EBF2 promotes the recruitment of beige adipocytes in white adipose tissue

Abstract

Objective: The induction of beige/brite adipose cells in white adipose tissue (WAT) is associated with protection against high fat diet-induced obesity and insulin resistance in animals. The helix-loop-helix transcription factor Early B-Cell Factor-2 (EBF2) regulates brown adipose tissue development. Here, we asked if EBF2 regulates beige fat cell biogenesis and protects animals against obesity.

Methods: In addition to primary cell culture studies, we used ​Ebf2 knockout mice and mice overexpressing EBF2 in the adipose tissue to study the necessity and sufficiency of EBF2 to induce beiging in vivo.

Results: We found that EBF2 is required for beige adipocyte development in mice. Subcutaneous WAT or primary adipose cell cultures from Ebf2 knockout mice did not induce Uncoupling Protein 1 (UCP1) or a thermogenic program following adrenergic stimulation. Conversely, over-expression of EBF2 in adipocyte cultures induced UCP1 expression and a brown-like/beige fat-selective differentiation program. Transgenic expression of Ebf2 in adipose tissues robustly stimulated beige adipocyte development in the WAT of mice, even while housed at thermoneutrality. EBF2 overexpression was sufficient to increase mitochondrial function in WAT and protect animals against high fat diet-induced weight gain.

Conclusions: Taken together, our results demonstrate that EBF2 controls the beiging process and suggest that activation of EBF2 in WAT could be used to reduce obesity.

Keywords: BAT, brown adipose tissue; Beige fat; Brown fat; EBF2; HFD, high fat diet; Obesity; RT, room temperature; TG, transgenic; TN, thermoneutrality; Thermogenesis; WAT, white adipose tissue; WT, wild type; epiWAT, epididymal white adipose tissue; ingWAT, inguinal white adipose tissue.

Graphical abstract

Figure 1

Ebf2 is required for in vivo beiging of ingWAT. (A) Confocal immunofluorescence imaging of whole-mount ingWAT tissue. EBF2 protein is present in wild type (WT) but not Ebf2 KO ingWAT. (B) H&E staining of ingWAT from WT and Ebf2 KO mice before (left panels) or after (right panels) injection with CL-316,243 to induce beiging. (C) Confocal immunofluorescence imaging of whole-mount ingWAT tissue. UCP1 protein is highly expressed in WT but not Ebf2 KO IngWAT following CL 316,243 injection. (D–G) Relative mRNA levels from in WT or Ebf2 knockout ingWAT before and after CL 316,243 injection as measured by RT-qPCR. Levels of (D)Ebf2, (E) the general adipose genes Pparϒ, AdipoQ and Fabp4, (F) the brown selective genes Ucp1, Cidea, Pgc1α, Pparα and Prdm16, and (G) the mitochondrial genes Cox7a1, Cox8b, Cycs. (n = 3). (H) Relative oxygen consumption of ingWAT from WT and Ebf2 KO mice. (n = 4). P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars represent SEM. Scale bars = 10 microns.

Figure 2

Ebf2 is required for beiging. (A) Relative EBF2 protein levels from WT primary ingWAT stromal vascular cells plus or minus rosi treatment as assayed by western blot. (B) Relative Ebf2 mRNA levels from WT or Ebf2 KO primary ingWAT stromal vascular cells before and after rosi treatment as measured by RT-qPCR. (C) Oil Red O staining of differentiated and rosi treated WT or Ebf2 KO primary ingWAT stromal vascular cells. (D–G) Relative mRNA levels from WT or Ebf2 KO primary ingWAT stromal vascular cells plus or minus rosi treatment as measured by RT-qPCR. Levels of (D) the general adipose genes Pparϒ and Fabp4, (E) the brown selective genes Ucp1 and Cidea, (F) the brown selective genes Pgc1α, Pparα and Prdm16, and (G) the mitochondrial genes Cox5b, Cox7a1, Cox8b. (H) Western blot to measure protein levels of UCP1 in differentiated and rosi treated WT or Ebf2 knockout primary ingWAT stromal vascular cells. NS = non-specific band loading control. (I) Relative Ucp1 and Pgc1α mRNA levels from WT or Ebf2 knockout primary ingWAT stromal vascular cells before and after Iso treatment as measured by RT-qPCR. P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars represent standard deviation.

Figure 3

EBF2-expression drives a brown fat/thermogenic profile in primary adipocytes. (A–C) Relative mRNA levels from control or retroviral EBF2-expressing primary ingWAT stromal vascular cells with or without rosi treatment as measured by RT-qPCR. Levels of (A) Ebf2, (B) the general adipose genes Pparϒ, AdipoQ and Fabp4, and (C) the brown selective genes Ucp1, Cidea, Pgc1α, Pparα and Prdm16, and (D) the mitochondrial genes Cox7a1, Cox8b, Cycs. (E) Western blot to measure protein levels of EBF2 and UCP1 levels in control or retroviral EBF2-expressing primary ingWAT stromal vascular cells following rosi treatment. P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars represent standard deviation.

Figure 4

Ectopic ingWAT expression of EBF2 induces beiging in vivo. (A) qPCR of relative Ebf2 mRNA levels from WT (white bar) and Fabp4-Ebf2 (black bar) mice in BAT, ingWAT and epiWAT (mice housed at room temperature). (n = 3). (B) Western blot to measure EBF2 protein levels in WT or Fabp4-Ebf2 (TG) mice in ingWAT, BAT and epiWAT (mice housed at room temperature). (C) H&E staining of ingWAT from WT and Fabp4-Ebf2 mice. (D) Confocal immunofluorescence imaging of UCP1 protein levels in ingWAT from WT or Fabp4-Ebf2 mice. (E–G)) Relative mRNA levels from WT and Fabp4-Ebf2 ingWAT as measured by RT-qPCR. Levels of (E) the general adipose genes Pparϒ, AdipoQ and Fabp4, (F) the brown selective genes Cidea, Pgc1α, Pparα and Prdm16 and (G) mitochondrial genes Cox7a1, Cox8b, Cycs. (H) Heat map of the top up-regulated and down-regulated genes in Fabp4-Ebf2 ingWAT compared to WT, with corresponding GO analysis. (I) Cross comparison between RNAseq data comparing WT and Fabp4-Ebf2 ingWAT gene expression and microarray comparing thermoneutral and cold exposed ingWAT. The majority of overlapping upregulated genes are related to mitochondrial function. (J) Relative UCP1 mRNA levels of WT (white bars) and Fabp4-Ebf2 (black bars) ingWAT from mice housed at thermoneutrality (TN, 30 °C) or room temperature (RT) as measured by RT-qPCR. (K) Relative oxygen consumption of ingWAT from WT and TG Fabp4-Ebf2 mice housed at room temperature, n = 4. (L) Body weight of wild type (square) and TG Fabp4-Ebf2 (circle) for mice housed at thermoneutrality on HFD beginning at 5 weeks of age. (n = 5). (M) Fat mass and lean weight mass of WT or TG Fabp4-Ebf2 (n = 5) mice after 25 weeks on HFD at TN. (N) Gross histology of ingWAT fat pads from WT or TG Fabp4-Ebf2 mice after 25 weeks on HFD at TN. P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars denote SEM. Scale bars = 10 microns.