Abstract WNIN/Ob the mutant obese rats (‘Sumo’) have been developed indigenously from the Wistar inbred stocks (> 80 yr old) of NIN animal facility. They are euglycemic and share several features common with the other well known obese rat models like Zucker and Koletsky rats. But there are unique characteristics exclusive to WNIN/Ob rats like the “kinky tails” (fusion of tail vertebrae at two to three places of the tail) and incomplete dominant mutation facilitating the development of three distinct phenotypes like lean [+/+], carrier [+/-] and obese [-/-].Apart from being obese, 60% of them developed tumors, 20% develop cataract, 90% of them have kidney abnormalities, and all of them are infertile.They also age faster with the average life span of being 1.5 years compared to 2.5-3 years seen in normal rats. Obesity reflects a state of imbalance between oxidative stress and anti-oxidative defense mechanism(s) and are unique to beta cells due to their inherent weak antioxidant systems compared to other organs making them highly vulnerable to the deleterious effects of oxidative stress. To investigate this, we analyzed the progression of the pancreatic beta cell dysfunction as an age dependent (35days/ 6months/12months) in phenotypes like lean [+/+], obese [-/-] and their parental control (C). Morphological analyses revealed that beta cell mass was significantly larger in obese, with evidence of increased apoptosis/EPR /HSP stress signals appreciable with age. Ultrastructural analysis of the pancreatic tissue showed insulin degranulation in obese animals along with low expression of Pdx-1 in the pancreatic tissue. In vitro, the primary islet cell cultures of obese showed four fold increase in the basal insulin secretion unlike lean and control. However, with high glucose challenge the islets from the obese were less responsive probably due to reduced expression of Glut-2. Taken together it appears that obesity induced oxidative stress and the impaired functional responses may account for the progressive impaired beta cell function found in diabetes. WNIN/Ob model system serves as a novel tool to study the cellular and molecular mechanism underlying obesity and Type 2 diabetes. They also provide opportunity to screen anti-obesity and anti-diabetes drugs both synthetic and natural in vivo.

Insulin secretion from pancreatic beta cells is stimulated by glucagon-like peptide-1 (GLP-1), a hormone that is released from enteroendocrine L cells of the distal intestine after the ingestion of a meal. GLP-1 mimetics (e.g., exenatide) and GLP-1 analogs (e.g., liraglutide) activate the beta-cell GLP-1 receptor (GLP-1R), and these compounds stimulate insulin secretion while also lowering levels of blood glucose in patients diagnosed with type 2 diabetes mellitus (T2DM). An additional option for the treatment of T2DM involves the administration of dipeptidyl peptidase-IV (DPP-IV) inhibitors (e.g., sitagliptin, vildagliptin). These compounds slow metabolic degradation of GLP-1, thereby raising post-prandial levels of circulating GLP-1 substantially. Investigational compounds that stimulate GLP-1 secretion also exist, and in this regard a noteworthy advance is the demonstration that small molecule GPR119 agonists (e.g., AR231453) stimulate L cell GLP-1 secretion while also directly stimulating beta-cell insulin release. In this talk, I will summarize what is currently known concerning the signal transduction properties of the beta-cell GLP-1R as they relate to insulin secretion. Emphasized are the cyclic AMP, protein kinase A, and Epac2 mediated actions of GLP-1 to regulate ATP-sensitive K+ channels, voltage-dependent K+ channels, TRPM2 cation channels, intracellular Ca2+ release channels, and Ca2+-dependent exocytosis. I will also discuss evidence that provides a conceptual framework with which to understand why GLP-1R agonists are less likely to induce hypoglycemia when they are administered for the treatment of T2DM.

