CHARACTERIZATION OF A NOVEL GLUCOSE-RESPONSIVE INSULIN-SECRETING CELL-LINE, BRIN-BD11, PRODUCED BY ELECTROFUSION

A novel insulin-secreting cell line (BRIN-BD11) was established after electrofusion of RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells, Wells of cell fusion mixture with insulin outp ut 5-10 times greater than parent RINm5F cells were subcultured with e ventual establishment of clones, including BRIN-BD11, Morphological st udies established that these cells grow as monolayers with epithelioid characteristics, maintaining stability in tissue culture for >50 pass ages, Culture of these cells for 24 h at 5.6-33.3 mmol/l glucose revea led a 1.8- to 2.0-fold increase of insulin output compared with 1.4 mm ol/l glucose, Dynamic insulin release was recorded in response to 16.7 mmol/l glucose, resulting in a rapid threefold insulin secretory peak followed by a sustained output slightly above basal, In acute 20-min tests, 4.2-16.7 mmol/l glucose evoked a stepwise two-to threefold stim ulation of insulin release, 3-Isobutyl-1-methylxanthine (1 mmol/l) ser ved to increase basal and glucose-stimulated insulin release, shifting the threshold from 4.4 to 1.1 mmol/l glucose, Stimulation of insulin secretion with 16.7 mmol/l glucose was abolished by manno-heptulose or diazoxide (15 or 0.5 mmol/l), In contrast, glyceraldehyde (10 mmol/l) and 25 mmol/l K+ evoked 1.7- to 9.0-fold insulin responses, L-Alanine (10 mmol/l) evoked a twofold secretory response, which was potentiate d 1.4-fold by increasing the Ca2+ concentration from 1.28 to 7.68 mmol /l, Forskolin (25 mu mol/l) and phorbol 12-myristate 13-acetate (10 nm ol/l) both increased insulin secretion in the presence of L-alanine (1 .4- and 1.8-fold, respectively), Western blotting confirmed that BRIN- BD11 cells expressed the GLUT2 glucose transporter, This, coupled with a high glucokinase/hexokinase ratio in the cells, confirms an intact glucose sensing mechanism, High-performance liquid chromatography anal ysis demonstrated that insulin was the major product secreted under st imulatory conditions, Collectively, these data indicate that the BRIN- BD11 cell line represents an important stable glucose-responsive insul in secreting beta-cell line for future studies.