Ablation of PPARγ in subcutaneous fat exacerbates age‐associated obesity and metabolic decline

Summary It is well established that aging is associated with metabolic dysfunction such as increased adiposity and impaired energy dissipation; however, the transcriptional mechanisms regulating energy balance during late life stages have not yet been fully elucidated. Here, we show that ablation of the nuclear receptor PPARγ specifically in inguinal fat tissue in aging mice is associated with increased fat tissue expansion and insulin resistance. These metabolic effects are accompanied by decreased thermogenesis, reduced levels of brown fat genes, and browning of subcutaneous adipose tissue. Comparative studies of the effects of PPARγ downregulation in young and mid‐aged mice demonstrate a preferential regulation of brown fat gene programs in inguinal fat in an age‐dependent manner. In conclusion, our study uncovers an essential role for PPARγ in maintaining energy expenditure during the aging process and suggests the possibility of targeting PPARγ to counteract age‐associated metabolic dysfunction.

cells in adult subjects in supraclavicular areas and the demonstration of their contribution to energy expenditure (Jespersen et al., 2013;Sharp et al., 2012;Wu et al., 2012), beige fat has become a potential target for the treatment of obesity and metabolic dysfunction.
In parallel with the increase in life expectancy observed over the last century (Christensen, Doblhammer, Rau & Vaupel, 2009), the incidence of obesity and metabolic dysfunction has also risen in the older population (Villareal, Apovian, Kushner & Klein, 2005). It has been shown recently that in addition to a decrease in BAT activity during the aging process, browning and beige fat cell function also decline late in life, potentially contributing to age-associated metabolic dysfunction (Nedergaard, Bengtsson & Cannon, 2010;Rogers, Landa, Park & Smith, 2012). Thus, the identification of molecular mechanisms involved in the acquisition of brown-like features in white fat during the aging process becomes an important step toward the development of treatments to counteract obesity and its metabolic consequences arising at late life stages.
The peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors is involved in the control of lipid and glucose homeostasis. PPARc, one of the members of this subfamily, is required for the development of all types of fat cells and functions as a regulator of both white and brown gene programs in adipocytes (Lefterova, Haakonsson, Lazar & Mandrup, 2014;Tontonoz & Spiegelman, 2008). Ectopic expression of PPARc in fibroblasts in vitro has been shown to drive adipogenesis (Mueller et al., 2002;Tontonoz, Hu & Spiegelman, 1994), and its selective ablation in fat leads to reduced adipose tissue mass and lipodystrophy (He et al., 2003;Jones et al., 2005;Wang, Mullican, DiSpirito, Peed & Lazar, 2013). In addition to the well-documented role in coordinating gene expression programs of adipocyte differentiation and lipid storage, PPARc has also been shown to directly bind to PPAR response element in promoters of brown fat-selective genes, such as UCP1, Cidea, and Elovl3, and to induce them transcriptionally (Sears, MacGinnitie, Kovacs & Graves, 1996;Viswakarma et al., 2007;Kobayashi & Fujimori, 2012). Furthermore, its ligands, such as the antidiabetic drug rosiglitazone, have been shown to modulate brown remodeling of white adipose tissues in rodents and to increase energy expenditure, indicating a critical role of PPARc in the browning process (Ohno, Shinoda, Spiegelman & Kajimura, 2012;Petrovic et al., 2009;Wilson-Fritch et al., 2004). Genomewide binding studies have recently permitted the identification of PPARc targets common to both white and brown fat tissues, as well as depot-selective ones (Siersbaek et al., 2012). Given the unique capability of iWAT to adapt to different metabolic states by inducing either white or brown-like gene programs, it is relevant to determine the contribution of PPARc to the execution of each of these opposing functions in iWAT during the aging process.
Recent studies using genetic mouse models have revealed the importance of browning of iWAT in protecting from obesity and its metabolic consequences (Harms & Seale, 2013). In particular, it has been shown that adipose tissue ablation of regulators involved in thermogenic programs, such as PRDM16 or PGC1a, affects iWAT function and leads to diet-induced obesity and metabolic alterations (Cohen et al., 2014;Kleiner et al., 2012). However, the analysis of the specific function of these regulators selectively in subcutaneous fat in vivo has been hindered by the lack of genetic methods to achieve iWAT-specific deletion. Previously reported animal models carrying PPARc ablation in every fat tissue generated by crossing PPARc-LoxP mice with either aP2-or adiponectin-Cre mice revealed impaired fat development and reduced fat mass (He et al., 2003;Jones et al., 2005;Wang et al., 2013). Given that none of the existing Cre-LoxP systems can permit the ablation of PPARc selectively in subcutaneous fat during late stages of life, it remains to be determined whether PPARc can specifically affect browning of subcutaneous tissue during the aging process.
Here, we achieved specific ablation or downregulation of PPARc In addition, to achieve knockdown of PPARc, we injected control or PPARc shRNA adenovirus into 12-month-old C57BL/6J mice (Figure 1a). As shown in Figure 1b Interestingly, we found no statistical difference in the levels of PPARc mRNA in the total population of macrophages present in iWAT after injection of sh control or shPPARc adenovirus (Figure 1d). To assess the differential efficiency in transduction of adenovirus in adipocytes and macrophages, we performed immunostaining using GFP and F4/80 antibodies in Ad-GFP-transduced iWAT and analyzed macrophages with single or double staining by confocal imaging (Fig. S1). The results of this analysis demonstrate that while adipocytes are transduced at a high percentage by GFP adenovirus (almost 100%), only a fraction (35.6 AE 14.7%) of macrophages present in fat tissue are also infected with GFP.

