IRS-1 is a major insulin receptor substrate which may play an important role in insulin signal transmission. The mRNA for IRS-1 in rat cells and tissues is about 9.5 kilobases (kb). Rat liver IRS-1 was stably expressed in Chinese hamster ovary (CHO) cells (CHO/IRS-1). Although its calculated molecular mass is 131 kDa, IRS-1 from quiescent cells migrated between 165 and 170 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRS-1 was phosphorylated strongly on serine residues and weakly on threonine residues before insulin stimulation. Insulin immediately stimulated tyrosine phosphorylation of IRS-1, and after 10-30 min with insulin its apparent molecular mass increased to 175-180 kDa. Expression of the human insulin receptor and rat IRS-1 together in CHO/IR/IRS-1 cells increased the basal serine phosphorylation of IRS-1 and strongly increased tyrosine phosphorylation during insulin stimulation. Purified insulin receptors directly phosphorylated baculovirus-produced IRS-1 exclusively on tyrosine residues. By immunofluorescence, IRS-1 was absent from the nucleus, but otherwise distributed uniformly before and after insulin stimulation. Some IRS-1 associated with the insulin receptor during insulin stimulation. In addition, a phosphatidylinositol 3'-kinase associated with IRS-1 during insulin stimulation, and this association was more sensitive to insulin in CHO cells overexpressing the insulin receptor (CHO/IR cells), more responsive to insulin to CHO/IRS-1 cells, and both sensitive and responsive in CHO/IR/IRS-1 cells. Similarly, insulin-stimulated DNA synthesis was more sensitive to insulin in CHO/IR cells, and more responsive in CHO/IRS-1 cells; however, insulin-stimulated DNA synthesis was sensitive but poorly responsive to insulin in CHO/IR/IRS-1 cells. Together, these results suggest that IRS-1 is a direct physiologic substrate of the insulin receptor and may play an important role in insulin signal transmission.

Growth factors stimulate the enzyme phosphatidylinositol (PI) 3-kinase in cells in culture. Insulin rapidly stimulates tyrosine phosphorylation of its endogenous substrate, insulin receptor substrate 1 (IRS-1), and in vitro IRS-1 associates with PI 3-kinase, thus activating the enzyme. We have examined whether insulin is capable of stimulating the PI 3-kinase pathway in two physiological target tissues for the actions of insulin in vivo, liver and skeletal muscle. After intraportal injection of insulin into anesthetized rats, there was a 2-fold stimulation of total hepatic PI 3-kinase activity in liver and muscle extracts and a 10- to 20-fold increase in PI 3-kinase activity immunoprecipitated with anti-IRS-1 antibodies. Stimulation of PI 3-kinase was accompanied by an association between this enzyme and IRS-1 as detected by immunoprecipitation of liver and muscle extracts with anti-IRS-1 antibodies and Western blotting with antibodies to the 85-kDa subunit of PI 3-kinase. Immunoprecipitation with anti-p85 antibodies and phosphotyrosine immunoblotting revealed no tyrosine phosphorylation of PI 3-kinase, but demonstrated co-precipitation of tyrosine-phosphorylated IRS-1, as well as another phosphotyrosine protein of approximately 135-140 kDa. Thus, IRS-1 phosphorylation plays a significant role in the activation of PI 3-kinase in vivo by insulin.

Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.

