Transcriptional control of brown fat determination by PRDM16

Abstract

Brown fat cells are specialized to dissipate energy and can counteract obesity; however, the transcriptional basis of their determination is largely unknown. We show here that the zinc-finger protein PRDM16 is highly enriched in brown fat cells compared to white fat cells. When expressed in white fat cell progenitors, PRDM16 activates a robust brown fat phenotype including induction of PGC-1alpha, UCP1, and type 2 deiodinase (Dio2) expression and a remarkable increase in uncoupled respiration. Transgenic expression of PRDM16 at physiological levels in white fat depots stimulates the formation of brown fat cells. Depletion of PRDM16 through shRNA expression in brown fat cells causes a near total loss of the brown characteristics. PRDM16 activates brown fat cell identity at least in part by simultaneously activating PGC-1alpha and PGC-1beta through direct protein binding. These data indicate that PRDM16 can control the determination of brown fat fate.

Figure 1. PRDM16 is expressed selectively in brown fat cells

(A) Real-time PCR analysis of PRDM16 mRNA expression in BAT relative to WAT; and its expression in the adipocyte fraction (Adip.) compared to the stromal vascular fraction (SV) of BAT (n= 6, mean ± SD). (B) PRDM16 mRNA expression in adipocytes from immortalized BAT cell lines (BAT1 and BAT2) and three WAT cell lines (3T3-F442A, 3T3-L1, C3H-10T1/2) (n=3–5 samples per cell line, mean ± SD). (C) PRDM16 mRNA levels during the differentiation of immortalized BAT cell lines (n=3 for each of 2 separate cell lines, mean ± SD). (D) Northern blot analysis of PRDM16 mRNA and control, 36B4 mRNA, in adult mouse tissues. (E) Expression of PRDM16, UCP1, PGC-1α and adiponectin in BAT after a 4°C cold exposure of mice for 4 hours (n=5 mice per group, mean ± SD) (rt: room temperature). * p < 0.05; ** p < 0.01.

Figure 2. PRDM16 expression induces the gene program of brown fat cells

(A) Oil-red-O staining of mature adipocytes (day 6) from PPARγ-deficient cells expressing retroviral PPARγ2 and either retroviral- PRDM16 or vector control (ctl). These adipocyte cultures were analyzed by real-time PCR for their expression of: differentiation markers common to WAT and BAT (A); brown fat cell-selective genes (as indicated) (B); brown fat- thermogenic genes (UCP1, deiodinase-d2 (dio-2), PGC-1α) with and without cAMP treatment (C); mitochondrial components (as indicated) (D); and white fat cell-selective markers (psat1, serpin3ak, resistin) (E). (n=3, mean ± SD). * p < 0.05; ** p < 0.01

Figure 3. PRDM16 stimulates mitochondrial biogenesis and uncoupled respiration

(A) Representative transmission electron micrographs of fibroblasts and mature adipocytes (day 6) from PPARγ-deficient cells expressing retroviral PPARγ2 and either retroviral- PRDM16 or vector control (ctl). (B) Comparison of mitochondrial volume densities from cells depicted in (A) (n= >20 micrographs per group, mean ± SD). (C, D) Total mitochondrial oxygen consumption and uncoupled respiration in mature adipocytes expressing either PRDM16 or control vector under basal conditions (n=4, mean ± SD) (C), or after stimulation with a cAMP analog for 12 hours (n=4, mean ± SD) (D). L: lipid droplet. * p < 0.05; ** p < 0.01

Figure 4. Differentiation of PRDM16-expressing cells into brown fat in vivo

(A) Immunohistochemistry for cidea protein expression in endogenous WAT and BAT, and in ectopic subcutaneous fat pads formed from fibroblasts expressing PPARγ2 and either vector control (ctl) or PRDM16. (B–D) 8-weeks after transplantation, ectopic fat pads were analyzed by real-time PCR for expression of: PRDM16 (B); differentiation markers common to white and brown fat (PPARγ and adiponectin) (C); brown fat-selective genes (UCP1, cidea, PGC-1α, elovl3, PPAR-α, endogenous PRDM16 (-3′UTR) and the white fat selective marker, resistin (D). (n=10 mice per cell line, mean ± SE). * p < 0.05; ** p < 0.01

