Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice (original) (raw)
Prdm16 is highly expressed in subcutaneous white adipocytes. We originally identified Prdm16 as a transcriptional regulator that is selectively expressed in interscapular BAT (iBAT) versus epidWAT of mice (53). Here we performed a more detailed analysis of Prdm16 expression levels in different depots of WAT from mice housed at a standard temperature (24°C). Importantly, Prdm16 mRNA was enriched by approximately 5-fold in 2 different subcutaneous WAT depots, inguinal WAT (ingWAT) and anterior subcutaneous WAT, relative to its levels in epidWAT and retroperitoneal WAT, 2 prominent intra-abdominal WAT depots in male mice (Figure 1A). The Prdm16 mRNA level in subcutaneous WAT was approximately half that in iBAT. Moreover, Western blot analysis showed that Prdm16 protein was present in ingWAT at about half its level in iBAT, and was not detectable in epidWAT (Figure 1B). Other brown adipocyte–selective (and Prdm16 target) genes, including Ucp1, Cidea, Cox8b, and Elovl3 (Figure 1C and Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI44271DS1), were also substantially enriched in subcutaneous WAT compared with their levels in epidWAT and retroperitoneal WAT. Conversely, Retn, a gene known to be preferentially expressed in white versus brown adipose cells in mice (37, 53, 57), was expressed at reduced levels in subcutaneous WAT compared with epidWAT and retroperitoneal WAT (Supplemental Figure 1A).
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.
WAT is composed of mature adipocytes as well as other cells in what is termed the stromal-vascular (SV) fraction; the SV fraction contains preadipose cells, fibroblasts, immune cells, and blood vessel–associated cells. To determine which cell populations in subcutaneous WAT expresses Prdm16, we measured Prdm16 mRNA levels in fractionated adipose tissues. Prdm16 mRNA was significantly enriched in the mature adipocyte component of ingWAT relative to its levels in the SV fraction. In fact, Prdm16 mRNA levels in subcutaneous adipocytes were similar to that found in mature brown fat cells from iBAT. As expected, Glut4 and Ucp1, genes known to be selectively expressed in mature adipocytes, were coenriched with Prdm16 in the adipocyte fraction relative to the SV fraction (Figure 1D). The relatively high levels of Ucp1 mRNA in the SV component of iBAT was due to the presence of a substantial number of brown adipocytes that do not contain enough lipids to float during the cell fractionation process. Prdm16, Ucp1, and other brown-selective markers were also induced during the in vitro differentiation of preadipocytes isolated from the SV fraction of ingWAT (Figure 1E and Supplemental Figure 1B), which indicates that a brown fat–like gene program is a cell-autonomous feature of subcutaneous adipocytes. Taken together, these results demonstrate that Prdm16 is expressed at very significant levels in mature subcutaneous adipocytes.
Transgenic expression of Prdm16 stimulates brown adipocyte development preferentially in subcutaneous WAT. We previously showed that transgenic expression of Prdm16 in all adipose depots (using the –5 kb aP2 promoter/enhancer; also referred to as Fabp4) caused no morphological or molecular changes in the iBAT or WAT of mice under basal conditions (53). However, we only examined visceral epidWAT in these studies. The depot-selective expression of Prdm16 in subcutaneous WAT (Figure 1) strongly suggested reexamining these mice, especially with regard to the subcutaneous WAT depots. As shown in Figure 2A, 10- to 14-week-old male aP2-Prdm16 transgenic mice fed a regular chow diet and housed at 24°C expressed similar amounts of Prdm16 mRNA in epidWAT, ingWAT, and iBAT. Strikingly, Prdm16 protein was elevated to a much greater extent in the ingWAT and iBAT compared with the epidWAT of these mice (Figure 2B). Ectopic expression of Prdm16 robustly increased the levels of several brown fat–selective genes, such as Ucp1, Cidea, and Ppargc1a, by approximately 5- to 10-fold in ingWAT, but not epidWAT (Figure 2C). In female transgenic mice, Prdm16 increased the levels of brown fat–selective genes in ingWAT, but not in periovarian (gonadal) WAT (Supplemental Figure 2). Importantly, the expression levels of these genes in the male or female iBAT, which was already quite high in WT animals, were not increased in aP2-Prdm16 mice. Furthermore, ectopic expression of Prdm16 did not increase the levels of adipocyte selective genes common to white and brown adipocytes, such as AdipoQ, Fabp4, or Glut4, in any male or female depot examined (Figure 2C and Supplemental Figure 2). Therefore, Prdm16 preferentially induces a brown fat–like gene program in subcutaneous WAT.
