Insulin-stimulated Tyrosine Kinase Activity Research Articles

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.

Insulin receptor : tyrosine kinase activity and insulin action

The first step in insulin action consists in binding of the hormone to specific cell surface receptors. This receptor displays two functional domains: an extracellular alpha-subunit containing the majority or the totality of the hormone binding site and an intracellular beta-subunit possessing insulin-stimulated tyrosine kinase activity. A general consensus has been reached in favour of the idea that this receptor enzymic function is essential for generation of the metabolic and growth-promoting effects of insulin. Concerning the mechanism of transmembrane signalling, we like to think that interaction of insulin with the receptor alpha-subunit triggers a conformational change, which is propagated to the beta-subunit and activates it. The active receptor kinase leads then to the phosphorylation of cellular protein substrates, which are likely to belong to two broad categories, those generating metabolic effects of insulin and those resulting in growth-promoting effects. The phosphorylated and active substrates then generate the final effects of insulin.

Insulin receptor binding and tyrosine kinase activity in liver and skeletal muscle from fasted rats.

Insulin binding and tyrosine kinase activity of the insulin receptor have been measured in the liver and muscles of rats fed or submitted to a 72-h-fasting. In both tissues, insulin binding increased in fasting rats. In liver, the ability of insulin to simulate receptor tyrosine kinase activity greatly unpaired during fasting, but remained unchanged in muscle. The change during fasting of the insulin-stimulated tyrosine kinase activity of the insulin receptor is specific to certain tissue.

Characterisation of insulin-like growth factor I receptor in skeletal muscles of normal and insulin resistant subjects.

The insulin-like growth factor I receptor and the activity of its associated tyrosine kinase activity were characterised in wheat germ agglutinin extracts from skeletal muscle biopsies from nine control and ten obese Type 2 (non-insulin-dependent) diabetic subjects, who had marked peripheral in vivo insulin resistance for glucose disposal and hyperinsulinaemia. In parallel studies, the concentration of the insulin receptor and its tyrosine kinase activity were examined in the biopsy extracts and compared to the findings for the insulin-like growth factor I receptor system. Specific binding sites for insulin-like growth factor I were detected. The receptor binding of insulin-like growth factor I was not changed in the obese diabetic subjects as compared to binding activity in the biopsies from the control subjects. The molecular weight of the insulin-like growth factor I receptor alpha subunit was similar in both groups (135 kDa). The insulin-like growth factor I stimulated tyrosine kinase activity was also similar for the two groups. In contrast, insulin binding activity was 30% less in the receptor extracts from the in vivo insulin resistant group when compared to the control group. Moreover, insulin-stimulated tyrosine kinase activity was reduced in the former group by 40% when the value was corrected for insulin binding. Thus, specific insulin-like growth factor I receptors are present in human skeletal muscle. These receptors are normal in insulin resistant obese Type 2 diabetic subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct modulation of insulin receptor protein tyrosine kinase by vanadate and anti-insulin receptor monoclonal antibodies

Sodium vanadate activates "in vitro" insulin receptor autophosphorylation and protein tyrosine kinase in a dose-dependent manner. Insulin receptor protein tyrosine kinase is directly activated also by the anti-insulin receptor beta subunit monoclonal antibody 18-44. We previously demonstrated that the anti-insulin receptor monoclonal antibody MA-10 decreases insulin-stimulated receptor protein tyrosine kinase activity "in vitro", without inhibiting insulin receptor binding. In this report we show that insulin receptor protein tyrosine kinase, activated by sodium vanadate or by monoclonal antibody 18-44, is inhibited by MA-10 antibody. These data suggest that insulin receptor protein tyrosine kinase activity can be either activated and inhibited through mechanisms different from insulin binding.

Influence of a small molecular weight proteinase inhibitor, gabexate mesilate (FOY), on insulin receptor function in vitro.

The effects of the low molecular weight serine proteinase inhibitor FOY (gabexate mesilate) upon insulin action was studied in three different experimental systems. Placenta membranes containing insulin receptors preincubated with FOY (10 microM) showed a reduction of insulin-stimulated tyrosine kinase activity (p less than 0.01). However, FOY (0.1-100 microM) did not affect the insulin-stimulated tyrosine kinase activity in a preparation of solubilized and partially purified insulin receptors from placental membranes. Isolated adipocytes were used to study the effect of FOY on intact cells. FOY neither altered the insulin induced inhibition of the catecholamine-stimulated lipolysis nor (at the low concentration of 0.1 mM) the stimulation of glucose transport by the hormone. High concentrations (0.5 mM) of FOY decreased the effect of insulin on the hexose transport.

