Len C. Harrison

Insulin receptors, receptor antibodies, and the mechanism of insulin action.

Publisher Summary Insulin exerts a wide spectrum of effects at the cellular level. These include effects at the membrane level, for example, the stimulation of glucose and amino acid uptake; effects on both membrane and cytoplasmic enzymes; and effects on protein synthesis, DNA synthesis, cell growth, and differentiation. This is the reason why unraveling the mechanism of insulin action has been difficult. This chapter describes insulin receptors, receptor antibodies, and the mechanism of insulin action. In addition, the effects of insulin vary both in time course and dose–response. Some effects of insulin, such as the stimulation of glucose transport, occur within seconds after hormone exposure, whereas others, such as the stimulation of DNA synthesis, require hours; effects on most cytoplasmic enzymes lie somewhere between these two extremes. It is well known that insulin stimulates glucose transport, and this effect persists in membrane vesicles even after the cell is broken. Several new probes have become available that have shed some light on the problem of insulin action. These include cultured cell systems, which are highly responsive to insulin; new methods for studying receptor structure; mutant cell lines that possess defects in either the insulin receptor or pathways of insulin action; and antibodies to the insulin receptor that block insulin binding and mimic insulin action.

Treatment by plasma exchange of a patient with autoantibodies to the insulin receptor.

WE recently described two groups of patients with severe insulin-resistant diabetes and acanthosis nigricans.1 In these patients the defect of insulin action could be correlated with markedly abnor...

Gliclazide Therapy Is Associated with Potentiation of Postbinding Insulin Action in Obese, Non-insulin-dependent Diabetic Subjects

Six obese, non-insulin-dependent diabetic subjects were studied before and 3 mo after treatment with the sulfonylurea gliclazide, 40–80 mg b.i.d. Fasting plasma glucose fell significantly from 13.4 ±1.6 (SEM) to 8.6 ±1.2 mmol/L, accompanied by a significant reduction from 40.6 ± 3.7 to 29.8 ± 2.8 mM · h of the plasma glucose response to 75 g oral glucose. Fasting plasma insulin showed a nonsignificant increase from 24.8 ± 2.0 to 31.3 ± 2.3 mU/L. The percent specific binding of tracer 125I-insulin to erythrocytes and monocytes did not change significantly (from 9.8 ± 1.7 to 8.5 ± 0.7 for erythrocytes and 1.7 ± 0.3 to 1.6 ± 0.4 for monocytes). Glucose utilization was measured at three levels of insulin infusion (40, 100, and 300 mU/ kg/h) by the euglycemic clamp technique. Overall there was a significant (P < 0.05) increase in the disappearance rate (Rd) and metabolic clearance rate (MCRg) for glucose at the two higher insulin infusion rates (MCRg: 3.3 ± 0.7 to 5.1 ± 0.7 and 5.9 ± 0.9 to 7.9 ± 0.9 ml/kg/min), but not at the lowest infusion rate (MCRg: 3.6 ± 0.8 to 3.3 ± 0.6). Thus, the chronic hypoglycemic effect of gliclazide in obese diabetic subjects was associated with an improvement in insulin-mediated glucose utilization at high plasma insulin concentrations. This enhanced effect of insulin after gliclazide treatment was not accompanied by increased monocyte or erythrocyte insulin binding, which suggests that it was due to potentiation of postbinding insulin-sensitive pathways.

Autoantibodies Against the Insulin Receptor Recognize the Insulin Binding Subunits of an Oligomeric Receptor

125I-insulin was specifically cross-linked to membranes of human cultured lymphocytes (IM-9 line) using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and autoradiography of this preparation revealed a major 125I-labeled band with an apparent mol wt of 130,000 and minor bands of about 300,000 and 95,000. Labeling of these bands was inhibited by incubation of membranes with either unlabeled insulin or autoantibodies to the insulin receptor. The bands were also observed after the 125I-insulin cross-linked preparation was solubilized and immunoprecipitated with a panel of autoantibodies to the insulin receptor. However, immunoprecipitation of the 125I-insulin-receptor complex was inhibited by preincubation with excess unlabeled insulin. Finally, 125I-Fab fragments of mol wt 50,000 prepared from anti-receptor antibodies and cross-linked to membranes were resolved into a major complex of mol wt 180,000 and a minor band of 125,000. Neither band was observed when 125I-Fab fragments were cross-linked to membranes in the presence of an excess of unlabeled insulin. These findings indicate that autoantibodies to the insulin receptor are directed against the insulin binding subunits of an oligomeric receptor.

