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Receptor-Dependent and Independent Degradation of Insulin by Isolated Swine Adipocytes at 37 C¹

The Pennsylvania State University², University Park 16802

Abstract

Insulin binding and degradation were measured at 37 C in isolated swine adipocytes. In preliminary experiments, binding decreased rapidly with increasing incubation time. This was associated with a marked increase in insulin degradation. Insulin binding was suppressed by some lots of bovine serum albumin (BSA), which suggests that some commercial preparations of BSA are contaminated with insulin-like molecules. In adipocyte suspensions, greater than 90% of the insulin degraded was due to a nonreceptor mediated process (i.e., insulin degrading activity present in the media). Despite the presence of insulin degrading activity in the media the cells were metabolically (as judged by lipogenic capacity and lactic dehydrogenase activity) and morphologically (greater than 98% excluded trypan blue) intact indicating that the cells were not leaking during the incubation. In subsequent experiments it was found that the specific step associated with transfer of cells during adipocyte isolation resulted in the release of insulin degrading activity. Implementation of a 30-min preincubation and washing sequence after adipocyte isolation removed the media insulin degrading activity, resulting in a marked reduction (70%) of insulin degradation by adipocyte suspensions. As a result of this modification, binding of tracer quantities of insulin attained steady-state binding conditions and maintained this for 2 h. These results demonstrate that techniques can be used to minimize nonreceptor mediated insulin degradation in adipocyte suspensions. As a result in vitro studies can be conducted that measure insulin binding and biological action in swine adipocytes at physiological temperatures.

¹ Authorized for publication as paper no. 6807 in the journal series of The Pennsylvania Agr. Exp. Sta. This project was supported in part by a BRSG Grant awarded by Biochemical Support Grant Program, Division of Research Resources, NIH. Portions of this research were presented in abstract form at the Amer. Soc. of Anim. Sci. meetings in Guelph, Ontario (August 8–11, 1982).

² Dept. of Dairy and Anim. Sci.