MicroRNA regulation of islet and enteroendocrine peptides: Physiology and therapeutic implications for type 2 diabetes.

The pathogenesis of type 2 diabetes (T2D) is associated with dysregulation of glucoregulatory hormones, including both islet and enteroendocrine peptides. Microribonucleic acids (miRNAs) are short noncoding RNA sequences which post transcriptionally inhibit protein synthesis by binding to complementary messenger RNA (mRNA). Essential for normal cell activities, including proliferation and apoptosis, dysregulation of these noncoding RNA molecules have been linked to several diseases, including diabetes, where alterations in miRNA expression within pancreatic islets have been observed. This may occur as a compensatory mechanism to maintain beta-cell mass/function (e.g., downregulation of miR-7), or conversely, lead to further beta-cell demise and disease progression (e.g., upregulation of miR-187). Thus, targeting miRNAs has potential for novel diagnostic and therapeutic applications in T2D. This is reinforced by the success seen to date with miRNA-based therapeutics for other conditions currently in clinical trials. In this review, differential expression of miRNAs in human islets associated with T2D will be discussed along with further consideration of their effects on the production and secretion of islet and incretin hormones. This analysis further unravels the therapeutic potential of miRNAs and offers insights into novel strategies for T2D management.

Enhancement of homocysteine toxicity to insulin-secreting BRIN-BD11 cells in combination with alloxan.

Previous studies have shown that homocysteine (HC) has a detrimental impact on insulin secretion and pancreatic beta cell function. The aim of the present study was to determine the role of reactive oxygen species (ROS) in the in vitro toxic effects of HC on insulin secretion and function of BRIN-BD11 insulin-secreting cells. In this study, insulin secretion from BRIN-BD11 cells was determined radioimmunologically, cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and glucokinase activity by a glucose phosphorylation assay following culture with HC plus alloxan (Alx). Treatment with HC resulted in concentration-dependent inhibition of insulin secretion induced by glucose and other insulinotropic agents. HC in combination with Alx resulted in a more pronounced decline in insulin secretion, including that induced by 20  mM alanine, by 43% (P<0.001) and 30  mM KCl by 60% (P<0.001), compared with control culture. The glucokinase phosphorylating capacity in cells cultured with HC plus Alx was significantly lower, compared with control cells. The cells also displayed a significant 84% (P<0.001) decline in cell viability. Prolonged, 72-h culture of insulin-secreting cells with HC followed by 18-h culture without HC did not result in full restoration of beta cell responses to insulinotropic agents. In vitro oxygen consumption was enhanced by a combination of Alx with HC. The study arrived at the conclusion that HC generates ROS in a redox-cycling reaction with Alx that explains the decline in viability of insulin-secreting cells, leading to reduced glucokinase phosphorylating ability, diminished insulin secretory responsiveness and cell death.

Acute and long-term effects of peroxisome proliferator-activated receptor-γ activation on the function and insulin secretory responsiveness of clonal beta-cells.

Thiazolidinediones (TZDs) are used as antidiabetic therapy. The purpose of the present study was to examine whether the TZD rosiglitazone has direct actions on pancreatic beta-cells that contribute to its overall effects. Effects of acute and prolonged (48 h) exposure to rosiglitazone, as a model glitazone compound, were assessed in clonal pancreatic BRIN-BD11 beta-cells maintained in standard, glucotoxic and lipotoxic cultures. In acute 20-min incubations, rosiglitazone (0.2-100 µM) did not alter basal or glucose-stimulated insulin secretion. However, rosiglitazone (6.25 µM) enhanced (p<0.001) the acute insulinotropic action of GLP-1. Prolonged exposure to 6.25 µM rosiglitazone in standard media had no effect on cell viability or cellular insulin content, but slightly reduced the insulin secretory response to glucose and alanine (p<0.05). Prolonged (48 h) exposure to glucotoxic or lipotoxic conditions reduced beta-cell viability (p<0.05), cellular insulin content (p<0.001 and p<0.05, respectively), and insulin release in response to glucose and a range of secretagogues. The adverse effect of lipotoxicity on beta-cell viability was prevented by concomitant exposure to 6.25 µM rosiglitazone. Culture with 6.25 µM rosiglitazone further decreased acute insulin release under glucotoxic conditions. However, when insulin secretion was expressed as percentage cellular insulin content, rosiglitazone (6.25 µM) significantly improved many of the adverse effects of gluco- and lipotoxic conditions on insulin secretory responsiveness. The results suggest that despite decrease in cellular insulin content TZDs exert direct beneficial effects on beta-cell viability and function during gluco- or lipotoxicity.