The thioredoxin reductase (TrxR) - thioredoxin (Trx) system is one of the major enzymatic cell defenses, providing protection from oxidative injury. It consists of NADPH and thioredoxin reductase (TrxR), which maintain thioredoxin (Trx) in a reduced state. Perturbing the [TrxR-Trx] system with a selective TrxR inhibitor, auranofin, induces oxidative stress by keeping Trx in its oxidized state. We have prepared Trx mimetic peptides consisting of a family of tri- and tetra-oligopeptides derived from the canonical CxxC motif of the Trx active site and a modified CxC motif. These Trx-mimetic compounds are N- and C-terminal-blocked peptides effectively restore CA secretion in bovine chromaffin cells, as monitored by amperometry in single cells. They also prevent the AuF-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase in both chromaffin and insulinoma cells, and protecting them from apoptosis. The Trx-mimetic peptides act intracellularly and are significantly more potent than the traditional antioxidants NAC, GSH, DTT, AD4 (NAC-amide), and ascorbic acid. In vivo, CB3, one of the Trx mimetic, was shown to attenuates allergic airway disease through negative regulation of MAPK p38 . Taken together, the CxxC and CxC peptides represent a new family of potent and selective redox compounds that could serve as potential candidates for prevention and treatment of oxidative-stress-related disorders such as asthma and diabetes.

Voltage-gated calcium channels (VGCC) are involved in a large variety of cellular Ca2+ signaling processes, including exocytosis, a Ca2+ dependent release of neurotransmitters and hormones. Great progress has been made in understanding the mode of action of VGCC in exocytosis, a process distinguished by two sequential yet independent Ca2+ binding reactions. First, Ca2+ binds at the selectivity filter, the EEEE motif of the VGCC, and second, subsequent to a brief and intense Ca2+ inflow to synaptotagmin, a vesicular protein. Inquiry into the functional and physical interactions of the channels with synaptic proteins has demonstrated that exocytosis is triggered during the initial Ca2+ binding at the channel pore, prior to Ca2+ entry. This model was proven experimentally, using an impermeable cation that replaced Ca2+ in islets and insulinoma cells and supported secretion without entry (Trus et al., 2007), and by an impermeable L-type calcium channel support depolarization-evoked secretion in chromaffin cells without Ca2+ entry (Hagalili et al., 2008; Marom et al., 2010; Atlas, 2010). Accordingly, a cycle of secretion begins by an incoming stimulus that releases vesicles from a releasable pool upon Ca2+ binding at the pore, and at the same time, the transient increase in [Ca2+]i primes a fresh set of non-releasable vesicles, to be fused by the next incoming stimulus. We propose a model, in which the Ca2+ binding at the EEEE motif and the consequent conformational changes in the channel are the primary event in triggering secretion, while synaptotagmin acts as a vesicle docking protein. Thus, the channel serves as the molecular On/Off signaling switch, where the predominance of a conformational change in Ca2+-bound channel provides for the fast secretory process. Trus et al., Biochemistry 2007 Dec 18;46(50):14461-7 Hagalili et al., 2008 Biochemistry 2008 Dec 30;47(52):13822-30 Marom et al., J Biol Chem. 2010 Mar 5;285(10):6996-7005 Atlas, Cell Signal. 2010 Nov;22(11):1597-603.

Neonatal diabetes (ND) with a monogenic inheritance pattern is invariably due to insulin deficiency, indicating that the genes involved are essential in beta cell development, maintenance or function. Even though the various forms of ND are rare, elucidating their molecular cause can provide insights very relevant to common forms of diabetes and to the normal function of the beta cell. About half of ND cases are transient and, in most cases, due to double expression of the imprinted (paternally expressed) PLAGL1 gene. A much rarer form, accompanied by intestinal atresia and absence of islets in an otherwise normal exocrine pancreas, was recently reported by us to be due to inactivating mutations of the RFX6 gene (Smith et al., Nature 2010). Objective: To evaluate the contribution of PLAGL1 and RFX6 to the function of the normal post-natal beta cell.