Effect of cyclic AMP-dependent protein kinase on insulin receptor tyrosine kinase activity.

To explain the insulin resistance induced by catecholamines, we studied the tyrosine kinase activity of insulin receptors in a state characterized by elevated noradrenaline concentrations in vivo, i.e. cold-acclimation. Insulin receptors were partially purified from brown adipose tissue of 3-week- or 48 h-cold-acclimated mice. Insulin-stimulated receptor autophosphorylation and tyrosine kinase activity of insulin receptors prepared from cold-acclimated mice were decreased. Since the effect of noradrenaline is mediated by cyclic AMP and cyclic AMP-dependent protein kinase, we tested the effect of the purified catalytic subunit of this enzyme on insulin receptors purified by wheat-germ agglutinin chromatography. The catalytic subunit had no effect on basal phosphorylation, but completely inhibited the insulin-stimulated receptor phosphorylation. Similarly, receptor kinase activity towards exogenous substrates such as histone or a tyrosine-containing copolymer was abolished. This inhibitory effect was observed with receptors prepared from brown adipose tissue, isolated hepatocytes and skeletal muscle. The same results were obtained on epidermal-growth-factor receptors. Further, the catalytic subunit exerted a comparable effect on the phosphorylation of highly purified insulin receptors. To explain this inhibition, we were able to rule out the following phenomena: a change in insulin binding, a change in the Km of the enzyme for ATP, activation of a phosphatase activity present in the insulin-receptor preparation, depletion of ATP, and phosphorylation of a serine residue of the receptor. These results suggest that the alteration in the insulin-receptor tyrosine kinase activity induced by cyclic AMP-dependent protein kinase could contribute to the insulin resistance produced by catecholamines.

Defects in insulin binding and autophosphorylation of erythrocyte insulin receptors in patients with syndromes of severe insulin resistance and their parents.

Genetic forms of severe insulin resistance are often characterized by alterations in binding and/or kinase properties of the insulin receptor. To evaluate whether alterations in insulin receptor kinase of erythrocytes can be used as genetic markers, we studied patients with two apparently inherited conditions of severe insulin resistance (leprechaunism and the type A syndrome of insulin resistance) and their families. In the two propositi, [125I]insulin binding to intact erythrocytes was decreased by 64% and 45%, respectively. This was primarily due to a decrease in receptor number and was found in intact cells and solubilization of the receptors. Similar insulin binding defects were found on monocytes. Insulin-stimulated tyrosine kinase activity of the solubilized receptor from erythrocytes was also decreased and to a similar extent as binding. Parents of neither patient had clinical manifestations of leprechaunism or the type A syndrome. Furthermore, no alterations in insulin receptor binding or kinase activity were found in erythrocytes from the mothers. Insulin binding in the father of the type A patient was also normal, whereas the father of the leprechaun had decreased receptor affinity. Receptors extracted from the both fathers' cells had a 40-60% decrease in maximal insulin-stimulated phosphorylation and significant rightward shifts of the insulin dose-response curves (ED50, 141 and 42 ng/mL, respectively; control ED50, 16 ng/mL). The finding of biochemical defects in insulin receptor kinase activity in clinically unaffected parents of patients suggests that these alterations may be useful genetic markers and more sensitive than insulin binding studies for studying pattern of inheritance of these diseases.

Polymyxin B selectively inhibits insulin effects on transport in isolated muscle.

Polymyxin B (PMB), a cyclic decapeptide antibiotic, inhibits the hypoglycemic effect of insulin in vivo. To elucidate the mechanism of PMB action, we have studied its effect in vitro on insulin-stimulated pathways in the mouse skeletal muscle. PMB, added to the incubation mixture, specifically inhibited insulin-stimulated 2-deoxyglucose transport and alpha-aminoisobutyric acid uptake in the isolated soleus muscle but did not affect the basal rates of transport (measured in the absence of insulin). PMB did not alter insulin binding and hexokinase activity. PMB effect was observed at all deoxyglucose concentrations tested, and PMB was also able to inhibit vanadate-stimulated glucose transport. By contrast, insulin activation of glycogen synthase was not prevented by PMB. Basal and maximally insulin-stimulated insulin receptor tyrosine kinase activity, tested in a cell-free system, was similar for both autophosphorylation and phosphorylation of exogenous substrates in the absence or in the presence of PMB. Furthermore, the insulin sensitivity of the kinase was increased in the presence of PMB. Our results suggest that the anti-insulin effect of PMB observed in vivo is due to an inhibition of insulin-stimulated glucose transport in the skeletal muscle perhaps through a specific blockade of the insulin-induced translocation of the glucose carriers.

