Engineering cultured insulin-secreting pancreatic B-cell lines

Despite many triumphs, a significant limitation of the usefulness of many of the available B-cell lines for the study of insulin secretion are either inappropriate or lack of responsiveness to glucose. Commonly employed cell lines generated prior to the 1990s following X-ray irradiation (RINm5F cells) or simian virus 40 B-cell transformation (HIT-T15 cells and BTC) fall into this category. More recent success has been achieved with the generation of INS-1 cells and MIN6 cells, but the production of these cell lines owes much to good fortune, dedication and hard work. In the present era, molecular biology techniques provide the opportunity to engineer novel pancreatic B-cell lines which possess many attributes of normal insulin-secreting cells. This review describes the electrofusion of normal NEDH rat pancreatic B-cells with immortal RINm5F cells to create three new glucose-responsive clonal insulin-secreting cells, designated BRIN-BG5, BRIN-BG7 and BRIN-BD11. These cell lines exhibit up to four-fold insulin-secretory responses to depolarization with 25 mmol/l K+, 7.68 mmol/l Ca2+, 10 mmol/l L-alanine, and activation of protein kinase C or adenylate cyclase with 10 nmol/l phorbol- 12-myristate-13-acetate or 25 micromol/l forskolin, respectively. The maximal insulin-secretory response of both BRIN-BG5 and BRIN-BG7 cells to glucose occurred at 8.4 mmol/l (1.9- and 1.8-fold increases, respectively). In contrast, 4.2-16.7 mmol/l glucose evoked a stepwise 2- to 3-fold of insulin release from BRIN-BD11 cells. The superior glucose responsiveness of BRIN-BD11 cells compared with BRIN-BG5 or BRIN-BG7 cells was associated with increased expression of GLUT-2 and a greater contribution of glucokinase to total glucose phosphorylating enzyme activity. Furthermore, BRIN-BD11 cells also showed appropriate responses to a diverse range of modulators of pancreatic B-cell function, including amino acids, neurotransmitters and sulphonylurea drugs. Collectively these observations indicate that genetic modification of insulin-secreting cells by electrofusion (or transfection with cDNA) offers a new avenue for generation of useful clonal glucose-responsive pancreatic B-cell lines for studies of insulin secretion and transplantation in insulin-dependent diabetes mellitus.