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.

Insulin degradation by isolated rat adipocytes was evaluated using gel filtration and a new technique of differential precipitation to fractionate the sample by molecular size using polyethylene glycol and trichloracetic acid. At 37 degrees C, 125I-insulin bound to adipocytes was rapidly degraded into small fragments or iodotyrosine. 125I-insulin in the medium was also degraded into iodotyrosine, as well as fragments intermediate in molecular weight between insulin and iodotyrosine. Lowering the temperature to 15 degrees C or adding bacitracin to the medium inhibited degradation in the medium but had little effect on cell-associated degradation. Methylamine, on the other hand, inhibited cell-associated degradation, but had little effect on the insulin degradation in the medium. Addition of methylamine or bacitracin or lowering of the temperature increased the amount of 125I-insulin bound to the cell and prolonged the steady-state of binding. Bacitracin also produced a slight shift to the left in the dose response curve for insulin-stimulated glucose oxidation. Methylamine increased basal glucose oxidation, but had no effect on insulin sensitivity as measured in the glucose oxidation bioassay. These data suggest that isolated adipocytes in vitro exhibit at least two distinct pathways of insulin degradation, a cell-associated pathway which can be inhibited by methylamine and a medium pathway which can be inhibited by bacitracin. Neither pathway, however, appears to be closely linked to insulin's ability to stimulate glucose metabolism in these cells.

It has been suggested that elevated levels of insulin or insulin-like growth factors (IGFs) play a role in the development of diabetic vascular complications. Previously, we have shown a differential response to insulin between vascular cells from retinal capillaries and large arteries with the former being much more insulin responsive. In the present study, we have characterized the receptors and the growth-promoting effect of insulinlike growth factor I (IGF-I) and multiplication-stimulating activity (MSA, an IGF-II) on endothelial cells and pericytes from calf retinal capillaries and on endothelial and smooth muscle cells from calf aorta. We found single and separate populations of high affinity receptors for IGF-I and MSA with respective affinity constants of 1 X 10(-9) M-1 and 10(-8) M-1 in all four cell types studied. Specific binding of IGF-I was between 7.2 and 7.9% per milligram of protein in endothelial cells and 9.1 and 10.4% in the vascular supporting cells. For 125I-MSA, retinal endothelial cells bound only 1.7-2.5%, whereas the aortic endothelial cells and the vascular supporting cells bound between 5.6 and 8.5% per milligram of protein. The specificity of the receptors for IGF-I and MSA differed, as insulin and MSA was able to compete with 125I-IGF-I for binding to the IGF-I receptors with 0.01-0.1, the potency of unlabeled IGF-I, whereas even 1 X 10(-6) M, insulin did not significantly compete with 125I-MSA for binding to the receptors for MSA. For growth-promoting effects, as measured by the incorporation of [3H]thymidine into DNA, confluent retinal endothelial cells responded to IGF-I and MSA by up to threefold increase in the rate of DNA synthesis, whereas confluent aortic endothelial cells did not respond at all. A similar differential of response to insulin between micro- and macrovascular endothelial cells was reported by us previously. In the retinal endothelium, insulin was more potent than IGF-I and IGF-I was more potent that MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In addition, insulin and IGF-I at 1 X 10(-6) and 1 X 10(-8) M, respectively, stimulated these cells to grow by doubling the number of cells as well. In all responsive tissues, the combination of insulin and IGFs were added together, no further increase in effect was seen. These data showed that vascular cells have insulin and IGF receptors, but have a differential response to these hormones. These differences in biological response between cells from retinal capillaries and large arteries could provide clues to understanding the pathogenesis of diabetic micro- and macroangiopathy.