Fig. 7.

Effects of S1P turnover and free fatty acids on mitochondrial network, stress, and metabolic activity in insulin-secreting INS1E cells. INS1E cells were incubated in the absence or presence of PA or OA (500 μM of each) for 24 h. Thereafter, (A) mitochondrial network was analyzed after incubation with Mitotracker Deep Red™, Bars: 5 μm, (B) the expression of proteins important for mitochondrial stress was measured by Western blot, (C) ATP content was measured by ATPlite assay, shown are Means ± SEM from n = 4 independent experiments, each conditions was measured in triplicates. (D) Cert1 protein expression was analyzed by Western blot and (E) cellular distribution and content of ceramide (green) was performed by immunofluorescence in parallel with the MitoTracker Deep Red™ staining (red), Bars: 10 μm. Arrows depict the areas of ceramide and mitochondria colocalization (yellow). Shown are representative pictures of n = 3–5 independent experiments. Densitometry analyses of protein expression normalized to β-actin are shown as Means ± SEM from n = 3–5 independent experiments. ANOVA followed by Bonferroni, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus untreated, #P < 0.05, versus INS1E-ctr cells treated in the same way.