The adipocytokines leptin, adiponectin, Nampt and NMN have no direct effects on beta-cell survival in INS-1E cells.

<p>INS-1E cells were kept under serum-free conditions 24 h before and during the 48 h experiment. (<b>A,B</b>) INS-1E cells were exposed to cytokines (<b>A</b>: IL-1β, IFN-γ or TNFα) or adipocytokines (<b>B</b>: adiponectin, leptin, Nampt, NMN) at the indicated concentrations for 48 h and cell viability was measured by WST-1 assay. Data are shown as means ±SEM of 3 independent experiments performed in triplicates. Statistical analyses were performed by one-way ANOVA with Bonferroni’s Multiple Comparison Test as posthoc test. <b>C,D</b>: INS-1E cells were exposed to adipocytokines (adiponectin 167 ng/ml, leptin 200 ng/ml, Nampt 2.5 ng/ml, NMN 100 µM) or a cytokine combination (10 ng/ml IL-1β+10 ng/ml IFN-γ) for 48 h. Cytotoxicity (<b>C</b>) was analyzed by measuring the release of adenylate kinase into the supernatant and (<b>D</b>) apoptosis was measured by FITC-annexinV (An) and propidium iodide (PI) staining and subsequent flow cytometric analysis of An-positive and double An/PI positive cells. Results were expressed relative to cells exposed to serum free medium (con) and as means ±SEM of three independent experiments performed in triplicates. <b>E,F</b>: INS-1E cells were exposed to a cytokine combination (IL/IF; 10 ng/ml IL-1β+10 ng/ml IFN-γ) (<b>E</b>) or 0.25 mM palmitate (pal) (<b>F</b>) for 48 h in the absence or presence of the adipocytokines (167 ng/ml adiponectin, 200 ng/ml leptin, 2.5 ng/ml Nampt) and induction of apoptosis was measured by An/PI staining and flow cytometric analysis. Data are shown as means ±SEM of triplicates of three independent experiments. Statistical analyses were performed by student´s t-test. G: INS-1E cells were exposed to the adipocytokines adiponectin (167 ng/ml), leptin (200 ng/ml) or Nampt (2.5 ng/ml) or a combination of camptothecin (2 µM) and etoposide (85 µM; CE, <b>upper and lower panel</b>) or a cytokine combination (10 ng/ml IL-1β+10 ng/ml IFN-γ, <b>middle and lower panel</b>). Western blot analyses were performed for full length and cleaved caspase-3 (<b>upper panel</b>), phospho-NF-κB p65 (Ser536) and NF-κB p65 (<b>middle panel</b>) and phospho-p53 (Ser15) (<b>lower panel</b>). GAPDH or beta-actin were used as loading control. All panels show one typical blot out of three independent experiments. *p<0.05 compared to untreated control.