27 results on '"McClenaghan, Neville H."'
Search Results
2. Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion
- Author
-
McClenaghan, Neville H., Barnett, Christopher R., Ah-Sing, Eric, Abdel-Wahab, Yasser H.A., O'Harte, Finbarr P.M., Yoon, Tai-Wook, Swanston-Flatt, Sara K., and Flatt, Peter R.
- Subjects
Pancreatic beta cells -- Evaluation -- Physiological aspects ,Insulin -- Physiological aspects -- Evaluation ,Health ,Evaluation ,Physiological aspects - 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 [...]
- Published
- 1996
3. ATP-Sensitive Potassium Channels and Efaroxan-Induced Insulin Release in the Electrofusion-Derived BRIN-BD 11 [Beta]-Cell Line
- Author
-
Chapman, Joanna C., McClenaghan, Neville H., Cosgrove, Karen E., Hashmi, Molly N., Shepherd, Ruth M., Giesberts, Aukje N., White, Stanley J., Ammala, Carina, Flatt, Peter R., and Dunne, Mark J.
- Subjects
Potassium channels ,Insulin -- Physiological aspects ,Health - Abstract
The properties of ATP-sensitive [K.sup.+] ([K.sub.ATP]) channels were explored in the electrofusion-derived, glucose-responsive, insulin-secreting cell line BRIN-BD11 using patch-clamp techniques. In intact cells, [K.sub.ATP] channels were inhibited by glucose, the [...]
- Published
- 1999
4. Molecular Mechanisms of Toxicity and Cell Damage by Chemicals in a Human Pancreatic Beta Cell Line, 1.1B4
- Author
-
Vasu, Srividya, McClenaghan, Neville H., and Flatt, Peter R.
- Published
- 2016
- Full Text
- View/download PDF
5. Cellular responses of novel human pancreatic β-cell line, 1.1B4 to hyperglycemia
- Author
-
Vasu, Srividya, McClenaghan, Neville H, McCluskey, Jane T, and Flatt, Peter R
- Abstract
The novel human-derived pancreatic β-cell line, 1.1B4 exhibits insulin secretion and β-cell enriched gene expression. Recent investigations of the cellular responses of this novel cell line to lipotoxicity and cytokine toxicity revealed similarities to primary human β cells. The current study has investigated the responses of 1.1B4 cells to chronic 48 and 72 h exposure to hyperglycemia to probe mechanisms of human β-cell dysfunction and cell death. Exposure to 25 mM glucose significantly reduced insulin content (p < 0.05) and glucokinase activity (p < 0.01) after 72 h. Basal insulin release was unaffected but acute secretory response to 16.7 mM glucose was impaired (p < 0.05). Insulin release stimulated by alanine, GLP-1, KCl, elevated Ca2+and forskolin was also markedly reduced after exposure to hyperglycemia (p < 0.001). In addition, PDX1 protein expression was reduced by 58% by high glucose (p < 0.05). Effects of hyperglycemia on secretory function were accompanied by decreased mRNA expression of INS, GCK, PCSK1, PCSK2, PPP3CB, GJA1, ABCC8,and KCNJ11. In contrast, exposure to hyperglycemia upregulated the transcription of GPX1,an antioxidant enzyme involved in detoxification of hydrogen peroxide and HSPA4, a molecular chaperone involved in ER stress response. Hyperglycemia-induced DNA damage was demonstrated by increased % tail DNA and olive tail moment, assessed by comet assay. Hyperglycemia-induced apoptosis was evident from increased activity of caspase 3/7 and decreased BCL2 protein. These observations reveal significant changes in cellular responses and gene expression in novel human pancreatic 1.1B4 β cells exposed to hyperglycemia, illustrating the usefulness of this novel human-derived cell line for studying human β-cell biology and diabetes.
- Published
- 2013
- Full Text
- View/download PDF
6. Role of islet structure and cellular interactions in the control of insulin secretion
- Author
-
Kelly, Catriona, McClenaghan, Neville H., and Flatt, Peter R.
