C R Kahn

125I-insulin binding to cultured human lymphocytes. Initial localization and fate of hormone determined by quantitative electron microscopic autoradiography.

Morphologic and biochemical studies indicate that the initial action of insulin is binding to a cell surface receptor. Whether further translocation of the hormone, or a product of the hormone, occurs is unclear and has not been investigated by direct means. To determine the fate of 125I-insulin bound to its receptor, we have examined the distribution of radioactivity by quantitative electron microscopic autoradiography. Cultured lymphocytes of the IM-9 cell line were incubated with 0.1 nM 125I-insulin at 15 degrees and 37 degreesC for incubation periods extending from 2 to 90 min. At 15 degreesC, grains localize to the plasma membane and there is no translocation as a function of time. At 37 degreesC, grains predominantly localize to the plasma membrane but there is a small shift in distribution to a distance of 300-700 nm from the plasma membrane. This small additional band component of irradiation extends to approximately to10--15% of the cell radius. When a morphometric analysis is applied to grains extending 300 nm and beyond from the plasma membrane, we find no preferential localization to any intracellular organelle. We interpret these data to indicate that in the cultured lymphocyte, labeled insulin initially localizes to the plasma membrane but as fuanction of time and increasing temperature there is a small but definite translocation of the hormone or a product of the hormone to a hihgly limited aea of the cell periphery.

Insulin-receptor antibodies mimic a late insulin effect.

AUTOANTIBODIES against the insulin receptor are found in patients with a rare form of extreme insulin resistance associated with acanthosis nigricans1. These antibodies are primarily polyclonal IgGs, compete with insulin for binding to its receptor2,3, immunoprecipitate solubilised insulin receptors4, and have been used as probes of insulin action. The acute action of the antibodies is to mimic many of insulins biological effects, particularly those which occur rapidly after hormone binding to its cell-surface receptors5–9. In addition, insulin exerts biological effects which appear more slowly. For example, insulin treatment of 3T3-L1 fatty fibroblasts increases the activity of lipo-protein lipase maximally after 2–4 d but is without effect during the first 4 h (refs 10, 11). Some investigators have suggested that these slower effects may be mediated by different mechanisms from those involved in the production of the acute effects12,13. We now report that autoantibodies against the insulin receptor mimic this long-term insulin effect on lipoprotein lipase.

The Insulin Receptor and Insulin of the Atlantic Hagfish: Extraordinary Conservation of Binding Specificity and Negative Cooperativity in the Most Primitive Vertebrate

The North Atlantic hagfish, a cyclostome that is representative of the most primitive vertebrates still alive, diverged from the other vertebrates about 500 million years ago. Hagfish insulin, which differs from porcine insulin in 18 (38%) of its amino acids, had a potency of 5-10% that of porcine insulin in stimulating glucose oxidation and deoxyglucose transport in rat adipocytes and was 5-10% as potent as porcine insulin in binding to insulin receptors on rat adipocytes and human (IM-9) lymphocytes. Like all other naturally occurring insulins, hagfish insulin accelerated the dissociation of 125I-porcine insulin from insulin receptors and the degree of the acceleration was related to its occupancy of the receptor. The insulin receptor of the hagfish erythrocyte showed the time, temperature, and pH dependence of binding and the negative cooperativity that are characteristic of all other insulin receptors. That the negative cooperativity is fully conserved in such an ancient insulin and receptor suggests that it is an important functional feature of this hormone-receptor system. The hagfish receptor showed the same absolute affinity and rank order of preference for insulins and insulin analogues (chicken > pork > proinsulin > guinea pig > desoctapeptide) found with other receptors of less primitive vertebrates, which supports the conclusion that the receptor for insulin is functionally better conserved evolutionary than the hormone. However, uniquely, hagfish insulin was more potent in binding to hagfish receptors than to mammalianreceptors; with all other species of insulins studied, the affinity of the hormone for homologous receptor was the same as for receptors of heterologous species.

In vitro lymphocyte proliferation response to therapeutic insulin components. Evidence for genetic control by the human major histocompatibility complex.

Genes in the major histocompatibility complex of mice and guinea pigs control immunologic responsiveness to insulins from other animal species. In order to determine if similar genetic control exists in man, we have examined lymphocyte proliferation responses to components of therapeutic insulins by employing lymphocytes from diabetic patients that receive insulin. Distinct groups of individuals demonstrated positive lymphocyte proliferative responses to beef insulin, beef and pork insulin, beef proinsulin, pork proinsulin, and protamine. Lymphocytes from the patient population were typed for the HLA-A, B, C, and DR antigens. An increased frequency of certain HLA antigens was found in those individuals that responded to the following therapeutic insulin components: beef, HLA-DR4; beef and pork, HLA-DR3; beef proinsulin, HLA-BW4, CW2, CW5, DR2, and DR5; protamine, HLA-CW3, CW5, and DR7. The results demonstrate that the human immune system recognized the structural differences between human and beef and/or pork insulin. These differences are two amino acids in the A chain, alpha loop, of beef insulin and the single terminal amino acid, alanine, which is common to pork and beef insulins. Positive responses to both beef proinsulin and pork proinsulin demonstrated the capability of restricted recognition of more complex proteins represented by the C-peptide in these insulin preparations. Lymphocyte proliferative responses to protamine were also restricted, which suggests a genetic control to this antigen. The association of these responses with HLA alloantigens strongly suggests that genes within the human major histocompatibility complex control recognition and lymphocyte response to therapeutic insulin components.

Virus-induced decrease of insulin receptors in cultured human cells.