Figure 5. PRDM16 activates PGC-1α and PGC-1β via direct binding

(A) The transcriptional activity of the -2 kb region of PGC-1α in response to PRDM16 or vector control expression in brown fat preadipocytes (n=3, mean ± SD). (B) PGC-1α promoter activity in response to PRDM16 or vector expression in PGC-1α-deficient cells (n=3, mean ± SD). (C) The transcriptional activity of Gal4-DNA binding domain (DBD) fusion proteins containing PGC-1α, PGC-1β, or PRC in response to PRDM16 or vector expression (n=3, mean ± SD). (D) Flag-PRDM16 and its associated proteins were immunoprecipitated from brown fat preadipocytes and analyzed by Western blot to detect PGC-1α and PGC-1β (E) GST fusion proteins containing different regions of PGC-1α were incubated with ³⁵S- labeled PRDM16 protein. ERR-α was used to demonstrate binding to the 1–190 and 200–350 regions of PGC-1α. (F) PGC-1α and PRDM16 were co-precipitated from cos7 cells that had been transfected with HA-PGC-1α and either wildtype (WT) or R998Q mutant PRDM16. The input was 2% of the cell lysate used for immunoprecipitation. (G) WT or R998Q mutant PRDM16 were expressed with PPARγ2 in PPARγ^−/− fibroblasts. After differentiation into adipocytes (day 6), real-time PCR was used to measure the mRNA expression of: brown fat-selective genes (as indicated); and resistin, a white fat selective gene (n=3, mean ± SD). * p < 0.05; ** p < 0.01

Figure 6. Knockdown of PRDM16 in brown fat cells ablates their brown fat characteristics

(A) PRDM16 mRNA levels in immortalized brown fat cells expressing shRNA targeted to PRDM16 or a scrambled (SCR) control- shRNA before (day 0) and after their differentiation (day 5) into adipocytes. (B) Gene expression in brown fat cells (day 5) expressing sh-PRDM16 or sh-SCR including: markers common to white and brown fat cells (aP2, PPARγ, adiponectin) and resistin, a white fat cell selective gene. (C) The differentiation-linked mRNA induction (day 0 to day 5) of brown fat- selective genes (as indicated) in sh-PRDM16 and sh-SCR expressing cells. (D–F) Gene expression in adipocytes (day 6) from sh-PRDM16 and sh-SCR expressing primary brown preadipocytes including mRNA levels of: PRDM16 (D); adiponectin, PPARγ and resistin (E); brown fat- selective genes (as indicated) (F). (G) Western blot analysis of UCP1 protein levels in primary brown fat cells expressing sh-PRDM16 or sh-SCR control with and without cAMP treatment. (H) mRNA levels of various mitochondrial components in adipocytes from sh-PRDM16 and sh-SCR expressing primary brown preadipocytes. (n= 3–5, mean ± SD). * p < 0.05; ** p < 0.01

Figure 7. Transgenic expression of PRDM16 in WAT depots induces the formation of BAT cells

(A) The fat-specific aP2 promoter/enhancer was used to express PRDM16 in WAT depots. Western blot analysis for PRDM16 protein expression in: non-transgenic, wildtype (wt) BAT; wt WAT; and WAT from two strains of aP2-PRDM16 transgenic mice (aP2-T1 and aP2-T2). POL-II protein expression was used to control for loading. (B) Expression of BAT-selective genes (as indicated) and resistin in WAT from wildtype (wt) and aP2-T1 transgenic mice. This gene set was also measured in WAT from wt, aP2-T1 and aP2-T2 mice that had been treated with CL 316, 243 (n= 7–10 mice per group, mean ± SE). (C) Immunohistochemistry for UCP1 protein (brown stain) in sections of WAT from wt and transgenic mice (T1 and T2) after treatment with CL 316, 243. * p < 0.05; ** p < 0.01