Effects of metformin on BRIN-BD11 beta-cell insulin secretory desensitization induced by prolonged exposure to sulphonylureas.

AIMS: Prolonged exposure of pancreatic beta-cells in vitro to the sulphonylureas tolbutamide and glibenclamide induces subsequent desensitization of insulinotropic pathways. Clinically, the insulin-sensitizing biguanide drug metformin is often administered alongside sulphonylurea as antidiabetic therapy. The present study examines the functional effects of metformin (200 µM) on tolbutamide- and glibenclamide-induced desensitisation. METHODS: Acute and prolonged (18 h) effects of exposure to tolbutamide and glibenclamide alone, or in the presence of metformin, were examined in insulin-secreting BRIN-BD11 cells. RESULTS: In acute 20 min incubations at 1.1 mM glucose, metformin increased (1.2-1.7-fold; p < 0.001) the insulin-releasing actions of tolbutamide and glibenclamide. At 16.7 mM glucose, metformin significantly enhanced glibenclamide-induced insulin release at all concentrations (50-400 µM) examined, but tolbutamide-stimulated insulin secretion was only augmented at higher concentrations (300-400 µM). Exposure for 18 h to 100 µM tolbutamide or glibenclamide significantly impaired insulin release in response to glucose and a broad range of insulin secretagogues. Concomitant culture with metformin (200 µM) prevented or partially reversed many of the adverse effects on K(ATP) channel dependent and independent insulinotropic pathways. Beneficial effects of metformin were also observed in cells exposed to glibenclamide for 18 h with significant improvements in the insulin secretory responsiveness to alanine, GLP-1 and sulphonylureas. The decrease of viable cell numbers observed with glibenclamide was reversed by co-culture with metformin, but cellular insulin content was depressed. CONCLUSIONS: The results suggest that metformin can prevent the aspects of sulphonylurea-induced beta-cell desensitization.

Detrimental actions of metabolic syndrome risk factor, homocysteine, on pancreatic beta-cell glucose metabolism and insulin secretion.

Elevated plasma homocysteine has been reported in individuals with diseases of the metabolic syndrome including vascular disease and insulin resistance. As homocysteine exerts detrimental effects on endothelial and neuronal cells, this study investigated effects of acute homocysteine exposure on beta-cell function and insulin secretion using clonal BRIN-BD11 beta-cells. Acute insulin release studies in the presence of various test reagents were performed using monolayers of BRIN-BD11 cells and samples assayed by insulin radioimmunoassay. Cellular glucose metabolism was assessed by nuclear magnetic resonance (NMR) analysis following 60-min exposure of BRIN-BD11 cell monolayers to glucose in either the absence or presence of homocysteine. Homocysteine dose-dependently inhibited insulin release at moderate and stimulatory glucose concentrations. This inhibitory effect was reversible at all but the highest concentration of homocysteine. 13C-glucose NMR demonstrated decreased labelling of glutamate from glucose at positions C2, C3 and C4, indicating that the tricarboxylic acid (TCA) cycle-dependent glucose metabolism was reduced in the presence of homocysteine. Homocysteine also dose-dependently inhibited insulinotropic responses to a range of glucose-dependent secretagogues including nutrients (alanine, arginine, 2-ketoisocaproate), hormones (glucagon-like peptide-1 (7-36)amide, gastric inhibitory polypeptide and cholecystokinin-8), neurotransmitter (carbachol), drug (tolbutamide) as well as a depolarising concentration of KCl or elevated Ca2+. Insulin secretion induced by activation of adenylate cyclase and protein kinase C pathways with forskolin and phorbol 12-myristate 13-acetate were also inhibited by homocysteine. These effects were not associated with any adverse action on cellular insulin content or cell viability, and there was no increase in apoptosis/necrosis following exposure to homocysteine. These data indicate that homocysteine impairs insulin secretion through alterations in beta-cell glucose metabolism and generation of key stimulus-secretion coupling factors. The participation of homocysteine in possible beta-cell demise merits further investigation.