- Abstract
Close cellular proximity and correct anatomical arrangement within islets are essential for normal patterns of insulin secretion. Thus, segregation of islets into single cells is associated with a dramatic decline in stimulus secretion-coupling and glucose-induced insulin release. Generation of pseudoislets from clonal islet cell lines provides a useful model to examine islet cell interactions and insulin secretion. Such studies have highlighted the functional importance of cell adhesion molecules and connexins. Pseudoislets comprising insulin-secreting cell lines have been shown to closely mimic primary islets in both size and morphology, displaying a significantly enhanced response to glucose, nutrients and drugs over equivalent monolayer cultures. Here, we consider the influence of islet structure and cellular interactions in the control of insulin secretion. The functional characteristics of pseudoislets derived from clonal beta-cell lines or a combination of alpha-, beta- and delta-cell lines are discussed in light of normal islet function and possible therapeutic application.
- Published
- 2011
- Full Text
- View/download PDF
7. Comparison of Insulin Release From MIN6 Pseudoislets and Pancreatic Islets of Langerhans Reveals Importance of Homotypic Cell Interactions
- Author
-
Kelly, Catriona, Guo, Hong, McCluskey, Jane T., Flatt, Peter R., and McClenaghan, Neville H.
- Abstract
Cellular communication is required for normal patterns of insulin secretion from cells. Experiments using isolated islets of Langerhans are hampered by lack of supply and the consuming isolation process. Pseudoislets comprising clonal cells have emerged as an alternative to study islet-cell interactions and insulin secretion. The current study compared MIN6 pseudoislets and freshly isolated mouse islets.
- Published
- 2010
- Full Text
- View/download PDF
8. Homocysteine-Induced Impairment of Insulin Secretion From Clonal Pancreatic BRIN-BD11 -Cells Is Not Prevented by Catalase
- Author
-
Patterson, Steven, Flatt, Peter R., and McClenaghan, Neville H.
- Abstract
Although detrimental effects of homocysteine attributed to homocysteine auto-oxidation and generation of hydrogen peroxide (H2O2) have been reported in various cell types, such actions have not been considered in pancreatic -cells. This study investigates the acute effects of homocysteine on -cell integrity and regulation, in particular, the role of H2O2generated by auto-oxidation.
- Published
- 2007
- Full Text
- View/download PDF
9. Actions of glucagon-like peptide-1 on KATPchannel-dependent and -independent effects of glucose, sulphonylureas and nateglinide
- Author
-
McClenaghan, Neville H, Flatt, Peter R, and Ball, Andrew J
- Abstract
This study examined the effects of glucagon-like peptide-1 (GLP-1) on insulin secretion alone and in combination with sulphonylureas or nateglinide, with particular attention to KATPchannel-independent insulin secretion. In depolarised cells, GLP-1 significantly augmented glucose-induced KATPchannel-independent insulin secretion in a glucose concentration-dependent manner. GLP-1 similarly augmented the KATPchannel-independent insulin-releasing effects of tolbutamide, glibenclamide or nateglinide. Downregulation of protein kinase A (PKA)- or protein kinase C (PKC)-signalling pathways in culture revealed that the KATPchannel-independent effects of sulphonylureas or nateglinide were critically dependent upon intact PKA and PKC signalling. In contrast, GLP-1 exhibited a reduced but still significant insulin-releasing effect following PKA and PKC downregulation, indicating that GLP-1 can modulate KATPchannel-independent insulin secretion by protein kinase-dependent and -independent mechanisms. The synergistic insulin-releasing effects of combinatorial GLP-1 and sulphonylurea/nateglinide were lost following PKA- or PKC-desensitisation, despite GLP-1 retaining an insulin-releasing effect, demonstrating that GLP-1 can induce insulin release under conditions where sulphonylureas and nateglinide are no longer effective. Our results provide new insights into the mechanisms of action of GLP-1, and further highlight the promise of GLP-1 or similarly acting analogues alone or in combination with sulphonylureas or meglitinide drugs in type 2 diabetes therapy.
