The expression of insulin-like growth factor (IGF) receptors at the cell surface and the changes in IGF responsiveness during differentiation were studied in the L6 skeletal muscle cell line. Throughout the entire developmental sequence, distinct receptors for IGF I and IGF II that differed in structure and peptide specificity could be demonstrated. During differentiation, both 125I-IGF I and 125I-IGF II binding to the L6 cells decreased as a result of a 3-4-fold reduction in receptor number, whereas 125I-insulin binding increased. Under nonreducing conditions, disuccinimidyl suberate cross-linked 125I-IGF I and 125I-IGF II to two receptor complexes with apparent Mr greater than 300,000 (type I) and 220,000 (type II). Under reducing conditions, the apparent molecular weight of the type I receptor changed to Mr 130,000 (distinct from the 120,000 insulin receptor) and the type II receptor changed to 250,000. IGF I and IGF II both stimulated 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake in the L6 cells with a potency close to that of insulin, apparently through interaction with their own receptors. The stimulatory effects of IGF II correlated with its affinity for the type II but not the type I IGF receptor, as measured by inhibition of affinity labeling, whereas the effects of IGF I correlated with its ability to inhibit labeling of the type I receptor. In spite of the decrease in type I and type II receptor number, stimulation of 2-deoxy-glucose and alpha-aminoisobutyric acid uptake by the two IGFs increased during differentiation.

Phosphotyrosine-containing proteins are minor components of normal cells which appear to be associated primarily with the regulation of cellular metabolism and growth. The insulin receptor is a tyrosine-specific protein kinase, and one of the earliest detectable responses to insulin binding is activation of this kinase and autophosphorylation of its beta-subunit. Tyrosine autophosphorylation activates the phosphotransferase in the beta-subunit and increases its reactivity toward tyrosine phosphorylation of other substrates. When incubated in vitro with [gamma-32P]ATP and insulin, the purified insulin receptor phosphorylates various proteins on their tyrosine residues. However, so far no proteins other than the insulin receptor have been identified as undergoing tyrosine phosphorylation in response to insulin in an intact cell. Here, using anti-phosphotyrosine antibodies, we have identified a novel phosphotyrosine-containing protein of relative molecular mass (Mr) 185,000 (pp185) which appears during the initial response of hepatoma cells to insulin binding. In contrast to the insulin receptor, pp185 does not adhere to wheat-germ agglutininagarose or bind to anti-insulin receptor antibodies. Phosphorylation of pp185 is maximal within seconds after exposure of the cells to insulin and exhibits a dose-response curve similar to that of receptor autophosphorylation, suggesting that this protein represents the endogenous substrate for the insulin receptor kinase.

Insulin and insulin-like growth factors (IGFs) have been implicated in the pathogenesis of diabetic retinopathy and peripheral vascular complications. Previously, we have shown that retinal capillary endothelial cells responded to insulin and IGFs for metabolic and growth effects, whereas aortic endothelial cells were not responsive. In contrast, vascular supporting cells from both retinal capillaries (i.e. pericytes) and aorta (i.e. smooth muscle cells) responded equally to insulin, IGF-I, and IGF-II. The structure and ligand specificities of the receptor for these peptides were studied by covalently cross-linking 125I-labeled peptide hormones to their respective receptors using disuccinimidyl suberate, followed by polyacrylamide gel electrophoresis and autoradiography. The binding subunit of the insulin receptor, alpha-subunit, for all cell types was found to have a mol wt 145,000 under reduced conditions. Labeling of this band was inhibited by 10(-9) M insulin, antiinsulin receptor antibodies, and 10(-8) M IGF-I, but not by multiplication-stimulating activity (IGF-II). The beta-subunit of the insulin receptor in endothelial cells was identified by its ability to be autophosphorylated when stimulated by insulin and was found to have a mol wt of 99,000. Covalent cross-linking of IGF-I to its receptor revealed a mol wt of 145,000, similar to that of insulin receptor, except that IGF-I was 100-fold more potent than insulin in competing with [125I]IGF-I for binding. [125I]IGF-II in all cells was cross-linked to receptor with mol wt of 260,000 and 230,000 under reduced and nonreduced conditions, respectively. IGF-I competed weakly with [125I]IGF-II, whereas insulin was ineffective. [125I]IGF-II also bound to the band with alpha mol wt of 135,000, which was inhibited by insulin, IGF-I, and IGF-II. In summary, receptors for insulin, IGF-I, and IGF-II on cells from micro- and macrovessels are biochemically similar to those in other cells. Interestingly, the finding of large numbers of IGF-I and IGF-II receptors on endothelial cells suggests that these growth factors play a physiological role and are involved in vascular complications associated with diabetes.