Function of a long-term, GLP-1-treated, insulin-secreting cell line is improved by preventing DPP IV-mediated degradation of GLP-1.

Glucagon-like peptide-1 (GLP-1) is an important insulinotropic hormone with potential in the treatment of type 2 diabetes. However, the short biological half-life of the peptide after cleavage by dipeptidylpeptidase IV (DPP IV) is a major limitation. Inhibition of DPP IV activity and the development of resistant GLP-1 analogues is the subject of ongoing research. In this study, we determined cell growth, insulin content, insulin accumulation and insulin secretory function of a insulin-secreting cell line cultured for 3 days with either GLP-1, GLP-1 plus the DPP IV inhibitor diprotin A (DPA) or stable N-acetyl-GLP-1. Native GLP-1 was rapidly degraded by DPP IV during culture with accumulation of the inactive metabolite GLP-1(9-36)amide. Inclusion of DPA or use of the DPP IV-resistant analogue, N-acetyl-GLP-1, improved cellular function compared to exposure to GLP-1 alone. Most notably, basal and accumulated insulin secretion was enhanced, and glucose responsiveness was improved. However, prolonged GLP-1 treatment resulted in GLP-1 receptor desensitization regardless of DPP IV status. The results indicate that prevention of DPP IV action is necessary for beneficial effects of GLP-1 on pancreatic beta cells and that prolonged exposure to GLP-1(9-36)amide may be detrimental to insulin secretory function. These observations also support the ongoing development of DPP-IV-resistant forms of GLP-1, such as N-acetyl-GLP-1.

13C NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and gamma-glutamyl cycle activity in a clonal pancreatic beta-cell line.

AIMS/HYPOTHESIS: Pancreatic islet cells and clonal beta-cell lines can metabolise L-glutamine at high rates. The pathway of L-glutamine metabolism has traditionally been described as L-glutamine-->L-glutamate-->2-oxoglutarate-->oxidation in TCA cycle following conversion to pyruvate. Controversially, the metabolism of D-glucose to L-glutamate in beta cells is not widely accepted. However, L-glutamate has been proposed to be a stimulation-secretion coupling factor in glucose-induced insulin secretion. We aimed to investigate the metabolism of glutamine and glucose by using (13)C NMR analysis. METHODS: BRIN-BD11 cells were incubated in the presence of 16.7 mmol/l [1-(13)C]glucose, 2 mmol/l [2-(13)C]L-glycine or 2 mmol/l [1,2-(13)C]glutamine in the presence or absence of other amino acids or inhibitors. After an incubation period the cellular metabolites were extracted using a PCA extract procedure. After neutralisation, the extracts were prepared for analysis using (13)C-NMR spectroscopy. RESULTS: Using (13)C NMR analysis we have shown that L-glutamine could be metabolised in BRIN-BD11 cells via reactions constituting part of the gamma-glutamyl cycle producing glutathione. Moderate or high activities of the enzymes required for these pathways of metabolism including glutaminase, gamma-glutamyltransferase and gamma-glutamylcysteine synthetase were observed. We additionally report significant D-glucose metabolism to L-glutamate. Addition of the aminotransferase inhibitor, aminooxyacetate, attenuated L-glutamate production from D-glucose. CONCLUSION/INTERPRETATION: We propose that L-glutamine metabolism is important in the beta cell for generation of stimulus-secretion coupling factors, precursors of glutathione synthesis and for supplying carbon for oxidation in the TCA cycle. D-glucose, under appropriate conditions, can be converted to L-glutamate via an aminotransferase catalysed step.

