We studied the structure of the insulin-receptor gene in normal individuals and in four unrelated patients with leprechaunism (Minn-1, Ark-1, Ark-2, Can-1) and four unrelated patients with the type A syndrome of insulin resistance, both disorders associated with genetic alterations in affinity, binding capacity, and kinase activity of the insulin receptor. Genomic cloning and Southern blot analysis indicate that the normal human insulin-receptor gene is greater than or equal to 150 kilobases long and consists of a minimum of 17 exons, 6 in the genomic region of the alpha-subunit and 11 in the region of the beta-subunit. Three of the patients, one with leprechaunism and two with type A syndrome, have decreases in insulin-receptor mRNA but on genomic blot analysis have no obvious abnormalities in the insulin-receptor gene. No distinctive pattern of restriction-fragment-length polymorphisms or evidence for major insertion or deletion mutations of the insulin-receptor gene was found in any of the patients. These data indicate that the insulin-receptor gene is greater than 35 times larger than coding regions and has a complex structure. Although leprechaunism and type A syndrome are most likely due to defects in the structure and expression of the insulin-receptor gene, they are likely to be associated with specific point mutations rather than major changes in gene structure.

Dexamethasone-induced changes in insulin and epidermal growth factor (EGF) receptor number, autophosphorylation, and kinase activity were studied in intact rat hepatocytes. Hepatocytes were freshly isolated from Sprague-Dawley rats treated with dexamethasone (1 mg/kg) for 4 days and from untreated littermates. Dexamethasone had no effect on insulin receptor number, while EGF receptor binding was slightly increased (21.3% vs. 17.2% binding/10(6) cells) after dexamethasone treatment. In hepatocytes from both control and dexamethasone-treated animals labeled with 32P, insulin induced tyrosine phosphorylation of the beta-subunit of the insulin receptor as well as of a 175K protein believed to be its endogenous substrate. The degree of phosphorylation of the insulin receptor was decreased 34% by dexamethasone treatment compared to the control value when studied in fasted animals. In contrast, phosphorylation was increased to a similar extent by dexamethasone treatment in fed animals. In addition, the beta-subunit of the insulin receptor extracted from dexamethasone-treated animals migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a slightly increased mobility compared to normal (89 +/- 1.2K vs. 92.5 +/- 0.4K). EGF induced tyrosine phosphorylation of its own receptor and of a 120K protein in intact hepatocytes. Their degree of phosphorylation was decreased by 30% as a result of dexamethasone treatment in the fasted animal and was unchanged in the fed animals. Our data indicate that glucocorticoids modulate insulin and EGF receptor kinase activity, but the nature of their effect depends on other factors, including the dietary state of the animal. These studies also suggest that postreceptor changes account for a major component of glucocorticoid-induced insulin resistance.

Insulin modifies cellular responsiveness to some hormones which operate via guanine nucleotide binding proteins (G-proteins); also, G-proteins have been implicated in some actions of insulin. Using pertussis toxin-catalyzed [32P]ADP-ribosylation of Gi as an index of G-protein conformation, we evaluated interaction of insulin receptors with G-proteins. In isolated rat liver plasma membranes, insulin treatment for 10 min inhibited [32P]ADP-ribosylation of Gi by 50%. This effect was half-maximal at 2 x 10(-8) M. A similar effect was observed with rat adipocyte plasma membranes with half-maximal effect at 1 x 10(-8) M. Pertussis toxin activity itself was uninfluenced by insulin, as ribosylation of tubulin or heat-treated bovine serum albumin was unaltered. Elevated Mg2+ diminished basal ADP-ribosylation, but insulin inhibition occurred at all Mg2+ levels between 0 and 1 mM. Insulin inhibition was independent of ATP (20 microM to 10 mM), and GTP (0-100 microM) concentrations. Because both protein kinase C and purified insulin receptor phosphorylate purified Gi in vitro, we examined Gi as a substrate for the insulin receptor tyrosine kinase in vivo. Triton-extracts of isolated rat hepatocytes which had been 32Pi labeled and treated with insulin were immunoprecipitated with a polyclonal anti-Gi antiserum. The dominant labeled phosphoprotein had a molecular weight of 42 kDa, consistent with the alpha-subunit of Gi, contained only phosphoserine, and was unaffected in its phosphorylation by insulin. These results indicate the existence of a novel pathway for physiological "cross-talk" between insulin and other hormones and further suggests that the insulin receptor may interact with regulatory G-proteins via biochemical mechanisms not directly involving the tyrosine kinase activity of the insulin receptor.