We have studied the effect of insulin stimulation on phosphotyrosine phosphatase (PTPase) activity in the well-differentiated rat hepatoma cell line Fao. PTPase activity was measured using a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation. Of the PTPase activity in Fao cells, 14% was in the cytosolic fraction, whereas 86% was in the particulate fraction; this latter fraction also had a 4-fold higher specific activity. Purification of the particulate fraction by lectin chromatography resulted in a 50% increase in specific activity, although this glycoprotein-rich fraction contained only 1.5% of the total activity. Both the cytosolic and particulate PTPase fractions were active toward the tyrosyl-phosphorylated insulin receptor in vitro. The activity of the particulate fraction but not the cytosolic fraction was inhibited by addition of a micromolar concentration of a phosphorylated peptide corresponding to residues 1142-1153 of the human insulin receptor sequence. By contrast, addition of the nonphosphorylated peptide even at millimolar concentration was without effect. Both PTPase fractions were inhibited by Zn+ at similar concentrations, whereas the cytosolic PTPase activity was 10-fold more sensitive to vanadate inhibition. Treatment of cells with 100 nM insulin increased PTPase activity in the particulate fraction by 40% and decreased activity in the cytosolic fraction by 35%. These effects occurred within 15 min and were half-maximal at 3-4 nM insulin. When assessed as total activity, the magnitude of the changes in PTPase activity in the particulate and cytosolic fractions could not be explained on the basis of a translocation of PTPases between the two pools.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin binding to its plasma membrane receptor stimulates a cascade of protein kinases and phosphatases which ultimately affects multiple processes in the membrane, cytosol, and nucleus of the cell, including transcription of specific genes. To gain insight into the relationship between the kinase cascade and the mechanism of insulin-induced nuclear events, we have studied the effect of insulin on the phosphorylation of DNA-binding nuclear proteins in differentiated NIH-3T3-F442A adipocytes. Insulin induced the phosphorylation of seven DNA-binding proteins: pp34, pp40, pp48, pp62, pp64, pp66, and pp72. The half-maximal response was observed at 10-30 min and reached its maximum at 60 min. The insulin-induced phosphorylation of each of these proteins was dose-dependent with ED50S of 2-10 nM. The phosphorylation of pp62, pp64, and pp72 took place on serine residues. On the basis of immunoprecipitation and immunoblotting experiments with anti-lamin antibodies, we found that the insulin-induced DNA-binding phosphoproteins pp62, pp64, pp66, and possibly pp48 were related to lamins A and C. The ED50 for insulin-stimulated lamin phosphorylation was approximately 10 nM, and phosphorylation was half-maximal at 30 min. The insulin-dependent phosphorylation of lamins and other DNA-binding proteins (pp34, pp40, and pp72) may play a mediatory role in the long-term effects of insulin.

Type 2 diabetes mellitus is characterised by resistance of peripheral tissues to insulin and a relative deficiency of insulin secretion. To find out which is the earliest or primary determinant of disease, we used a minimum model of glucose disposal and insulin secretion based on intravenous glucose tolerance tests to estimate insulin sensitivity (SI), glucose effectiveness (ie, insulin-independent glucose removal rate, SG), and first-phase and second-phase beta-cell responsiveness in normoglycaemic offspring of couples who both had type 2 diabetes. 155 subjects from 86 families were followed-up for 6-25 years. More than 10 years before the development of diabetes, subjects who developed the disease had lower values of both SI (mean 3.2 [SD 2.4] vs 8.1 [6.7] 10(-3) I min-1 pmol-1 insulin; p 0.0001) and SG (1.6 [0.9] vs 2.3 [1.2] 10(-2) min-1, p 0.0001) than did those who remained normoglycaemic). For the subjects with both SI and SG below the group median, the cumulative incidence of type 2 diabetes during the 25 years was 76% (95% confidence interval 54-99). By contrast, no subject with both SI and SG above the median developed the disease. Subjects with low SI/high SG or high SI/low SG had intermediate risks. Insulin secretion, especially first phase, tended to be increased rather than decreased in this prediabetic phase and was appropriate for the level of insulin resistance. The development of type 2 diabetes is preceded by and predicted by defects in both insulin-dependent and insulin-independent glucose uptake; the defects are detectable when the patients are normoglycaemic and in most cases more than a decade before diagnosis of disease.

