In the present study, we used a homogenous population of human -cells with the aim to investigate effects and the underlying mechanisms of fatty acids on IAPP and insulin expression and secretion from insulin-producing -cells

In the present study, we used a homogenous population of human -cells with the aim to investigate effects and the underlying mechanisms of fatty acids on IAPP and insulin expression and secretion from insulin-producing -cells. Materials and methods Cell culture and in vitro exposure Human being EndoC-H1 cells were cultured as previously described (22). protein (TXNIP), reduced insulin mRNA manifestation and glucose-induced insulin secretion, as well as increased mRNA manifestation and secretion. Further, these effects were self-employed of fatty acid oxidation, but abolished in response to GPR40 inhibition/downregulation. In human being islets both a high glucose concentration and palmitate advertised improved IAPP mRNA levels, resulting in an augmented IAPP/insulin mRNA percentage. This was paralleled by elevated IAPP/insulin protein secretion and content material ratios. Conclusions Addition of exogenous palmitate to human being -cells improved the IAPP/insulin manifestation ratio, an effect contributed to by activation of GPR40. These findings may be relevant to our understanding of the islet amyloid formation process. studies show that insulin helps prevent IAPP aggregation (18), and it may be that a switch in IAPP/insulin percentage, rather than an increase of IAPP per se, is important for amyloid formation. Amyloidogenic forms of IAPP have BI-9627 been shown to result in Nlrp3 inflammasome activation (19), activating caspase-1-mediated cleavage of pro-IL-1 into adult IL-1 (20). Further, monocyte-derived macrophages from diabetic patients display significantly elevated cleaved caspase-1 and launch of IL-1 following treatment with IAPP (21). Therefore, it is possible that amyloid deposits, promoted by an increased IAPP/insulin percentage, initiate the islet inflammatory reactions observed, BI-9627 which may further deteriorate -cell function. Despite the possible part of IAPP BI-9627 in -cell failure and T2DM, the effects of fatty acids on -cell IAPP manifestation and launch are far from well characterized. In addition, studies Mouse monoclonal to FBLN5 previously carried out possess in many cases utilized rodent -cells/islets. In the present study, we used a homogenous human population of human being -cells with the aim to investigate effects and the underlying mechanisms of fatty acids on IAPP and insulin manifestation and secretion from insulin-producing -cells. Materials and methods Cell tradition and in BI-9627 vitro exposure Human being EndoC-H1 cells were cultured as previously explained (22). Mouse insulinoma (MIN6) cells were cultured in 25?mmol/L glucose DMEM supplemented with 15% FBS. Palmitate (sodium salt, Sigma-Aldrich) exposure media were supplemented with 2% fatty acid free BSA (Roche). During incubations with palmitate serum-free medium was utilized for MIN6 cells. KRBH buffer contained 115?mmol/L NaCl, 24?mmol/L NaHCO3, 5?mmol/L KCl, 1?mmol/L MgCl2, 1?mmol/L CaCl2, 0.2% BSA, and 10?mmol/L HEPES. Human being pancreatic islets were kindly provided by Professor Olle Korsgren (Division of Radiology, Oncology and Clinical Immunology, Uppsala University Hospital, Uppsala, Sweden), through the Uppsala facility for the isolation of human being islets from Scandinavian brain-dead individuals. After isolation, the islets were cultured free-floating in Sterilin dishes in CMRL 1066 medium (ICN Biomedicals, Costa Mesa, CA, USA) comprising 5.6?mmol/L glucose, 10% fetal calf serum, and 2?mmol/L L-glutamine for 1C5 days, and then subsequently transferred to the same tradition conditions as those utilized for palmitate exposure of EndoC-H1 cells. All cells were kept at 37?C inside a humidified atmosphere with 5% CO2. Etomoxir was from Sigma-Aldrich. GPR40 antagonist (GW1100) was from Calbiochem. The PKC inhibitor Bisindolylmaleimide (GF109203X) and the PKD inhibitor CID755673 were from Tocris Bioscience (Bristol, UK). Propidium iodide staining and circulation cytometry A total of 105 EndoC-H1 cells were plated and pre-cultured as explained above in 48-well plates for 24C72?h. The cells were then cultured for numerous time points with or without 1.5?mmol/L palmitate +2% BSA. Cell figures and cell viability were determined by incubation with 5 g/mL propidium iodide for 10?min, followed by trypsinization and circulation cytometry analysis using a FacsCalibur instrument (BD). Hormone secretion to the tradition medium or during batch incubation EndoC-H1 cells were plated BI-9627 at a denseness of 150,000 cells/500 L and cultivated in 48-well plates for 24?h. Cells were then cultured with or without 1.5?mmol/L palmitate, in the presence/absence of 28?mmol/L glucose for an additional 72?h. For analysis of hormone secretion, cells were pre-incubated for 30?min with 0.5?mmol/L glucose KRBH buffer/0.2% BSA, followed by 0.5?mmol/L/15?mmol/L glucose for 2?h. Islets were similarly exposed to 1.5?mmol/L palmitate, in the presence/absence of 28?mmol/L glucose for 72?h. For analysis of hormone secretion, islets were incubated for 30?min with 2?mmol/L glucose KRBH buffer/0.2% BSA, followed by 20?mmol/L glucose +1.5?mM palmitate for 30?min. Buffers and cell lysates were analyzed for insulin and IAPP material using an ultrasensitive human being insulin ELISA (Mercodia). IAPP concentrations were analyzed using a human.