Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion

Neville McClenaghan, CR Barnett, E AhSing, Yasser Abdel-Wahab, Finbarr O'Harte, TW Yoon, SK SwanstonFlatt, Peter Flatt

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Abstract

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 threefold 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 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 potentiated 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 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.
LanguageEnglish
Pages1132-1140
JournalDiabetes
Volume45
Issue number8
Publication statusPublished - Aug 1996

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Insulin-Secreting Cells
Insulin
Glucose
Cell Line
Glyceraldehyde
Islets of Langerhans
Alanine
Biphasic Insulins
Diazoxide
Glucokinase
1-Methyl-3-isobutylxanthine
New England
Hexokinase
Facilitative Glucose Transport Proteins
Cell Fusion
Colforsin
Acetates
Clone Cells
Cell Culture Techniques
Western Blotting

Cite this

McClenaghan, Neville ; Barnett, CR ; AhSing, E ; Abdel-Wahab, Yasser ; O'Harte, Finbarr ; Yoon, TW ; SwanstonFlatt, SK ; Flatt, Peter. / Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion. In: Diabetes. 1996 ; Vol. 45, No. 8. pp. 1132-1140.
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abstract = "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 threefold 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 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 potentiated 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 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.",
author = "Neville McClenaghan and CR Barnett and E AhSing and Yasser Abdel-Wahab and Finbarr O'Harte and TW Yoon and SK SwanstonFlatt and Peter Flatt",
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McClenaghan, N, Barnett, CR, AhSing, E, Abdel-Wahab, Y, O'Harte, F, Yoon, TW, SwanstonFlatt, SK & Flatt, P 1996, 'Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion', Diabetes, vol. 45, no. 8, pp. 1132-1140.

Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion. / McClenaghan, Neville; Barnett, CR; AhSing, E; Abdel-Wahab, Yasser; O'Harte, Finbarr; Yoon, TW; SwanstonFlatt, SK; Flatt, Peter.

In: Diabetes, Vol. 45, No. 8, 08.1996, p. 1132-1140.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion

AU - McClenaghan, Neville

AU - Barnett, CR

AU - AhSing, E

AU - Abdel-Wahab, Yasser

AU - O'Harte, Finbarr

AU - Yoon, TW

AU - SwanstonFlatt, SK

AU - Flatt, Peter

PY - 1996/8

Y1 - 1996/8

N2 - 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 threefold 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 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 potentiated 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 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.

AB - 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 threefold 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 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 potentiated 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 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.

M3 - Article

VL - 45

SP - 1132

EP - 1140

JO - Diabetes

T2 - Diabetes

JF - Diabetes

SN - 0012-1797

IS - 8

ER -