Comparison of the secretory properties of four insulin-secreting cell lines.

The insulin-secretory responsiveness of four popular and widely used insulin-secreting cells lines (RINm5F, HIT-T15, INS-1 and BRIN-BD11 cells) to a range of stimuli including glucose, amino acids, neurotransmitters, peptide hormones and sulphonylureas was studied. Differences were seen in the pattern of responsiveness of the cell lines to the various modulators of insulin release. While these studies revealed that INS-1 cells had the highest insulin content, only BRIN-BD11 cells exhibited a significant step-wise insulin secretory response to increasing glucose concentrations. BRIN-BD11 cells also showed pronounced insulin responses to leucine, KIC, L-arginine, L-alanine and palmitic acid. All the cell lines tested gave significant responses to the neurotransmitters carbachol and glibenclamide with increased insulin release. A comparison was made between the functional characteristics of the various cell lines with those of freshly isolated rat islets. This illustrated the general value of each cell line as a model for studies of insulin secretion. Electrofusion-derived BRIN-BD11 cells appeared to closely mimic the glucose sensitivity and overall secretory performance of normal rat islets.

Effects of cytotoxic agents on functional integrity and antioxidant enzymes in clonal beta-cells.

Effects of cytotoxic agents and hydrogen peroxide were examined using pancreatic BRIN-BD11 cells and the parental insulinoma RINm5F cell line. Cell viability was determined using the MTT colorimetric assay and the TUNEL assay was used to assess apoptosis and acridine orange assay was used to determine levels of apoptosis versus necrosis. RT-PCR studies were employed to investigate the effects of the toxins on the expression of antioxidative enzymes, superoxide dismutase (SOD), glutathionine peroxidase (GPX) and catalase (CAT). Streptozotocin, hydrogen peroxide, alloxan and ninhydrin exerted time- and concentration-dependent toxic effects on BRIN-BD11 and RINm5F cells. RT-PCR showed that 90 minutes exposure of BRIN-BD11 cells or RINm5F cells to 5 mM ninhydrin down regulates SOD, GPX and CAT antioxidative enzymes. Glutathionine peroxidase gene expression was also down regulated in both types of cell by hydrogen peroxide. There were no significant differences in antioxidant gene expression after exposure to the other toxins under the conditions employed. TUNEL assay revealed that streptozotocin (8 mM) and hydrogen peroxide (125 microM) had no significant effect on the number of cells undergoing apoptosis. However after exposure to ninhydrin (5 mM) almost 100% of the non-viable BRIN-BD11 cells and around 50% of the RINm5F cells were dying by apoptosis. With the BRIN-BD11 cells there was around a 30% increase in the number of apoptotic cells compared with 50% in the RINm5F cells after exposure to alloxan (16 mM). The results indicate multiple effects of cytotoxic agents on functional integrity and antioxidant enzyme gene expression in clonal beta-cells.

Functional examination of microencapsulated bioengineered insulin-secreting beta-cells.

Clonal insulin-secreting BRIN-BD11 cells engineered by electrofusion were encapsulated inside natrium alginate beads and cultured in RPMI 1640 culture media. Acute insulin secretory responses to glucose and amino acids were compared between microencapsulated cells and non-encapsulated cells maintained in monolayer culture. Encapsulated cells exhibited a 1.5-fold, 2.9-fold and 4.2-fold increase (P< 0.001) in insulin release in response to 16.7 mmol/l glucose, 10 mmol/l L-arginine and 10 mmol/l L-alanine respectively. Insulin output by non-encapsulated cells was approximately 30% greater but the relative magnitudes of responses were similar. This is the first study to demonstrate the stability of cellular engineered insulin-secreting cells encapsulated in alginate beads, illustrating the utility of this approach for cellular engineering and potential transplantation in diabetes.