- Published
- 2006
- Full Text
- View/download PDF
10. Glutamine regulates expression of key transcription factor, signal transduction, metabolic gene, and protein expression in a clonal pancreatic β-cell line
- Author
-
Corless, Mary, Kiely, Aoife, McClenaghan, Neville H, Flatt, Peter R, and Newsholme, Philip
- Abstract
We have investigated the effects of prolonged exposure (24 h) to the amino acid l-glutamine, on gene and protein expression using clonal BRIN-BD11 β-cells. Expression profiling of BRIN-BD11 cells was performed using oligonucleotide microarray analysis. Culture for 24 h with 10 mM l-glutamine compared with 1 mM resulted in substantial changes in gene expression with 148 genes upregulated more than 1.8-fold, and 18 downregulated more than 1.8-fold, including many genes involved in cellular signaling, metabolism, gene regulation, and the insulin-secretory response. Subsequent functional experiments confirmed that l-glutamine increased the activity of the Ca2+regulated phosphatase calcineurin and the transcription factor Pdx1. Additionally, we demonstrated that β-cell-derived l-glutamate was released into the extracellular medium at high rates. As calcineurin is a regulator of the glutamate N-methyl-d-aspartate (NMDA) receptor activity, we investigated the action of NMDA on nutrient-induced insulin secretion, and demonstrated suppressed insulin release. These observations indicate important long-term effects of l-glutamine in regulating β-cell gene expression, signaling, and secretory function.
- Published
- 2006
- Full Text
- View/download PDF
11. Deleterious Effects of Supplementation with Dehydroepiandrosterone Sulphate or Dexamethasone on Rat Insulin-Secreting Cells Under In Vitro Culture Condition
- Author
-
Liu, Hui-Kang, Green, Brian D., McClenaghan, Neville H., McCluskey, Jannie T., and Flatt, Peter R.
- Abstract
Dehydroepiandrosterone (DHEA) and glucocorticoids are steroid hormones synthesised in the adrenal cortex. Administration of DHEA, its sulphate derivative, DHEAS, and more controversially dexamethasone (DEX), a synthetic glucocorticoid, have beneficial effects in diabetic animals. Cultivating BRIN-BD11 cells for 3 days with either DHEAS (30 μM) or DEX (100 nM), reduced total cell number and reduced cell viability and cellular insulin content. DHEAS-treated cells had poor glucose responsiveness and regulated insulin release, coupled with reduced basal insulin release. In contrast, DEX-treated cells lacked responsiveness to glucose and membrane depolarisation, and both protein kinase A (PKA) and protein kinase C (PKC) secretory pathways were desensitised. Therefore, we conclude that this steroid hormone and synthetic glucocorticoid are not beneficial to pancreatic β-cells in vitro.
- Published
- 2006
- Full Text
- View/download PDF
12. L-Alanine induces changes in metabolic and signal transduction gene expression in a clonal rat pancreatic β-cell line and protects from pro-inflammatory cytokine-induced apoptosis
- Author
-
Cunningham, Grainne A., Mcclenaghan, Neville H., Flatt, Peter R., and Newsholme, Philip
- Abstract
Acute effects of nutrient stimuli on pancreatic β-cell function are widely reported; however, the chronic effects of insulinotropic amino acids, such as L-alanine, on pancreatic β-cell function and integrity are unknown. In the present study, the effects of prolonged exposure (24 h) to the amino acid L-alanine on insulin secretory function, gene expression and pro-inflammatory cytokine-induced apoptosis were studied using clonal BRIN-BD11 cells. Expression profiling of BRIN-BD11 cells chronically exposed to L-alanine was performed using oligonucleotide microarray analysis. The effect of alanine, the iNOS (inducible nitric oxide synthase) inhibitor NMA (NG-methyl-L-arginine acetate) or the iNOS and NADPH oxidase inhibitor DPI (diphenylene iodonium) on apoptosis induced by a pro-inflammatory cytokine mix [IL-1β (interleukin-1β), TNF-α (tumour necrosis factor-α) and IFN-γ (interferon-γ)] was additionally assessed by flow cytometry. Culture for 24 h with 10 mM L-alanine resulted in desensitization to the subsequent acute insulin stimulatory effects of L-alanine. This was accompanied by substantial changes in gene expression of BRIN-BD11 cells. Sixty-six genes were up-regulated >1.8-fold, including many involved in cellular signalling, metabolism, gene regulation, protein synthesis, apoptosis and the cellular stress response. Subsequent functional experiments confirmed that L-alanine provided protection of BRIN-BD11 cells from pro-inflammatory cytokine-induced apoptosis. Protection from apoptosis was mimicked by NMA or DPI suggesting L-alanine enhances intracellular antioxidant generation. These observations indicate important long-term effects of L-alanine in regulating gene expression, secretory function and the integrity of insulin-secreting cells. Specific amino acids may therefore play a key role in β-cell function in vivo.
