Correction to: Islet-intrinsic effects of CFTR mutation.

Unfortunately, due to a tagging error, Dr Fiona N. Manderson Koivula's name is shown incorrectly as Koivula FN on PubMed. Her name appears correctly in the html and pdf versions of the paper.

Islet-intrinsic effects of CFTR mutation.

Cystic fibrosis-related diabetes (CFRD) is the most significant extra-pulmonary comorbidity in cystic fibrosis (CF) patients, and accelerates lung decline. In addition to the traditional view that CFRD is a consequence of fibrotic destruction of the pancreas as a whole, emerging evidence may implicate a role for cystic fibrosis transmembrane-conductance regulator (CFTR) in the regulation of insulin secretion from the pancreatic islet. Impaired first-phase insulin responses and glucose homeostasis have also been reported in CF patients. CFTR expression in both human and mouse beta cells has been confirmed, and recent studies have shown differences in endocrine pancreatic morphology from birth in CF. Recent experimental evidence suggests that functional CFTR channels are required for insulin exocytosis and the regulation of membrane potential in the pancreatic beta cell, which may account for the impairments in insulin secretion observed in many CF patients. These novel insights suggest that the pathogenesis of CFRD is more complicated than originally thought, with implications for diabetes treatment and screening in the CF population. This review summarises recent emerging evidence in support of a primary role for endocrine pancreatic dysfunction in the development of CFRD. Summary • CF is an autosomal recessive disorder caused by mutations in the CFTR gene • The vast majority of morbidity and mortality in CF results from lung disease. However CFRD is the largest extra-pulmonary co-morbidity and rapidly accelerates lung decline • Recent experimental evidence shows that functional CFTR channels are required for normal patterns of first phase insulin secretion from the pancreatic beta cell • Current clinical recommendations suggest that insulin is more effective than oral glucose-lowering drugs for the treatment of CFRD. However, the emergence of CFTR corrector and potentiator drugs may offer a personalised approach to treating diabetes in the CF population.

Differential molecular and cellular responses of GLP-1 secreting L-cells and pancreatic alpha cells to glucotoxicity and lipotoxicity.

Knowledge of the effects of glucotoxic and lipotoxic environments on proglucagon producing intestinal L cells and pancreatic alpha cells is limited compared with pancreatic beta cells. This study compares the in vitro responses of these cell types to hyperglycaemia and hyperlipidaemia. Glucose (30 mM) and palmitate (0.5mM) reduced GLUTag and MIN6 cell viability while alpha TC1 cells were sensitive only to lipotoxicity. Consistent with this, Cat mRNA expression was substantially higher in GLUTag and alpha TC1 cells compared to MIN6 cells. Glucose and palmitate reduced GLUTag cell secretory function while hypersecretion of glucagon was apparent from alpha TC1 cells. Glucose exposure increased transcription of Cat and Sod2 in MIN6 and GLUTag cells respectively while it decreased transcription of Cat and Gpx1 in alpha TC1 cells. Palmitate increased transcription of Cat and Sod2 in all three cell lines. Upregulation of antioxidant enzyme expression by palmitate was accompanied by an increase in Nfkb1 transcription, indicative of activation of defence pathways. Lipotoxicity activated ER stress response, evident from increased Hspa4 mRNA level in GLUTag and MIN6 cells. Glucose and palmitate-induced DNA damage and apoptosis, with substantially smaller effects in alpha TC1 cells. Thus alpha cells are resistant to gluco- and lipotoxicity, partly reflecting higher expression of genes involved in antioxidant defence. In contrast, intestinal L cells, like beta cells, are prone to gluco- and lipotoxicity, possibly contributing to abnormalities of GLP-1 secretion in type 2 diabetes.

Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4.