Comparative functional study of clonal insulin-secreting cells cultured in five commercially available tissue culture media.

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.

Specific desensitization of sulfonylurea- but not imidazoline-induced insulin release after prolonged tolbutamide exposure.

Functional effects of prolonged exposure to the sulfonylurea, tolbutamide, were examined in the clonal electrofusion-derived BRIN-BD11 cell line. In acute 20-min incubations, 50-400 microM tolbutamide stimulated a dose-dependent increase (P < 0.01) in insulin release at both non-stimulatory (1.1 mM) and stimulatory (8.4 mM) glucose. Culture with 100 microM tolbutamide (18 hr) caused a marked (67%) decrease in subsequent insulin-secretory responsiveness to acute challenge with 200 microM tolbutamide, though notably, tolbutamide culture exerted no influence on 200 microM efaroxan-induced insulin secretion. Duration of exposure (3-18 hr) to 100 microM tolbutamide in culture also time-dependently influenced subsequent responsiveness to acute tolbutamide challenge, with progressive 47-58% decreases from 6-18 hr (P < 0.001). Similarly, 6- to 18-hr culture with 100 microM efaroxan specifically desensitized efaroxan-induced insulin release. Tolbutamide- and efaroxan-induced desensitization exhibited a time-dependent reversibility, with a sustained return to full insulin-secretory responsiveness by 12 hr. Notably, 18-hr culture with tolbutamide or efaroxan did not significantly affect insulinotropic responses to 16.7 mM glucose, 10 mM 2-ketoisocaproic acid, 10 mM alanine, 10 mM arginine, or 30 mM KCl. Diverse inhibitory actions of tolbutamide or efaroxan culture on late events in stimulus-secretion coupling reveal that drug desensitization is both a specific and important phenomenon. As such, the model system described could prove an important tool in determining the complex modes of action of established and novel clinically useful insulinotropic compounds.

Differential acute and long term actions of succinic acid monomethyl ester exposure on insulin-secreting BRIN-BD11 cells.

Esters of succinic acid are potent insulin secretagogues, and have been proposed as novel antidiabetic agents for type 2 diabetes. This study examines the effects of acute and chronic exposure to succinic acid monomethyl ester (SAM) on insulin secretion, glucose metabolism and pancreatic beta cell function using the BRIN-BD11 cell line. SAM stimulated insulin release in a dose-dependent manner at both non-stimulatory (1.1 mM) and stimulatory (16.7 mM) glucose. The depolarizing actions of arginine also stimulated a significant increase in SAM-induced insulin release but 2-ketoisocaproic acid (KIC) inhibited SAM induced insulin secretion indicating a possible competition between the preferential oxidative metabolism of these two agents. Prolonged (18 hour) exposure to SAM revealed decreases in the insulin-secretory responses to glucose, KIC, glyceraldehyde and alanine. Furthermore, SAM diminished the effects of non-metabolized secretagogues arginine and 3-isobutyl-1-methylxanthine (IBMX). While the ability of BRIN-BD11 cells to oxidise glucose was unaffected by SAM culture, glucose utilization was substantially reduced. Collectively, these data suggest that while SAM may enhance the secretory potential of non-metabolized secretagogues, it may also serve as a preferential metabolic fuel in preference to other important physiological nutrients and compromise pancreatic beta cell function following prolonged exposure.

Desensitization of sulphonylurea- and nutrient-induced insulin secretion following prolonged treatment with glibenclamide.

