Edward R Arquilla

Modulation of 125I-insulin degradation by receptors in liver plasma membranes.

Abstract This study investigated the interactions of insulin with receptors in a purified mouse liver plasma membrane. Two processes were found when insulin was incubated with the liver plasma membranes at 30°C; 1) the binding of insulin to these membranes and 2) the degradation of insulin by one or more associated insulinases or proteases. These two activities were physically separated, analyzed, and it was found that bound insulin is a substrate for insulin degradation. In addition, the rate of degradation of free insulin in solution was found to be 1.1% degraded insulin/available insulin/min in a 30 minute period. This rate is compared to 25.3% degraded insulin/available insulin/min under similar conditions when insulin is bound to the plasma membranes. Thus, insulin binding was shown to aid in its degradation. These results suggest that a specific binding by at least one of the receptors for insulin orients the molecule such that its degradation is facilitated.

The effect of antisera to insulin, 2-deoxyglucose-induced hyperglycemia, and starvation on wound healing in normal mice.

Wound healing was examined in normal C57B/6 male mice treated with antiserum to insulin or 2-deoxyglucose (2-DG) and in mice starved for 33 h. Hyperglycemia was induced after antiserum or 2-DG treatment; the blood glucose was lowered in the starved mice when compared with controls. Small dermal wounds were made in the ears of the mice 1 h after the initial injection of antisera or 2-DG. the starved group were wounded after 25 h of fasting. All animals were biopsied 8 h later. The wounds were examined by light microscopy and wound components (capillaries, fibroblasts, PMNs, collagen, and edema) were quantitated by lineal point analysis. Mice treated with antisera to insulin and mice starved for 33 h had an impaired healing response; the mice treated with 2-DG had a response similar to controls. These results suggest that hyperglycemia, per se, or the production of any toxic metabolites from high blood glucose levels could not alone induce the poor healing response. The depressed response in the antiserum-treated and starved mice may be due to the decreased availability of insulin to the wound tissues. These data support the hypotheses that insulin is a necessary component for an adequate wound healing response. In addition to a role in glucose transport and metabolism, insulin may also promote cellular growth.

Naturally occurring insulin autoantibodies in neonates of normal pregnancies and their relationship to insulinemia and birth weight

OBJECTIVE The objectives of this study were to determine whether insulin autoantibodies are present in umbilical cord blood from normal pregnancies, determine whether cord blood insulin autoantibody levels correlate with respective maternal levels at delivery, determine whether cord blood insulin autoantibody levels are related to cord blood or maternal insulin levels, and to determine what relationship neonatal birth weight has with either cord blood insulin autoantibody and insulin levels or maternal insulin autoantibody and insulin levels. STUDY DESIGN Paired umbilical cord and maternal serum samples were taken from 70 normal subjects at delivery. Measurements of serum insulin autoantibody (competitive charcoal radiobinding assay) and insulin (radioimmune inhibition assay) levels were performed. Multiple linear regression analysis and paired t tests were used for data analyses. RESULTS Neonatal insulin autoantibody levels (120 nU/ml) were more than two times higher than maternal levels (49 nU/ml) (p < 0.001). No correlation was observed between neonatal and maternal insulin autoantibody levels (r = 0.14, p = 0.25). A positive correlation of both neonatal and maternal insulin with birth weight was observed (r = 0.28, p < 0.02; and r = 0.36, p < 0.01, respectively). CONCLUSIONS These results suggest that the insulin autoantibody levels in fetal cord blood are not related to maternal levels in normal uncomplicated pregnancies. In addition, insulin levels in both maternal and neonatal circulations were positively correlated with increased birth weight in the normal pregnancies studied.

Differences in Humoral Insulin-Antibody Response Among Inbred Lou/M Rats and Epitope Presentation Differences in ELISA and Radioimmune Titration

Insulin antibodies were not detected in an insulin-capture enzyme-linked immunosorbent assay (ELISA), but they were easily detected in an insulin-copolymer-capture ELISA. Thus, there is a high degree of steric hindrance because of the proximity of the epitopes on the insulin monomer. This is circumvented by substituting an insulin copolymer for insulin in the capture ELISA. A regression analysis comparing the titers of 28 Lou/M rat insulin antiserums measured by liquid-phase radioimmune titration (RIT) with titers obtained in the direct insulin ELISA was not significant (P >.05). Thus, epitopes on insulin available and/or masked for antibody binding in the RIT differ from those available and/or masked in the direct insulin ELISA. As more of the epitopes become available when an insulin copolymer is substituted for monomeric insulin in the ELISAs, a significant positive correlation (P <.05) with the RIT was observed with these 28 insulin antiserums. Twenty-five percent (7 of 28) of these antiserums contained more antibodies that bound to epitopes available in the ELISAs that were masked in the RIT. Conversely, two antiserums contained more antibodies that bound to epitopes that were available in the RIT but were masked in the ELISAs. Thus, the amount of insulin antibodies measured in a given antiserum can vary substantially, depending on which epitopes are made available or are masked in the particular antibody-titration method used. These results demonstrate that the humoral immune response to insulin among inbred Lou/M rats can vary in insulin-antibody levels as well as the epitopes on insulin to which the antibodies bind.

