Fig 2: Prdm16 is directly targeted by miR-149-3p.(a) Heat map showing the relative expression of miRNAs that changed in the opposite direction in inguinal adipose tissues of fasted and cold exposed mice. Each sample comprised a pool of inguinal adipose tissues from four animals. Each column depicts an individual miRNA. Each row depicts the miRNA expression in fasted or cold exposed samples relative to the expression in control mice. The fold change for the samples is colour-coded according to the key. (b) Relative expression level of miRNA-149-3p normalized to snRNAU6 measured by quantitative real-time PCR in inguinal, epididymal, mesenteric and brown adipose tissue from cold-exposed and fasted male mice, or in inguinal adipose tissue from fasted female mice (n=8). (c) Putative miRNA target sites of miR-149-3p within the 3'-UTR of Prdm16. (d) Bioinformatic prediction of miR-149-3p target sites and free energy values within the 3'-UTRs of mouse and human Prdm16. (e) Relative luciferase activity in HEK293T cells transfected with plasmid reporter constructs containing the 3'-UTR or mutated 3'-UTR of Prdm16, co-transfected with mimic-miR-149-3p (n=6). (f,g) Prdm16 mRNA (f) and protein (g) levels in two-day differentiated inguinal SV cells infected with adenovirus expressing a shRNA targeted to Prdm16 or a scrambled control shRNA (sh-Nc), co-transfected with scramble or miR-149-3p mimic. (h,i) PRDM16 mRNA (h) and protein (i) levels in two-day differentiated inguinal SV cells infected with adenovirus expressing a shRNA targeted to Prdm16 or a scrambled control shRNA (sh-Nc), co-transfected with scramble or miR-149-3p anti-miRs. (j) Relative miR-149-3p expression level in different tissues of mice measured by RT–PCR (n=8). BAT; brown adipose tissue; Epi, epididymal; Ing, inguinal; Mes, mesenteric. The data represent the mean±s.e.m. *P<0.05; **P<0.001; (Student's t-test).

Fig 3: Fasting induces a functional visceral-like switch in ingWAT.(a) Transmission electron microscopy of inguinal adipose tissue in the three groups of mice. Scale bar, 5µm. (b) Mitochondrial numbers in inguinal adipose tissue sections from male mice (n=5). (c) O2 consumption in inguinal, epididymal and mesenteric white adipose tissue from the three groups of mice (n=6). (d) Normalized expression of mitochondrial component genes in inguinal, epididymal and mesenteric adipose tissue in fasted mice compared with that in male control mice (n=8). (e) Normalized thermogenic genes in inguinal adipose tissues from the ad libitum and 24 h-fasted male and female mice (n=8). (f–h) Quantification of UCP1 protein in inguinal subcutaneous adipose tissue from ad libitum-fed and, 24-h-fasted male mice. (f) Representative images of sections from UCP1 immunohistochemistry. Scale bar, 100 µm. (g) Quantification of UCP1+ signals per field (h), and percentage of UCP1+ cells in sections (n=5). (i–k) Analysis of Prdm16 mRNA (n=8) (i) and protein levels (j,k) in the inguinal adipose depots from ad libitum fed and 24 h-fasted male mice. GAPDH was used as an internal control. BAT; brown adipose tissue; Epi, epididymal; Ing, inguinal; Mes, mesenteric. The data represent the mean±s.e.m. *P<0.05; **P<0.001; (Student's t-test).

Fig 4: Prdm16 is not required for maintaining sympathetic parenchymal innervation in mature iWAT.(A) Control (Cre-) and iPrdm16KO (Cre+) mice housed at RT (23°C) were kept on a regular chow diet until 8 weeks of age before being switched to a doxycycline-containing chow diet for 4 weeks. Control and iPrdm16KO mice were either maintained at RT (23°C) or exposed to cold (8°C) for 2 days at the end of doxycycline treatment. (B) Normalized gene expression of inguinal regions from control and iPrdm16KO (adult) mice exposed to RT or cold, n = 3–5. Data are normalized to control RT group, presented as mean + SEM, and analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. * denotes p<0.05 iKO (adult) vs. control samples at RT. # denotes p<0.05 iKO (adult) vs. control samples at cold. & denotes p<0.05 cold- vs. RT-exposed control samples. (C–F) Representative optical sections of iWAT from (C–D) control and (E–F) iPrdm16KO (adult deletion) mice maintained at RT, immunolabeled with TH. (C and E) Images of the dorsolumbar region. (D and F) Images of the inguinal region. (G) High-magnification optical section of the boxed region in (D). (H) High-magnification optical section of the boxed region in (F). Scale bars are indicated. (I–J) Quantification of sympathetic parenchymal innervation in inguinal regions with total neurite length normalized to (I) regional volume or (J) adipocyte density. N = 3 biological replicates per genotype were analyzed. Average neurite density from four to eight randomly selected tissue volumes (technical replicates) contributes to neurite density measurement of one biological sample. * denotes p<0.05 analyzed by Student’s t test.

Fig 5: Depletion of Appbp2 promotes fat thermogenesis.a, The experimental scheme for identifying the substrate receptor for PRDM16. b, Silver-stained gel of immuno-purified Flag-Cul2 complex in inguinal adipocytes. c, Relative mRNA levels of indicated genes in differentiated inguinal adipocytes expressing a scrambled control shRNA (Control) or shRNAs targeting respective substrate receptors. n = 3 per group. d, Relative mRNA levels of Appbp2 in inguinal adipocytes stably expressing a scrambled control shRNA (Control) or two distinct shRNAs targeting Appbp2 (#1, #2). n = 3 per group. e, Oil-Red-O staining of inguinal adipocytes stably expressing a scrambled control shRNA (Control) or shRNAs targeting Appbp2 (#1, #2). Scale bar, 200 µm. f, Relative mRNA levels of indicated genes in inguinal adipocytes stably expressing a scrambled control shRNA (Control) or shRNAs targeting Appbp2 (Upper: sh-Appbp2 #1; Lower: sh-Appbp2 #2) in the presence or absence of forskolin (cAMP). n = 3 per group. g, Relative mRNA levels of indicated genes in control and Appbp2 KD primary inguinal adipocytes. n = 4 per group. h, The protein interaction between APPBP2 and PRDM16. *NS, non-specific band. i, The protein interaction between Cul2 and wild-type (WT) or L13A mutant form of APPBP2. Representative results in b, e, h, i from two independent experiments. Gel source data are presented in Supplementary Fig. 1. c, d, f, g, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by one-way ANOVA followed by Dunnett’s test (c, d) or unpaired Student’s t-test (f, g). Source data