Viral infections may produce abnormalities in carbohydrate metabolism in normal subjects and profound changes in glucose homeostasis in insulin-dependent diabetics. Using an in vitro radio-receptor assay with 125I-labeled insulin and human-amnion (WISH) cells, the effect of viral infections on insulin receptors was examined. Both herpes simplex virus and vesicular stomatitis virus produced a 50% decrease in insulin binding. There was no evidence that this decrease was due to degradation of insulin. On quantitative analysis, this decrease in binding was found to be the result of a decrease in receptor concentration with no change in receptor affinity. The decrease in receptors occurred between 4 and 12 h, at the time viral antigens were being inserted into the plasma membrane of infected cells. Because the t 1/2 of insulin receptors in uninfected cells was between 14 and 24 h, the decrease in insulin receptors cannot be explained solely by virus-induced shut-off of macromolecular synthesis. Moreover, viruses such as encephalomyocarditis that do not insert new antigens into the plasma membrane, did not cause changes in the number of insulin receptors. The most likely explanation is that virus-induced changes in the plasma membrane altered or displaced insulin receptors. It is concluded that the insulin receptor assay is a sensitive and quantitative method for studying the effect of viral infections on cell membranes. These data also suggest that abnormalities in glucose metabolism associated with some viral infections may be due, in part, to changes in the concentration of insulin receptors.

The Immune Response to Insulin in Man: Interaction of HLA Alloantigens and the Development of the Immune Response

In both mice and guinea pigs, the immunologic response to insulin has been demonstrated to be under immune response (Ir) gene control. In the present study, we have attempted to identify Ir genes to insulin in man by correlating HLA allotype with immunologic response to insulin. Three groups of patients were studied: (1) 117 diabetic patients with a history of exaggerated immunologic response to insulin (insulin allergy, resistance, or systemic reactions); (2) 95 insulin-taking diabetics without clinically significant immune response; and (3) 109 nondiabetic controls. Determination of HLA alloantigen frequencies in the “insulin allergic” population and the nonallergic diabetics revealed only modest increases of HLA-Bw44 (29% versus 16%, P < 0.05) and -DR7 (28% versus 15%, P < 0.05). Studies of linkage disequilibrium, however, revealed interesting associations involving HLA-A2, -Bw44, and -DR7 in the diabetic with insulin allergy that were not present in the nonallergic diabetic. Combinations of any two or all three of these antigens were observed in 39% of diabetics with insulin allergy but in only 3% of nonallergic diabetics. An intermediate frequency (23%) was observed in the normal population. Thus, if an individual with any combination of HLA-A2, -Bw44, and -DR7 develops diabetes and receives insulin therapy, there is over a 90% probability he will exhibit a clinically significant immune reaction to insulin (calculated relative risk = 20.6). These data suggest that in man, as in the mouse, the immune response to insulin is under genetic control, but this may involve interactions of several genes in the major histocompatibility (MHC) complex, or a locus in linkage with these genes that is not measured by current techniques.

Subpopulations of antibodies directed against evolutionarily conserved regions of the insulin molecule in insulin-treated patients.

SummaryIn the present study, we attempted to define possible subpopulations of antibodies which theoretically could be directed against evolutionarily conserved regions of the insulin molecule in sera from insulin-treated diabetic patients using a variety of labelled and unlabelled insulins which differ widely in structure but are very similar in functional properties. Ten high titre human insulin antisera from patients treated with mixed beef-pork insulin were examined. All sera were found to bind 125I-pork insulin better than labelled chicken insulin which bound better than labelled fish insulin. Detailed studies were conducted with four of the antisera using the pork and fish tracers. With two of the antisera, a subpopulation of antibody could be detected with 125I-fish insulin which had similar affinity for both fish and pork insulin, but reacted much less well with guinea pig insulin and the desoctapeptide derivative of porcine insulin. Based on the known properties of these four insulins, the data provide suggestive evidence consistent with the hypothesis that there are subpopulations of antibodies recognizing regions on the insulin molecule that are well conserved, possibly the region involved in the formation of insulin dimers or receptor binding.

The Binding of Insulin to Mouse Leucocytes During Viral Infections

SummaryThe effect of viral infections on insulin binding in vivo was evaluated by measuring the binding of 125I-insulin to several different tissues. We found that splenic leucocytes from mice infected with either the diabetogenic (D) or non-diabetogenic (B) variants of encephalomyocarditis virus, herpes simplex virus, or lactic dehydrogenase virus showed up to a 130% increase in insulin binding. As much as a 300% increase in the binding of 125I-insulin to splenic leucocytes was observed in mice given bacterial lipopolysaccharide. In neither virus-infected nor lipopolysaccharide-treated mice was there any substantial change in insulin receptors on thymocytes, liver membranes, or peripheral erythrocytes. Thus, the increased binding of insulin appears to be limited to leucocytes and does not appear to represent a generalized metabolic alteration. These experiments suggest that during infection, the binding of insulin to leucocytes, which is widely used to measure insulin receptors, may not always accurately reflect the insulin receptor status of other tissues.

Autoantibodies to the Insulin Receptor and Insulin-Resistant Diabetes

Autoantibodies to the insulin receptor are a rare cause of insulin-resistant diabetes, but when they occur they produce a profound clinical syndrome. These antibodies block insulin binding, immunoprecipitate solubilized insulin receptors, and their acute effect is to mimic the biological effects of insulin. However, prolonged exposure of cells to these antibodies produces a state of insulin resistance. Since the antigen to which the antibody is directed is relatively well-characterized, many of the observations in this syndrome can serve as a model for elucidating molecular mechanisms in other diseases with antibodies against membrane components. The autoantibodies to the insulin receptor have also provided valuable probes in the study of insulin receptor structure and insulin action.