BACKGROUND: Molecular mechanisms of toxicity and cell damage were investigated in the novel human beta cell line, 1.1B4, after exposure to proinflammatory cytokines - IL-1ß, IFN-γ, TNF-α. METHODS: MTT assay, insulin radioimmunoassay, glucokinase assay, real time reverse transcription PCR, western blotting, nitrite assay, caspase assay and comet assay were used to investigate mechanisms of cytokine toxicity. RESULTS: Viability of 1.1B4 cells decreased after 18h cytokine exposure. Cytokines significantly reduced cellular insulin content and impaired insulin secretion induced by glucose, alanine, KCl, elevated Ca(2+), GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca(2+) and PDX1 protein expression were significantly reduced by cytokines. mRNA expression of genes involved in secretory function - INS, GCK, PCSK2 and GJA1 was downregulated in cytokine treated 1.1B4 cells. Upregulation of transcription of genes involved in antioxidant defence - SOD2 and GPX1 was observed, suggesting involvement of oxidative stress. Cytokines also upregulated transcriptions of NFKB1 and STAT1, which was accompanied by a significant increase in NOS2 transcription and accumulation of nitrite in culture medium, implicating nitrosative stress. Oxidative and nitrosative stresses induced apoptosis was evident from increased % tail DNA, DNA fragmentation, caspase 3/7 activity, apoptotic cells and lower BCL2 protein expression. CONCLUSIONS: This study delineates molecular mechanisms of cytokine toxicity in 1.1B4 cells, which agree with earlier observations using human islets and rodent beta cells. GENERAL SIGNIFICANCE: This study emphasizes the potential usefulness of this cell line as a human beta cell model for research investigating autoimmune destruction of pancreatic beta cells.

Pseudoislet formation enhances gene expression, insulin secretion and cytoprotective mechanisms of clonal human insulin-secreting 1.1B4 cells.

We have studied the effects of cell communication on human beta cell function and resistance to cytotoxicity using the novel human insulin-secreting cell line 1.1B4 configured as monolayers and pseudoislets. Incubation with the incretin gut hormones GLP-1 and GIP caused dose-dependent stimulation of insulin secretion from 1.1B4 cell monolayers and pseudoislets. The secretory responses were 1.5-2.7-fold greater than monolayers. Cell viability (MTT), DNA damage (comet assay) and apoptosis (acridine orange/ethidium bromide staining) were investigated following 2-h exposure of 1.1B4 monolayers and pseudoislets to ninhydrin, H2O2, streptozotocin, glucose, palmitate or cocktails of proinflammatory cytokines. All agents tested decreased viability and increased DNA damage and apoptosis in both 1.1B4 monolayers and pseudoislets. However, pseudoislets exhibited significantly greater resistance to cytotoxicity (1.5-2.7-fold increases in LD50) and lower levels of DNA damage (1.3-3.4-fold differences in percentage tail DNA and olive tail moment) and apoptosis (1.3-1.5-fold difference) compared to monolayers. Measurement of gene expression by reverse-transcription, real-time PCR showed that genes involved with insulin secretion (INS, PDX1, PCSK1, PCSK2, GLP1R and GIPR), cell-cell communication (GJD2, GJA1 and CDH1) and antioxidant defence (SOD1, SOD2, GPX1 and CAT) were significantly upregulated in pseudoislets compared to monolayers, whilst the expression of proapoptotic genes (NOS2, MAPK8, MAPK10 and NFKB1) showed no significant differences. In summary, these data indicate cell-communication associated with three-dimensional islet architecture is important both for effective insulin secretion and for protection of human beta cells against cytotoxicity.

Molecular determinants and intracellular targets of taurine signalling in pancreatic islet ß-cells.

AIM: Despite its abundance in pancreatic islets of Langerhans and proven antihyperglycemic effects, the impact of the essential amino acid, taurine, on islet ß-cell biology has not yet received due consideration, which prompted the current studies exploring the molecular selectivity of taurine import into ß-cells and its acute and chronic intracellular interactions. METHODS: The molecular aspects of taurine transport were probed by exposing the clonal pancreatic BRIN BD11 ß-cells and primary mouse and human islets to a range of the homologs of the amino acid (assayed at 2-20 mM), using the hormone release and imaging of intracellular signals as surrogate read-outs. Known secretagogues were employed to profile the interaction of taurine with acute and chronic intracellular signals. RESULTS: Taurine transporter TauT was expressed in the islet ß-cells, with the transport of taurine and homologs having a weak sulfonate specificity but significant sensitivity to the molecular weight of the transporter. Taurine, hypotaurine, homotaurine, and ß-alanine enhanced insulin secretion in a glucose-dependent manner, an action potentiated by cytosolic Ca2+ and cAMP. Acute and chronic ß-cell insulinotropic effects of taurine were highly sensitive to co-agonism with GLP-1, forskolin, tolbutamide, and membrane depolarization, with an unanticipated indifference to the activation of PKC and CCK8 receptors. Pre-culturing with GLP-1 or KATP channel inhibitors sensitized or, respectively, desensitized ß-cells to the acute taurine stimulus. CONCLUSION: Together, these data demonstrate the pathways whereby taurine exhibits a range of beneficial effects on insulin secretion and ß-cell function, consistent with the antidiabetic potential of its dietary low-dose supplementation.

