Fig. 5: In vivo Dio3os expression activates brown adipogenesis and thermogenesis, improving energy expenditure and glucose tolerance.

a C57BL/6 J female mice at 7 days of age (n = 5) were injected with adeno-associated virus type 8 (AAV8)-Dio3os-CMV or AAV8-scramble-CMV (1.5 × 10¹¹ vg per mouse) into interscapular brown fat (BAT) at 22 °C. Mice were euthanized 5 weeks post-injection at 22 °C. b, c Body weight (b) and BAT mass (c) after AAV injections (n = 5). d mRNA expression of Dio3os and Dio3 in interscapular BAT at 5 weeks post-injection (n = 5). e T3 concentration (e) in BAT (n = 5). f, g Immunoblotting measuring protein contents of D3, PRDM16, PGC-1a, and UCP-1 in BAT (n = 5). β-tubulin was as a loading control. h H&E staining and immunohistochemical staining of mitochondria (mito-tracker, green) and UCP-1 (red) in BAT (n = 5). Scale bar 150 µm (H&E) and 250 µm (immunostaining). i Heatmap displaying mRNA expression of brown adipogenic and mitochondrial biogenic genes in BAT (n = 5). j PRDM16 immunoprecipitation in measuring PGC-1a and CtBP1 complex in isolated BAT-SVFs. PRDM16, PGC-1a, and CtBP1 were detected by immunoblotting. IgG was used as a negative control (n = 5). k, l Oxygen consumption (k) and energy expenditure (l) of mice at 5-week post AAV injections (n = 5). Metabolic data were regressed to body mass according to guidelines of ANCOVA NIDDK MCCP tool. m, n Mice were exposed to 4 °C and surface body temperature (m) and core body temperature (24 h in cold) (n) (n = 5). Data are presented as mean ± s.e.m. Unpaired Student t test with two-tailed distribution was used for data analyses.