- Published
- 2005
- Full Text
- View/download PDF
13. Long-Term Beneficial Effects of Vanadate, Tungstate, and Molybdate on Insulin Secretion and Function of Cultured Beta Cells
- Author
-
Liu, Hui-Kang, Green, Brian D., McClenaghan, Neville H., McCluskey, Jane T., and Flatt, Peter R.
- Abstract
The ultratrace elements vanadate, tungstate, and molybdate exhibit significant antihyperglycemic effects in both type 1 and 2 diabetic animals, but possible effects on the function of pancreatic beta cells are understudied. In the present study, clonal BRIN BD11 cells were cultured for 3 days with each ultratrace element to establish doses lacking detrimental effects on viable beta cell mass. Vanadate treatment (4 μmol/L) had no effect on cellular insulin content but improved glucose-induced insulin secretory responsiveness. However, insulin secretion mediated by PKA and PKC activation was desensitized in vanadate-treated cells. Culture with tungstate (300 μmol/L) and molybdate (1 mmol/L) increased cellular insulin content and enhanced basal insulin release and the responsiveness to glucose and a wide range of other secretagogues. These observations suggest significant effects of ultratrace elements on pancreatic beta cells that may contribute to their antihyperglycemic action.
- Published
- 2004
14. Arachidonic acid, palmitic acid and glucose are important for the modulation of clonal pancreatic β-cell insulin secretion, growth and functional integrity
- Author
-
DIXON, Gordon, NOLAN, John, McCLENAGHAN, Neville H., FLATT, Peter R., and NEWSHOLME, Philip
- Abstract
Insulin-resistant states such as obesity can result in an increase in the function and mass of pancreatic β-cells, so that insulin secretion is up-regulated and Type II diabetes does not develop. However, expansion of β-cell mass is not indefinite and may well decrease with time. Changes in circulating concentrations of nutritional factors, such as fatty acids and/or glucose, may lead to a reduction in β-cell mass in vivo. Few previous studies have attempted to explore the interplay between glucose, amino acids and fatty acids with respect to β-cell mass and functional integrity. In the present study, we demonstrate that culture of clonal BRIN-BD11 cells for 24 h with the polyunsaturated fatty acid arachidonic acid (AA) increased β-cell proliferation and enhanced alanine-stimulated insulin secretion. These effects of AA were associated with significant decreases in the cellular consumption of D-glucose and L-alanine as well as decreased rates of production of nitric oxide and ammonia. Conversely 24 h exposure to the saturated fatty acid palmitic acid (PA) was found to decrease β-cell viability (by increasing apoptosis), increase the intracellular concentration of triacylglycerol (triglyceride), while inhibiting alanine-stimulated insulin secretion. These effects of PA were associated with significant increases in D-glucose and L-glutamine consumption as well as nitric oxide and ammonia production. However, L-alanine consumption was decreased in the presence of PA. The effects of AA, but not PA, were additionally dependent on glucose concentration. These studies indicate that AA may have a critical role in maintaining the appropriate mass and function of islet β-cells by influencing rates of cell proliferation and insulin secretion. This regulatory effect may be compromised by high circulating levels of glucose and/or PA, both of which are elevated in Type II diabetes and may impact upon dysfunctional and apoptotic intracellular events in the β-cell.
- Published
- 2004
- Full Text
- View/download PDF
15. Comparative Functional Study of Clonal Insulin-Secreting Cells Cultured in Five Commercially Available Tissue Culture Media
- Author
-
Hamid, Muhajir, McCluskey, Jane T., McClenaghan, Neville H., and Flatt, Peter R.
- Abstract
The electrofusion-derived rat insulin-secreting cell line BRIN-BD11 was cultured in five different commercially available media to determine the optimum medium for the in vitro maintenance of such clonal cell lines. Cells were cultured in RPMI-1640, DMEM, McCOY'S, F-12K, or MEM culture medium supplemented with 10% (v/v) fetal bovine serum and antibiotics (100 U/ml penicillin and 0.1 g/L streptomycin). Insulin secretion studies performed after 10 days revealed RPMI-1640 to be the best performing medium in terms of insulin secretory responsiveness to a range of stimuli including glucose, l-alanine, l-arginine, carbachol, and glibenclamide. Insulin release was significantly decreased (p < 0.01 to p < 0.05) in all other media compared to RPMI-1640. Only the cells cultured in RPMI-1640 and DMEM showed a significant glucose-induced insulin secretory response (p < 0.01 and p < 0.05). McCOY'S gave the next best result followed by F-12K and MEM. After the 10-day culture period, the highest insulin content was found in cells cultured in RPMI-1640 and DMEM with significantly lower levels of insulin in cells cultured in McCOY'S, F-12K, and MEM (p < 0.01 to p < 0.001). RPMI-1640 was used for further studies to investigate the effects of 5.6 – 16.7 mmol/L glucose in culture on the secretory responsiveness of BRIN-BD11 cells. Significant responses to a number of nonglucidic secretagogues were seen following culture at 5.6 and 16.7 mmol/L glucose, although responsiveness was less than after culture with 11.1 mmol/L glucose. At 16.7 mmol/L glucose culture, glucose-stimulated insulin release was abolished.