Effects of lipotoxicity on a novel insulin-secreting human pancreatic ß-cell line, 1.1B4.

The novel insulin-secreting human pancreatic ß-cell line, 1.1B4, demonstrates stability in culture and many of the secretory functional attributes of human pancreatic ß-cells. This study investigated the cellular responses of 1.1B4 cells to lipotoxicity. Chronic 18-h exposure of 1.1B4 cells to 0.5 mm palmitate resulted in decreased cell viability and insulin content. Secretory responses to classical insulinotropic agents and cellular Ca2+ handling were also impaired. Palmitate decreased glucokinase activity and mRNA expression of genes involved in secretory function but up-regulated mRNA expression of HSPA5, EIF2A, and EIF2AK3, implicating activation of the endoplasmic reticulum stress response. Palmitate also induced DNA damage and apoptosis of 1.1B4 cells. These responses were accompanied by increased gene expression of the antioxidant enzymes SOD1, SOD2, CAT and GPX1. This study details molecular mechanisms underlying lipotoxicity in 1.1B4 cells and indicates the potential value of the novel ß-cell line for future research.

Taurine rescues pancreatic ß-cell stress by stimulating α-cell transdifferentiation.

The semi-essential ubiquitous amino acid taurine has been shown to alleviate obesity and hyperglycemia in humans; however, the pathways underlying the antidiabetic actions have not been characterized. We explored the effect of chronic taurine exposure on cell biology of pancreatic islets, in degenerative type 1-like diabetes. The latter was modeled by small dose of streptozotocin (STZ) injection for 5 days in mice, followed by a 10-day administration of taurine (2% w/v, orally) in the drinking water. Taurine treatment opposed the detrimental changes in islet morphology and ß-/α-cell ratio, induced by STZ diabetes, coincidentally with a significant 3.9 ± 0.7-fold enhancement of proliferation and 40 ± 5% reduction of apoptosis in ß-cells. In line with these findings, the treatment counteracted an upregulation of antioxidant (Sod1, Sod2, Cat, Gpx1) and downregulation of islet expansion (Ngn3, Itgb1) genes induced by STZ, in a pancreatic ß-cell line. At the same time, taurine enhanced the transdifferentiation of α-cells into ß-cells by 2.3 ± 0.8-fold, echoed in strong non-metabolic elevation of cytosolic Ca2+ levels in pancreatic α-cells. Our data suggest a bimodal effect of dietary taurine on islet ß-cell biology, which combines the augmentation of α-/ß-cell transdifferentiation with downregulation of apoptosis. The dualism of action, stemming presumably from the intra- and extracellular modality of the signal, is likely to explain the antidiabetic potential of taurine supplementation.

NKCC transport mediates the insulinotropic effects of taurine and other small neutral amino acids.

AIMS: Despite its high concentration in pancreatic islets of Langerhans and broad range of antihyperglycemic effects, the route facilitating the import of dietary taurine into pancreatic ß-cell and mechanisms underlying its insulinotropic activity are unclear. We therefore studied the impact of taurine on beta-cell function, alongside that of other small neutral amino acids, L-alanine and L-proline. MAIN METHODS: Pharmacological profiling of insulin secretion was conducted using clonal BRIN BD11 ß-cells, the impact of taurine on the metabolic fate of glucose carbons was assessed using NMR and the findings were verified by real-time imaging of Ca2+ dynamics in the cytosol of primary mouse and human islet beta-cells. KEY FINDINGS: In our hands, taurine, alanine and proline induced secretory responses that were dependent on the plasma membrane depolarisation, import of Ca2+, homeostasis of K+ and Na+ as well as on cell glycolytic and oxidative metabolism. Taurine shifted the balance between the oxidation and anaplerosis towards the latter, in BRIN BD11 beta-cells. Furthermore, the amino acid signalling was significantly attenuated by inhibition of Na+-K+-Cl- symporter (NKCC). SIGNIFICANCE: These data suggest that taurine, like L-alanine and L-proline, acutely induces glucose-dependent insulin-secretory responses by modulating electrogenic Na+ transport, with potential role of intracellular K+ and Cl- in the signal transduction. The acute action delineated would be consistent with antidiabetic potential of dietary taurine supplementation.