The autoimmune attack by T cells on insulin-producing pancreatic beta cells results in type 1 diabetes (T1D) in both humans and NOD mice. T cells participate in all phases of the disease from its initiation, termed, insulitis, to its subsequent effector phase, when beta cells are selectively destroyed by both direct and indirect cytolysis. How these rogue T cells escape both central and peripheral tolerance is not fully understood, but genetic and environmental factors alter both the tempo and overall incidence of T1D. One of the major features of NOD diabetes is that both CD4+ and CD8+ T cells are required for the development of T1D, and for robust transfer into disease-free NOD recipients. Naïve islet-reactive T cells are stimulated by islet-antigen-containing dendritic cells (DC) that drain from the pancreas and accumulate in the pancreatic lymph nodes (PLN). CD4+ T cells are activated by DC presenting islet antigens on MHC class II (I-Ag7), while CD8+ T cells are activated by cross-presentation of islet antigens on MHC class I (Kd, Db). Antigen presented by these DC appears to originate from degranulating and apoptotic beta cells. Thus, peripheral tolerance for both CD4+ and CD8+ T cells is broken by islet-antigen-loaded DC. This process is somewhat paradoxical; while DC are seen as crucial APC during times of inflammation, the clearance of apoptotic self cells by DC is generally considered to be non-inflammatory, even tolerogenic. Yet, under certain situations acquisition of antigen from apoptotic cells by DC can be pro-inflammatory and can lead to the priming of self-reactive T cells; this appears to be the case for apoptotic pancreatic beta cell antigens in T1D-prone animals. Which DC subset is responsible for breaking peripheral tolerance and how this process unfolds mechanistically are not well understood. Recently, we found that in NOD mice a rather heterogeneous subset of CD11c+PDCA-1- conventional DC (cDC) are responsible for acquiring, processing and presenting beta cell antigen in vivo. This subset is characterized not only by high expression of CD11c, but also by the lack of expression of PDCA-1 and CD8α and a rather broad expression of CD11b. We found that the ablation of this subset in vivo resulted in the loss of CD4+ T cells activation, insulitis and diabetes. Moreover, only these cDC could restore in vivo antigen presentation and diabetes. While we initially termed these cells myleoid DC (mDC) based on their broad expression of CD11b, we have since determined that this subset includes not only CD11b+DC but also a recently-described novel subset of CD11c+CD11b-/loCD8α- DC that acquired small particulate fragments of antigen from apoptotic cells in vivo, and unlike other DC subsets, were uniquely capable of processing and presenting this antigen in an immunogenic and non-tolerogenic manner to both CD4+ and CD8+ T cells. These DC were termed merocytic DC (mcDC) since they stored particles of apoptotic cells in discrete, punctate vesicles in their cytoplasm, meros (μεροσ, particle in Greek). These mcDC are more numerous in NOD mice, and antigen-loaded mcDC rescue CD8+ T cells from peripheral anergy and deletion, and stimulate islet-reactive CD4+ T cells. When purified from the pancreatic lymph nodes of overtly diabetic NOD mice, mcDC can break peripheral T cell tolerance to beta cell antigens in vivo and induce rapid onset T cell-mediated T1D in young NOD mouse. Thus, the mcDC subset appears to represent the long-sought critical antigen presenting cell responsible for breaking peripheral tolerance to beta cell antigen in vivo.

