Processing and release of human proinsulin-cleavage products into culture media by different engineered non-endocrine cells: a specific assessment by capillary electrophoresis.
The aim of this study was to compare the metabolic pathway to mature insulin through the intermediate forms (32-33 split, 65-66 split, des31,32 and des64,65) in human or murine cells engineered for the release of wild-type human proinsulin and in a genetically mutated one, in the search for a new approach for an insulin-dependent diabetes mellitus cure by gene therapy. Primary human fibroblasts, myoblasts and stabilized cell lines (HepG2 and NIH3T3) were transduced either with a retroviral vector coding for wild-type proinsulin or for a genetically mutated one, carrying cleavage sites sensitive to furin. The pattern of all the proinsulin cleavage products released into the cell culture supernatants was analyzed by capillary electrophoresis. 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 primary myoblasts released all the intermediate forms and a limited amount of mature insulin. All the cells transduced with a furin-sensitive proinsulin 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 difference in the processing and release of immature and mature insulin forms, pointing to human hepatic cells as the most efficacious. Capillary electrophoresis provided on-line and in a single run a complete overview of the proinsulin metabolic pathway in different cells.
Arcelloni
C
C
Laboratory of Separative Techniques, Department of Laboratory Medicine, Scientific Institute H S Raffaele and School of Medicine, Milan, Italy. arcelloni.cinzia@hsr.it
Falqui
L
L
Martinenghi
S
S
Stabilini
A
A
Pontiroli
A E
AE
Paroni
R
R
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000379:methods; D005347:Fibroblasts; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009206:Myocardium / Q000166:cytology; D009862:Online Systems; D011384:Proinsulin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D011384:Proinsulin