Functional effects of prolonged exposure to the sulphonylurea glibenclamide were examined in a popular clonal pancreatic beta-cell line, denoted as BRIN-BD11. In acute 20-min incubations, 200 microM of tolbutamide or glibenclamide stimulated insulin release from non-depolarized and depolarized cells, which was dramatically reduced following 18-h culture with 100 microM glibenclamide. Sulphonylurea desensitization in non-depolarized cells was reversed following 6-36-h subsequent culture in the absence of glibenclamide. However, desensitization of insulinotropic effects of sulphonylureas in depolarized cells following glibenclamide culture and associated decline in cellular insulin content was not fully reversible. Culture with 100 microM glibenclamide also markedly reduced the acute insulinotropic actions of glucose, L-alanine, L-arginine, 2-ketoisocaproic acid (KIC) and KCl. These effects were almost completely reversed following 18-h culture in the absence of the sulphonylurea.

Stimulation of insulin secretion in clonal BRIN-BD11 cells by the imidazoline derivatives KU14r and RX801080.

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 m m) glucose concentration, both KU14R (50-200 microm;P< 0.01 to P< 0.001) and RX801080 (50-200 microm;P< 0.01 to P< 0.001) were found to dose-dependently stimulate insulin secretion. The imidazoline efaroxan (200 microm) stimulated insulin secretion (P< 0.001) from BRIN-BD11 cells. This insulinotropic effect was significantly augmented by KU14R (100-200 microm;P< 0.01 to P< 0.001) and RX801080 (200 microm;P< 0.05). Insulin secretion from BRIN-BD11 cells was also stimulated by the novel guanidine derivative BTS 67 582 (200 microm;P< 0.001). This secretagogue action was augmented both by KU14R (25-200 microm;P< 0.001) and by RX801080 (25-200 microm;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.

Culture and function of electrofusion-derived clonal insulin-secreting cells immobilized on solid and macroporous microcarrier beads.

In view of the advantages of the bulk production of clonal pancreatic beta cells, an investigation was made of the growth and insulin secretory functions of an electrofusion-derived cell line (BRIN-BD11) immobilized on a solid microcarrier, cytodex-1 or a macroporous microcarrier, cultispher-G. For comparison, similar tests were performed using BRIN-BD11 cells present in single cell suspensions or allowed to form pseudoislets. Similar growth profiles were recorded for each microcarrier with densities of 4.4 x 10(5) +/- 0.3 cells/ml and 4.2 x 10(5) +/- 0.2 cells/ml achieved using cytodex-1 and cultispher-G, respectively. Cell viability began to decline on day 5 of culture. Insulin concentration in the culture medium reached a peak of 26 +/- 2.0 ng/ml and 24 +/- 2.2 ng/ml for cells grown on cytodex-1 and cultispher-G, respectively. Cells grown on both types of microcarrier showed a significant 1.5-1.8-fold acute insulin-secretory response to 16.7 mmol/l glucose. L-alanine (10 mmol/l) and L-arginine (10 mmol/l) also induced significant 3 4 fold increases of insulin release. BRIN-BD11 cells immobilized on cytodex-1 or cultispher-G out-performed single cell suspensions and pseudoislets in terms of insulin-secretory responses to glucose and amino acids. A 1.3-fold, 2.2-fold and 1.7-fold stimulation of insulin secretion was observed for glucose, L-alanine and L-arginine respectively in single cell suspensions. Corresponding increases for pseudoislets were 1.6-1.8-fold for L-alanine and L-arginine, with no significant response to glucose alone. These data indicate the utility of micro-carriers for the production of functioning clonal beta cells.

Induced desensitization of the insulinotropic effects of antidiabetic drugs, BTS 67 582 and tolbutamide.

