Aims/hypothesis Glucose and incretins regulate beta cell function, gene expression and insulin exocytosis via calcium and cAMP. Prolonged exposure to elevated glucose (also termed glucotoxicity) disturbs calcium homeostasis, but little is known about cAMP signalling. We therefore investigated long-term effects of glucose on this pathway with special regard to the incretin glucagon-like peptide 1 (GLP-1). Methods We exposed INS-1E cells and rat or human islets to different levels of glucose for 3 days and determined functional responses in terms of second messengers (cAMP, Ca 2+ ), transcription profiles, activation of cAMP-responsive element (CRE) and secretion by measuring membrane capacitance. Moreover, we modulated directly the abundance of a calcium-sensitive adenylyl cyclase (ADCY8) and GLP-1 receptor (GLP1R).Results GLP-1-or forskolin-mediated increases in cytosolic calcium, cAMP-levels or insulin secretion were largely reduced in INS-1E cells cultured at elevated glucose (>5.5 mmol/l). Statistical analysis of transcription profiles identified cAMP pathways as major targets regulated by glucose. Quantitative PCR confirmed these findings and unravelled marked downregulation of the calcium-sensitive adenylyl cyclase ADCY8 also in rat and in human islets. Re-expression of ADCY8, but not of the GLP1R, recovered GLP-1 signalling in glucotoxicity in INS-1E cells and in rat islets. Moreover, knockdown of this adenylyl cyclase showed that GLP-1-induced cAMP generation, calcium signalling, activation of the downstream target CRE and direct amplification of exocytosis by cAMP-raising agents (evaluated by capacitance measurement) proceeds via ADCY8. Conclusions/interpretation cAMP-mediated pathways are modelled by glucose, and downregulation of the calcium-B. Roger and J. Papin contributed equally to this study. Electronic supplementary material The online version of this article

a b s t r a c tSynaptotagmins are two C 2 domain-containing transmembrane proteins. The function of calciumsensitive members in the regulation of post-Golgi traffic has been well established whereas little is known about the calcium-insensitive isoforms constituting half of the protein family. Novel binding partners of synaptotagmin 11 were identified in b-cells. A number of them had been assigned previously to ER/Golgi derived-vesicles or linked to RNA synthesis, translation and processing. Whereas the C2A domain interacted with the Q-SNARE Vti1a, the C2B domain of syt11 interacted with the SND1, Ago2 and FMRP, components of the RNA-induced silencing complex (RISC). Binding to SND was direct via its N-terminal tandem repeats. Our data indicate that syt11 may provide a link between gene regulation by microRNAs and membrane traffic. Structured summary of protein interactions:Syt11C2A physically interacts with Vti1a by pull down (View interaction) Syt11C2B physically interacts with SND1, PDIA6, Vti1b, Vti1a, Ago2 and FMRP by pull down (View interaction) syt11C2B binds to SND1 by filter binding (View interaction) Syt11C2B physically interacts with EIF3A, PDIA6, NPM1, EIF3B, NCL, RS3, RS3A, CBR1, ANP32B, LOC683961, SET, SND1, TBB2C, RS10 and RS18 by pull down (View interaction) SND1 physically interacts with Ago2 by anti bait coip (View interaction)