An Introduction to Receptors and Receptor Disorders

Summary In the last decade reliable methods have been introduced to quantitate and characterize receptors for hormones and other biologically interesting ligands. Applied initially to hormone receptors on malignant cells and to androgen-resistant and insulin-resistant states, these methods have led to the identification of many disease processes where the receptor plays an important role. This includes not only endocrine-related diseases but also neurological, metabolic, infectious, and immune disorders as well. Note. On the basis of biological and chemical data, it appears that the 40 to 50 different hormones found in a given organism each evolved from a much smaller number of primordial hormones. Presumably, for the peptide hormones the gene for the primordial hormone was duplicated as was the gene for its cell surface receptor; the new hormone-receptor pair evolved to establish a second signaling system. The affinity of each hormone for its specific receptor was high but in many cases the ligand retained some finite (albeit low) affinity for the other receptor. Thus, teleologically, greater diversity was paid for by a small loss in specificity. The weak affinity of one hormone for the receptor of a closely related hormone forms the basis for specificity spillover. The modest impairment of specificity in endocrine systems, its postulated origins, and suggested consequences may have significant counterparts in other areas of biology where diversity coexists with modest degeneracies in specificity, e.g., the large series of trypsin-like proteases that regulate multiple extracellular events such as clotting and kinin generation; neurotransmitters and related drugs; cyclase and the nucleotide-stimulated kinases; autoantibodies and prostaglandins.

Diabetes is Associated with Autoimmunity in the New Zealand Obese (NZO) Mouse

The New Zealand Obese (NZO) mouse was studied as a potential model for autoimmune diabetes. NZO mice develop obesity, glucose intolerance, and insulin resistance, and have low-titer IgM antibodies to the insulin receptor. It is shown that they have circulating antibodies to both native DNA and denatured, singlestranded DNA. The antibody levels are higher in females, and, up to 6 mo of age, are comparable to those found in the related NZB × NZW F1 (NZB/W) mouse, a model for systemic lupus erythematosus. After 6 mo of age the antibody levels in NZO mice fall toward normal, in contrast to the persistently elevated levels in NZB/W mice. NZB/W mice are known to succumb to immune complex-mediated proliferative glomerulonephritis before 1 yr of age, whereas NZO mice survive. NZO kidneys exhibit light microscopic features of both diabetic and lupus nephropathies: glomerular proliferation, mesangial deposits, mild basement membrane thickening, glomerulosclerosis, eosinophilic nodules in some glomeruli, occasional hyalinization of the glomerular arterioles, and healing arteriolar inflammation. These changes are associated with glomerular deposition of immunoglobulin, especially IgM, in a granular pattern on fluorescent staining. The NZO mouse, therefore, has evidence of a generalized immune disorder and provides a model for studying the relationship between autoimmunity, obesity, and diabetes.

Structural differences between insulin and somatomedin-C/insulin-like growth factor-1 receptors revealed by autoantibodies to the insulin receptor

Abstract Five sera containing autoantibodies to the insulin receptor were used to compare the immunological characteristics of the insulin and the somatomedin-C/insulin-like growth factor-1 receptors of Triton-solubilized human placental membranes. Complete immunoprecipitation of [ 125 I] insulin-labelled receptors was achieved using all five antisera. Three antisera precipitated 90, 65 and 40% of [ 125 I] insulin-like growth factor-1-labelled receptors while the other two caused less than 7% immunoprecipitation. These results are consistent with the view that insulin and insulin-like growth factor-1 receptors are separate molecules which although structurally similar, possess a significant degree of immunologic non-identity.

