Characteristics of BRIN-BG5 and BRIN-BG7, two novel glucose-responsive insulin-secreting cell lines produced by electrofusion

Abstract

Two hybrid insulin-secreting cell lines (BRIN-BG5 and BRIN-BG7) were established by the novel approach of electrofusing RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells. Cells were selected from the fusion mixture on the basis of insulin output. Wells showing five to ten times greater insulin output than parental RINm5F cells were selected, subcultured and cloned. Clonal BRIN-BG5 and BRIN-G7 cells grow as monolayers with epithelial morphology. The differences in doubling time of 28 and 20 h respectively were associated with morphological differences; the growth pattern and insulin content of each cell line remaining stable for over 50 passages. In acute 20-min tests, both cell lines showed peak secretory responses (1.9- and 1.8-fold respectively) to 8.4 mmol/l glucose. Membrane depolarization with 25 mmol/l K+ evoked 3.7- and 3.9-fold increases in insulin output. L-Alanine (10 mmol/l) also served to promote 2.4- and 1.6-fold increases in insulin release respectively. Increasing the Ca2+ concentration from 1.28 to 7.68 mmol/l potentiated this effect by 1.8- and 1.5-fold. Incubation with forskolin (25 mumol/l) or phorbol-12-myristate 13-acetate (10 nmol/l), in the presence of L-alanine, similarly enhanced the secretory effect on BRIN-BG5 and BRIN-BG7 cells by 1.3- to 2.1-fold and 1.2- to 1.5-fold respectively. The presence of a functional glucose-sensing mechanism in both cell lines was confirmed by the demonstration of the glucose transporter GLUT-2 and measurement of glucokinase activity. These functional properties suggest that insulin-secreting BRIN-BG5 and BRIN-BG7 cells represent two useful glucose-responsive cell lines for future studies of the function of the pancreatic B-cell.

Cellular models for beta‐cell function and diabetes gene therapy

“…Another useful approach is to use electrofusion to combine the functional properties of freshly isolated islet beta‐cells with a partner cell line that can confer immortality to the beta‐cells (Table ). One such example that has lent itself to a significant number of studies is the glucose‐responsive insulin‐secreting BRIN‐BD11 cell line which, like slightly inferior clones BRIN‐BG5 and BRIN‐BG7, was produced by the electrofusion of RINm5F cells, a subclone of RIN with freshly isolated rat beta‐cells . Of these, the BRIN‐BD11 cell line in particular has shown highly desirable characteristics for a clonal beta‐cell line, having retained near‐normal beta‐cell functionality and responsiveness to glucose and other modulators of pancreatic insulin secretion .…”

“…Following previous success with the use of electrofusion technology to create the BRIN‐BD11 and related rat beta‐cell lines (Table ), similar technology has been applied to immortalize human pancreatic beta‐cells. Three electrofusion‐derived human beta‐cell lines have been described in literature by McCluskey et al, namely 1.1B4, 1.1E7 and 1.4E7 (Table ).…”

Physiological and pharmacological regulation of insulin release: insights offered through exploitation of insulin‐secreting cell lines

“…Electrofusion of normal pancreatic b cells with RINm5F cells might be expected to yield hybrid cells which obtain immortality from the established cell line and appropriate features of insulin synthesis and secretion from the normal b cells. Using this approach with selection of immortal hybrids on the basis of high insulin output, three novel clonal pancreatic b cell lines were established and designated BRIN-BG5, BRIN-BG7 and BRIN-BD11 ( [74,75]; table 1). Primary characterization studies revealed that each clone shows subtle morphological differences coupled with different patterns and magnitudes of responsiveness to glucose and other key regulators of b cell function [74,75].…”

“…Using this approach with selection of immortal hybrids on the basis of high insulin output, three novel clonal pancreatic β cell lines were established and designated BRIN‐BG5, BRIN‐BG7 and BRIN‐BD11 (74,75]; Table 1). Primary characterization studies revealed that each clone shows subtle morphological differences coupled with different patterns and magnitudes of responsiveness to glucose and other key regulators of β cell function[74, 75]. Of these, BRIN‐BD11 cells most closely mirror the characteristics of the parental β cell.…”

Physiological regulation of the pancreatic β‐cell: functional insights for understanding and therapy of diabetes

“…As such, electrofusion‐derived hybrid cells should obtain immortality from the RINm5F fusion partner and intact features of insulin biosynthesis and secretion from the normal parental pancreatic β‐cells. Adopting this novel approach, three clonal rodent cell lines were established following electrofusion of New England Deaconess hospital (NEDH) rat β‐cells with immortal RINm5F cells, originally derived from the transplantable NEDH rat insulinoma (McClenaghan et al 1996 a , c ). Procedures adopted for the isolation of these three clonal β‐cells (denoted BRIN‐BD11, BRIN‐BG5 and BRIN‐BG7) are described in detail elsewhere (McClenaghan et al 1996 a , c ), but the BRIN nomenclature comes from the B‐cell‐RIN cell fusion, with each cell line cloned from well B into three wells: D11, G5 and G7.…”