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  • Author: S. K. Swanston-Flatt x
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P. R. Flatt, M. G. DeSilva, S. K. Swanston-Flatt, C. J. Powell, and V. Marks

ABSTRACT

The effects of repeated s.c. transplantation of clonal insulin-secreting RINm5F cells in NEDH rats on tumour morphology, insulin–glucose homeostasis and the function of RINm5F cells re-established in culture were examined after maintenance in vivo for periods of up to 308 days. Transplantation of RINm5F cells for ten consecutive passages consistently produced a single encapsulated vascularized tumour associated with the development in recipient rats of hyperinsulinaemia, hypoglycaemia and eventual neuroglycopenic coma. At the tenth passage, tumour-bearing rats exhibited a markedly enhanced rate of insulinstimulated glucose uptake by 19 days, with evidence of a large and variable insulin response to i.p. glucose. Survival was 22 ± 2 days, and resected RINm5F cell tumours exhibited weak immunostaining for insulin in scattered cells, with strands of fibrous tissue separating clusters of tumour cells many of which had distinct polarity. There was no detectable immunostaining for glucagon, somatostatin or pancreatic polypeptide. The insulin content and insulin secretory output of RINm5F cells re-established in culture after 20, 146, 259 or 308 days propagation in vivo were generally enhanced compared with non-passaged RINm5F cells. The magnitude of the effect was not appreciably affected by the duration of maintenance in vivo, but it was critically dependent upon the subsequent period of culture in vitro. Thus, whereas 2-day cultured RINm5F cells from the eighth tumour passage exhibited a greater than 100–fold increment of insulin content and release, with enhanced secretory responsiveness to leucine, arginine, theophylline, K + (25 mmol/l) or Ca2+ (7·6 mmol/l), RINm5F cells cultured for a further 19 days had almost completely lost the attributes resulting from 259 days of maintenance in vivo. The results indicate substantial enhancement of the functional capabilities of RINm5F cells in vivo, and suggest that the resulting tumours afford a novel model in NEDH rats of responsive trabecular-type insulinomas.

J. Endocr. (1988) 118, 429–437

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N H McClenaghan, C R Barnett, F P M O'Harte, S K Swanston-Flatt, E Ah-Sing, and P R Flatt

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 μmol/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 glucoseresponsive cell lines for future studies of the function of the pancreatic B-cell.

Journal of Endocrinology (1996) 148, 409–417