Aims/hypothesis Nutrient homeostasis requires integration of signals generated by glucose metabolism and hormones. Expression of the calcium-stimulated adenylyl cyclase ADCY8 is regulated by glucose and the enzyme is capable of integrating signals from multiple pathways. It may thus have an important role in glucose-induced signalling and glucose homeostasis. Methods We used pharmacological and genetic approaches in beta cells to determine secretion and calcium metabolism. Furthermore, Adcy8 knockout mice were characterised. Results In clonal beta cells, inhibitors of adenylyl cyclases or their downstream targets reduced the glucose-induced increase in cytosolic calcium and insulin secretion. This was reproduced by knock-down of ADCY8, but not of ADCY1. These agents also inhibited glucose-induced increase in cytosolic calcium and electrical activity in primary beta cells and similar effects were observed after ADCY8 knock-down.Moreover, insulin secretion was diminished in islets from Adcy8 knockout mice. These mice were glucose intolerant after oral or intraperitoneal administration of glucose whereas their levels of glucagon-like peptide-1 remained unaltered. Finally, we knocked down ADCY8 in the ventromedial hypothalamus to evaluate the need for ADCY8 in the central regulation of glucose homeostasis. Whereas mice fed a standard diet had normal glucose levels, high-fat diet exacerbated glucose intolerance and knock-down mice were incapable of raising their plasma insulin levels. Finally we confirmed that ADCY8 is expressed in human islets. Conclusions/interpretations Collectively, our findings demonstrate that ADCY8 is required for the physiological activation of glucose-induced signalling pathways in beta cells, for glucose tolerance and for hypothalamic adaptation to a high-fat diet via regulation of islet insulin secretion.Electronic supplementary material The online version of this article (doi:10.1007/s00125-014-3445-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Aims/hypothesis Calcium plays an important role in the process of glucose-induced insulin release in pancreatic beta cells. These cells are equipped with a double system responsible for Ca 2+ extrusion-the Na/Ca exchanger (NCX) and the plasma membrane Ca 2+ -ATPase (PMCA). We have shown that heterozygous inactivation of NCX1 in mice increased glucose-induced insulin release and stimulated beta cell proliferation and mass. In the present study, we examined the effects of heterozygous inactivation of the PMCA on beta cell function. Methods Biological and morphological methods (Ca 2+ imaging, Ca 2+ uptake, glucose metabolism, insulin release and immunohistochemistry) were used to assess beta cell function and proliferation in Pmca2 (also known as Atp2b2) heterozygous mice and control littermates ex vivo. Blood glucose and insulin levels were also measured to assess glucose metabolism in vivo. Results Pmca (isoform 2) heterozygous inactivation increased intracellular Ca 2+ stores and glucose-induced insulin release. Moreover, increased beta cell proliferation, mass, viability and islet size were observed in Pmca2 heterozygous mice. However, no differences in beta cell glucose metabolism, proinsulin immunostaining and insulin content were observed. Conclusions/interpretation The present data indicates that inhibition of Ca 2+ extrusion from the beta cell and its subsequent intracellular accumulation stimulates beta cell function, proliferation and mass. This is in agreement with our previous results observed in mice displaying heterozygous inactivation of NCX, and indicates that inhibition of Ca 2+ extrusion mechanisms by small molecules in beta cells may represent a new approach in the treatment of type 1 and type 2 diabetes.

An important feature of type 2 diabetes is a decrease in β-cell mass. Therefore, it is essential to find new approaches to stimulate β-cell proliferation. We have previously shown that heterozygous inactivation of the Na+/Ca2+ exchanger (isoform 1; NCX1), a protein responsible for Ca2+ extrusion from cells, increases β-cell proliferation, mass, and function in mice. Here, we show that Ncx1 inactivation also increases β-cell proliferation in 2-year-old mice and that NCX1 inhibition in adult mice by four small molecules of the benzoxyphenyl family stimulates β-cell proliferation both in vitro and in vivo. NCX1 inhibition by small interfering RNA or small molecules activates the calcineurin/nuclear factor of activated T cells (NFAT) pathway and inhibits apoptosis induced by the immunosuppressors cyclosporine A (CsA) and tacrolimus in insulin-producing cell. Moreover, NCX1 inhibition increases the expression of β-cell–specific genes, such as Ins1, Ins2, and Pdx1, and inactivates/downregulates the tumor suppressors retinoblastoma protein (pRb) and miR-193a and the cell cycle inhibitor p53. Our data show that Na+/Ca2+ exchange is a druggable target to stimulate β-cell function and proliferation. Specific β-cell inhibition of Na+/Ca2+ exchange by phenoxybenzamyl derivatives may represent an innovative approach to promote β-cell regeneration in diabetes and improve the efficiency of pancreatic islet transplantation for the treatment of the disease.

These observations could be of fundamental importance in the improvement of LA columns/systems engineering and in developing new therapeutic approaches regarding dyslipidemia and associated pathologies such as atherosclerosis and type 2 diabetes.

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