Androgen Receptor is a Negative Regulator of PRDM16 in Beige Adipocyte

Figures

• 

  Figure 1. Establishment of a luciferase‐based PRDM16 reporter knock‐in mouse model. A) Schematic diagram of the strategy for generating PRDM16‐Luc KI mice (KI). B) Relative luciferase activity (RLA) in interscapular BAT. C57BL/6J mice were used as the wild‐type (WT) (n = 6/group). C) Live imaging of luminescence in WT and KI mice. D) Western blot of Luciferase, UCP1, and PRDM16 in WT and KI mice. E–G) RLA (E), qPCR analysis of Luciferase (F), and Prdm16 mRNA expressions (G) in adipose tissues (n = 5/group). H,I) 8‐week‐old male PRDM16‐Luc KI mice were housed at room temperature or cold for 3 days and the luciferase activity (H) and protein levels of UCP1 and PRDM16 (I) in iWAT were measured (n = 5/group). J,K) RLA (J) and western blotting (K) in primary iWAT stromal vascular cells (SVC) from KI mice during the differentiation process (n = 3/group). Data are presented as mean ± SEM; statistical significances between groups were assessed by two‐sided unpaired Student's t‐test (B,H) and one‐way ANOVA (E,F,G,J); *p < 0.05, **p < 0.01.

• 

  Figure 2. Prdm16 is negatively correlated with androgen receptor (Ar) in adipose tissue. A) Schematic diagram of clonal analysis in SVC from iWAT of PRDM16‐Luc KI mice. B) RLA of 64 clonal cell lines. C–F) Prdm16H igh and Prdm16L ow cells were subjected to RNA‐Seq. C) Heatmap showing beige‐related genes. D) Volcano plot of all the transcription factors (TF) (Prdm16H igh versus Prdm16L ow). E) ENCODE and ChEA TF enrichment analysis in significantly downregulated genes. F) Venn diagram of downregulated TFs and enriched consensus TFs in (E). G) mRNA levels of Ar in SVC clones (n = 3/group). H,I) Protein expression of PRDM16 and AR in SVC clones (n = 3/group) (H) and in adipose tissues (I). J) mRNA expression of Ar in adipose tissues (n = 5/group). K) Ar mRNA expression in mouse adipose tissues housed at ambient or cold temperature (n = 5/group). Data are presented as mean ± SEM; statistical significances between groups were assessed by two‐sided unpaired Student's t‐test (G), one‐way ANOVA (J), and two‐way ANOVA (K); *p < 0.05, **p < 0.01.

• 

  Figure 3. Prdm16 expression is sex dimorphic in human and mouse adipose tissue. A,B) Relative PRDM16 (A) and AR (B) mRNA expression in human omental adipose tissue. (male n = 52, female n = 64). C) Pearson correlation plot of AR and PRDM16 mRNA in human omental adipose tissue (n = 116). D,E) PRDM16 mRNA expression in mature adipocytes and adipose tissue precursor cells (ASPC) were extracted from single nuclei sequencing (sn‐Seq) data from human visceral white adipose tissue. D) PRDM16 mRNA expression in adipocytes of human visceral adipose tissue of both sexes (male n = 3, female n = 7). E) PRDM16 mRNA expression in ASPCs of human visceral adipose tissue of both sexes (male n = 3, female n = 7). F) mRNA expression of Prdm16 in iWAT from male and female mice (n = 4/group). G) Representative western blot result of PRDM16 and AR in mouse iWAT of different sexes. Data are presented as mean ± SEM; statistical significances between groups were assessed by two‐sided unpaired Student's t‐test; *p < 0.05, **p < 0.01. ns, not significant. RPM, reads per million mapped reads.

