Long-term exposure of rat hepatoma cells to insulin results in a total desensitization of the cells to the action of the hormone. This is characterized by changes in binding and post-binding steps of insulin action, including a decrease in the number and phosphorylation of receptors, and a major modification in receptor oligomerization.

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.

Insulin initiates its action by binding to a glycoprotein receptor on the surface of the cell. This receptor consists of an alpha-subunit, which binds the hormone, and a beta-subunit, which is an insulin-stimulated, tyrosine-specific protein kinase. Activation of this kinase is believed to generate a signal that eventually results in insulin's action on glucose, lipid, and protein metabolism. The growth-promoting effects of insulin appear to occur through activation of receptors for the family of related insulin-like growth factors. Both genetic and acquired abnormalities in the number of insulin receptors, the activity of the receptor kinase, and the various post-receptor steps in insulin action occur in disease states leading to tissue resistance to insulin action.

The insulin receptor is a tyrosine-specific protein kinase. Upon binding of the hormone, the kinase is activated resulting in autophosphorylation of the receptor. This kinase activity has been postulated to be an early step in the transmembrane signaling produced by insulin. To evaluate the physiologic relevance of receptor phosphorylation, we have studied insulin binding and autophosphorylation properties using cells from an individual with a variant of the Type A syndrome of severe insulin resistance and acanthosis nigricans. Erythrocytes and cultured fibroblasts from this individual exhibited normal or near normal 125I-insulin binding. Receptors extracted from erythrocytes with Triton X-100 also exhibited normal 125I-insulin binding and competition curves. Despite this, receptors extracted from both erythrocytes and fibroblasts showed a 50% decrease in insulin-stimulated autophosphorylation. Partially purified receptors from the patient's fibroblasts also exhibited a 40% decrease in their ability to phosphorylate exogenous substrates. These data suggest that the insulin resistance in this syndrome is due to a genetic abnormality which impairs insulin receptor phosphorylation and kinase activity and further support the possible role of receptor phosphorylation and kinase activity in insulin action.

The effect of the tumor-promoting agent phorbol 12-myristate 13-acetate (PMA) on insulin receptors and insulin action was studied in rat hepatoma cells in culture. PMA (0.1-1.0 micrograms/ml) did not affect insulin binding either acutely or chronically but inhibited insulin stimulation of glycogen synthase and tyrosine aminotransferase. PMA (1 microgram/ml) stimulated the phosphorylation of the beta subunit of insulin receptor purified from [32P]phosphate-labeled Fao cells by 1.3-fold in the absence of insulin. In contrast, insulin-stimulated phosphorylation in the presence of PMA was reduced. Phosphoamino acid analysis of the beta subunit after PMA stimulation revealed an increase of both phosphoserine and phosphothreonine residues, whereas insulin stimulated primarily phosphorylation of tyrosine and serine residues. Insulin stimulation of cells after PMA treatment revealed a decrease in phosphotyrosine when compared to cells stimulated by insulin alone. Tryptic peptide mapping of the beta subunit by a two-dimensional chromatographic/electrophoretic separation revealed nine phosphopeptides from the cells treated with PMA. Insulin stimulated phosphorylation at six new sites in the receptor, three of which appeared to be similar to those in PMA-treated cells. This report shows that phorbol esters stimulate insulin receptor phosphorylation, inhibit insulin-induced receptor phosphorylation and insulin action, and suggest a physiologic relation between insulin action and the calcium-activated and phospholipid-dependent protein kinase C.

We characterized insulin receptors on a human lymphoblastoid cell line (IM-9) and studied their regulation using anti-receptor antibodies and fluorescence flow cytometry. The fluorescence intensity distribution of insulin receptors on cells was determined by incubating the cells with one of three different anti-receptor antisera (human serum B-9 containing polyclonal autoantibodies, serum from a rabbit with polyclonal antibodies, and a monoclonal antibody to the receptor produced in mouse hybridomas), followed by incubation with an appropriate fluorescein isothiocyanate-labeled second antibody and analysis on an Epics-V flow cytometer. All three anti-receptor antibodies specifically labeled the insulin receptors. The monoclonal antibody showed the highest level of labeling. Treatment of cells with proteolytic enzymes, such as trypsin or chymotrypsin, produced a dose-dependent loss of 125I-labeled insulin (125I-insulin) binding but a relatively small decrease in the binding of anti-receptor antibodies, suggesting that most antibody binding occurred in domains other than the insulin binding site. Treatment with glycosidic enzymes, such as neuraminidase and beta-galactosidase did not affect the binding of 125I-insulin, and fluorescence was actually enhanced by about 20% in the beta-galactosidase-treated cells. Exposure of IM-9 cells to insulin resulted in a reduction in the number of insulin receptors. Analysis of the down-regulated cells by immunofluorescence revealed a complete correlation between the percent binding of 125I-insulin and percent peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical peak by fluorescence analysis. Exposure of IM-9 cells to anti-receptor antibodies at 37 degrees C for 16 hr also produced a down-regulation in the number of insulin receptors. Incubation with human antiserum B-9 caused a 95% loss of both 125I-insulin binding and peak fluorescence, while the monoclonal antibody resulted in a 50% loss of receptors. Incubation of cells with anti-receptor antibodies for 2 hr at 4 degrees C did not produce any receptor loss; however, the human anti-receptor antisera B-2 and B-9 inhibited the binding of the monoclonal anti-receptor antibody by about 50%, suggesting that these antisera contained autoantibodies directed at the monoclonal antibody binding site. These data indicate that insulin receptors can be regulated by both insulin and anti-receptor antibody and demonstrate the utility of immunofluorescence and flow cytometry as a tool for the study of the insulin receptor.