- Published
- 2001
- Full Text
- View/download PDF
16. Stimulation of insulin secretion in clonal BRIN-BD11 cells by the imidazoline derivatives KU14r and RX801080
- Author
-
Ball, Andrew J., Flatt, Peter R., and McClenaghan, Neville H.
- Abstract
The imidazoline derivatives KU14R and RX801080 have each been reported to antagonize imidazoline-stimulated insulin secretion. This study investigated the effects of a range of concentrations of both KU14R and RX801080 on insulin secretion from the clonal pancreatic beta cell line, BRIN-BD11. In the presence of a stimulatory (8.4 mm) glucose concentration, both KU14R (50–200 μm;P< 0.01 to P< 0.001) and RX801080 (50–200 μm;P< 0.01 to P< 0.001) were found to dose-dependently stimulate insulin secretion. The imidazoline efaroxan (200 μm) stimulated insulin secretion (P< 0.001) from BRIN-BD11 cells. This insulinotropic effect was significantly augmented by KU14R (100–200 μm;P< 0.01 to P< 0.001) and RX801080 (200μm;P< 0.05). Insulin secretion from BRIN-BD11 cells was also stimulated by the novel guanidine derivative BTS 67 582 (200 μm;P< 0.001). This secretagogue action was augmented both by KU14R (25–200 μm;P< 0.001) and by RX801080 (25–200μm;P< 0.05 to P< 0.001). It is concluded that, rather than acting as antagonists of imidazoline-induced insulin secretion, the imidazoline derivatives KU14R and RX801080 are themselves potent insulinotropic agents.
- Published
- 2000
- Full Text
- View/download PDF
17. Induced desensitization of the insulinotropic effects of antidiabetic drugs, BTS 67 582 and tolbutamide
- Author
-
McClenaghan, Neville H, Ball, Andrew J, and Flatt, Peter R
- Abstract
Acute and chronic mechanisms of action of novel insulinotropic antidiabetic drug, BTS 67 582 (1,1‐dimethyl‐2‐(2‐morpholinophenyl)guanidine fumarate), were examined in the stable cultured BRIN‐BD11 cell line.BTS 67 582 (100–400 μM) stimulated a concentration‐dependent increase (P<0.01) in insulin release at both non‐stimulatory (1.1 mM) and stimulatory (8.4 mM) glucose.Long‐term exposure (3–18 h) to 100 μMBTS 67 582 in culture time‐dependently decreased subsequent responsiveness to acute challenge with 200 μMBTS 67 582 or 200 μMtolbutamide at 12–18 h (P<0.001). Similarly 3–18 h culture with the sulphonylurea, tolbutamide (100 μM), also effectively suppressed subsequent insulinotropic responses to both BTS 67 582 and tolbutamide.Culture with 100 μMBTS 67 582 or 100 μMtolbutamide did not affect basal insulin secretion, cellular insulin content, or cell viability and exerted no influence on the secretory responsiveness to 200 μMof the imidazoline, efaroxan.While 18 h BTS 67 582 culture did not affect the insulin‐releasing actions (P<0.001) of 16.7 mMglucose, 10 mMarginine, 30 mMKCl, 25 μMforskolin or 10 nMphorbol‐12‐myristate 13‐acetate (PMA), significant inhibition (P<0.001) of the insulinotropic effects of 10 mM2‐ketoisocaproic acid (KIC) and 10 mMalanine were observed.These data suggest that BTS 67 582 shares a common signalling pathway to sulphonylurea but not imidazoline drugs. Desensitization of drug action may provide an important approach to dissect sites of action of novel and established insulinotropic antidiabetic agents.
