This study explored the role of irisin as a new pancreatic β-cell secretagogue and survival factor and its potential role in the communication between skeletal muscle and pancreatic β-cells under lipotoxic conditions. Recombinant irisin stimulated insulin biosynthesis and glucose-stimulated insulin secretion (GSIS) in a PKA-dependent manner and prevented saturated fatty acid-induced apoptosis in human and rat pancreatic β-cells, as well as in human and murine pancreatic islets, via AKT/BCL2 signaling. Treatment of myotubes with 0.5 mmol/L palmitate for 4 h, but not with oleate, promoted an increase in irisin release in the culture medium. Moreover, increased serum levels of irisin were observed in mice fed with a high-fat diet. Mouse serum rich in irisin and the conditioned medium from myotubes exposed to palmitate for 4 h significantly reduced apoptosis of murine pancreatic islets and insulin-secreting INS-1E cells, respectively, and this was abrogated in the presence of an irisin-neutralizing antibody. Finally, in vivo administration of irisin improved GSIS and increased β-cell proliferation. In conclusion, irisin can promote β-cell survival and enhance GSIS and may thus participate in the communication between skeletal muscle and β-cells under conditions of excess saturated fatty acids.

Aims/hypothesis The mechanisms of the protective effects of exendin-4 on NEFA-induced beta cell apoptosis were investigated. Methods The effects of exendin-4 and palmitate were evaluated in human and murine islets, rat insulin-secreting INS-1E cells and murine glucagon-secreting alpha-TC1-6 cells. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting or immunofluorescence, respectively. Small interfering (si)RNAs for Ib1 and Gpr40 were used. Cell apoptosis was quantified by two independent assays. Insulin release was assessed with an insulin ELISA. Results Exposure of human and murine primary islets and INS-1E cells, but not alpha-TC1-6 cells, to exendin-4 inhibited phosphorylation of the stress kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and prevented apoptosis in response to palmitate. Exendin-4 increased the protein content of islet-brain 1 (IB1), an endogenous JNK blocker; however, siRNA-mediated reduction of IB1 did not impair the ability of exendin-4 to inhibit JNK and prevent apoptosis. Exendin-4 reduced G-protein-coupled receptor 40 (GPR40) expression and inhibited palmitateinduced phosphorylation of mitogen-activated kinase kinase (MKK)4 and MKK7. The effects of exendin-4 were abrogated in the presence of the protein kinase A (PKA) inhibitors, H89 and KT5720. Knockdown of GPR40, as well as use of a specific GPR40 antagonist, resulted in diminished palmitateinduced JNK and p38 MAPK phosphorylation and apoptosis. Furthermore, inhibition of JNK and p38 MAPK activity prevented palmitate-induced apoptosis. Conclusions/interpretation Exendin-4 counteracts the proapoptotic effects of palmitate in beta cells by reducing GPR40 expression and inhibiting MKK7-and MKK4-dependent phosphorylation of the stress kinases, JNK and p38 MAPK, in a PKA-dependent manner.

Glucagon-like peptide-1 and its analogs may preserve pancreatic beta-cell mass by promoting resistance to cytokine-mediated apoptosis. The mechanisms of TNFalpha-induced apoptosis and of its inhibition by exendin-4 were investigated in insulin-secreting cells. INS-1 and MIN6 insulinoma cells were exposed to 20 ng/ml TNFalpha, with or without pretreatment with 10 nm exendin-4. Treatment with TNFalpha increased c-Jun N-terminal protein kinase (JNK) phosphorylation 2-fold, reduced inhibitor-kappaBalpha (IkappaBalpha) protein content by 50%, induced opposite changes in caspase-3 and Bcl-2 protein content, and increased cellular apoptosis. Moreover, exposure to TNFalpha resulted in increased serine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-2 and reduced basal and insulin-induced Akt phosphorylation. However, in the presence of a JNK inhibitor, TNFalpha-induced apoptosis was diminished and serine phosphorylation of IRS proteins was prevented. When cells were pretreated with exendin-4, TNFalpha-induced JNK and IRS-1/2 serine phosphorylation was markedly reduced, Akt phosphorylation was increased, caspase-3 and Bcl-2 protein levels were restored to normal, and TNFalpha-induced apoptosis was inhibited by 50%. This was associated with a 2-fold increase in IRS-2 expression levels. A similar ability of exendin-4 to prevent TNFalpha-induced JNK phosphorylation was found in isolated pancreatic human islets. The inhibitory effect of exendin-4 on TNFalpha-induced JNK phosphorylation was abrogated in the presence of the protein kinase A inhibitor H89. In conclusion, JNK activation mediates TNFalpha-induced apoptosis and impairment of the IRS/Akt signaling pathway in insulin-secreting cells. By inhibiting JNK phosphorylation in a PKA-dependent manner, exendin-4 counteracts TNFalpha-mediated apoptosis and reverses the inhibitory events in the IRS/Akt pathway, resulting in promotion of cell survival.

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Aims/hypothesis The role of the redox adaptor protein p66 Shc as a potential mediator of saturated fatty acid (FA)-induced beta cell death was investigated. Methods The effects of the FA palmitate on p66 Shc expression were evaluated in human and murine islets and in rat insulinsecreting INS-1E cells. p66 Shc expression was also measured in islets from mice fed a high-fat diet (HFD) and from human donors with different BMIs. Cell apoptosis was quantified by two independent assays. The role of p66 Shc was investigated using pancreatic islets from p66 Shc−/− mice and in INS-1E cells with knockdown of p66 Shc or overexpression of wildtype and phosphorylation-defective p66 Shc . Production of reactive oxygen species (ROS) was evaluated by the dihydroethidium oxidation method. Results Palmitate induced a selective increase in p66 Shc protein expression and phosphorylation on Ser 36 and augmented apoptosis in human and mouse islets and in INS-1E cells. Inhibiting the tumour suppressor protein p53 prevented both the palmitate-induced increase in p66 Shc expression and beta cell apoptosis. Palmitate-induced apoptosis was abrogated in islets from p66 Shc−/− mice and following p66 Shc knockdown in INS-1E cells; by contrast, overexpression of p66 Shc , but not that of the phosphorylation-defective p66 Shc mutant, enhanced palmitate-induced apoptosis. The pro-apoptotic effects of p66 Shc were dependent upon its c-Jun N-terminal kinasemediated phosphorylation on Ser 36 and associated with generation of ROS. p66 Shc protein expression and function were also elevated in islets from HFD-fed mice and from obese/ overweight cadaveric human donors. Conclusions/interpretation p53-dependent augmentation of p66 Shc expression and function represents a key signalling response contributing to beta cell apoptosis under conditions of lipotoxicity.