Tumour formation and insulin secretion by clonal RINm5F cells following repeated subcutaneous transplantation in NEDH rats

Flatt, Peter R.; Desilva, Mark G.; Swanston-Flatt, Sara K.; Powell, C.J.

doi:10.1677/joe.0.1180429

Cited by 10 publications

(8 citation statements)

References 23 publications

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“…The glucose responsiveness exhibited by the BRIN-BD11 cell line indicates that these cells do not suffer from the inherent defect recorded with parental RINm5F cells (21,22,50,51). Collectively these studies demonstrate that BRIN-BD11 cells have inherited many key attributes of the normal pancreatic (B-cell and that they represent a particularly useful cell line for future research into pancreatic p-cell function.…”

Section: Discussionmentioning

confidence: 75%

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Characterization of a Novel Glucose-Responsive Insulin-Secreting Cell Line, BRIN-BD11, Produced by Electrofusion

McClenaghan¹,

Barnett²,

Ah-Sing

et al. 1996

Diabetes

276

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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 output 5-10 times greater than parent RINm5F cells were subcultured with eventual establishment of clones, including BRIN-BD11. Morphological studies established that these cells grow as monolayers with epithelioid characteristics, maintaining stability in tissue culture for > 50 passages. Culture of these cells for 24 h at 5.6-33.3 mmol/l glucose revealed a 1.8- to 2.0-fold increase of insulin output compared with 1.4 mmol/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 three-fold stimulation of insulin release. 3-Isobutyl-1-methylxanthine (1 mmol/l) served 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 mannoheptulose 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 potentiated 1.4-fold by increasing the Ca2+ concentration from 1.28 to 7.68 mmol/l. Forskolin (25 mumol/l) and phorbol 12-myristate 13-acetate (10 nmol/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 analysis demonstrated that insulin was the major product secreted under stimulatory conditions. Collectively, these data indicate that the BRIN-BD11 cell line represents an important stable glucose-responsive insulin-secreting beta-cell line for future studies.

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Section: Discussionmentioning

confidence: 75%

“…The characteristics of parental RINm5F cells used for electrofusion have been described by the authors in detail elsewhere (21,22). These cells exhibit a low insulin content and basal insulin release, together with glucose unresponsiveness or even inhibition of insulin secretion in acute incubations.…”

mentioning

confidence: 99%

Characterization of a Novel Glucose-Responsive Insulin-Secreting Cell Line, BRIN-BD11, Produced by Electrofusion

McClenaghan¹,

Barnett²,

Ah-Sing

et al. 1996

Diabetes

276

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“…their cellular insulin content compared with only 9% from BRIN-BG7 cells. Both cells had basal levels of insulin secretion substantially higher than the very modest output characteristic of the parental RINm5F cell (Flatt et al , 1988.…”

Section: Discussionmentioning

confidence: 87%

“…Cells were gently washed in 10 ml Hank's balanced salt solution (HBSS) prior to detachment from the culture flasks by incubation with 0-025% (w/v) trypsin/EDTA (Gibco) and immediately utilized. The RINm5F cells used in these electrofusion studies have been extensively characterized elsewhere (Flatt et al , 1988. These cells have been noted to have a low basal insulin release, coupled with a glucose unresponsiveness or even inhibition of insulin secretion in acute incubations with increasing glucose concentrations.…”

Section: Methodsmentioning

confidence: 99%

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

McClenaghan

Barnett²,

O’Harte³

et al. 1996

Journal of Endocrinology

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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.

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“…The radiation-induced insulin secreting RINm5FceIl line has been used in several studies on cytoioxicity to beta cells (Flatter at.. 1987), Tcell specificity for beta cells (Prud'homme cr ai, 1986;van Vliet et at.. 1989) and beta cell specificity in transplantation (Flatt et at.. 1988). In these studies it was demonstrated that RINm5F cells express antigens relevant for autoimmune cell-mediated reactivity in type !…”

Section: Introductionmentioning

confidence: 99%

A delayed-type hypersensitivity skin-test system using the insulinoma cell line RINm5F to monitor beta cell-specific cellular autoimmune reactivity in the spontaneously diabetic BB/O rat

Voorby

Jeucken

Drexhage

1990

Clinical and Experimental Immunology

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SUMMARYIslet-specific autoimmune re;ictivity (humoral and cell-media ted) is the basis for the insulitis process of type I diabetes mellitus. In Ihis report n delaycd-lype hypersensitivity (DTH) skin test was used to monitor the presence of an is let-specific cell-mediated autoimmune component in BB/O rats. The BB/O rat is a strain characterized by the spontaneous development of type I diabetes. Intact RINm5F cells as well as a RINm5FccII nietnbrane preparation were used as DTH skin test antigens. Rats of different ages and disease stages were tested in the e:tr with the insulinoma eell line and its cell membrane preparation. As control antigens, the (ibroblast cell line .WI and a cell membrane preparation made thereof were used. The DTH reaction system showed a positive cell-mediated reactivity in BB/O rats for membrane-bound RINmSF cell antigens, and not for the control fibroblast 3YI cell membrane determinants. The true DTH character of the skin test was established by the titne-course ofthe reaction (maximum at 24 h). the histopathology (infiltration by dendritic cells, lymphocytesandmacrophages), and the possibility to transfer the reaction with spleen cells and lymph node cells. The DTH test towards RINm5F cells showed the highest prevalence of positivity (lOO'^ii) in BB/O rats around the onset of diabetes (3 weeks before to .1 weeks after the onset of glucosuria). The prevalence of DTH positivity was 56% in the period ofmore than 3 weeks before the onset of glucosuria. In BB/O rats with a duration of glucosuria of more than . weeks, the prevalence of positivity was around 60-70''/l).

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Tumour formation and insulin secretion by clonal RINm5F cells following repeated subcutaneous transplantation in NEDH rats

Cited by 10 publications

References 23 publications

Characterization of a Novel Glucose-Responsive Insulin-Secreting Cell Line, BRIN-BD11, Produced by Electrofusion

Characterization of a Novel Glucose-Responsive Insulin-Secreting Cell Line, BRIN-BD11, Produced by Electrofusion

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

A delayed-type hypersensitivity skin-test system using the insulinoma cell line RINm5F to monitor beta cell-specific cellular autoimmune reactivity in the spontaneously diabetic BB/O rat

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