- Published
- 2000
- Full Text
- View/download PDF
18. Drug-Induced Desensitization of Insulinotropic Actions of Sulfonylureas
- Author
-
Ball, Andrew J., McCluskey, Jane T., Flatt, Peter R., and McClenaghan, Neville H.
- Abstract
KATP-channel-dependent and KATP-channel-independent insulin-releasing actions of the sulfonylurea, tolbutamide, were examined in the clonal BRIN-BD11 cell line. Tolbutamide stimulated insulin release at both nonstimulatory (1.1 mM) and stimulatory (16.7 mM) glucose. Under depolarizing conditions (16.7 mM glucose plus 30 mM KCl) tolbutamide evoked a stepwise KATP channel-independent insulinotropic response. Culture (18 h) with tolbutamide or the guanidine derivative BTS 67 582 (100 μM) markedly reduced (P < 0.001) subsequent responsiveness to acute challenge with tolbutamide, glibenclamide, and BTS 67 582 but not the imidazoline drug, efaroxan. Conversely, 18 h culture with efaroxan reduced (P < 0.001) subsequent insulinotropic effects of efaroxan but not that of tolbutamide, glibenclamide, or BTS 67 582. Culture (18 h) with tolbutamide reduced the KATP channel-independent actions of both tolbutamide and glibenclamide. Whereas culture with efaroxan exerted no effect on the KATP channel-independent actions of sulfonylureas, BTS 67 582 abolished the response of tolbutamide and inhibited that of glibenclamide. These data demonstrate that prolonged exposure to tolbutamide desensitizes both KATP-channel-dependent and -independent insulin-secretory actions of sulfonylureas, indicating synergistic pathways mediated by common sulfonylurea binding site(s).
- Published
- 2000
- Full Text
- View/download PDF
19. Metabolic and KATPChannel–Independent Actions of Keto Acid Initiators of Insulin Secretion
- Author
-
McClenaghan, Neville H and Flatt, Peter R
- Abstract
Insulin-releasing effects of 2-ketobutyric acid (KB), 2-ketoisocaproic acid (KIC), 2-keto-3-methylvaleric acid (KMV), and 3-phenylpyruvic acid (PP) were examined by using clonal beta cells. Whereas KIC, KMV, and PP dose-dependently initiated insulin secretion and potentiated the effects of 4.2–16.7 m Mglucose, equimolar KB was without effect. Transport inhibition by using 10 m Mvaline, isoleucine, 2-cyano-3 hydroxycinnamate or 2-cyano-4 hydroxycinnamate, or metabolic inhibition by 15 m Mmannoheptulose, 5 m Msodium azide, 5 m Msodium cyanide, or removal of HCO3reduced the secretory effects of KIC, KMV, and PP. Whereas Kdepletion reduced keto acid–induced insulin output, depolarizing concentrations of l-leucine and l-arginine potentiated the keto acid–induced effects. Under depolarizing conditions (25 m MKCl and 16.7 m Mglucose), 10 m MKIC, KMV, or PP induced insulin secretion, suggesting KATPchannel–independent actions. Furthermore, the KATPchannel opener diazoxide reduced, but did not abolish, the keto acid–induced effects. However, voltage-dependent Ca2channel blockade with verapamil or removal of extracellular Ca2abolished keto acid–induced insulin release. Collectively, these results indicate that KIC, KMV, and PP initiate insulin secretion at least partially independently of KATPchannel activity, through both mitochondrial metabolism and regulation of Ca2influx.
- Published
- 2000
20. Engineering cultured insulin-secreting pancreatic B-cell lines
- Author
-
McClenaghan, Neville H. and Flatt, P. R.