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.
The activation of brown adipose–selective genes in the subcutaneous WAT of aP2-Prdm16 animals was accompanied by a profound morphological transformation toward a BAT-like phenotype. Specifically, the ingWAT of aP2-Prdm16, but not WT littermates, contained numerous clusters of Ucp1-expressing, multilocular adipocytes under basal conditions (Figure 3, A–D). However, Ucp1-expressing adipocytes were not detected in the epidWAT from transgenic or WT mice (Figure 3, E–G). Furthermore, we did not observe any discernible morphological changes in the iBAT between transgenic and WT mice (Supplemental Figure 3). Interestingly, aP2-Prdm16 mice ingWAT contained 5 times more tyrosine hydroxylase–positive (TH+), sympathetic parenchymal nerve fibers than that of WT littermates. The increase in sympathetic nerve fibers was depot selective, since we did not observe any change in the number of TH+ parenchymal fibers between the epidWAT of transgenic and WT animals. Together, these results indicate that Prdm16 can drive a full program of brown adipocyte–like development, obvious at both morphological and molecular levels, in subcutaneous WAT under basal conditions.
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.
Protection against high-fat diet–induced weight gain in aP2-Prdm16 mice. In light of the striking effect of Prdm16 action in subcutaneous WAT depots, the transgenic animals were subjected to metabolic analyses in response to both regular chow and high-fat diet. On regular chow, aP2-Prdm16 mice and their WT littermates did not display any notable difference in daily energy balance, but aP2-Prdm16 mice had a slightly increased lean mass (Supplemental Figure 4). During high-fat feeding, aP2-Prdm16 mice gained significantly less weight than their age- and sex-matched WT littermates (Figure 4A). Body composition analyses after 16 weeks of high-fat diet showed that transgenic animals had gained substantially less fat, resulting in a higher lean/fat mass ratio compared with WT animals (Figure 4B). The suppressed weight gain in the aP2-Prdm16 animals was not associated with decreased food intake (Figure 4C). However, we observed higher energy expenditure during both day and night cycles in these mice (Figure 4C). Importantly, energy expenditure was assessed after short-term high-fat feeding (7 days), before the occurrence of changes in body weight or composition that could potentially confound these calculations (58, 59). Physical activity was also comparable between aP2-Prdm16 and WT animals (Supplemental Figure 5). Taken together, these data strongly suggest that the aP2-Prdm16 mice were protected from obesity on a high-fat diet because of a significant increase in energy expenditure not linked to physical activity.
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.
Diet-induced obesity is frequently associated with glucose intolerance and progressive metabolic dysfunction. Notably, aP2-Prdm16 animals displayed a marked increase in glucose tolerance relative to WT animals, assayed after 16 weeks of high-fat feeding (Figure 4D). High-fat–fed aP2-Prdm16 mice were also more insulin sensitive, as determined by insulin tolerance tests (Figure 4E). As was observed in chow-fed animals, Ucp1-expressing fat cells (both multilocular and unilocular) were readily detected in the ingWAT of aP2-Prdm16 mice, but not in WT mice, after 16 weeks of consuming a high-fat diet (Figure 4F). The WAT of high-fat–fed aP2-Prdm16 animals also expressed higher levels of brown fat–selective genes relative to WT mice (Figure 4F). Interestingly, high-fat diet promoted a significant induction in the levels of thermogenic genes in epidWAT of aP2-Prdm16 animals (Figure 4F); this effect was not seen in chow-fed cohorts. Importantly, the levels of these thermogenic genes were not altered by the aP2-Prdm16 transgene in the iBAT, even after 16 weeks of high-fat diet. These results suggest that the elevated energy dissipation in aP2-Prdm16 animals is not due to superactivation of the endogenous classical BAT. Rather, it is more likely to have occurred via the thermogenic program expressed in the remodeled brown-like subcutaneous adipose. Together, these observations demonstrate that increased Prdm16 action in adipose tissue raises energy expenditure to counteract weight gain and drive glucose disposal. These actions appear to occur mainly in the subcutaneous WAT.
