We have studied the relationship between glucose uptake rate and Glut 1 and Glut 4 protein and mRNA levels per fat cell in lean (FA/FA) and obese (fa/fa) Zucker rats at 5, 10, and 20 wk of age, and after induction of acute diabetes with streptozotocin. 5 wk obese rats exhibit insulin hyperresponsive glucose uptake, whereas 20 wk obese rats show insulin resistant glucose uptake. The relative abundance of Glut 1 and Glut 4 mRNA and protein per equal amount of total RNA and total membrane protein, respectively, is lower in adipocytes from obese rats. However, at all ages the enlargement of fat cells from obese rats is accompanied by a severalfold increase in total RNA and total membrane protein per cell. Thus, on a cellular basis, mRNA and protein levels of Glut 4 increases in young obese rats and gradually declines as a function of age. Basal glucose uptake is increased severalfold in fat cells from obese rats, and in parallel Glut 1 expression per cell in obese rats is two- to threefold increased over lean rats at all ages. Acute diabetes in 20 wk obese rats causes a profound downregulation of glucose uptake and a concomitant reduction of both Glut 1 and Glut 4 protein levels. Thus, changes in Glut 4 expression are a major cause of alteration in insulin-stimulated glucose uptake of adipocytes during evolution of obesity and diabetes in Zucker rats.

We have investigated the structural and functional properties of two mutant insulin receptors in which Cys647 and Cys682,683,685 have been replaced with Ser (IRS647 and IRS682,683,685, respectively). Compared with the wild-type receptor (IRWT), both mutant receptors displayed altered sensitivities to dithiothreitol with respect to insulin binding and reduction of oligomeric forms. Subunit composition of the oligomeric forms of the receptors as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-labeled receptors indicated that Cys682,683,685 are required for normal heterotetrameric structure and that Cys647 plays a major role in the normal covalent association of the alpha- and beta-subunits. Under nonreducing conditions, the affinity-labeled IRS647 migrated, almost exclusively, as a 230-kDa species which appeared to represent an alpha 2 form of the receptor. Furthermore, Chinese hamster ovary cells expressing IRS647 did not exhibit basal or insulin-stimulated autophosphorylation, suggesting that Cys647 is also required for signal transduction.

The intrinsic tyrosyl kinase activity of the insulin receptor is regulated by a balance between insulin-induced receptor autophosphorylation, which stimulates the receptor kinase, and enzymatic dephosphorylation of the receptor, which deactivates its kinase activity. The cellular protein-tyrosine phosphatase (PTPase) enzymes responsible for reversing the activated state of the insulin receptor have not been characterized. Our laboratory is interested in identifying and cloning the specific PTPase(s) that regulate the phosphorylation state of the insulin receptor. This chapter will summarize the design and results of our initial molecular cloning studies to identify specific PTPases in insulin-sensitive tissues that may have a potential physiological role in insulin action and clinical insulin resistance.