Structure of the Human Erythrocyte Insulin Receptor

The structure of the insulin receptor in intact human erythrocytes was defined using the techniques of disuccinimidyl suberate (DSS) cross-linking of 125I-insulin and surface [125I]iodination followed by receptor immunoprecipitation. In contrast to a recent report, we found the erythrocyte insulin receptor to be similar in structure to that in classic target tissues for insulin, consisting of at least three species of molecular weight approximately 295,000, 265,000, and 245,000, containing disulfide-linked subunits of molecular weight approximately 130,000 and 95,000. The interconversion of the three oligomeric forms could mediate changes in receptor affinity as postulated in other tissues. The 95,000 subunit was detected by immunoprecipitation only if surface iodination was performed in a Tris/Hepes buffer using lodogen and not if phosphate-buffered saline or lactoperoxidase iodination was used. These findings indicate that the lack of a bioeffect of insulin in erythrocytes is not explained by a gross defect in the structure of their insulin receptors. The apparent identity of the insulin receptor structure in erythrocytes and insulin target tissues provides a firmer basis for the use of erythrocytes in some circumstances to reflect insulin receptor status.

Insulin sensitivity of adipose tissue in vitro and the response to exogenous insulin in obese human subjects

The effect of varying concentrations of insulin on 1-14C-glucose conversion to 14CO2 was measured in subcutaneous adipose tissue samples obtained from 16 obese human subjects (10 nondiabetic, 6 diabetic). An index of insulin sensitivity in vitro, Kins, was calculated as the concentration of insulin stimulating one-half maximal 14CO2 production. An index of insulin sensitivity in vivo, Kitt, was calculated as the rate constant for decrease in blood glucose after rapid intravenous administration of 0.05 U/kg insulin. There was, over-all, a significant correlation between Kins and Kitt, indicating that insulin sensitivity of 1-14C-glucose oxidation by adipose tissue in vitro reflects the general state of sensitivity of glucose metabolism to insulin in vivo in obese human subjects. The mean values for both Kins and Kitt in the nondiabetic subjects were significantly different from those in the diabetic subjects, indicating greater sensitivity to insulin in the former group. The nondiabetic group was also distinguished by a significantly greater plasma insulin:blood glucose ratio in the oral glucose tolerance test. These results support the view that tissue insulin sensitivity as well as pancreatic beta cell response play an important role in determining glucose tolerance in obesity.

A Postbinding Inhibitor of Insulin Action: Increased Concentrations in the Plasma of Non-insulin-dependent Diabetic Subjects

“Postreceptor” insulin resistance in persons with non-insulin-dependent diabetes (NIDDM) could be due to an intrinsic defect in insulin-sensitive pathways or to the action of a circulating inhibitor. Since evidence for the former is lacking, we have addressed the question of a circulating inhibitor by examining the effect of plasma and plasma extracts from NIDDM subjects on the lipogenic response of rat adipocytes to insulin. A majority (77%) of plasma samples (1:20 dilution) from unselected, treated NIDDM subjects (N = 69) inhibited insulin-stimulated conversion of 3-3H-glucose to 3H-lipid in rat adipocytes to a greater extent than did control samples (N = 24). The mean ± SD inhibition by NIDDM plasma (81 ± 21%) was significantly greater (P < 0.01) than by control plasma (50 ± 14%). Diabetic and, to a lesser degree, control plasma both caused a significant decrease in the maximal response of lipogenesis to insulin. Inhibitory activity was extracted into acid/ethanol, present in the flow of a Seppak C18 column, heat-stable (56°C for 30 min [plasma], 80°C for 30 min [acid/ethanol]), resistant to proteases, and dialyzable through 1000-dalton-mol wt exclusion dialysis tubing. The inhibition by NIDDM plasma or partially purified inhibitor could not be explained by the presence of insulin antibodies, insulin receptor antibodies, other inhibitors of insulin binding, or the concentrations of known counterregulatory factors. There was no correlation between inhibitory activity and plasma glucose (r = 0.26), insulin (r = 0.33), C-peptide (r = 0.26), or HbA1c(r = 0.26). Our findings suggest that a humoral inhibitor of postreceptor insulin action is present in increased concentrations in the plasma of NIDDM subjects. The nature of this inhibitory activity and its pathophysiologic significance deserve further investigation.