The pancreas is a richly innervated organ and several neurotransmitters potently enhance insulin and glucagon secretion from the pancreatic islet. The peptidergic neurotransmitters PACAP (pituitary adenylate cyclase activating polypeptide) and VIP (vasoactive intestinal polypeptide) both potently enhance insulin and glucagon secretion from pancreatic islets. Both neurotransmitters activate G protein coupled receptors in the PACAP receptor family comprising the PAC1, VPAC1 and VPAC2 receptors. All three receptors have been detected in pancreatic islets and we have therefore evaluated a series of VPAC2 receptor selective peptides to see if there is an opportunity to selectively enhance insulin secretion without affecting glucagon secretion by obtaining VPAC2 receptor selectivity. We have generated a series of peptides that have a more than 20-fold improved potency at VPAC2 receptors over the natural ligands VIP or PACAP and better than 1000-fold binding selectivity for VPAC2 receptors over PAC1 and VPAC1 receptors. From this series of VPAC2 receptor selective peptides, we proceeded to using P17 (a cyclisized VIP analogue) and P104 (a linear VIP analogue) to address if the beneficial effects on insulin secretion could be separated from the enhancement of glucagon secretion seen with VIP or PACAP. Both analogues potently (3-4 fold) enhanced glucose-dependent insulin secretion, were capable of protecting islet cells from cytokines-induced apoptosis and, in difference to PACAP, failed to stimulate glucagon secretion from rat islets. VPAC2 receptor selective analogues were also able to enhance insulin secretion from human islets up to 2-fold. In vivo both P17 and P104 enhanced insulin secretion in response to either an i.v. glucose bolus (P104) or to a steady-rate i.v. glucose infusion (P17) by a factor similar to what was observed in the isolated islet. In order to assess the risk that VPAC2 receptors desensitize when subjected to chronic stimulation the P17 analogue was given for 72h followed (2.6 or 8 µg/kg/h) by a glucose challenge (i.v. glucose infusion; 5 and 15 mg/kg/min). At both infusion rates the compound was able to enhance insulin secretion to the glucose infusion by approximately 4-fold over vehicle. In order to assess the safety of this analogue a second study was performed with P17 given continuously for 72h at rates of 8 and 27 µg/kg; which would correspond to at least 3 and 10x the maximally efficacious dose for insulin secretion. No adverse events were observed, however, plasma triglycerides and free fatty acids were dose-dependently reduced after the 72h infusion. Both doses reduced food intake by 20% and 50% for 8 and 27 µg/kg/h, respectively. No signs of liver damage or perturbations of plasma GH, corticosterone, prolactin or total cholesterol levels were observed. The P104 analogue was furthermore capable of enhancing insulin secretion by 3-4-fold in diabetic GK rats in the intravenous glucose tolerance test (IVGTT) at doses of 3 and 10 µg/kg i.v. In man VIP has a half-life of 1-2 minutes and in general peptides of this size, 4-5 kDa, have elimination half-lives of 30 minutes even if they are stable to peptidases since they are cleared by the kidney. Kidney clearance is dictated by compound size and molecular weight. We have therefore conjugated a 40 kDa polyethyleneglycol (PEG) to the C-terminus of the peptide to increase the in vivo half-life of the peptide analogue. Using an analogue of P104 we found that PEGylation results in a 50-100-fold loss of in vitro potency but exposure levels are increased by a similar factor and the elimination half-lives went from 10-15 minutes to 10-20 hours. The PEGylated analogue was capable of increasing insulin secretion 3 to 4-fold 18h after 0.1 mg/kg had been given subcutaneously in rats. In summary, VPAC2 receptor selective peptides may enhance insulin secretion without malevolent effects on glucagon secretion, reduce plasma lipids in vivo and are efficacious in both normal and diabetic rodents. Combine with the observed reductions in food intake and these compounds could represent a novel class of anti-diabetic compounds.

Abdominal obesity constitutes a high risk factor for insulin resistance and hyperinsulinemia, which are signs of metabolic syndrome (MS). Fat tissue development depends on genetic and environmental factors. We analyzed the effects of a high-sucrose diet on pancreatic beta cell function in young adult Wistar rats; a model without a special susceptibility to become obese. Young adult male Wistar rats were treated with 20% sucrose in drinking water for 8 or 24 weeks. At the end of this period we analyzed parameters that characterize MS; specifically, abdominal obesity, arterial tension, and insulin and triglyceride levels in plasma. We also explored possible mechanisms that lead to an increased insulin secretion.

Increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) in the β-cells triggers insulin exocytosis. Among the Ca2+ channels present in the plasma membrane, the transient receptor potential (TRP) channels receptors are currently of great interest. The mechanisms by which the extracellular adenosine diphosphate ribose (ADPr) increases the [Ca2+]i is unknown. Our aims were to study the roles of the TRP channels in the tolbutamide induced [Ca2+]i increase and to identify the surface receptor that is activated by ADPr. We used S5 cells, a highly differentiated rat insulinoma cell line, as a model for β-cells. Single cell ratiometric microfluorometry was used to measure the [Ca2+]i changes in the Fura-2 loaded cells. Tolbutamide increased [Ca2+]i in the form of oscillations. After tolbutamide increased [Ca2+]i, capsazepine, a potent blocker of the transient receptor potential vanilloid subtype 1 (TRPV1) channel was added to the β-cells, which reduced the tolbutamide-induced [Ca2+]i increase. capsazepine, N-(p-Amylcinnamoyl) anthranilic acid (ACA), TRPM2 channel blocker, and triphenyl phosphine oxide (TPPO), TRPM5 channel blocker were tested for their effect on potassium chloride (KCl) induced [Ca2+]i response. These blockers did not inhibit the KCl induced [Ca2+]i increase. Adenosine diphosphate ribose (ADPr) increased [Ca2+]i in the form of initial transient peak followed by an elevated plateau. Application of ADPr shortly after a prior application and washout of Adenosine diphosphate (ADP) elicited only small [Ca2+]i increase indicating desensitization of the receptor involved. 2´deoxy-N6-methyladenosine 3´5´bis-phosphate (MRS2179), and chloro N6-methyl-(N)-methanocarba 2´deoxyadenosine 3´5´ bis-phosphate (MRS2279), two selective inhibitors of P2Y1 receptor, abolished the ADPr-induced [Ca2+]i increase. Tolbutamide closes ATP sensitive potassium (KATP) channels. Our results demonstrate that besides the closure of the KATP channels, inward cation currents carried by Ca2+ through the TRPV1 channel are necessary for depolarization to the threshold for the activation of the voltage gated calcium channels (VGCC) to increase the [Ca2+]i. Our results also show that ADPr increases [Ca2+]i by activating the P2Y1 receptor.

