Rapid Publications Early Detection of Degraded A14- l-lnsulin in Human Fibroblasts by the Use of High Performance Liquid Chromatography

We studied the metabolism of A14l-insulin in intact human fibroblasts using high performance liquid chromatography (HPLC) to detect and separate its early degradation products. The high resolving power of HPLC enabled us to separate what has been considered "intact insulin" by Sephadex G-50 chromatography or TCA precipitability into two additional peaks that had decreased biochemical properties with respect to immunoprecipitability and receptor binding but not decreased TCA precipitability. We conclude that human fibroblast is capable of metabolizing insulin within 2 min at 37°C into intermediate molecules that can be detected by HPLC but not by TCA precipitability or molecular sieve chromatography. DIABETES 32:474-477, May 1983. used either TCA precipitability and/or molecular sieve chromatography to assess the intactness of insulin. Therefore, we used intact human fibroblasts to study cell-associated insulin degradation using not only TCA precipitability and molecular sieve chromatography but also a more sensitive method of separation by high performance liquid chromatography (HPLC) to isolate the intermediate products for studying the biochemical properties in regard to TCA precipitability, immunoprecipitability, and liver membrane binding. Our studies indicate that neither TCA precipitability nor molecular sieve chromatography is sensitive enough to detect early product formation of cell-associated insulin degradation. We found HPLC to be the most useful tool to separate the products of initial insulin degradation in intact human fibroblasts.