Insulin receptors from rat hepatoma cells (Fao) and human placenta were partially purified by detergent solubilization and lectin purification. The insulin receptor preparations were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under reducing or nonreducing conditions. The proteins were transferred to nitrocellulose paper and the electrophoretic blots were treated with human anti-receptor autoantibodies, rabbit antibody to purified insulin receptor, or a monoclonal antibody to human insulin receptor. The nitrocellulose paper was then treated with 125I protein A or 125I second antibody followed by autoradiography. The rabbit polyclonal antiserum and one of the human autoantibodies recognized both the alpha (Mr = 135,000) and beta (Mr = 95,000) subunits after transfer from a SDS gel to nitrocellulose paper. On transfers from nonreduced gels, several high-molecular species were labeled ranging from Mr = 200,000 to Mr = 330,000. Similar high-molecular bands of the receptor were seen if highly purified human placental receptor, as well as partially purified receptor from rat or human origin, were used. As little as 0.1-0.5 microgram of pure receptor could be detected by this technique. Treatment of the receptor with neuraminidase (50 mU/ml) before gel electrophoresis resulted in a 50% increase in intensity of intact receptor and about a 70% increase in the labeling of the alpha-subunit of the receptor, but no change in labeling of the beta-subunit. The monoclonal antibody used, as well as two other human autoantibodies, did not recognize the receptor after transfer to nitrocellulose paper.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously reported that prolonged incubations of Fao cells, a cell line derived from the well-differentiated Reuber H35 rat hepatoma, with 10(-6) M insulin, induced a decrease in receptor number (down-regulation), an increase in receptor affinity for insulin, and a loss of insulin's biological effect (desensitization). In the present study, we have investigated the relationship between these changes in insulin binding and action and changes in the structure of the insulin receptor. Intact cells were surface labeled with Na125I and lactoperoxidase, and the 125I-labeled insulin receptor was immunoprecipitated using specific antibodies and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of gels done under reducing conditions demonstrated the alpha (Mr = 135,000) and the beta (Mr = 95,000) subunits of the receptor. In nonreduced gels, free insulin receptor subunits were observed as well as four higher molecular weight bands with Mr = 210,000, 270,000, 350,000, and 520,000. Two-dimensional gel electrophoresis revealed that these bands correspond to alpha-beta heterodimer, alpha 2 homodimer, and two alpha-beta oligomers of high molecular weights, respectively. Cross-linking of 125I-insulin to intact cells with disuccinimidyl suberate revealed bands of Mr = 125,000, 210,000, 250,000 and 320,000, indicating that most of the forms of the receptor could bind insulin. After incubation with 10(-6) M insulin for 24 h, Fao cells revealed a marked decrease of the four oligomeric forms of the receptor, with little change in the level of the free alpha and beta subunits. A similar decrease of the oligomeric forms of the insulin receptor and an increase in the free subunits was observed when normal Fao cells are treated with 7 mM dithiothreitol. In dithiothreitol-treated cells, 125I-insulin binding was increased and this increase was accounted for by a change in affinity. In contrast to Fao cells, down-regulation of the insulin receptor in IM-9 lymphocytes occurs without a change in receptor affinity. In these cells, surface labeling revealed a decrease in total receptors after down-regulation, but not change in the proportion of the oligomeric forms to the free subunits of the receptor. These data suggest the following in Fao hepatoma cells. In the native state, the insulin receptor consists of free alpha and beta subunits and several kinds of disulfide-linked oligomers of these subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Familial hyperproinsulinemia is a genetic disorder characterized by elevated plasma levels of proinsulin-like material. In two previously described kindreds this has been shown to be due to a structural abnormality in the proinsulin molecule. We have identified a third family with hyperproinsulinemia in which there appeared to be a different defect. The propositus, a 12-year-old girl, had borderline glucose intolerance and markedly elevated immunoreactive-insulin levels on oral glucose-tolerance testing. Gel filtration of plasma revealed that 66 per cent of circulating insulin immunoreactivity was accounted for by the proinsulin-like components. Two of four siblings, the father, and the paternal grandfather also had elevated fasting insulin immunoreactivity in the presence of normal plasma glucose concentrations and elevated levels of proinsulin-like material. In vitro tryptic digestion of plasma proinsulin-like material from an affected family member revealed that proinsulin was converted to insulin in a manner indistinguishable from that in the control. Similarly, proinsulin and insulin exhibited normal activity in a radioreceptor assay. These findings suggest that the proinsulin molecule in this family was normal and that hyperproinsulinemia was due to a defect in the conversion of proinsulin to insulin.