Pancreatic islet microencapsulation represents an attractive therapeutical approach to type 1 Diabetes mellitus (T1DM), however, maintenance of ß-cell function, which is tightly bound to islet structure, remains a major problem. Employing basic, pre-clinical and clinical research the Cell and Molecular Therapy Center (NUCEL) is aiming at improving the current islet transplantation procedure to render it safer, more effective and applicable to a wider range of patients. Despite significant improvement in islet transplantation after the Edmonton Protocol, T1DM patients are bound to take life-long immunosuppressive drugs, which are closely associated to several side effects. NUCEL and its first spin-off company, CellProtect Biotechnology, developed and patented a new alginate-based biopolymer formulation which is suitable for islet/cell encapsulation. This innovative biopolymer (BioProtect, patent pending), based on the islet dependence on extracellular matrix components, eliminates the need for immunosuppression and allows encapsulated islets to survive for longer periods of time, with significant improvement in beta-cells viability and function, due to the addition of bioactive compounds to the biomaterial formulation. The problem of organ scarcity can also be mitigated, since encapsulation allows the safe use of other beta-cell/islet sources, such as porcine islets and pre-differentiated stem cells. Hyperglycemic streptozotocin-induced DM1 mice received a xenotransplant of Wistar rat islets microencapsulated in BioProtect, achieving normoglycemia in a few days post-surgery. After 200 days 80% of the animals still remained euglycemic, with some of these mice remaining normoglycemic for over 300 days. OGTTs have shown that the BioProtect biomaterial allowed the response to the high glucose challenge to be similar to that observed for non-diabetic animals. Therefore, BioProtect constitutes a potential biomaterial for transplantation of microencapsulated islets into DM1 patients and, possibly, of other cell types for this and other diseases.

A new therapy with first-in-class nuclear import modifier, which averts the loss of beta cell function in NOD mice, has a significant potential for translation (Moore DJ et al. 2010. In vivo islet protection by a nuclear import inhibitor in a mouse model of type 1 diabetes PLoS ONE 5(10): e13235. Doi: 10.1371/journal pone. 0013235.). Our studies have demonstrated that in autoimmune inflammation, such as Type 1 diabetes, in vivo delivery of a nuclear import modifier (NIM), also known as cell-penetrating cSN50 peptide, to non-obese diabetic (NOD) mice protected pancreatic islets from autoimmune inflammation following a 2- day course of intense treatment. Remarkably, this treatment resulted in a diabetes-free state for one year without the need for insulin therapy and the lack of apparent side effects. NIM eliminates islet-attacking autoreactive T and B lymphocytes, restores lost tolerance, and protects islets from death caused by inflammation-driven apoptosis. Whereas NIM restores tolerance to islet antigens in NOD mice for at least one year, its mechanism remains to be elucidated. We hypothesize that NIM-mediated inhibition of pro-inflammatory nuclear signaling augments regulatory T cell (Treg) function by leading to their increased survival, enhanced Treg action, enhanced effector cell sensitivity, and diminished antigen-presenting cells activity and/or Th17 cell generation. Further development of NIM as a lead compound offers a unique opportunity to use it for T1D monotherapy when existing beta cell mass is not completely wiped out. Thus, NIM monotherapy offers high translational value for T1D and, potentially, for T2D as a part of dysregulated metabolic inflammation syndrome.