Development and functional characterization of insulin-releasing human pancreatic beta cell lines produced by electrofusion.

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells.

INTRODUCTION: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. METHODS: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5mM glucose) or DMEM (25mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. RESULTS: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P<0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P<0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. CONCLUSIONS: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

Acute and long-term effects of metformin on the function and insulin secretory responsiveness of clonal ß-cells.

Functional effects of acute and prolonged (48 h) exposure to the biguanide drug metformin were examined in the clonal pancreatic ß-cell line, BRIN-BD11. Effects of metformin on prolonged exposure to excessive increased concentrations of glucose and palmitic acid were also assessed. In acute 20-min incubations, 12.5-50 µm metformin did not alter basal (1.1 mm glucose) or glucose-stimulated (16.7 mm glucose) insulin secretion. However, higher concentrations of metformin (100-1000 µm) increased (1.3-1.5-fold; p<0.001) insulin release at basal glucose concentrations, but had no effect on glucose-stimulated insulin secretion. There were no apparent acute effects of metformin on intracellular Ca²(+) concentrations, but metformin enhanced (p<0.05 to p<0.01) the acute insulinotropic actions of GIP and GLP-1. Exposure for 48 h to 200 µm metformin improved aspects of ß-cell insulin secretory function, whereas these benefits were lost at 1 mm metformin. Prolonged glucotoxic and lipotoxic conditions impaired ß-cell viability and insulin release in response to glucose and to a broad range of insulin secretagogues. Concomitant culture with 200 µm metformin partially reversed many of the adverse effects of prolonged glucotoxic conditions. However, there were no beneficial effects of metformin under prolonged culture with elevated concentrations of palmitic acid. The results suggest that metformin exerts direct effects on ß-cell viability, function and survival that could contribute to the use of this agent in the treatment of type 2 diabetes.

The role of glucagon- and somatostatin-secreting cells in the regulation of insulin release and beta-cell function in heterotypic pseudoislets.

BACKGROUND: Pseudoislet studies have concentrated on single beta-cell lines or a combination of insulin and glucagon-secreting cells, overlooking the potential role of somatostatin in insulin release. This study sought to evaluate a heterotypic pseudoislet model containing insulin- (MIN6), glucagon- (αTC1.9) and somatostatin (TGP52)-secreting cells of mouse origin and to compare these pseudoislets with traditional monolayer preparations. METHODS: Cellular viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays), proliferation (5-bromo-2-deoxyuridine ELISA), hormone content and functional insulin release in response to a variety of stimuli were measured. Differential expression of E-cadherin, connexin 36 and connexin 43 was assessed by reverse transcriptase-polymerase chain reaction and Western blot to determine a possible role for adherens in insulin release from these pseudoislets. RESULTS: All pseudoislet cells displayed reduced proliferation coupled with an increase in cell death which may contribute to their static size in culture. While MIN6 and TGP52 cells expressed E-cadherin and showed sustained or improved hormone content when configured as pseudoislets, αTC1.9 lacked E-cadherin and contained less glucagon following pseudoislet formation. MIN6 and αTC1.9 cells expressed connexin 36, but not connexin 43 and TGP52 cells expressed connexin 43 only. In the presence of Alanine, Arginine and glucagon-like peptide-1, heterotypic pseudoislet cultures secreted levels of insulin that were comparable to that of MIN6 pseudoislets. In addition, pseudoislets comprising all three cell lines released more insulin into the surrounding culture medium than MIN6 pseudoislets when studied over a 1-week period. CONCLUSIONS: The current model may prove useful in studying the role of islet cell interactions in the release of insulin from pancreatic islets.

Nutrient regulation of insulin secretion and beta-cell functional integrity.