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 microM) 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 microM BTS 67 582 in culture time-dependently decreased subsequent responsiveness to acute challenge with 200 microM BTS 67 582 or 200 microM tolbutamide at 12 - 18 h (P<0.001). Similarly 3 - 18 h culture with the sulphonylurea, tolbutamide (100 microM), also effectively suppressed subsequent insulinotropic responses to both BTS 67 582 and tolbutamide. Culture with 100 microM BTS 67 582 or 100 microM tolbutamide did not affect basal insulin secretion, cellular insulin content, or cell viability and exerted no influence on the secretory responsiveness to 200 microM of the imidazoline, efaroxan. While 18 h BTS 67 582 culture did not affect the insulin-releasing actions (P<0.001) of 16.7 mM glucose, 10 mM arginine, 30 mM KCl, 25 microM forskolin or 10 nM phorbol-12-myristate 13-acetate (PMA), significant inhibition (P<0.001) of the insulinotropic effects of 10 mM 2-ketoisocaproic acid (KIC) and 10 mM alanine 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.

Drug-induced desensitization of insulinotropic actions of sulfonylureas.

K(ATP)-channel-dependent and K(ATP)-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 K(ATP) channel-independent insulinotropic response. Culture (18 h) with tolbutamide or the guanidine derivative BTS 67 582 (100 microM) 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 K(ATP) channel-independent actions of both tolbutamide and glibenclamide. Whereas culture with efaroxan exerted no effect on the K(ATP) 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 K(ATP)-channel-dependent and -independent insulin-secretory actions of sulfonylureas, indicating synergistic pathways mediated by common sulfonylurea binding site(s).

Engineering of a glucose-responsive surrogate cell for insulin replacement therapy of experimental insulin-dependent diabetes.

Glucose responsiveness in the millimolar concentration range is a crucial requirement of a surrogate pancreatic beta cell for insulin replacement therapy of insulin-dependent diabetes. Novel insulin-secreting GK cell clones with millimolar glucose responsiveness were generated from an early-passage glucose-unresponsive RINm5F cell line. This line expressed constitutively both the K(ATP) channel and the GLUT2 glucose transporter; but it had a relative lack of glucokinase. Through overexpression of glucokinase, however, it was possible to generate glucose-responsive clones with a glucokinase-to-hexokinase ratio comparable to that of a normal pancreatic beta cell. This aim, on the other hand, was not achieved through overexpression of the GLUT2 glucose transporter. Raising the expression level of this glucose transporter into the range of rat liver, without correcting the glucokinase-to-hexokinase enzyme ratio, did not render the cells glucose responsive. These glucokinase-overexpressing RINm5F cells also stably maintained their molecular and insulin secretory characteristics in vivo. After implantation into streptozotocin diabetic immunodeficient rats, glucokinase-overexpressing cells retained their insulin responsiveness to physiological glucose stimulation under in vivo conditions. These cells represent a notable step toward the future bioengineering of a surrogate beta cell for insulin replacement therapy in insulin-dependent diabetes mellitus.

Metabolic and K(ATP) channel-independent actions of keto acid initiators of insulin secretion.

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 mM glucose, equimolar KB was without effect. Transport inhibition by using 10 mM valine, isoleucine, 2-cyano-3 hydroxycinnamate or 2-cyano-4 hydroxycinnamate, or metabolic inhibition by 15 mM mannoheptulose, 5 mM sodium azide, 5 mM sodium cyanide, or removal of HCO3 reduced the secretory effects of KIC, KMV, and PP. Whereas K+ depletion reduced keto acid-induced insulin output, depolarizing concentrations of L-leucine and L-arginine potentiated the keto acid-induced effects. Under depolarizing conditions (25 mM KCI and 16.7 mM glucose), 10 mM KIC, KMV, or PP induced insulin secretion, suggesting K(ATP) channel-independent actions. Furthermore, the K(ATP) channel opener diazoxide reduced, but did not abolish, the keto acid-induced effects. However, voltage-dependent Ca2+ channel blockade with verapamil or removal of extracellular Ca2+ abolished keto acid-induced insulin release. Collectively, these results indicate that KIC, KMV, and PP initiate insulin secretion at least partially independently of K(ATP) channel activity, through both mitochondrial metabolism and regulation of Ca2+ influx.