We have previously reported on the purification, characterization, and biological significance of insulin-degrading enzyme (IDE) from pig and rat skeletal muscle. In the present study, we have investigated the detection and the HPLC separation of degradation products of native insulin from the reaction of monocomponent porcine insulin with affinity-purified pig IDE. Insulin was degraded by IDE in a time- and dose-dependent manner. Eight peaks (peaks I through VIII) appeared after 1 h of incubation, and peak V was identified as insulin. Among seven peaks representing degradation products, peak VI appeared most rapidly at 30 sec of incubation, increased until 10 min, and then decreased after 15 min of incubation; and six degradation products other than peak VI were not detected within 15 min of incubation, suggesting that peak VI was an initial degradation product of insulin produced by IDE and converted into relatively low molecular weight products as incubation time increased. The generation of peak VI may be due to cleavage at a peptide bond between the interchain disulfide bonds of the A or B chain. Subsequently, the split insulin derivative (peak VI) was evidently further degraded to relatively low molecular weight intermediates, such as peaks III and IV, peaks II and VIII, or peaks I and VII, because these pairs of peaks appeared and were degraded concomitantly. The peptide products designated as peaks IV, VI, VII, and VIII had both immunoprecipitability by antiinsulin antibodies and binding capacity to IM-9 lymphocytes, whereas the less hydrophobic intermediates (peaks I, II, and III) did not have these activities. Since some of these peptides have insulin-like properties, amino acid analysis of these products may enable us to identify not only the splitting position of insulin by IDE but also the site of the hormone for receptor binding.

Non-obese diabetic mice display a syndrome with dramatic clinical and pathological features similar to those of Type 1 (insulin-dependent) diabetes in man. Circulating autoantibodies to the surface of islet cells were demonstrated in some of these mice by a protein A radioligand assay. To produce monoclonal antibodies to islet cell surface antigens, therefore, we took the spleens of non-obese diabetic mice, transferred the spleen cells into non-immunized recipient mice, which were made immunologically incompetent by a large dose of X-irradiation, and then fused their lymphocytes with FO mouse myeloma cells. After screening the resultant hybrids, one stable hybridoma (3A4) that produced a monoclonal antibody (IgG1) specifically bound to the surface of islet cells was obtained. The purified monoclonal antibody was bound to the surface of transplantable Syrian golden hamster insulinoma cells sevenfold more than control antibody. Adsorption of the antibody on mouse spleen lymphocytes or thymocytes resulted in only a slight decrease in 125I-protein A binding to insulinoma cells. This antibody also reacted with the surface of mouse and rat islet cells, but not with that of rat spleen cells or hepatocytes. A spectrophotometric assay for peroxidase activity demonstrated that six times more peroxidase bound to insulinoma cells incubated with the antibody than to cells treated with control antibody. Furthermore, this antibody could be visually detected in the immunoenzymatic labelling of the surface of insulinoma cells. In summary, we have developed a novel method of producing monoclonal antibodies to the surface of islet cells for probing into the pathogenesis of Type 1 diabetes.

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We previously reported on the degradation of monocomponent porcine insulin by affinity-purified pig skeletal muscle insulin-degrading enzyme (IDE) and on the detection and HPLC separation of the initial degradation product (peak VI). Using relatively high concentration of insulin, peak VI appeared rapidly at 30 sec of incubation, whereas other peaks were not detected within 5 min of incubation. Performate oxidation studies suggested that peak VI is composed of a cleaved A-chain and an intact B-chain. To assess whether the initial degradation product of insulin generated by IDE preserves biological properties, we analyzed several insulin-like activities of peak VI. It had a hypoglycemic effect on rats. In vitro, it bound to the insulin receptors of rat adipocytes and stimulated glucose oxidation there. It also strengthened insulin receptor kinase activity in insulin receptors from rat liver and human placenta. Its biological potency, however, was 1/40th to 1/160th that of insulin itself. This is probably due to reduced affinity for the insulin receptor, since it had 2.5% of insulin's ability to both bind to the receptor and stimulate glucose oxidation. Moreover, peak VI had all of insulin's agonistic effect on glucose oxidation when used at a higher concentration. On the other hand, cross-linking analysis suggested that peak VI preserves almost the same affinity for IDE as does insulin. These results suggest that pig skeletal muscle IDE may cleave peptide bonds within the A-chain early in insulin degradation, generating peak VI; this then serves as the next substrate of IDE while exerting reduced insulin-like activity, and peak VI is converted to several relatively low mol wt products.

The kinetic changes of insulin receptors and cell surface insulin degrading enzyme were examined in Bri-7 cultured human lymphocytes after preincubation with or without insulin. The concentration of cell surface insulin degrading enzyme was determined by immunoenzymatic labeling method using a polyclonal antiserum to insulin degrading enzyme. In Bri-7 cells preincubated with 10(-10) to 10(-5) mol/l insulin for 18 h, the surface insulin receptors and insulin degrading enzyme decreased progressively as a function of the concentration of insulin in the preincubation medium. The surface insulin receptors and insulin degrading enzyme of cells preincubated with 10(-6) mol/l insulin were decreased to 25 and 35% of the control respectively. In Bri-7 cells preincubated with 10(-6) mol/l insulin for 30 min to 18 h, the loss of surface insulin degrading enzyme was slightly slower than that of the receptors; however, the curves were essentially parallel to each other. Thus, the treatment of Bri-7 cells with insulin caused down-regulation of insulin receptors in a dose- and time-dependent manner. Cell surface insulin degrading enzyme also decreased simultaneously. A combination of several insulin degradation assays (trichloroacetic acid precipitation, gel filtration and receptor rebinding) demonstrated that cell surface bound insulin remained intact, and that the degradation in Bri-7 cells seemed to be a limiting proteolysis of insulin. Furthermore, by the receptor rebinding method insulin degrading activity in cells after preincubation with 10(-6) mol/l insulin (19.6 +/- 4.6%) was decreased, although not significantly, as compared with cells after preincubation without insulin (24.6 +/- 4.8%).(ABSTRACT TRUNCATED AT 250 WORDS)