Effect of IgG Subclasses on In Vivo Bioavailability and Metabolic Fate of Immune-Complexed Insulin in Lewis Rats

The bioavailability, distribution, and metabolic fate of 125l-labeled insulin complexed to antibodies in guinea pig antiserum, purified guinea pig lgG1, lgG2, a mixture of lgG1 and lgG2, and homologous Lou/m rat antiserum were studied in inbred Lewis rats. 125l-insulin complexed to purified guinea pig lgG2 antibodies was rapidly cleared from the blood and sequestered in increasing amounts with time in the liver. Large amounts of the 125l-insulin complexed to guinea pig lgG1 antibodies remained in the blood for at least 30 min. The bioavailability of 125l-insulin bound to lgG1 and lgG2 antibodies was inhibited for at least 30 min because significantly less was available for rapid binding to insulin receptors on hepatocytes and renal tubular cells and its subsequent rapid degradation. The bioavailability of 125l-insulin was further decreased when bound to antibodies in native guinea pig antiserum or a mixture of lgG1 and lgG2 antibodies compared with the 125l-insulin complexed to either purified lgG1 or lgG2 antibodies alone. The 125l-insulin bound to antibodies in native guinea pig antiserum or a mixture of lgG1 and lgG2 antibodies was distributed invivo in a manner reflecting the relative concentrations of the lgG1 and lgG2 antibodies present. The bioavailability, distribution, and metabolic fate of 125l-insulin in immune complexes prepared with homologous Lou/m rat insulin antiserum was qualitatively similar to that observed with immune complexes prepared with guinea pig insulin antiserum. It appears that the Lewis rat can be used as an in vivo model to study the bioavailability, distribution, and metabolic fate of insulin bound to xenogenic or homologous insulin antibodies.

Induction of Hyperglycemia with Insulin Antibodies to B-Chain Determinants

Insulin antibodies measured by a radioimmune method (ABR) are significantly better inducers of hyperglycemia than are insulin antibodies measured by an immune hemolysis method (ABH) when injected intraperitoneally into mice. The ability to induce hyperglycemia by an insulin antiserum can be predicted by the titer of ABR measured. ABR interact in vitro with determinants severely perturbed on nickel-insulin, partially perturbed on proinsulin and desasparaginedesalanine insulin, and unaffected on zinc-insulin or zinc-free monocomponent insulin. ABH, on the other hand, interact in vitro with determinants severely perturbed on proinsulin and desasparagine-desalanine insulin but stabilized on nickel-insulin and zinc-insulin. Since the connecting peptide of proinsulin is probably in apposition to the A-chain residues on the solvent surface, the more effective reaction of proinsulin with ABR than with ABH is submitted as evidence that ABR are directed toward residues on the B-chain surface of insulin. Because ABR are more effective inducers of hyperglycemia than are ABH, it is proposed that the degree of hyperglycemia induced by antibodies in vivo is a result of interactions with determinants on the B-chain surface of insulin. These results support the possibility that insulin in vivo is more accessible for interaction with antibodies directed to the B-chain of insulin. It is also possible that ABR, which are directed to B-chain determinants, are of higher affinity than is the affinity between insulin and receptors or that the active site of insulin for maintaining euglycemia includes the B-chain surface residues.

Effect of Isologous and Autologous Insulin Antibodies on In Vivo Bioavailability and Metabolic Fate of Immune-Complexed Insulin in Lou/M Rats

The in vivo bioavailability, distribution, and metabolic fate of 125I-labeled insulin complexed to isologous and autologous antibodies were studied in inbred Lou/M rats. There was an impaired bioavailability of the 125I-insulin bound to the isologous and autologous antibodies. Very little of the 125I-insulin in these immune complexes could bind to insulin receptors on hepatocytes or renal tubular cells and be degraded, because the amounts of 125I from degraded 125I-insulin in the blood or secreted into the stomach were markedly attenuated in both cases for at least 30 min after injection. There was a simultaneous accumulation of 125I-insulin immune complexes in the liver and the kidneys of Lou/M rats injected with ,25I-insulin complexed with isologous antibodies or when insulin-immunized Lou/M rats were injected with 125I-insulin during the same interval. The impaired bioavailability of immune-complexed insulin and altered distribution of radioactivity due to the accumulation of immune complexes in the liver and kidney were also observed in previous experiments in which Lewis rats were injected with xenogenic guinea pig and homologous insulin antibodies. These observations are therefore submitted as evidence that the Lou/M rat is a valid model in which to study the bioavailability of insulin immune complexed to isologous, homologous, and xenogenic antibodies and the metabolic fate of the respective insulin-antibody immune complexes.

Immunology of Islet Cells

Interest in the immunology of pancreatic islets has been sparked by the numerous clinical and experimental observations that an autoimmune mechanism is centrally involved in the pathogenesis of type I diabetes. Although much of this evidence is reviewed by Gepts and LeCompte (Chapter 16, this volume), it will be necessary to discuss possible immune mechanisms involved in experimental and clinical type I diabetes in this chapter.

PHAGOCYTOSIS AND CHEMOTAXIS OF MACROPHAGES FROM NORMAL AND DIABETIC MICE

SummaryExperiments were designed to study the chemotaxis and phagocytic functions of mouse peritoneal exudate macrophages from normal and chronic diabetic mice. There was no difference in the total number of white blood cells (WBCs), or the total number of any one type of leukocyte (i.e., macrophage, neutrophil) migrating into the casein-induced peritoneal exudate of normal and diabetic mice.In vitro phagocytosis of heat-killedCandida albicans by peritoneal induced macrophages was also similar in both of these groups of mice. No correlation between the percent phagocytosis and blood glucose level within each group was observed. Therefore, the attenuated humoral and cellular immune response which has been previously observed in diabetes cannot be attributed to defects in chemotaxis or phagocytosis as studied using mouse peritoneal macrophages.