Research Paper Volume 7, Issue 10 pp 869—881

Mitochondrial Hormesis links nutrient restriction to improved metabolism in fat cell


Figure 3. Fragmentation and altered functionality occurs in mitochondria of white and beige adipose cells after starvation. (A-C) Mitochondrial fragmentation assessed by analyzing mitochondrial morphology through confocal microscopy in cells transfected with mitochondrial Ds-Red fluorescent protein (A) or by analyzing the content of Drp1, Fis1 and OPA1 through Western blot (B, C). (D) Fragmented mitochondria detected through confocal microscopy after co-staining with Drp1 and TOMM20 antibodies. (E) Mitochondrial fragmentation assessed as described in (B) in crude mitochondria (n=4 mice per group). (F-K) Mitochondrial amount and membrane potential quantified by MitoTracker Green (F, I) and MitoTracker Red CMX-ROS (G, J) by cytofluorimetric analysis, respectively. Polarized mitochondria determined by calculating Red-to-Green ratio (MTR/MTG) (H, K). (L) Polarized mitochondria detected through confocal microscopy after staining with MitoTracker Green and MitoTracker Red CMX-ROS. Co-localization points indicate polarized mitochondria. (M-O) Polarographic recording of oxygen consumption (M) and cheminoluminescent assay of ATP content (N) under NR and 1h after nutrient refill (Re-feed) with complete culture medium. Mitochondrial uncoupling determined by calculating the ratio between O2 consumption and ATP production (O). Bar graphs are expressed as mean ±S.D. (n=3; *p<0.05; **p<0.01 vs CM; °p<0.05). Actin, TOMM20, H2B and catalase staining served as loading controls or for assessing the purity of cell protein fractions. NR: nutrient restriction; CM: complete medium.