Phosphorylation of the insulin receptor was studied in intact well differentiated hepatoma cells (Fao) and in a solubilized and partially purified receptor preparation obtained from these cells by affinity chromatography on wheat germ agglutinin agarose. Tryptic peptides containing the phosphorylation sites of the beta-subunit of the insulin receptor were analyzed by reverse-phase high performance liquid chromatography. Phosphoamino acid content of these peptides was determined by acid hydrolysis and high voltage electrophoresis. Separation of the phosphopeptides from unstimulated Fao cells revealed one major and two minor phosphoserine-containing peptides and a single minor phosphothreonine-containing peptide. Insulin (10(-7) M) increased the phosphorylation of the beta-subunit of the insulin receptor 3- to 4-fold in the intact Fao cell. After insulin stimulation, two phosphotyrosine-containing peptides were identified. Tyrosine phosphorylation reached a steady state within 20 s after the addition of insulin and remained nearly constant for 1 h. Under our experimental conditions, no significant change in the amount of [32P]phosphoserine or [32P]phosphothreonine associated with the beta-subunit was found during the initial response of cells to insulin. When the insulin receptor was extracted from the Fao cells and incubated in vitro with [gamma-32P]ATP and Mn2+, very little phosphorylation occurred in the absence of insulin. In this preparation, insulin rapidly stimulated autophosphorylation of the receptor on tyrosine residues only and high performance liquid chromatography analysis of the beta-subunit digested with trypsin revealed one minor and two major phosphopeptides. The elution position of the minor peptide corresponded to that of the major phosphotyrosine-containing peptide obtained from the beta-subunit of the insulin-stimulated receptor labeled in vivo. In contrast, the elution position of one of the major phosphopeptides that occurred during in vitro phosphorylation corresponded to the minor phosphotyrosine-containing peptide phosphorylated in vivo. The other major in vitro phosphotyrosine-containing peptide was not detected in vivo. Our results indicate that: tyrosine phosphorylation of the insulin receptor occurs rapidly following insulin binding to intact cells; the level of tyrosine phosphorylation remains constant for up to 1 h; the specificity of the receptor kinase or accessibility of the phosphorylation sites are different in vivo and in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

To characterize the carbohydrate moieties of the insulin receptor on IM-9 lymphocytes, the cells were surface iodinated and solubilized, and the insulin receptors were precipitated with anti-receptor antibody. The precipitates were resuspended, subjected to either enzymatic digestion or chemical treatment with trifluoromethanesulfonic acid and the relative mobilities of the alpha and beta subunits before and after treatment were analyzed by polyacrylamide gel electrophoresis and autoradiography. The results indicate that the alpha subunit possesses primarily N-linked carbohydrate which is both complex (Endoglycosidase F sensitive) and polymannose (Endoglycosidase H sensitive). The beta subunit also contains polymannose oligosaccharide units and has, in addition, a substantial amount of carbohydrate which is removed by chemical treatment but is not susceptible to Endoglycosidase F, suggesting the presence of O-linked saccharides. The apparent molecular weights of the core protein of the mature alpha and beta subunits as determined by gel electrophoresis following complete deglycosylation are 98 kDa and 80 kDa, respectively.

Anti-phosphotyrosine antibody and anti-insulin receptor antibody were used to study insulin-stimulated phosphorylation of the beta-subunit of the insulin receptor in [32P]orthophosphate-labeled Fao hepatoma cells. Without insulin, the receptor contained both phosphoserine and phosphothreonine and could be immunoprecipitated with anti-receptor antibody but not with the anti-phosphotyrosine antibody. After incubation of these cells with insulin, both antibodies immunoprecipitated the phosphorylated receptor. The beta-subunit of the receptor precipitated with anti-phosphotyrosine antibody from cells stimulated with insulin (100 nM) for 1 min contained predominantly phosphotyrosine, whereas, after 10 min with insulin, the amounts of phosphotyrosine and phosphoserine were nearly equal. These results suggest that insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the beta-subunit. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibody followed by precipitation of the remaining proteins with anti-receptor antibody suggests that insulin receptors which contain phosphoserine in the basal state are tyrosine phosphorylated more slowly than the dephosphorylated receptors or not at all after the addition of insulin. The beta-subunit of the insulin receptor was the major phosphorylated protein precipitated by the anti-phosphotyrosine antibody from insulin-stimulated Fao cells. These results confirm our notion that insulin initially stimulated tyrosine autophosphorylation and subsequently serine phosphorylation of the insulin receptor in intact cells and suggests that this sequence of reactions occurs faster on receptors that are dephosphorylated before the incubation with insulin.

Although reovirus infection may lead to changes in endocrine function in vivo, little is known about the precise interaction of reovirus with endocrine cells. In this study we have examined the effects of reovirus infection on two types of endocrine cells, GH4C1 cells and RINm5F cells. Both type 1 reovirus and type 3 reovirus infect the two cells lines and appear to grow equally well. Viral replication occurred within the first 24 h following infection after which viral titers remained stable for 3 days. By 48-72 h after viral infection, substantial cytopathic effects were noted in RINm5F cells infected with both type 1 and type 3 reovirus. In GH4C1 cells, type 3 reovirus was most effective in producing cell death, and type 1 reovirus was significantly less cytotoxic despite a similar viral titer. Only type 1 reovirus caused a specific inhibition of overall protein and DNA synthesis, and this occurred only in the RINm5F cells. Over the time course studied, GH4C1 cells successfully infected with type 1 reovirus demonstrated no cytopathic effects, and only minimal alterations in cellular function were noted. Intracellular insulin content and insulin secretion, a "luxury function" of the RINm5F cells, were also surprisingly well maintained in the first 48 h after viral infection. In addition, virally infected cells were able to respond to glyceraldehyde, an insulin secretagogue, although the response appeared to be somewhat blunted compared to that of control cells. These results suggest that viral infection of endocrine cells results in specific alterations that depend on the nature of the infecting virus. In addition, the cellular environment of the host cell may be an important determinant in the outcome of viral infection.