Prdm16 is a cell-autonomous regulator of thermogenesis in subcutaneous adipocytes. The transgenic experiments above showed that elevated Prdm16 expression in WAT can drive a program of adaptive thermogenesis in vivo. However, a key question is whether isolated subcutaneous adipocytes require Prdm16 for thermogenic gene expression and respiratory function. To answer this question, we knocked down expression of Prdm16 in primary cultured adipocytes from subcutaneous WAT of WT mice using a shRNA expressed from an adenovirus. Adenoviral vectors expressing a control scrambled sequence or Prdm16 shRNA (sh-Prdm16) were used to infect subconfluent SV cells from ingWAT, which contain a high proportion of adipocyte precursors. These SV cultures were induced to undergo adipogenesis 2 days after adenovirus transduction. As shown in Figure 5A, endogenous Prdm16 mRNA levels were very efficiently decreased by the sh-Prdm16–expressing vector in adipocytes. Moreover, the adenoviral vectors (which also expressed GFP) were taken up and expressed by greater than 90% of adipocytes derived from primary inguinal preadipose cells (Figure 5B). Knockdown of Prdm16 did not interfere with adipogenesis per se, since control and Prdm16-depleted cells underwent a similar extent of morphological differentiation, as shown by Oil-Red-O staining for lipid accumulation (Figure 5C). Control and Prdm16-depleted adipocyte cultures also expressed equivalent levels of general adipocyte-related mRNAs like Fabp4 and AdipoQ (Figure 5C). However, suppression of Prdm16 expression completely blocked the differentiation-linked expression of most brown fat–selective genes, including Ucp1, Cidea, and Cox8b (Figure 5D). This decrease in Prdm16 levels also blunted the activation of thermogenic genes Ucp1 and Ppargc1a in response to the synthetic catecholamine isoproterenol. Western blot analysis indicated that Ucp1 protein expression was almost completely ablated by loss of Prdm16 in both untreated and isoproterenol-stimulated adipocytes (Figure 5E). To determine whether Prdm16 is required for the expression of brown fat–selective genes in mature fat cells or whether it functions during the process of differentiation, we knocked down Prdm16 in adipocytes 7 days after induction of differentiation. Loss of Prdm16 from fully differentiated adipocytes caused a dramatic reduction in the expression of brown fat–related genes like Ucp1, Cidea, Ppargc1a, and Cox8b without affecting the levels of general fat markers Fabp4 and AdipoQ (Supplemental Figure 6). These results indicate that Prdm16 is required in mature subcutaneous fat cells for the full activation of a brown fat–like gene program.
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.
Next, to examine whether Prdm16 is required for the respiratory activity of the isolated primary subcutaneous adipocytes, we measured oxygen consumption in control and Prdm16-depleted cells using a Clark electrode. sh-Prdm16–expressing subcutaneous adipocyte cultures had a significantly lower rate of uncoupled (state IV) respiration under unstimulated conditions. Upon treatment with isoproterenol, control cultures underwent a dramatic increase in uncoupled respiration, whereas adipocytes with reduced Prdm16 had a severely blunted response. Moreover, Prdm16-depleted cells exhibited lower maximal respiration than control cells after treatment with the uncoupler CCCP (Figure 5F). These results demonstrate that Prdm16 stimulates uncoupled respiration in a β-agonist–dependent manner in subcutaneous adipocytes.
Finally, we tested the in vivo requirement for Prdm16 expression in the β-adrenergic–induced activation of a brown fat gene program in WAT. _Prdm16_-deficient mice have a cleft palate and die perinatally, thus precluding analysis of _Prdm16_-deficient WAT (60). We have therefore studied the phenotype of WAT from 3-month-old male WT and Prdm16+/– heterozygous littermates after 3 days of treatment with the β3-selective adrenergic agonist CL316,243. As shown in Figure 5G, there were many fewer clusters of multilocular brown fat–like areas in the ingWAT of Prdm16+/– compared with WT mice. At the molecular level, approximately 50% reduction in Prdm16 mRNA levels causes a significant and specific reduction in the expression of brown fat–selective genes like Ucp1 and Cidea in WAT, especially in the ingWAT depot (Figure 5H). There was a near 8-fold decrease in Ucp1 levels in subcutaneous ingWAT from Prdm16+/– compared with WT animals. Conversely, Retn, a white fat–enriched gene, tended to be increased in the ingWAT of Prdm16+/– relative to WT mice. Interestingly, the expression of BAT-selective genes in the inguinal depot was much more sensitive to Prdm16 dosage compared with iBAT, at least under β3-agonist–stimulated conditions. These results reveal a genetic requirement for Prdm16 in the β3-adrenergic–stimulated induction of a brown fat phenotype within WAT.