To study the role of membrane lipids in signal transduction by the insulin receptor, we have studied the effect of phospholipase C (Clostridium perfringens) and a phosphatidylinositol-specific phospholipase (Staphylococcus aureus) on insulin binding, a function of the alpha-subunit, and tyrosine kinase activity, a function of the beta-subunit in IM-9 lymphocytes and NIH 3T3 fibroblasts transfected with the human insulin receptor. Treatment of the cells with phospholipase C at concentrations up to 3.4 U/ml did not affect specific insulin binding, but reduced insulin-stimulated receptor phosphorylation by 50%. This effect of phospholipase C was observed within 10 min of treatment and occurred with no change in the basal level of phosphorylation. Pre-treatment of cells with insulin for 5 min prior to enzyme addition prevented any change in kinase activity. Insulin-stimulated phosphorylation of pp 185, the presumed endogenous substrate for the insulin receptor kinase, was also reduced following phospholipase C treatment, with an almost complete loss of insulin stimulation after exposure of cells to enzyme at concentrations as low as 0.6 U/ml. In contrast to these effects of phospholipase C on intact cells, receptor autophosphorylation was not affected in insulin receptors purified on wheat germ agglutinin-agarose from phospholipase C treated cells. Likewise, the phospholipase C effect was reduced by the addition of phosphatidylcholine, but not by the addition of the protease inhibitors, aprotinin and phenylmethylsulfonyl fluoride, to the incubation indicating its dependence on phospholipid hydrolysis. Treatment of cells with the phosphatidylinositol-specific phospholipase C did not affect any of the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the phosphatidylinositol 3-kinase (PtdIns 3-kinase) in insulin-stimulated Chinese hamster ovary (CHO) cells expressing normal (CHO/IR) and mutant human insulin receptors. Insulin stimulation of CHO/IR cells results in an increase in PtdIns 3-kinase activity associated with anti-phosphotyrosine (alpha PY) immunoprecipitates, which has been previously shown to correlate with the in vivo production of PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (Ruderman, N., Kapeller, R., White, M.F., and Cantley, L.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). Stimulation was maximal within 1 min and showed a dose response identical to that of insulin receptor autophosphorylation. The PtdIns 3-kinase also associated with the insulin receptor in an insulin-stimulated manner, as approximately 50% of the total alpha PY-precipitable activity could be specifically immunoprecipitated with anti-insulin receptor antibody. Mutant insulin receptors displayed variable ability to stimulate the PtdIns 3-kinase, but in all cases the presence of PtdIns 3-kinase in alpha PY immunoprecipitates correlated closely with the tyrosyl phosphorylation of the endogenous substrate pp185. In CHO cells expressing a kinase-deficient mutant (IRA1018), there was no observable insulin stimulation of PtdIns 3-kinase activity in alpha PY immunoprecipitates and no tyrosyl phosphorylation of pp185. Substitution of Tyr1146 in the insulin receptor regulatory region with phenylalanine partially impaired receptor autophosphorylation, pp185 phosphorylation, and insulin-stimulated increases in alpha PY-precipitable PtdIns 3-kinase activity. In contrast, a deletion mutant lacking 12 amino acids from the juxtamembrane region (IR delta 960) displayed normal in vivo autophosphorylation but failed to stimulate the PtdIns 3-kinase or phosphorylate pp185. Finally, a mutant receptor from which the C-terminal 43 amino acids had been deleted (IR delta CT) exhibited normal insulin-stimulated autophosphorylation, pp185 phosphorylation, and stimulation of the PtdIns 3-kinase activity in alpha PY immunoprecipitates. These data suggest that the PtdIns 3-kinase is itself a substrate of the insulin receptor kinase or associates preferentially with a substrate. A comparison of the biological activities of the mutant receptors with their activation of the PtdIns 3-kinase furthermore suggests that the PtdIns 3-kinase may be linked to insulin's ability to regulate DNA synthesis and cell growth.

The exact relationship between EGF-stimulated tyrosine phosphorylation, induction of the cellular proto-oncogenes c-myc and c-fos, and DNA synthesis remains uncertain. Madin-Darby Canine Kidney (MDCK) cells possess EGF receptor sites with high binding capacity, and in contrast to A431 cells, respond to EGF by increasing DNA synthesis. Following EGF stimulation of intact MDCK cells, there was a rapid and marked increase in the autophosphorylation of the EGF receptor. This was associated with an increase in the tyrosine phosphorylation of a 120 kDa phosphoprotein believed to be an endogenous substrate of this receptor kinase. The ED50 for stimulation of phosphorylation of pp120 was approximately 0.05 nM versus 1.0 nM for receptor autophosphorylation, consistent with amplification of signalling at this step in EGF action. Stimulation of DNA synthesis occurred after 12 to 24 hours and revealed even further amplification with an ED50 of about 0.1 nM. Intermediate between these events was a time-dependent activation of c-fos and c-myc gene expression. However, the ED50 for these processes was approximately 10 nM, indicating a relatively lower sensitivity of EGF for stimulation of proto-oncogene expression. Tyrphostin (RG 50864), a compound reported to inhibit specifically the EGF receptor kinase, completely blocked EGF stimulation of proto-oncogene induction. Interestingly, under the same experimental conditions, EGF receptor autophosphorylation was decreased only 60%. These data, along with the dose-response studies, indicate that proto-oncogene induction requires near maximal stimulation of EGF receptor autophosphorylation. They also suggest that, in MDCK cells, the EGF dependent induction of the c-fos and c-myc genes is not strictly correlated to the extent of EGF receptor autophosphorylation or EGF-stimulated DNA synthesis, and that EGF stimulation of DNA synthesis likely involves additional rate-limiting intermediate steps.