Events in the natural history of diabetic nephropathy (including the onset of persistent proteinuria and end-stage renal failure) were studied in a cohort of 292 patients with juvenile-onset type I diabetes who were followed for 20 to 40 years. The risk of persistent proteinuria increased rapidly between the fifth and 15th years of diabetes and declined thereafter. This pattern suggests that susceptibility to this complication was limited to a subset of patients and was exhausted over time. Patients with the most frequent severe hyperglycemia (the highest quartile) during the first 15 years of diabetes had a risk of persistent proteinuria that was four and a half times higher than that for those with the least frequent hyperglycemia (the lowest quartile). Patients whose diabetes was diagnosed in the 1930s had twice the risk of persistent proteinuria as those in whom the condition was diagnosed in later decades. Once persistent proteinuria appeared, progression to renal failure almost always followed. Half reached this stage within 10 years, and the interval for progression did not vary according to sex, frequency of hyperglycemia, or calendar year of diagnosis of diabetes. This period, however, was significantly shorter (eight versus 14 years) for patients whose diabetes was diagnosed after puberty than for those who were younger at onset. In conclusion, the development of diabetic nephropathy consists of at least two stages. The onset of proteinuria, although related to the level of exposure to hyperglycemia, appears to be influenced by genetic and/or environmental factors. The second stage, progression to renal failure, seems to be influenced by processes related to maturation or aging.

Anesthetized rats were treated with saline, antiinsulin receptor serum, or antiinsulin serum, and the biodistribution of high pressure liquid chromatography-purified 123I-Tyr A14-insulin was studied by scintillation scanning. Time activity curves over organs of interest were calibrated by sacrificing the rats at the end of the experiment and directly determining the radioactivity in the blood, liver, and kidneys. Saline-treated rats exhibited normal insulin biodistribution. The highest concentration of 123I-insulin was found in the liver, and reached 30% of total injected dose between 3 and 5 min after injection. After this peak, activity rapidly decreased with a t1/2 of 6 min. Activity of 123I-insulin in kidney showed a more gradual rise and fall and was approximately 15% of injected dose at its maximum. In rats treated with antiinsulin antiserum, insulin biodistribution was markedly altered. Peak liver activity increased with increasing antibody concentration with up to 90% of injected dose appearing in the liver. In addition, there was no clearance of the liver 123I-insulin over 30 min. Autoradiographic studies demonstrated that in contrast to the normal rats in which radioactivity was associated with hepatocytes, in rats passively immunized with anti-insulin serum, 125I-insulin was associated primarily with the Kuppfer cells. In contrast, antibodies to the insulin receptor markedly inhibited 123I-insulin uptake by the liver. Kidney activity increased, reflecting the amount of free 123I-insulin that reached this organ. This is similar to the pattern observed when insulin receptors are saturated with a high concentration of unlabeled insulin. Thus, both insulin antibodies and anti-receptor antibodies alter the distribution of insulin, but with very different patterns. The use of 123I-insulin and scintillation scanning allows one to study specific alterations in insulin distribution in animal models of insulin-resistant states, and should also be useful in human disease states.

The primary approach for the characterization of the insulin receptor has been through the study of its interaction with 125I-labeled insulin. Recently, we demonstrated that insulin receptors can also be identified by flow cytometry using antibodies to the receptor. In the present study, we characterized the insulin receptor on human lymphoblastoid cells (IM-9) and studied its regulation using insulin and antiinsulin antibodies as a probe for flow cytometry. The mean peak fluorescence of the cells treated with insulin followed by antiinsulin serum was 30-50 U above the control value. There was a close correlation between [125I]insulin binding and peak fluorescence. Fish insulin, which has about 50% the affinity of porcine insulin for the insulin receptor but does not bind to antiinsulin antibodies, did not enhance antiinsulin antibody binding, but competed for the pork insulin-antiinsulin antibody complexes in a dose-dependent manner. Exposure of IM-9 cells to insulin or antireceptor antibodies resulted in reduction in the number of insulin receptors. Cells down-regulated with 10(-6) M insulin or a monoclonal antibody to the insulin receptor had 40% of the [125I]insulin binding of the control cells and 40-50% of the peak fluorescence when insulin-antiinsulin was the probe for the immunofluorescence studies. Cells down-regulated with human autoantibodies to the receptor had 4% [125I]insulin binding and 10% peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical fluorescent peak. These date indicate that flow cytometry with insulin-antiinsulin antibody complexes provides a new approach to the measurement of insulin receptors, since it provides direct measurement of the occupied receptor.