Fig 2: (a) Expression patterns of PRDM16 were responsive to treatment dose. 3 T3-L1 adipocytes were treated with either 0.5% DMSO, 1 µM ROSI or 25–150 µg/ml DBB for 4, 7 or 10 days respectively, followed by PRDM16 primary antibody labelling and FITC-conjugated secondary antibody conjugation. Image acquisition was performed using the IN Cell 2200 Analyzer and all fluorescent images were analysed using the IN Cell Developer software. * denotes statistically significance (ANOVA P < 0.05) as compared to DMSO (vehicle) after 4 days of treatment, ? denotes statistically significance (ANOVA P < 0.05) as compared to the vehicle after 7 days of treatment, and # indicates statistically significance (ANOVA P < 0.05) as compared to the vehicle after 10 days of treatment. (b) Western blot analysis was performed to detect the PRDM16 protein levels in 3 T3-L1 and C2C12 cells treated with 0.5% DMSO, 1 µM ROSI or 50–200 µg/ml DBB for 7 days. Beta actin was served as the loading control. (c) Quantification of PRDM16 protein normalised by beta actin and represented by fold expression

Fig 3: Diagram summaries that maternal obesity impairs thermogenesis and energy expenditure of brown adipose tissue (BAT), predisposing female offspring to obesity and metabolic dysfunctions.Dio3os, a maternally imprinted long-coding RNA, has DNA hypermethylation in its promoter, which has an oocyte origin. The hypermethylation attributes to Dio3os inactivation in offspring BAT, which activates Dio3 and reduces thyroid hormone T3 action, leading to impaired PRDM16 activity and thermogenesis. Mouse BAT receiving AAV vector expressing Dio3os prevents MO female offspring from diet-induced obesity and glucose intolerance, providing a pharmaceutic opportunity to improve the metabolic health of offspring and intervene in a vicious maternal-daughter obesity cycle.

Fig 4: Dio3os activation in BAT improves MO female offspring 4 °C-cold resistance, and energy expenditure and glucose tolerance at 30 °C.a After 4 °C exposure for 4 days, BAT mass in 8-week-old normal and MO female offspring administrated with PBS, AAV8-scramble-CMV or AAV8-Dio3os-CMV viral particle in BAT (1.5 × 1011 vg per mouse, n = 6). b Heatmap displaying mRNA expression of brown thermogenic and mitochondrial biogenic genes in BAT after 4 °C exposure (n = 6). mRNA expression was normalized to 18 S rRNA. c Glucose tolerance of offspring at 4 °C (n = 6). d, e Interscapular surface (d) and rectal body temperature (e) in offspring at 4 °C (n = 6). f–j Normal and MO female neonates were administrated with PBS, AAV-scramble, or AAV-Dio3os in BAT (1.5 × 1011 vg per mouse) and further acclimated to 30 °C for 5-wk (n = 5). f Immunoblotting measuring PRDM16, UCP-1, and PGC-1a proteins in offspring BAT at 30 °C (n = 5). ß-tubulin was used as a loading control. g BAT, inguinal- and gonadal-WAT mass in offspring at 30 °C (n = 5). h Immunostaining of UCP-1 and mito-tracker in BAT, and H&E staining of BAT, inguinal- and gonadal-WAT (n = 5). Scale bar 250 µm in immunostaining and 200 µm in H&E. i Blood glucose concentration after intraperitoneal glucose administration in offspring for glucose tolerance test at 30 °C (n = 5). j, k O2 consumption (j) and energy expenditure (k) in offspring at 30 °C (n = 5). Metabolic data were regressed to body mass according to guidelines of ANCOVA NIDDK MCCP tool. Data are presented as mean ± s.e.m. Two-way ANOVA with Bonferroni post hoc analysis was used in data analysis.

Fig 5: Maternal exercise induces DNA demethylation of the Prdm16 promoter through apelin/a-KG axis.(A) mRNA expression of Prdm16 of fetal BAT from M-Ctrl and M-Ex (n = 6). Expression was normalized by ?Ct values. (B) Diagram showing three regions in the Prdm16 promoter. (C, D, and H) 5mC and 5hmC enrichment fold of fetal BAT (ME study, C; APN study, H) and adult offspring challenged with HFD (D) (n = 6). (E) a-KG to 2-hydroxyglutarate (2-HG) ratio of fetal iBAT from ME or APN study (n = 6). (F, G, I, and J) Z scores of mRNA level of ten-eleven translocation (Tet) and IDH families in the fetal iBAT of APN study (I and J) or ME study (F and G) (n = 6). Expression was normalized by ?Ct values. (K) Working model: DNA demethylation stimulated by a-KG/AMPK/maternal exercise–induced apelin. Data are mean ± SEM, and each dot represents one litter. *P < 0.05, **P < 0.01, and ***P < 0.001 in M-Ctrl versus M-Ex or PBS versus APN by two-tailed Student’s t test (A to J).