Aim: We investigated the function of activin receptor like kinase 7 (ALK7) on glucose-stimulated insulin secretion in pancreatic islets. Methods: Batch incubation and perfusion study are employed to evaluate insulin secretion function in isolated pancreatic islets from ALK7 wildtype and knockout mice. Effects of ligands or an inhibitor of ALK7 on insulin secretion are investigated by batch incubation. The expressions of the genes related to glucose sensing mechanism in pancreas were determined by real-time PCR. ATP assay is performed in pancreatic islets. Results: Islets lacking ALK7 or activin B show increased insulin secretion during the second phase of GSIS and increased glucose-mediated ATP generation. Conclusions: Our data suggest that ALK7 plays an important role in regulating the functional plasticity of pancreatic islets.

Introduction: To improve the success of pancreatic islet transplantation, the reestablishment of this environment is necessary to achieve optimal condition for restore and improvement of islet viability before transplantation. The purpose of this study was investigating the effect of four different components of extracellular matrix, on rat islets of langerhans adhesion, morphology, viability and function during in vitro culture. Material and method: Isolated islets from male adult rat were seeded onto the dishes coated with 6.25 mg/cm2 of collagen I, collagen IV, laminin and fibronectin. BSA coated dishes considered as a control. Morphological studies of 72-h attached islets were performed using optical microscopy. Islets were stained with Annexin-V-FITC apoptosis detection Kit after 7 days of culture, and four separated cell population (viable, necrotic, late apoptotic and early apoptotic) detected by flow cytometry analysis. Moreover, cultured Islets were also processed for assessment of insulin secretion by the glucose challenge test (3.3 mM glucose and 16.7 mM glucose) using ELISA. Real time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed in order to evaluate islet specific genes expression (Ins1, Ins2, Gcg, Glut2, Nkx6.1) in the cultured islets for 7 days. Results: The islets attached to all ECMs and this was significantly more in comparison to BSA group. After 72-h culture, Islets in fibronectin and collagen IV lost their spherical shape as a result of migrating cells around the islet and start to exhibit monolayer feature. In contrast, Islets in laminin and collagen I reserve their spherical shape similar to the freshly isolated cells. Additionally, the viability of islets was more on ECM Early apoptotic cell population in fibronectin group showed significant increase among other groups. (n=3, P <0.05). In our study not only, there were no significant differences in mRNA expression of Ins1, Ins2, Gcg, Glut2, Nkx6.1between groups but also none of the groups were responsive to glucose stimulation after7 days. (n=3, P <0.05). Conclusion: According to our results, although laminin, fibronectin, collagen IV and collagen I can preserve Islets viability after 7 days culture, but only ones cultured on laminin and collagen I are able to maintain their spherical shape. Our results suggest that laminin and collagen I can provide suitable ECM components to reach optimum attachment and cell viability for islets 2D culture. This study provides a basis for the future establishment rat pancreatic islet of langerhans of culture with optimum condition. Keywords: Islets of Langerhans, Extracellular matrix, Function, Viability

Background: Single nucleotide polymorphisms (SNPs) in the promoter region of CRYAB gene have been associated with in multiple sclerosis. CRYAB gene encodes alpha-B-crystallin, a member of small heat shock protein, was also reported as a potential autoimmune target. In this study we investigated whether SNPs in the promoter region of CRYAB gene were also important in the etiology of type 1 diabetes. Methods: Genotyping of SNPs in the promoter region of CRYAB gene was performed in a Swedish cohort contains 447 subjects diagnosed as T1D patients and 356 non T1D controls. Three SNPs was included in this study, CRYAB-652 A>G (rs762550), -650 C>G (rs2234702) and -249 C>G (rs14133), the first two SNPs is not included in previous genome wide association studies. Results: CRYAB-650 (rs2234702)*C allele was significantly more frequent in patients than in controls (OR =1.48, Pc=0.03). CRYAB-650*C allele was associated with IAA positivity (OR=8.17, Pc<0.0001) and IA-2A positivity (OR=2.14, Pc=0.005) in T1D patients. This association with IAA was amplified by high-risk HLA carrier state (OR=10.6, P<0.0001). We were not able to find any association between CRYAB-650 and other autoantibody positivity (GADA, IA-2A and ICA). No association was found between the other two SNPs and T1D. Conclusions: In our studied population (Swedish), CRYAB-650 showed association with T1D, CRYAB-650 *C allele conferred to T1D susceptibility. CRYAB-650 was associated with autoantibody (IAA) positivity in T1D patients, especially in those who carried T1D susceptible HLA haplotype.

