Processing and release of human proinsulin-cleavage products into culture media by different engineered non-endocrine cells: a specific assessment bycapillary electrophoresis

The aim of this study was to compare the metabolic pathway to mature insuli n through the intermediate forms (32-33 split, 65-66 split, des31,32 and de s64,65) in human or murine cells engineered for the release of wild-type hu man proinsulin and in a genetically mutated one, in the search for a new ap proach for an insulin-dependent diabetes mellitus cure by gene therapy. Primary human fibroblasts, myoblasts and stabilized cell lines (HepG2 and N 1H3T3) were transduced either with a retroviral vector coding for wild-type : proinsulin or for a genetically mutated one, carrying cleavage sites sens itive to furin. The pattern In of all the proinsulin cleavage products rele ased into the cell culture supernatants was analyzed by capillary electroph oresis sis. All the cells transduced with the wild-type gene released intact proinsulin . HepG2 released a considerable amount of 65-66 split and des64,65, while p rimary myoblasts released all the intermediate forms and a limited amount o f mature insulin. All the cells transduced with a furin-sensitive proinsuli n gene released a higher amount of mature insulin (23-59% conversion yield) than the cells expressing wild-type proinsulin, whereas the total insulin was nearly constant. Only primary cells released all the cleavage products. Screening a wide variety of non-endocrine cells has revealed a large differ ence in the processing and release of immature and mature insulin forms, po inting to human hepatic cells as the most efficacious. Capillary electropho resis provided on-line and in a single run a complete overview of the proin sulin metabolic pathway in different cells.