- Abstract
Abstract: Despite many triumphs, a significant limitation of the usefulness of many of the available B-cell lines for the study of insulin secretion are either inappropriate or lack of responsiveness to glucose. Commonly employed cell lines generated prior to the 1990s following X-ray irradiation (RINm5F cells) or simian virus 40 B-cell transformation (HIT-T15 cells and BTC) fall into this category. More recent success has been achieved with the generation of INS-1 cells and MIN6 cells, but the production of these cell lines owes much to good fortune, dedication and hard work. In the present era, molecular biology techniques provide the opportunity to engineer novel pancreatic B-cell lines which possess many attributes of normal insulin-secreting cells. This review describes the electrofusion of normal NEDH rat pancreatic B-cells with immortal RINm5F cells to create three new glucose-responsive clonal insulin-secreting cells, designated BRIN-BG5, BRIN-BG7 and BRIN-BD11. These cell lines exhibit up to four-fold insulin-secretory responses to depolarization with 25 mmol/l K
+ , 7.68 mmol/l Ca2+ , 10 mmol/l l-alanine, and activation of protein kinase C or adenylate cyclase with 10 nmol/l phorbol-12-myristate-13-acetate or 25 μmol/l forskolin, respectively. The maximal insulin-secretory response of both BRIN-BG5 and BRIN-BG7 cells to glucose occurred at 8.4 mmol/l (1.9- and 1.8-fold increases, respectively). In contrast, 4.2–16.7 mmol/l glucose evoked a stepwise 2- to 3-fold of insulin release from BRIN-BD11 cells. The superior glucose responsiveness of BRIN-BD11 cells compared with BRIN-BG5 or BRIN-BG7 cells was associated with increased expression of GLUT-2 and a greater contribution of glucokinase to total glucose phosphorylating enzyme activity. Furthermore, BRIN-BD11 cells also showed appropriate responses to a diverse range of modulators of pancreatic B-cell function, including amino acids, neurotransmitters and sulphonylurea drugs. Collectively these observations indicate that genetic modification of insulin-secreting cells by electrofusion (or transfection with cDNA) offers a new avenue for generation of useful clonal glucose-responsive pancreatic B-cell lines for studies of insulin secretion and transplantation in insulin-dependent diabetes mellitus.- Published
- 1999
- Full Text
- View/download PDF
21. Hexose Recognition by Insulin-Secreting BRIN-BD11 Cells
- Author
-
McClenaghan, Neville H., Gray, Alison M., Barnett, Christopher R., and Flatt, Peter R.
- Abstract
Clonal BRIN-BD11 cells were produced by electrofusion of NEDH rat islet B-cells with immortal RINm5F cells. Western blotting analysis revealed that unlike RINm5F, novel BRIN-BD11 cells expressed high levels of the glucose transport protein GLUT-2, coupled with a rapid and sustained uptake of D-glucose, significantly greater than RINm5F after only 5 min (p<0.05). Whereas BRIN-BD11 cells expressed a high glucokinase:hexokinase ratio with 1.4–2.0 fold and 1.4–1.7 fold stepwise stimulation of insulin secretion with 4.2–16.7 mM D-glucose and D-mannose respectively, RINm5F had a lower glucokinase:hexokinase ratio (p<0.001) and were notably unresponsive to D-glucose and D-mannose. Unlike RINm5F cells, BRIN-BD11 were unresponsive to other hexoses, with RINm5F only responding to D-galactose (p<0.05). BRIN-BD11 cells should be useful for studies of nutrient-induced insulin secretion.
- Published
- 1996
- Full Text
- View/download PDF
22. Na+Cotransport by Metabolizable and Nonmetabolizable Amino Acids Stimulates a Glucose-Regulated Insulin-Secretory Response
- Author
-
McClenaghan, Neville H., Barnett, Christopher R., and Flatt, Peter R.
- Abstract
The involvement of Na+in insulin-secretory responses to metabolizable and nonmetabolizable amino acids known to be cotransported with Na+, were examined using islet-derived BRIN-BD11 cells. At stimulatory (16.7 mM) glucose, 10 mM ofl-alanine, α-aminoisobutyric acid (AIB) orl-proline stimulated 1.3- to 10.4-fold (p< 0.01) insulin-secretory responses. In each case, these effects were significantly greater than those observed at nonstimulatory (1.1 mM) glucose (p< 0.01). While, tetrodotoxin blockade of voltage-dependent Na+channels exerted no significant effect on insulin release, Na/K pump blockade with ouabain significantly promoted the amino acid-induced effects (p< 0.05). Replacement of extracellular Na+with equimolarN-methyl-d-glucamine+and omission of extracellular K+or Ca2+were all effective in removing the actions of each amino acid, confirming the critical role of ionic fluxes in the secretory responses to these amino acids. Collectively these results demonstrate that metabolizable and nonmetabolizable amino acids can induce glucose-dependent insulin-secretory responses by modulating electrogenic Na+transport.
- Published
- 1998
- Full Text
- View/download PDF
23. Insulin‐releasing action of the novel antidiabetic agent BTS 67 582
- Author
-
McClenaghan, Neville H., Flatt, Peter R., and Bailey, Clifford J.
