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.

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.

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.

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.

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.

Implanting 1.1B4 human ß-cell pseudoislets improves glycaemic control in diabetic severe combined immune deficient mice.

AIM: To investigate the potential of implanting pseudoislets formed from human insulin-releasing ß-cell lines as an alternative to islet transplantation. METHODS: In this study, the anti-diabetic potential of novel human insulin releasing 1.1B4 ß-cells was evaluated by implanting the cells, either as free cell suspensions, or as three-dimensional pseudoislets, into the subscapular region of severe combined immune deficient mice rendered diabetic by single high-dose administration of streptozotocin. Metabolic parameters including food and fluid intake, bodyweight and blood glucose were monitored throughout the study. At the end of the study animals were given an intraperitoneal glucose tolerance test. Animals were then culled and blood and tissues were collected for analysis. Insulin and glucagon contents of plasma and tissues were measured by insulin radioimmunoassay and chemiluminescent enzyme-linked immunosorbance assay respectively. Histological analyses of pancreatic islets were carried out by quantitative fluorescence immunohistochemistry staining. RESULTS: Both pseudoislet and cell suspension implants yielded well vascularised ß-cell masses of similar insulin content. This was associated with progressive amelioration of hyperphagia (P < 0.05), polydipsia (P < 0.05), body weight loss (P < 0.05), hypoinsulinaemia (P < 0.05), hyperglycaemia (P < 0.05 - P < 0.001) and glucose tolerance (P < 0.01). Islet morphology was also significantly improved in both groups of transplanted mice, with increased ß-cell (P < 0.05 - P < 0.001) and decreased alpha cell (P < 0.05 - P < 0.001) areas. Whereas mice receiving 1.1B4 cell suspensions eventually exhibited hypoglycaemic complications, pseudoislet recipients displayed a more gradual amelioration of diabetes, and achieved stable blood glucose control similar to non-diabetic mice at the end of the study. CONCLUSION: Although further work is needed to address safety issues, these results provide proof of concept for possible therapeutic applicability of human ß-cell line pseudoislets in diabetes.

Improved antioxidative defence protects insulin-producing cells against homocysteine toxicity.

Homocysteine (HC) is considered to play an important role in the development of metabolic syndrome complications. Insulin-producing cells are prone to HC toxicity and this has been linked to oxidative stress. However, the exact mechanisms remain unknown. Therefore it was the aim of this study to determine the nature of reactive oxygen species responsible for HC toxicity. Chronic exposure of RINm5F and INS1E insulin-producing cells to HC decreased cell viability and glucose-induced insulin secretion in a concentration-dependent manner and led to a significant induction of hydrogen peroxide generation in the cytosolic, but not the mitochondrial compartment of the cell. Cytosolic overexpression of catalase, a hydrogen peroxide detoxifying enzyme, provided a significant protection against viability loss and hydrogen peroxide generation, while mitochondrial overexpression of catalase did not protect against HC toxicity. Overexpression of CuZnSOD, a cytosolic superoxide dismutating enzyme, also protected against HC toxicity. However, the best protection was achieved in the case of a combined overexpression of CuZnSOD and catalase. Incubation of cells in combination with alloxan resulted in a significant increase of HC toxicity and an increase of hydrogen peroxide generation. Overexpression of CuZnSOD or catalase protected against the toxicity of HC plus alloxan, with a superior protection achieved again by combined overexpression. The results indicate that HC induces oxidative stress in insulin-producing cells by stimulation of superoxide radical and hydrogen peroxide generation in the cytoplasm. The low antioxidative defence status makes the insulin-producing cells very vulnerable to HC toxicity.

Molecular Mechanisms of Toxicity and Cell Damage by Chemicals in a Human Pancreatic Beta Cell Line, 1.1B4.

OBJECTIVES: Mechanisms of toxicity and cell damage were investigated in novel clonal human pancreatic beta cell line, 1.1B4, after exposure to streptozotocin, alloxan, ninhydrin, and hydrogen peroxide. METHODS: Viability, DNA damage, insulin secretion/content, [Ca]i, and glucokinase/hexokinase, mRNA expression were measured by MTT assay, comet assay, radioimmunoassay, fluorometric imaging plate reader, enzyme-coupled photometry, and real-time polymerase chain reaction, respectively. RESULTS: Chemicals significantly reduced 1.1B4 cell viability in a time/concentration-dependent manner. Chronic 18-hour exposure decreased cellular insulin, glucokinase, and hexokinase activities. Chemicals decreased transcription of INS, GCK, PCSK1, PCSK2, and GJA1 (involved in secretory function). Insulin release and [Ca]i responses to nutrients and membrane-depolarizing agents were impaired. Streptozotocin and alloxan up-regulated transcription of genes, SOD1 and SOD2 (antioxidant enzymes). Ninhydrin and hydrogen peroxide up-regulated SOD2 transcription, whereas alloxan and hydrogen peroxide increased CAT transcription. Chemicals induced DNA damage, apoptosis, and increased caspase 3/7 activity. Streptozotocin and alloxan decreased transcription of BCL2 while increasing transcription of BAX. Chemicals did not affect transcription of HSPA4 and HSPA5 and nitrite production. CONCLUSIONS: 1.1B4 cells represent a useful model of human beta cells. Chemicals impaired 1.1B4 cell secretory function and activated antioxidant defense and apoptotic pathways without activating endoplasmic reticulum stress response/nitrosative stress.

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.

Evaluation of the role of N-methyl-D-aspartate (NMDA) receptors in insulin secreting beta-cells.

