Insulin binding and processing was studied in monolayer cultures of bovine aortic endothelial cells. Specific 125I-insulin binding was both time and temperature dependent. Maximum binding at 37 degrees C occurred at 90 min, and was 3.8%/mg protein and, at 15 degrees C, 7%/mg protein at 4 h. 125I-insulin was crosslinked to its receptor using disuccinimidyl suberate (DSS), and the structure of the receptor complex was identified by SDS-polyacrylamide gel electrophoresis and autoradiography; a major band with Mr = 145,000 was identified, which corresponds to the alpha-subunit of the insulin receptor reported in other tissues. Receptor-bound insulin was internalized, and both the rate and the amount of internalization were temperature dependent. The rate of internalization was slowest at 4 degrees C, and fastest at 37 degrees C, and the maximum amount of 125I-insulin internalized in 120 min was 16% at 4 degrees C, 45% at 15 degrees C, and 81% at 37 degrees C. Despite the high rate of internalization, endothelial cells do not appear to degrade insulin significantly, as determined by gel chromatography and TCA solubility (7% at 4 h) of media-associated radioactivity. In addition, the majority of internalized insulin (75%) was released by 60 min, largely as intact insulin. Chloroquine treatment at high concentration did not exert any major effect on insulin binding or degradation within the first 60 min, but thereafter produced a marked increase in cell-associated radioactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.

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.

To examine the role of heritable factors in insulin-dependent diabetes mellitus (IDDM), we studied the incidence of IDDM in the offspring of patients with the disease who were identified by the medical records of the Joslin Diabetes Center from 1928 to 1939. We found 187 survivors who, after the diagnosis of IDDM, had produced 419 offspring for whom information about diabetes status was available. By the age of 20, 6.1 per cent of the offspring of the 88 men had diabetes; in contrast, only 1.3 per cent of the offspring of the 99 women had the disease by the age of 20 (P less than 0.05). Daughters and sons of the men with IDDM were affected equally (there were insufficient numbers of affected offspring of diabetic women to permit determination of whether the sexes were equally affected). We conclude that IDDM is transmitted less frequently to the offspring of diabetic women than to those of diabetic men. More study is required to determine whether this difference reflects a genetic mechanism or, instead, selective perinatal loss of the affected offspring of diabetic mothers.

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 have studied the reversibility of insulin receptor phosphorylation to establish the relation between this autophosphorylation reaction and the initiation of insulin action and between dephosphorylation and the termination of insulin effects in cells. In cultured Fao hepatoma cells labeled with 32PO4(3-), insulin increased 5-fold the phosphorylation of the beta-subunit of the insulin receptor at serine, threonine, and tyrosine residues. Addition of anti-insulin antiserum to cells incubated with insulin caused dissociation of insulin from the receptor and concurrent dephosphorylation of the beta-subunit. 32PO4(3-) associated with the insulin-stimulated receptor could be decreased by the addition of sodium phosphate to the medium but with a slower time course. Insulin stimulated phosphorylation of insulin receptor purified partially on immobilized wheat germ agglutinin. This reaction utilized [gamma-32P] ATP and occurred exclusively on tyrosine residues. Addition of unlabeled ATP caused a decrease in the amount of PO4(3-) associated with the receptor. Insulin-stimulated phosphorylation was also observed if the receptors were further purified by immunoprecipitation with anti-insulin receptor antibody prior to the phosphorylation reaction; however, addition of unlabeled ATP to this system did not chase the labeled 32PO4(3-) from the beta-subunit. These data are consistent with the notion that phosphorylation and dephosphorylation of the insulin receptor parallel the onset and termination of insulin action. Phosphatase activity involved in the dephosphorylation of the insulin receptor appears to be a glycoprotein because it was retained after partial purification of the receptor on wheat germ agglutinin-agarose; however, this phosphatase activity is distinct from the insulin receptor because it was not retained after immunoprecipitation of the receptor with anti-insulin receptor antibodies.

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.

Antibodies to the insulin receptor have provided important experimental probes of receptor structure and function. In the present study, we have characterized the insulin receptor on human lymphoblastoid cell lines using polyclonal and monoclonal anti-receptor antibodies and fluorescence flow cytometry. The cell lines were derived by Epstein-Barr virus transformation of peripheral mononuclear leucocytes from normal subjects or patients with disorders that affect the insulin receptor. Fluorescence analysis revealed a high level of specific fluorescence on lymphoid cell lines from normal individuals (mean peak fluorescence 30-50 units above the control) and was similar to the labelling of the spontaneously transformed lymphoblastoid cell line IM-9. Transformed cells from patients with syndromes of insulin resistance, such as the Rabson Mendenhall syndrome, leprechaunism and the type A syndrome of insulin resistance and acanthosis nigricans, exhibited little or no specific fluorescence. In all cases, there was an unimodal distribution of receptors on cells. In addition, there was a good correlation between specific binding of 125I-insulin and percentage peak fluorescence. The data indicate that fluorescence flow cytometry can be used to study the distribution of insulin receptor on different cell lines and to study cells derived from patients with disease states.

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 studied the effects of chronic exposure to insulin on the binding and the biologic activity of the hormone using a well-differentiated cell line (Fao) derived from the Reuber H35 rat hepatoma. Prolonged incubation (24 h) with 10(-6) M insulin produced a 20-25% decrease in binding of tracer concentrations (2 X 10(-11) M) of 125I-insulin, and a leftward shift of the curve for inhibition by unlabeled insulin. Scatchard analysis of the binding data revealed that a 75-80% decrease in the number of binding sites had occurred in the insulin-treated cells, but was accompanied by an increase in apparent receptor affinity. Kinetic studies suggested negative cooperativity in insulin binding and indicated that the change in affinity was accounted for by a decrease in the rate of dissociation. Both the decrease in receptor number and the increase in affinity were dependent on time, temperature, and the insulin concentration during the treatment period. Both effects were also blocked by cycloheximide, suggesting that they required new protein synthesis. Plasma membranes isolated from downregulated cells retained both the change in receptor number and affinity. Anti-receptor antibodies present in two human sera (B-2 and B-9) inhibited 125I-insulin binding in downregulated cells with equal or slightly greater sensitivity than in control cells. The changes in insulin binding were accompanied by changes in insulin's biologic effects in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)