Pancreatic beta-cells are often referred to as "fuel sensors" as they continually monitor and respond to dietary nutrients, under the modulation of additional neurohormonal signals, in order to secrete insulin to best meet the needs of the organism. beta-cell nutrient sensing requires metabolic activation, resulting in production of stimulus-secretion coupling signals that promote insulin biosynthesis and release. The primary stimulus for insulin secretion is glucose, and islet beta-cells are particularly responsive to this important nutrient secretagogue, It is important to consider individual effects of different classes of nutrient or other physiological or pharmacological agents on metabolism and insulin secretion. However, given that beta-cells are continually exposed to a complex milieu of nutrients and other circulating factors, it is important to also acknowledge and examine the interplay between glucose metabolism and that of the two other primary nutrient classes, the amino acids and fatty acids. It is the mixed nutrient sensing and outputs of glucose, amino and fatty acid metabolism that generate the metabolic coupling factors (MCFs) involved in signaling for insulin exocytosis. Primary MCFs in the beta-cell include ATP, NADPH, glutamate, long chain acyl-CoA and diacylglycerol and are discussed in detail in this article.

Overexpression of the malate-aspartate NADH shuttle member Aralar1 in the clonal beta-cell line BRIN-BD11 enhances amino-acid-stimulated insulin secretion and cell metabolism.

In the present study, we have investigated the effects of the transduction with recombinant adenovirus AdCA-Aralar1 (aspartate-glutamate carrier 1) on the metabolism, function and secretory properties of the glucose- and amino-acid-responsive clonal insulin-secreting cell line BRIN-BD11. Aralar1 overexpression increased long-term (24 h) and acute (20 min) glucose- and amino-acid-stimulated insulin secretion, cellular glucose metabolism, L-alanine and L-glutamine consumption, cellular ATP and glutamate concentrations, and stimulated glutamate release. However, cellular triacylglycerol and glycogen contents were decreased as was lactate production. These findings indicate that increased malate-aspartate shuttle activity positively shifted beta-cell metabolism, thereby increasing glycolysis capacity, stimulus-secretion coupling and, ultimately, enhancing insulin secretion. We conclude that Aralar1 is a key metabolic control site in insulin-secreting cells.

Prolonged L-alanine exposure induces changes in metabolism, Ca(2+) handling and desensitization of insulin secretion in clonal pancreatic beta-cells.

Acute insulin-releasing actions of amino acids have been studied in detail, but comparatively little is known about the beta-cell effects of long-term exposure to amino acids. The present study examined the effects of prolonged exposure of beta-cells to the metabolizable amino acid L-alanine. Basal insulin release or cellular insulin content were not significantly altered by alanine culture, but acute alanine-induced insulin secretion was suppressed by 74% (P<0.001). Acute stimulation of insulin secretion with glucose, KCl or KIC (2-oxoisocaproic acid) following alanine culture was not affected. Acute alanine exposure evoked strong cellular depolarization after control culture, whereas AUC (area under the curve) analysis revealed significant (P<0.01) suppression of this action after culture with alanine. Compared with control cells, prior exposure to alanine also markedly decreased (P<0.01) the acute elevation of [Ca(2+)](i) (intracellular [Ca(2+)]) induced by acute alanine exposure. These diminished stimulatory responses were partially restored after 18 h of culture in the absence of alanine, indicating reversible amino-acid-induced desensitization. (13)C NMR spectra revealed that alanine culture increased glutamate labelling at position C4 (by 60%; P<0.01), as a result of an increase in the singlet peak, indicating increased flux through pyruvate dehydrogenase. Consistent with this, protein expression of the pyruvate dehydrogenase kinases PDK2 and PDK4 was significantly reduced. This was accompanied by a decrease in cellular ATP (P<0.05), consistent with diminished insulin-releasing actions of this amino acid. Collectively, these results illustrate the phenomenon of beta-cell desensitization by amino acids, indicating that prolonged exposure to alanine can induce reversible alterations to metabolic flux, Ca(2+) handling and insulin secretion.

Cystic Fibrosis-Related Diabetes: Pathophysiology and Therapeutic Challenges.

