surface insulin binding sites up to approximately 5-fold through recruitment within the plasma membrane. This was completely abolished by the presence of 4 mM-N6-monobutyryl cAMP (a non-hydrolysable cAMP analogue) or 1 microM-isoprenaline, without any apparent change in receptor internalization. Insulin-stimulated receptor tyrosine kinase activity was attenuated by the cAMP analogue only if the exposure of the adipocytes was prolonged to 60 min. The cellular sensitivity to insulin, assessed as 3-O-methylglucose uptake, was markedly decreased by the cAMP analogue, and this could be attributed to the impaired cell-surface binding. However, evidence for post-receptor interactions between cAMP and insulin was also found: an impairment of maximal insulin-stimulated 3-O-methylglucose transport and a delay in the rate of activation of the glucose transport system by insulin. In conclusion, these data demonstrate that beta-adrenergic stimulation and elevated cAMP levels markedly impair the ability of insulin to enhance cell-surface insulin-binding capacity. This novel interaction may be an important mechanism for the cellular insensitivity to insulin produced by cAMP.

Evidence for the lack of spare high-affinity insulin receptors in skeletal muscle.

In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full occupancy of high-affinity binding sites. Therefore our data provide evidence for a lack of spare high-affinity insulin receptors in skeletal muscle.

Mechanism of Defective Insulin-Receptor Kinase Activity in NIDDM: Evidence for Two Receptor Populations

We used anti-insulin-receptor and anti-phosphotyrosine antibodies to elucidate the mechanism of decreased insulin-receptor tyrosine kinase activity observed in subjects with non-insulin-dependent diabetes mellitus (NIDDM). Lectin-purified insulin receptors were labeled with 125I-labeled NAPA-DP-insulin and autophosphorylated in the presence of 500 microM unlabeled ATP. Immunoprecipitation occurred in 43 +/- 8% of the autophosphorylated, 125I-labeled receptors from nondiabetic subjects with anti-phosphotyrosine antibodies in contrast to 100% immunoprecipitation with anti-insulin-receptor antibodies. Anti-phosphotyrosine antibodies immunoprecipitated only 14 +/- 6% of NIDDM receptors (P less than .05 vs. nondiabetic receptors). A significant correlation existed between maximal insulin-stimulated receptor tyrosine kinase activity and the proportion of receptors immunoprecipitated by anti-phosphotyrosine antibodies (r = .76, P less than .01). These results suggest that human adipocytes contain two distinct receptor populations, both of which bind insulin but only one of which is capable of insulin-stimulated tyrosine phosphorylation. In nondiabetic subjects, 40-50% of the receptors that bind insulin are capable of insulin-stimulated tyrosine autophosphorylation. The proportion of receptors that bind insulin but are incapable of insulin-stimulated tyrosine autophosphorylation is increased in NIDDM; the magnitude of this increase correlated with the magnitude of the decrease in kinase activity.