Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice

Abstract

The white adipose organ is composed of both subcutaneous and several intra-abdominal depots. Excess abdominal adiposity is a major risk factor for metabolic disease in rodents and humans, while expansion of subcutaneous fat does not carry the same risks. Brown adipose produces heat as a defense against hypothermia and obesity, and the appearance of brown-like adipocytes within white adipose tissue depots is associated with improved metabolic phenotypes. Thus, understanding the differences in cell biology and function of these different adipose cell types and depots may be critical to the development of new therapies for metabolic disease. Here, we found that Prdm16, a brown adipose determination factor, is selectively expressed in subcutaneous white adipocytes relative to other white fat depots in mice. Transgenic expression of Prdm16 in fat tissue robustly induced the development of brown-like adipocytes in subcutaneous, but not epididymal, adipose depots. Prdm16 transgenic mice displayed increased energy expenditure, limited weight gain, and improved glucose tolerance in response to a high-fat diet. shRNA-mediated depletion of Prdm16 in isolated subcutaneous adipocytes caused a sharp decrease in the expression of thermogenic genes and a reduction in uncoupled cellular respiration. Finally, Prdm16 haploinsufficiency reduced the brown fat phenotype in white adipose tissue stimulated by β-adrenergic agonists. These results demonstrate that Prdm16 is a cell-autonomous determinant of a brown fat-like gene program and thermogenesis in subcutaneous adipose tissues.

Figure 1. Expression of Prdm16 and a brown fat–like gene program in subcutaneous adipocytes.

(A and B) Analysis of Prdm16 mRNA (A) and protein levels (B) in the indicated adipose depots from 12-week-old male WT mice. epid, epidWAT; RP, retroperitoneal WAT; ant SC, anterior (forelimb level) subcutaneous WAT; ing, ingWAT (subcutaneous). (C) mRNA levels of brown fat–selective (and Prdm16 target) genes (Ucp1, Cidea, and Cox8b) in adipose depots from A. (D) mRNA levels of Prdm16, Glut4 (mature adipocyte marker), and Ucp1 (brown adipocyte selective) in the SV and adipocyte fractions of epidWAT, ingWAT, and iBAT. (E) Prdm16 and Ucp1 mRNA levels during the in vitro differentiation of primary preadipocytes (from the SV fraction) of ingWAT. Values are mean ± SD (n = 4–6 mice per group). *P < 0.05, **P < 0.01.

Figure 2. Transgenic expression of Prdm16 induces a thermogenic gene program in subcutaneous WAT.

(A–C) The aP2 promoter/enhancer was used to drive ectopic Prdm16 expression in all adipose depots. (A) Real-time PCR analysis of Prdm16 mRNA levels in the epidWAT, ingWAT, and iBAT of 10- to 14-week-old male WT and aP2-Prdm16 mice fed a regular chow diet. (B) Western blot analysis of Prdm16 protein levels in adipose depots from mice in A. Pol-II was used as a loading control. (C) mRNA levels of brown fat–selective genes (Ucp1, Cidea, and Ppargc1a) and general adipocyte markers (AdipoQ, Fabp4, and Glut4) in the indicated adipose depots from WT and aP2-Prdm16 mice. Values are mean ± SEM (n = 8 mice per group). **P < 0.01 vs. WT.

Figure 3. Prdm16 stimulates the development of brown-like adipocytes in subcutaneous WAT.

(A–G) Immunohistochemistry for Ucp1 protein (brown stain) in sections of ingWAT (A–D) and epidWAT (E–G) from 10- to 14-week-old male WT (A and E) and aP2-Prdm16 (B–D, F, and G) mice. (D) High-magnification and representative image of ingWAT from a transgenic animal. Arrowheads depict Ucp1 immunopositive cells that have a unilocular morphology typical of white adipocytes. (H and I) Immunohistochemistry for TH protein in samples of ingWAT from WT (H) and aP2-Prdm16 (I) animals as described above. Original magnification, ×20 (A–C and E–G); ×100 (D, H, and I). (J) Quantification of TH-expressing nerve fibers in WT and transgenic ingWAT. Values are mean ± SEM (n = 20 fields per sample in each of 3 animals per group). **P < 0.01 vs. WT.

Figure 4. aP2-Prdm16 mice are protected from obesity and metabolic dysfunction upon high-fat feeding.

(A) Body weights of WT and aP2-Prdm16 mice during 10-week time course of high-fat feeding. (B) MRI was used to analyze body composition (fat and lean mass) in WT and aP2-Prdm16 mice after 16 weeks of high-fat diet. (C) Energy expenditure and food intake was measured for 72 hours in individually housed WT and aP2-Prdm16 mice after 1 week of high-fat diet. Energy expenditure is reported as VO₂/mouse/hour. (D) Glucose tolerance test. Blood glucose levels were measured in 16-week high-fat diet–fed WT or aP2-Prdm16 mice after an overnight fast (time 0) and at the indicated times after intraperitoneal injection of glucose. (E) Insulin tolerance test. Blood glucose levels were measured after an overnight fast (time 0) and at the indicated times after an intraperitoneal injection of insulin in mice from D. (F) Immunohistochemistry for Ucp1 (brown stain) protein in ingWAT from WT and aP2-Prdm16 mice after 16 weeks of high-fat diet. The boxed region is shown at higher magnification at right. Ucp1-expressing multilocular and unilocular fat cells are indicated by the arrow and arrowhead, respectively. Original magnification, ×20 (left and middle); ×100 (right). mRNA levels of brown fat–selective genes (Ucp1 and Cidea) were determined in epidWAT, ingWAT, and iBAT from WT and aP2-Prdm16 animals after 16 weeks of high-fat diet. Significance between curves was determined by 2-way ANOVA. Values are mean ± SEM (n = 16 mice per group per experiment). *P < 0.05, **P < 0.01 vs. WT.

Figure 5. Prdm16 is required for expression of a thermogenic gene program in subcutaneous adipocytes.

(A–G) Subcutaneous preadipocytes in the SV fraction of inguinal fat from WT mice were transduced with adenovirus expressing a shRNA targeted to Prdm16 or a scrambled control shRNA (ctl). These cultures were then induced to differentiate in vitro into adipocytes. (A) Prdm16 mRNA levels (with and without isoproterenol stimulation, as indicated). (B) GFP was expressed from adenoviral shRNA vectors, and its expression was used to reveal control shRNA– and sh-Prdm16–transduced adipocytes. (C) Oil-Red-O staining (red) for lipid accumulation. mRNA levels of general adipocyte markers (Fabp4 and AdipoQ) were also determined. (D) mRNA levels of brown fat–selective genes (Ucp1, Cidea, Cox8b, and Ppargc1a). (E) Western blot analysis for Prdm16 and Ucp1 protein. (F) Oxygen consumption, assayed using a Clark-type electrode. Oligomycin (ATPase inhibitor) and CCCP (chemical uncoupler) were added to cells to measure the rates of uncoupled and maximal respiration, respectively. (G and H) WT Prdm16^+/+ and heterozygous Prdm16^+/– littermates were treated with CL316,243 for 3 days. (G) H&E staining of inguinal adipose tissue. (H) mRNA levels of Prdm16, brown fat–selective genes Ucp1 and Cidea, and Retn in epidWAT, ingWAT, and iBAT. Original magnification, ×20 (B, C, and G). Values are mean ± SD (n = 3–5). *P < 0.05; **P < 0.01.