Intrinsic differences of insulin receptor kinase activity in red and white muscle.

The sensitivity and responsiveness of glucose uptake and glycogen synthesis to insulin are 3-4-fold greater in red than in white skeletal muscle (James, D. E., Jenkins, A. B., and Kraegen, E. W. (1985) Am. J. Physiol. 248, E567-E574). In the present study, the insulin receptor tyrosine kinase activity has been examined in red and white muscle of rats. Partially purified insulin receptors were obtained from muscle following solubilization in detergent, ultracentrifugation, and lectin affinity chromatography. Total insulin receptor number per gram of tissue was slightly higher in red (30%) than in white muscle. In contrast, basal and insulin-stimulated autophosphorylation, normalized for receptor number, were 2.3-fold higher in red muscle. A similar difference was observed in the ability of partially purified receptors to phosphorylate the exogenous substrate polyglutamate/tyrosine. The integrity of the insulin receptor preparation in the two fiber types was identical as determined by affinity cross-linking of [125I-TyrB26]insulin to the receptor. Mixing partially purified receptors from red and white muscle resulted in an additive response for exogenous substrate phosphorylation, suggesting that the difference in tyrosine kinase activity was not due to the presence of an inhibitor or activator. The results suggest that there are differences in the insulin receptors of red and white muscles that lead to discordance in their basal and insulin-stimulated intrinsic tyrosine kinase activity. The correlation between these differences and insulin action in red and white muscle supports the concept that the insulin receptor tyrosine kinase activity is involved in the initiation of insulin action.

Tyrosine Kinase Activity of the Insulin Receptor of Patients with Type A Extreme Insulin Resistance: Studies with Circulating Mononuclear Cells and Cultured Lymphocytes

The syndrome of type A insulin resistance in nonobese women is characterized by hyperinsulinemia, resistance to exogenous insulin, acanthosis nigricans, polycystic ovaries, and masculinization. Insulin binding to intact circulating monocytes and cultured Epstein-Barr virus-transformed B-lymphocytes derived from these patients is decreased in some patients but normal in others. Insulin receptors consist of two subunits; the alpha-subunit contains the insulin-binding site, and the beta-subunit possesses an insulin-sensitive tyrosine-specific protein kinase activity. Insulin binding to circulating monocytes was decreased in five patients, suggesting a decreased number of alpha-subunits on the surface of cells from the patients with type A insulin resistance. In the present work, we demonstrated that there is a proportional decrease in the function of the beta-subunit (i.e. tyrosine kinase activity) in cells from these subjects. In one patient, insulin binding to circulating monocytes was normal, and the insulin-stimulated tyrosine kinase activity of the receptors was normal as well. In separate studies, using cultured Epstein-Barr virus-transformed lymphocytes from the same six patients with type A extreme insulin resistance, the results were similar, in that the functions of the alpha- and beta-subunits of the receptor from these cells correlated. Though heterogeneity among the six patients with type A extreme insulin resistance at the level of the kinase activity of their insulin receptors was demonstrated, it does not appear that a selective defect in beta-subunit phosphorylation per se can be implicated in the mechanisms of insulin resistance of these patients. These findings are distinct from our previously reported patient with normal binding and very low insulin-stimulated phosphorylation of the beta-subunit of the receptor of circulating monocytes, in whom it was speculated that selective reduction in beta-subunit phosphorylation was responsible for insulin resistance.

Defect in phosphorylation of insulin receptors in cells from an insulin-resistant patient with normal insulin binding.

Mononuclear blood cells were obtained from a patient with type A insulin resistance. The cells showed a normal ability to bind iodine 125-labeled insulin. Analysis of solubilized insulin receptors from the patient's cells revealed a defect in insulin-stimulated tyrosine kinase activity, which is closely associated with the receptor itself. The enzyme failed to phosphorylate the insulin receptor and showed a markedly reduced ability to phosphorylate exogenously added substrates. It appears that receptors from this insulin-resistant patient have a defect distal to the insulin-binding site (the alpha subunit of the receptor). The defect could be located in the beta subunit, which has an adenosine triphosphate-binding site, or in another receptor component that transfers a signal of insulin binding into kinase activity. This dissociation between the normal binding and the defective protein kinase component of the insulin receptor represents the first biochemical defect of the receptor distal to ligand binding.

Substrate depletion in different types of muscle and in liver during prolonged running.

Substrate depletion in different types of muscle and in liver during prolonged running.