Fig. 3: Excess autophagy in adipocytes promotes age-related hepatic steatosis.

a Immunoblotting to detect Rubicon and p62 in eWAT depots of 3-, 12- and 18-month-old control mice. n = 4 mice. b Relative mRNA expression of Rubicon and p62 in eWAT depots of 3-, 12- and 18-month-old control mice. n = 6 mice. c Ex vivo autophagic flux assay based on LC3-II degradation in the eWAT depots of mice of the indicated ages and genotypes. n = 4 mice. Body weight (d) and eWAT weight (e) of mice of the indicated ages and genotypes. 12-month control, n = 21; 12-month Rubicon^ad−/−, n = 16; 12-month Atg5^ad−/−, n = 19; 12-month Rubicon^ad−/−;Atg5^ad−/−, n = 20; 18-month control; n = 18; 18-month Rubicon^ad−/−, n = 23; 18-month Atg5^ad−/−, n = 18; 18-month Rubicon^ad−/−;Atg5^ad−/−, n = 13. Representative images of H&E staining of eWAT (f) and liver (k) sections from mice of the indicated ages and genotypes. Scale bars, 50 μm. n = 5 mice with similar results. Distribution of adipocyte area in eWAT sections from 12- (g) and 18- (h) month-old mice in (f). n = 5 mice. Quantification of mean adipocyte area is shown in the graphs at right. Liver triglyceride (i) and cholesterol (j) levels in mice of the indicated ages and genotypes. n = 14 mice, except for 3-month-old mice (n = 5). Aged mice were maintained on an NCD. Quantification data are shown in the graphs at the right of each blot. Error bars indicate means ± SEM. Data were analysed by two-tailed Student’s t test (d, e, g, h), one-way ANOVA followed by Tukey’s test (a–c) or Dunnett’s test (i, j). P value from top to bottom and left to right: 0.0014, 0.0004, 0.0079, 0.0081 (a), 0.0039, 0.0237, 0.0753, 0.9927 (b), 0.0365, 0.0136 (c), 0.0432, 0.0293, 0.6662, 0.8681 (d), 0.0362, 0.0157, 0.1353, 0.5337 (e), 0.0047 (g), 0.0093 (h), 0.0077, 0.0148, 0.0430, 0.0125 (i), 0.0145, 0.0009, 0.0242, <0.0001, <0.0001, 0.0007 (j). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. N.S. not significant.