Offspring of women with insulin-dependent diabetes mellitus (IDDM) have a lower risk of developing IDDM than offspring of men with IDDM (1). To determine whether the risk of diabetes in offspring of diabetic mothers has changed after dramatic improvements in perinatal survival of these infants, we undertook a follow-up study of 1602 pregnancies of 739 women with IDDM who were patients at the Joslin Diabetes Center. Improvements in perinatal survival were abrupt rather than gradual. During the two decades before 1961, perinatal mortality was stable around 23%. After a sudden drop in 1961, it stabilized around 14% until 1975, when it was brought down to 4%, where it has remained. Of the 1391 offspring who survived the neonatal period, IDDM has developed in 21, a cumulative risk of 2.1 +/- 0.5% (SE) by age 20 yr. This is one-third the risk previously reported for offspring of fathers with IDDM and is independent of the calendar time of the births (1). The risk of diabetes in offspring of diabetic mothers is increased in young mothers and is otherwise independent of risk factors for perinatal mortality in this series. We conclude that there is no evidence that selective loss of diabetes-susceptible fetuses in perinatal deaths is a mechanism for the lower incidence of IDDM in the offspring of mothers with IDDM than in those of fathers with IDDM. The principal alternative mechanism is that exposure in utero to an affected mother can protect a fetus from developing IDDM later in life. Induction of immunologic tolerance to the autoantigens of the beta-cells is a plausible mechanism for this protective effect.

Insulin receptor function was examined in cultured skin fibroblasts from three patients with leprechaunism (Ark-1, Minn-1, and Can-1), a rare syndrome of severe insulin resistance and neonatal growth retardation. All three patients cell lines demonstrated insulin binding less than 15% of control. This was primarily due to reduced affinity of the receptor in Can-1 and due to reduced number of receptors in the other two cell lines (Ark-1 and Minn-1). When expressed as a fraction of total insulin bound, the percentage of cell-associated insulin internalized and degraded did not differ between the patient cell lines and the controls. However, chloroquine, which inhibited degradation by 50% in the control cells, had no effect in the cells from the patients. When normalized to insulin binding, insulin receptor autophosphorylation was normal in cells from Can-1, but reduced in those of Ark-1 and Minn-1. In contrast, the receptor-associated tyrosine kinase activity toward exogenous substrates was decreased in all three patient cell lines. These results suggest that leprechaunism is a biochemically heterogenous disease associated with a variety of alterations in receptor function. Cells from Ark-1 and Minn-1 exhibit parallel alterations in receptor autophosphorylation and kinase activity. Cells from Can-1 demonstrate normal receptor autophosphorylation but reduced kinase activity, thus displaying a unique form of a mutant insulin receptor. Despite reduced kinase activity, all three cell lines exhibit normal rates of insulin internalization, but decreased lysosomal-mediated degradation. Our data imply that receptor autophosphorylation and kinase activity may be regulated separately and that kinase activity may be linked to insulin degradation, but not necessarily internalization.

To evaluate the synthesis and initial processing of the insulin receptor precursor, we compared cell-free translation of rat liver poly(A)+ RNA in a reticulocyte lysate system with metabolically labeled rat hepatoma (Fao) cells. In in vitro translation assays, the primary L-[35S]cysteine-labeled products of rat liver mRNA specifically immunoprecipitable with insulin receptor antiserum were two closely migrating polypeptides with a Mr range of 160,000-164,000 (n = 7). This is similar to the size predicted by the insulin receptor cDNA sequence. When heterologous microsomal membranes were included in the cell-free system to process newly synthesized proteins co-translationally, the receptor precursors migrated as larger species of 180 +/- 2 kDa (n = 3). For comparison, when Fao cells were treated with tunicamycin to block core N-glycosylation and pulse-labeled with L-[35S]methionine, two closely migrating precursors were labeled that co-migrated with the unprocessed in vitro translation products (approximately 160 kDa). Pulse labeling of Fao cells in the absence of tunicamycin revealed receptor precursor species of 188 and 198 kDa that rapidly disappeared (t1/2 = 54 min) as the receptor subunits were observed. Thus, the initial products of insulin receptor mRNA translation are two approximately 162-kDa polypeptides that are rapidly processed in intact cells and can only be observed by in vitro studies or by using inhibitors of core glycosylation. Insulin proreceptor species can also be partially glycosylated during cell-free translation by added microsomal membranes. This is the first description of cell-free translation of the insulin proreceptor in a system that will allow detailed characterization of the earliest steps in insulin receptor biogenesis.

Tyrosyl phosphorylation is implicated in the mechanism of insulin action. Mutation of the beta-subunit of the insulin receptor by substitution of tyrosyl residue 960 with phenylalanine had no effect on insulin-stimulated autophosphorylation or phosphotransferase activity of the purified receptor. However, unlike the normal receptor, this mutant was not biologically active in Chinese hamster ovary cells. Furthermore, insulin-stimulated tyrosyl phosphorylation of at least one endogenous substrate (pp185) was increased significantly in cells expressing the normal receptor but was barely detected in cells expressing the mutant. Therefore, beta-subunit autophosphorylation was not sufficient for the insulin response, and a region of the insulin receptor around Tyr-960 may facilitate phosphorylation of cellular substrates required for transmission of the insulin signal.