- Abstract
BTS 67 582 (1,1‐dimethyl‐2‐(2‐morpholinophenyl)guanidine fumarate) is a novel antidiabetic agent with a short‐acting insulin‐releasing effect. This study examined its mode of action in the clonal B‐cell line BRIN‐BD11.BTS 67 582 increased insulin release from BRIN‐BD11 cells in a concentration‐dependent manner (10−8to 10−4M) at both non‐stimulating (1.1 mM) and stimulating (16.7 mM) concentrations of glucose.BTS 67 582 (10−4M) potentiated the insulin‐releasing effect of a depolarizing concentration of K+(30 mM), whereas the K+channel openers pinacidil (400 μM) and diazoxide (300 μM) inhibited BTS 67 582‐induced release.Suppression of Ca+channel activity with verapamil (20 μM) reduced the insulin‐releasing effect of BTS 67 582 (10−4M).BTS 67 582 (10−4M) potentiated insulin release induced by amino acids (10 mM), and enhanced the combined stimulant effects of glucose plus either the fatty acid palmitate (10 mM), or agents which raise intracellular cyclic AMP concentrations (25 μMforskolin and 1 mMisobutylmethylxanthine), or the cholinoceptor agonist carbachol (100 μM).Inhibition of glucose‐stimulated insulin release by adrenaline or noradrenaline (10 μM) was partially reversed by BTS 67 582 (10−4M).These data suggest that the insulin‐releasing effect of BTS 67 582 involves regulation of ATP‐sensitive K+channel activity and Ca2+influx, and that the drug augments the stimulant effects of nutrient insulin secretagogues and agents which enhance adenylate cyclase and phospholipase C. BTS 67 582 may also exert insulin‐releasing effects independently of ATP‐sensitive K+channel activity.
- Published
- 1998
- Full Text
- View/download PDF
24. Molecular Characterization of the Glucose-Sensing Mechanism in the Clonal Insulin-Secreting BRIN-BD11 Cell Line
- Author
-
McClenaghan, Neville H., Elsner, Matthias, Tiedge, Markus, and Lenzen, Sigurd
- Abstract
BRIN-BD11 cells represent a novel insulin-secreting cell line generated by electrofusion. Molecular characterization of these cells demonstrated the presence of mRNA and protein for the two key elements of the beta cell glucose-sensing system, GLUT2 and glucokinase. While levels of GLUT2 expression and 3-O-methyl-d-glucose equilibration were similar for both the BRIN-BD11 cell line and the parental control RINm5F cells, glucokinase expression was substantially higher in BRIN-BD11 cells. Expression of the two-component KATPchannel complex, KIR6.2 and SUR1, was similar in both cells. However, while control RINm5F cells were completely unresponsive to glucose, BRIN-BD11 cells responded to physiological millimolar concentrations of this hexose sugar. These studies strongly suggest that the glucose-sensing ability of insulin-secreting cells is largely dictated by the level of glucokinase, as opposed to GLUT2, expression. Thus, BRIN-BD11 cells expressing the key attributes of the normal beta cell provide an interesting model for elucidation of regulatory principles of beta cell function.
- Published
- 1998
- Full Text
- View/download PDF
25. Secretory properties of clonal insulin-secreting BRIN-BG5 cells, produced by electrofusion
- Author
-
McClenaghan, Neville H., Barnett, Christopher R., Ah-Sing, Eric, Swanston-Flatt, Sara K., and FLATT, PETER R.
- Published
- 1996
- Full Text
- View/download PDF
26. Mechanisms of action of cationic amino acids on insulin-secreting BRIN-BD11 cells
- Author
-
McCLENAGHAN, NEVILLE H., BARNETT, CHRISTOPHER R., and FLATT, PETER R.
- Published
- 1997
- Full Text
- View/download PDF
27. Effect of glucose and amino acids on insulin-secretion from a novel pancreatic B-cell line produced by electrofusion
- Author
-
McCLENAGHAN, NEVILLE H., YOON, TAI-WOOK, BARNETT, CHRISTOPHER R., WILSON, ALISON M., ABDEL-WAHAB, YASSER H. A., SWANSTON-FLATT, SARA, AH-SING, ERIC, and FLATT, PETER R.
- Published
- 1994
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.