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Characteristics of High Molecular Weight Insulins in Insulinoma Patients*^*

Washington University School of Medicine, Department of Internal Medicine, Division of Metab olism 660 S. Euclid, St. Louis, Missouri 63110

These studies were undertaken to characterize the high molecular weight immunoreactive insulins in plasma and tumor extracts of two patients with insulinoma, and to determine whether they are biosynthetic precursors of proinsulin. Plasma was chromatographed on a Sephadex G-50 column and fractions assayed for IRI. Three peaks were observed, one in the void volume region (6%), a peak in the proinsulin region (25%), and the largest peak comigrating with insulin standard (69%). Insulinoma slices were incubated for 4 h and high molecular weight IRI was observed in chromatographed extracts of the tumor, as well as in extracts of the incubation medium, which comprised up to 3% of the total IRI.

Gel filtration chromatography of a pre-operative fasting plasma from the second patient revealed a heterogeneous distribution of IRI, with approxi: mately 53% in the high molecular weight region. At surgery an insulinoma was removed. Slices were incubated for 4 h with ¹⁴C-amino acids and extracts chromatographed. ¹⁴C-labeled protein was observed n the V₀, and in the proinsulin and insulin regions. These ¹⁴C-labeled proteins were precipitated with anti-insulin serum, and it was determined that whereas 60–90% of ¹⁴C-protein in the proinsulininsulin region was precipitated, virtually none of the ¹⁴C-protein in the V_o was immunoprecipitated with anti-insulin serum. The specific activities (CPM precipitated by anti-insulin serumµU IRI) were 1, 11.4, and 3.1 for the V_o proinsulin, and insulin regions, respectively. These results suggest that proinsulin is synthesized first, followed by insulin, and then the high molecular weight IRI. The high molecular weight IRI is not a biosynthetic precursor of proinsulin. Rechromatography of the high molecular weight IRI in the presence of 8M urea dissociated 90% into smaller immunoreactive components which chromatographed in the proinsulin and insulin region. It was concluded that high molecular weight IRI's are present in plasma, tumor extracts, and incubation medium from insulinoma patients. These high molecular weight IRI's are not biosynthetic precursors, but either aggregates of proinsulin and insulin, or insulin bound to larger proteins. These studies do not rule out the existence in humans of a smaller rapidly-turning over precursor to proinsulin similar to the pre-proinsulin discovered in a rat insulinoma by Chan et al.

^* Presented in part at the 58th Annual Meeting of the Endocrine Society, June, 1976.

Supported in part by the Diabetes and Endocrinology Center Grant #AM 17904 and Insulin Biosynthesis Grant #AM 16746 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland, and by the Diabetic Children's Welfare Association, Inc., St. Louis, Missouri.

Received June 29, 1977.