Insulin secretion from the pancreatic beta cell is controlled by a variety of modulatory pathways and glucose-induced insulin secretion can be amplified by KATP channel independent pathways. These amplification pathways may utilise an increase in intracellular cAMP that acts in PKA-dependent or PKA-independent manner. The whole-cell patch-clamp simultaneously with slow photo-release of caged Ca2+ was used to assess the role of these two cAMP-dependent amplifying pathways in Ca2+ -dependent exocytosis in mouse pancreatic beta cells. In control cells exposed to slow Ca2+ uncaging the membrane capacitance (Cm) increased in a reproducible Ca2+- dependent manner. The Ca2+-dependency of the rate of the initial Cm change followed the saturation kinetics with half-maximal value (EC50) of 2.9 ± 0.2 µM. After clamping the cytosol at 200 µM cAMP or inclusion of a specific PKA activator the Ca2+-dependency of the initial Cm change has been shifted to significantly lower EC50. Alternatively the specific activation of Epac2 increased the rate of exocytosis without changing the sensitivity to [Ca2+]i. Our findings suggest that cAMP modulates the rate of exocytosis in pancreatic beta cells mainly through PKA-dependent pathway by sensitizing the insulin releasing machinery to [Ca2+]i while Epac2 may contribute to enhance the rates of secretory vesicle fusion and regulates the activity of different ion channels as previously reported.

We have studied whether functional TRPV1 channels exist in the INS-1E cells, a cell type used as a model for beta cells, and in primary beta cells from rat and human. The effects of the TRPV1 agonists capsaicin and AM404 on the intracellular free Ca2+ concentration ([Ca2+]i) in INS-1E cells were studied by fura-2 based microfluorometry. Capsaicin increased [Ca2+] i in a concentration-dependent manner, and the [Ca2+] i increase was dependent on extracellular Ca2+. AM404 also increased [Ca2+]i in the INS-1E cells. Capsazepine, a specific antagonist of TRPV1, completely blocked the capsaicin- and AM404-induced [Ca2+]i increases. Capsaicin did not increase [Ca2+]i in primary beta cells from rat and human. Whole cell patch clamp configuration was used to record currents that were inhibited by capsazepine. Western blot analysis detected TRPV1 proteins in the INS-1E cells and the human islets. Immunohistochemistry was used to study the expression of TRPV1, but no TRPV1 protein immunoreactivity was detected in the human islets or the human insulinoma cells. We conclude that the INS-1E cells, but not the primary beta cells, express functional TRPV1 channels.

Objective: L-type calcium channels are essential for pancreatic beta-cell function and they have been well studied in rodents. Here, we set out to determine which L-type channel isoform is operative in human pancreatic beta-cells and how it may be regulated in physiology and disease. Research design and methods: Cav1.3 expression was compared in rat islets, INS1 832/13 cells and human islets using quantitative PCR and microarrays respectively and we assessed Cav1.3 dependent insulin release in human islets. Phenotype/genotype associations of three single nucleotide polymorphisms (SNPs) were investigated in 8987 non-diabetic and 3298 type 2 diabetic subjects from Finland and Sweden. We used RNAi techniques to study the effect of Cav1.3 knockdown on insulin release in INS1 832/13 cells and on exocytosis in human beta-cells. Results: We find Cav1.3 expression being the major L-type isoform in FACS sorted human beta-cells on mRNA level with protein also mainly localizing to beta-cells within islets. Cav1.3 mRNA is less abundant in diabetic islets compared to controls and this reduction in expression coincides with decreased human islet insulin release in vitro. Cav1.3 knockdown reduced glucose stimulated insulin secretion in INS1 832/13 cells and exocytosis in human beta-cells respectively. The C allele of the CACNA1D gene SNP rs312480 was associated with reduced mRNA expression of the channel in vitro and decreased insulin secretion in vivo, whereas rs312486/G and rs9841978/G both associated with type 2 diabetes. Furthermore, we find high glucose L-type calcium channel dependently to stimulate Cav1.3 expression in human islets contributing to increased intracellular calcium levels. Conclusion: The L-type Ca2+ channel Cav1.3 is important for appropriate glucose induced insulin secretion and deregulated expression and common variants in CACNA1D might contribute to type 2 diabetes.