The possibility that antagonism of N-methyl-D-aspartate (NMDA) receptors represent a novel drug target for diabetes prompted the current studies probing NMDA receptor function in the detrimental actions of homocysteine on pancreatic beta-cell function. Cellular insulin content and release, changes in membrane potential and intracellular Ca(2+) and gene expression were assessed following acute (20min) and long-term (18h) exposure of pancreatic clonal BRIN-BD11 beta-cells to known NMDA receptor modulators in the absence and presence of cytotoxic concentrations of homocysteine. As expected, acute or long-term exposure to homocysteine significantly suppressed basal and secretagogue-induced insulin release. In addition, NMDA reduced glucose-stimulated insulin secretion (GSIS). Interestingly, the selective NMDA receptor antagonist, MK-801, had no negative effects on GSIS. The effects of the NMDA receptor modulators were largely independent of effects on membrane depolarisation and increases of intracellular Ca(2+). However, combined culture of the NMDA antagonist, MK-801, with homocysteine did enhance intracellular Ca(2+) levels. Actions of NMDA agonists/antagonists and homocysteine on signal transduction pathways were independent of changes in cellular insulin content, cell viability, DNA damage or expression of key beta-cell genes. Taken together, the data support a role for NMDA receptors in controlling pancreatic beta-cell function. However, modulation of NMDA receptor function was unable to prevent the detrimental beta-cell effects of homocysteine.

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.

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.

Responses of GLP1-secreting L-cells to cytotoxicity resemble pancreatic ß-cells but not α-cells.

Little is known about responses of intestinal L-cells to chemical or cytokine-mediated attack and how these compare with pancreatic ß- or α-cells. Administration of streptozotocin to mice induced severe diabetes, islet lymphocytic infiltration, increased α-cell proliferation and decreased numbers of ß- and L-cells. In vitro, streptozotocin and cytokines reduced cell viability with higher lethal dose 50 values for α-TC1 cells. mRNA expression of Glut2 was lower and Cat was greater in GLUTag and α-TC1 cells compared with MIN6 cells. Cytotoxins affected the transcription of genes involved in secretion in GLUTag and MIN6 cells. They are also involved in upregulation of antioxidant defence enzymes, transcription of NfκB and Nos2, and production of nitrite in all cell types. Cytotoxin-induced DNA damage and apoptosis were apparent in all cells, but α-TC1 cells were less severely affected. Thus, responses of GLP1-secreting L-cells to cytotoxicity resemble ß-cells, whereas α-cells are resistant due to differences in the expression of genes involved in cytotoxicity or antioxidant defence.

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.

Cellular responses of novel human pancreatic ß-cell line, 1.1B4 to hyperglycemia.

The novel human-derived pancreatic ß-cell line, 1.1B4 exhibits insulin secretion and ß-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 ß 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 ß-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 Ca (2+) 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 ß cells exposed to hyperglycemia, illustrating the usefulness of this novel human-derived cell line for studying human ß-cell biology and diabetes.

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.

Physiological concentrations of interleukin-6 directly promote insulin secretion, signal transduction, nitric oxide release, and redox status in a clonal pancreatic ß-cell line and mouse islets.

Interleukin-6 (IL6) has recently been reported to promote insulin secretion in a glucagon-like peptide-1-dependent manner. Herein, the direct effects of IL6 (at various concentrations from 0 to 1000 pg/ml) on pancreatic ß-cell metabolism, AMP-activated protein kinase (AMPK) signaling, insulin secretion, nitrite release, and redox status in a rat clonal ß-cell line and mouse islets are reported. Chronic insulin secretion (in µg/mg protein per 24  h) was increased from 128·7±7·3 (no IL6) to 178·4±7·7 (at 100  pg/ml IL6) in clonal ß-cells and increased significantly in islets incubated in the presence of 5·5  mM glucose for 2  h, from 0·148 to 0·167±0·003  ng/islet. Pretreatment with IL6 also induced a twofold increase in basal and nutrient-stimulated insulin secretion in subsequent 20 min static incubations. IL6 enhanced both glutathione (GSH) and glutathione disulphide (GSSG) by nearly 20% without changing intracellular redox status (GSSG/GSH). IL6 dramatically increased iNOS expression (by ca. 100-fold) with an accompanying tenfold rise in nitrite release in clonal ß-cells. Phosphorylated AMPK levels were elevated approximately twofold in clonal ß-cells and mouse islet cells. Calmodulin-dependent protein kinase kinase levels (CaMKK), an upstream kinase activator of AMPK, were also increased by 50% after IL6 exposure (in ß-cells and islets). Our data have demonstrated that IL6 can stimulate ß-cell-dependent insulin secretion via direct cell-based mechanisms. AMPK, CaMKK (an upstream kinase activator of AMPK), and the synthesis of nitric oxide appear to alter cell metabolism to benefit insulin secretion. In summary, IL6 exerts positive effects on ß-cell signaling, metabolism, antioxidant status, and insulin secretion.

Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility.

Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human ß-cell line. Structural pseudoislets formed readily over 3-7 days in culture using ultra-low-attachment plastic, attaining a static size of 100-200 µm in diameter, corresponding to ~6000 ß cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 1·7- to 12·5-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human ß cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.

Directed differentiation of progenitor cells towards an islet-cell phenotype.

Exogenous insulin administration and oral anti-diabetic drugs are the primary means of treating diabetes. However, tight glycaemic control, with its inherent risk of hypoglycaemia, is required to prevent the microvascular and macrovascular complications of the disease. While islet or pancreas transplantations offer a longer-term cure, their widespread application is not possible, primarily because of a lack of donor tissue, the burden of life-long immunosuppression, and eventual graft rejection. The rapid increase in the incidence of diabetes has promoted the search for alternative cell-based therapies. Here we review recent advances in the directed differentiation of both endocrine and non-endocrine progenitors towards an islet-like phenotype.