• 

  Figure 4. PRDM16 expression and white adipocyte beiging are inhibited by androgen receptor. A–C) PRDM16‐Luc KI mice were treated with DHT or vehicle for 7 days. A) Luminescence images and B) relative luminescence in mouse adipose tissues (B: n = 5/group). C) qPCR of Ucp1 mRNA expression in adipose tissues (n = 5/group). D,E) Male C57BL/6J mice were treatment with DHT or vehicle and housed at 6 °C for 7 days. D) qPCR of beige markers and E) immunohistochemical staining of PRDM16 in iWAT. Scale bar: 100 µm. F) Prdm16 mRNA expression in iWAT SVCs from Prdm16‐Luc KI mice treated with DHT for 24 h (n = 4/group). G) iWAT SVCs from PRDM16‐Luc KI mice with doxycycline (Dox)‐inducible Ar expression were established. The cells with Dox (AR) or without Dox (ctr) were treated with DHT (1 µm) or vehicle for 24 h before luminescence was determined (n = 3/group). H–M) iWAT SVCs from wild‐type mice were differentiated to beige adipocytes and treated with DHT for 24 h. Relative mRNA levels of Prdm16 (H) and Ucp1 (I) in adipocytes (n = 4/group). J) PRDM16 and UCP1 protein expression in adipocytes. K) Immunofluorescence staining of UCP1 in beige adipocytes. L,M) Oxygen consumption rate (OCR) was measured by…

• 

  Figure 5. Androgen inhibits white adipocyte beiging via suppression of Prdm16 expression. A–C) iWAT SVCs were transfected with a Dox‐inducible Prdm16 cassette (PRDM16) and differentiated to beige adipocytes, followed by treatment with DHT (1 µm) or vehicle for 24 h. A) mRNA expression of Ucp1 (n = 5/group). B,C) OCR in adipocytes (n = 5/group). D,E) iWAT SVCs expressing scrambled shRNA or shRNA targeting Prdm16 were differentiated to beige adipocytes and treated with DHT (1 µm) or vehicle for 24 h. mRNA expression of D) Prdm16 and E) Ucp1 (n = 3/group). F–I) SVCs and mature beige adipocytes were subjected to CUT&Tag analysis for AR. F,G) Heat map illustrating the signal intensity of AR‐binding. The x‐axis represents read densities within 5‐kb region around the peak summit; the y‐axis represents each predicted binding site. H,I) Genome browser tracks showing enrichment of reads in Prdm16 (H) and Ucp1 loci (I). Data are presented as the mean ± SEM; statistical significances between groups were assessed by two‐way ANOVA; *p < 0.05, **p < 0.01.

• 

  Figure 6. Adipocyte‐selective Ar gene knockout enhances WAT beiging in mice. 8‐week‐old male WT and AKO mice were subjected to cold exposure for 3 days. A) OCR in WAT of WT and AKO mice (n = 5/group). B) Representative images of HE staining in iWAT and eWAT. Scale bar: 50 µm. C–F) RNAseq analysis of iWAT and eWAT of WT and AKO mice. C,D) KEGG pathway analysis of the RNAseq data of iWAT (C) and eWAT (D). E,F) Heatmap of beiging genes in iWAT (E) and eWAT (F). G,H) qPCR analysis of the thermogenic genes in iWAT (G) and eWAT (H) (n = 5/group). Data are presented as the mean ± SEM; statistical significances between groups were assessed by two‐way ANOVA (A) and two‐sided unpaired Student's t‐test (G,H); *p < 0.05, **p < 0.01.

• 

  Figure 7. Adipocyte‐selective overexpression of AR blunts WAT beiging in mice. A) Schematic diagram of AR OE mice generation. A CAG promoter‐LoxP‐stop codon‐LoxP‐human AR orf cassette was knocked into the Rosa26 locus. The mice were mated with AdipoQ‐Cre transgenic mice to obtain the adipocyte selective AR overexpressing mice (AR OE). The AR KI mice without Cre were used as the WT control. B) Adipose depots from 8‐weeks‐old male AR OE and WT mice were isolated for validation of AR expression. Western blotting of AR expression in adipose depots of the WT and AR OE mice. C–F) 8‐week‐old male WT and AR OE mice were housed at cold temperature for 3 days. C,D) qPCR analysis of the thermogenic genes in iWAT (C) and eWAT (D) (n = 5/group). E) OCR in WAT (n = 5/group). F) Representative images of HE staining of iWAT and eWAT. Scale bar: 50 µm. Data are presented as the mean ± SEM; statistical significances between groups were assessed by two‐sided unpaired Student's t‐test (C,D) and two‐way ANOVA (E); *p < 0.05, **p < 0.01. G) Hypothetical diagram of the study.