Insulin stimulates tyrosine phosphorylation of the insulin receptor and of an endogenous substrate of approximately 185 kd (insulin receptor substrate 1 or IRS-1) in most cell types. Tyrosine phosphorylation of insulin receptor and of IRS-1 have been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the study presented here, the levels and phosphorylation state of the insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo have been examined in spontaneously hypertensive rats (SHR) by immunoblotting with antipeptide antibodies to insulin receptor and IRS-1 and antiphosphotyrosine antibodies. It was found that the levels of insulin receptor and IRS-1 protein in liver and muscle are similar in controls (Wistar-Kyoto rats) and SHR. By contrast, there is a decrease in autophosphorylation in the liver and muscle of SHR and a parallel decrease in phosphorylation of IRS-1. These data indicate that reduced insulin receptor kinase activity and reduced substrate phosphorylation may play an important role in the impaired insulin action in the hypertensive rat.

We have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (Tyr1158, Tyr1162, and Tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. A single-Phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. The double-Phe replacements showed a 60-70% reduction, and substitution of all 3 tyrosine residues with Phe or Ser reduced insulin-stimulated tyrosine autophosphorylation by greater than 80%. Phosphopeptide mapping each mutant revealed that all remaining tyrosine autophosphorylation sites were phosphorylated normally following insulin stimulation, and no new sites appeared. The single-Phe mutants showed insulin-stimulated kinase activity toward a synthetic peptide substrate of 50-75% when compared with wild-type receptor kinase activity. Insulin-stimulated kinase activity was further reduced in the double-Phe mutants and barely detectable in the triple-Phe mutants. In contrast to the wild-type receptor, all of the mutant receptor kinases showed a significant reduction in activation following in vitro insulin-stimulated autophosphorylation. When studied in intact Chinese hamster ovary cells, insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of the cellular substrate pp185 in the single-Phe and double-Phe mutants was progressively lower with increased tyrosine replacement and did not exceed the basal levels in the triple-Phe mutants. However, all the mutant receptors, including the triple-Phe mutant, retained the ability to undergo insulin-stimulated Ser and Thr phosphorylation. Thus, full activation of the insulin receptor tyrosine kinase is dependent on insulin-stimulated Tris phosphorylation of the kinase domain, and the level of autophosphorylation in the kinase domain provides a mechanism for modulating insulin receptor kinase activity following insulin stimulation. By contrast, insulin stimulation of receptor phosphorylation on Ser and Thr residues by cellular serine/threonine kinases can occur despite markedly reduced tyrosine autophosphorylation.

The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp 185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Anti-receptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.

To examine the role of the transmembrane domain (TM) of the insulin receptor in insulin-induced receptor kinase activation, we prepared four mutated insulin receptors: 1) a Val938----Asp substitution (IR/TMv----D), 2) insertion of a 3-amino acid repeat (Val938-Phe939-Leu940) (IR/TM+3), or the entire TM was replaced by the corresponding domain of either the 3) platelet-derived growth factor (PDGF) receptor (IR/TMPDGFR) or 4) c-neu/erbB2 proto-oncogene product (IR/TMc-neu). Each mutant receptor was stably expressed in Chinese hamster ovary cells, assessed by fluorescence-activated cell sorting, insulin binding, and biosynthetic labeling. All mutant receptors exhibited normal affinity for insulin. Pulse-chase experiments showed that each proreceptor was processed into alpha- and beta-subunits, although the rate of IR/TMV----D conversion was reduced approximately 3-fold. With IR/TMPDGFR, IR/TMV----D, and IR/TM+3 basal and insulin-stimulated levels of autophosphorylation and tyrosine kinase activation were normal, both in wheat germ agglutinin (WGA)-purified receptor preparations and intact cells. By contrast, following WGA purification or isolation of crude membranes, IR/TMc-neu was a constitutively active autokinase and substrate kinase in vitro. However, in intact cells insulin-stimulated autophosphorylation and kinase activity appeared normal. We conclude that although there is considerable latitude in acceptable structure, residues within the insulin receptor transmembrane domain can play a functional role in regulation of insulin receptor tyrosine kinase activity.