Figure 4. RalA knockout in white adipocytes increases mitochondrial activity and fatty acid oxidation via preventing obesity-induced mitochondrial fission in iWAT.

a, OCR was measured in differentiated primary adipocytes (n = 8). Vertical arrows indicate injection ports of indicated chemicals, b, ¹⁴C-palmitic acid oxidation in differentiated primary adipocytes under basal condition (n = 3–4). ASM: acid-soluble metabolites, c, Representative confocal images (n = 3) of live primary and immortalized adipocytes stained with TMRM (red) and BODIPY (green). Scale bar = 15 μm. d, Representative electron microscope (EM) images of iWAT from CD-fed and HFD-fed Rala^f/f and Rala^AKO mice (n = 3). Red arrow indicates fissed mitochondria; blue arrow indicates elongated mitochondria. Scale bar = 1 μm (CD) or = 500 nm (HFD). e, Representative EM images of WT and RalA KO immortalized adipocytes (n = 3). blue arrow indicates elongated mitochondria; asterisk indicates lipid droplet. Scale bar = 2 μm. f, g, Histogram (f) and violin plot (g) of maximal mitochondrial length in immortalized adipocytes (n = 100–180). Violin plot is presented as violin: 25^th to 75^th percentile, and whiskers: min to max. The data (a, b) are shown as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t-test (b, g), two-way ANOVA alone (f) or with Bonferroni’s multiple comparison as post-test (a).