Cystic fibrosis-related diabetes (CFRD) is among the most common extrapulmonary co-morbidity associated with cystic fibrosis (CF), affecting an estimated 50% of adults with the condition. Cystic fibrosis is prevalent in 1 in every 2500 Caucasian live births and is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutated CFTR leads to dehydrated epithelial surfaces and a build-up of mucus in a variety of tissues including the lungs and pancreas. The leading cause of mortality in CF is repeated respiratory bacterial infections, which prompts a decline in lung function. Co-morbid diabetes promotes bacterial colonisation of the airways and exacerbates the deterioration in respiratory health. Cystic fibrosis-related diabetes is associated with a 6-fold higher mortality rate compared with those with CF alone. The management of CFRD adds a further burden for the patient and creates new therapeutic challenges for the clinical team. Several proposed hypotheses on how CFRD develops have emerged, including exocrine-driven fibrosis and destruction of the entire pancreas and contrasting theories on the direct or indirect impact of CFTR mutation on islet function. The current review outlines recent data on the impact of CFTR on endocrine pancreatic function and discusses the use of conventional diabetic therapies and new CFTR-correcting drugs on the treatment of CFRD.

Short-term CFTR inhibition reduces islet area in C57BL/6 mice.

Cystic fibrosis-related diabetes (CFRD) worsens CF lung disease leading to early mortality. Loss of beta cell area, even without overt diabetes or pancreatitis is consistently observed. We investigated whether short-term CFTR inhibition was sufficient to impact islet morphology and function in otherwise healthy mice. CFTR was inhibited in C57BL/6 mice via 8-day intraperitoneal injection of CFTRinh172. Animals had a 7-day washout period before measures of hormone concentration or islet function were performed. Short-term CFTR inhibition increased blood glucose concentrations over the course of the study. However, glucose tolerance remained normal without insulin resistance. CFTR inhibition caused marked reductions in islet size and in beta cell and non-beta cell area within the islet, which resulted from loss of islet cell size rather than islet cell number. Significant reductions in plasma insulin concentrations and pancreatic insulin content were also observed in CFTR-inhibited animals. Temporary CFTR inhibition had little long-term impact on glucose-stimulated, or GLP-1 potentiated insulin secretion. CFTR inhibition has a rapid impact on islet area and insulin concentrations. However, islet cell number is maintained and insulin secretion is unaffected suggesting that early administration of therapies aimed at sustaining beta cell mass may be useful in slowing the onset of CFRD.

Homocysteine and other structurally-diverse amino thiols can alter pancreatic beta cell function without evoking cellular damage.

Homocysteine and related amino thiols, homocysteic acid, cysteic acid, homocysteine sulphinic acid and cysteine sulphinic acid have been labelled as neurotoxins. Homocysteine thiolactone, a metabolic derivative of homocysteine, is cytotoxic to endothelial cells and other cell lineages. Since pancreatic beta cells share many phenotypic similarities with neuronal cells, the present study uses clonal pancreatic BRIN-BD11 cells to investigate possible detrimental effects of these amino thiols on insulin secretion and pancreatic beta cell function. Insulin secretion was concentration-dependently inhibited at both basal (1.1 mM) and stimulatory (16.7 mM) glucose by homocysteine, homocysteine thiolactone and homocysteine sulphinic acid. Cysteic acid concentration-dependently inhibited insulin secretion at 16.7 mM glucose. Cell viability was not compromised by any of the amino thiols. Insulin secretory responses to alanine were inhibited by homocysteine, homocysteine thiolactone, homocysteic acid and cysteic acid. Insulin secretion in the presence of elevated Ca(2+) and forskolin were lowered by all amino thiols, except homocysteic acid. The secretory responsiveness to PMA, GLP-1 and KCl were only impaired in the presence of homocysteine and homocysteine thiolactone. These findings indicate that homocysteine, homocysteine thiolactone and, to a lesser extent, other amino thiols cause dysfunctional insulin secretion from pancreatic beta cells.

Pro-inflammatory cytokines increase glucose, alanine and triacylglycerol utilization but inhibit insulin secretion in a clonal pancreatic beta-cell line.

We have investigated the effects of prolonged exposure (24 h) to pro-inflammatory cytokines on beta-cell metabolism and insulin secretion using clonal BRIN-BD11 beta cells. Addition of IL-1beta, tumour necrosis factor-alpha and IFN-gamma (at concentrations that did not induce apoptosis) inhibited chronic (24 h) and acute stimulated levels of insulin release (by 59 and 93% respectively), increased cellular glucose and alanine consumption, and also elevated lactate and glutamate release. However, ATP levels and cellular triacylglycerol were decreased while glutathione was increased. We conclude that sub-lethal concentrations of pro-inflammatory cytokines appear to shift beta-cell metabolism away from a key role in energy generation and stimulus-secretion coupling and towards a catabolic state which may be related to cell defence.