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.

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Spleen cells from non-obese diabetic mice were found to generate low interleukin 2 production and cell proliferation in response to concanavalin A. However, some of non-obese diabetic mice maintained in the same environment preserved their responsiveness to this T cell mitogen. Non-obese diabetic mice at every age had a higher percentage of Thyl.2, L3T4, and Lyt2-positive spleen cells than did control mice, suggesting that the dysfunction of spleen cells did not depend on the number of T cells or the ratio of these subpopulations. Evidence for macrophage-mediated suppression participating in the deficient function of splenic lymphocytes in this mouse model of insulin-dependent diabetes includes: 1) the restoration of mitogen-induced interleukin 2 production after the macrophages have been depleted by silica absorption form spleen cells; 2) the complete suppression of the cell proliferation by thioglycollate-stimulated peritoneal exudate cells from non-obese diabetic and control mice, and the partial suppression by spleen macrophages from non-obese diabetic mice; 3) the reversal of the suppression of interleukin 2 production by the prostaglandin synthetase inhibitor indomethacin (0.1-1 microgram/ml); 4) the partial suppression of interleukin 2 production, conversely, by the exogenous prostaglandins E1 and E2 (2.5 x 10(-6) mol/l). These results indicate that the activated macrophages existing among the spleen cells suppress the response of splenic T cells to concanavalin A. This impairment may contribute to the pathogenesis of insulin-dependent diabetes in non-obese diabetic mice.

Some results on surface modification of Si and graphite with highly charged ions (HCIs) are presented. Modified surfaces were observed using scanning tunneling microscopy. Crater-like structure with a diameter in nm region is formed on a Si(111)-(7x7) surface by the incidence of a single HCI. The protrusion structure is formed on a highly oriented pyrolytic graphite surface on the other hand, and the structure becomes an active site for molecular adsorption. A new, intense HCI source and an experimental apparatus are under development in order to process and observe aligned nanostructures created by the impact of collimated HCI beam.

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.