| Decreased energy expenditure and brown fat gene expression in mice with PPARc deficiency in iWAT
We next compared the metabolic performance of control mice with that of mice with PPARc deficiency in iWAT. As shown in Figure 4a

| DISCUSSION
It has long been noted that during aging, obesity and metabolic dysfunction often ensues; however, to date, the transcriptional switches turned on during the aging process and responsible for the metabolic changes observed late in life have not yet been fully characterized.
Given the importance of PPARc in fat tissue biology, in this study we sought to determine the role of PPARc in aging-associated metabolic decline. Through the use of two adenoviral-based in vivo methodologies, we have provided for the first time evidence to support a novel and critical requirement of PPARc for the maintenance of browning programs in subcutaneous tissue during aging. Given the recent demonstration that beige fat cells interspersed in inguinal fat tissue expend energy via creatine metabolism (Kazak et al., 2015), it is of interest to assess whether the effects of PPARc reported here involve alternative futile cycles in addition to classical thermogenic pathways.
The results of our studies showing that PPARc deficiency selectively in subcutaneous fat during aging is associated with increased adiposity are surprising given that they are sharply in contrast with the lipodystrophic phenotype and the impairment in adipose tissue expansion previously reported in aP2-and adiponectin-driven fatspecific PPARc KO mice (He et al., 2003;Jones et al., 2005;Wang et al., 2013) and in young mice with decreased PPARc levels selectively in iWAT ( Figure 5). The striking dissimilarity between the effects of PPARc ablation on fat tissue reported in published aP2and adiponectin-driven knockout models and in our study of young mice may be due to the differences in the spatiotemporal conditions of PPARc ablation, given that PPARc deletion was previously achieved in every fat depot during development and in adult mice (He et al., 2003;Jones et al., 2005;Wang et al., 2013), while here PPARc levels are selectively reduced in subcutaneous fat tissue in mid-aged mice. It is conceivable that the animal model tested here may have allowed the specification of the select fat depot and life stage in which one of the two opposed PPARc functions, adipogenic and thermogenic, is predominant. Given that it has been recently shown that PPARc gene target selection is dictated by depot-selective coregulators, such as TLE3 and Prdm16, which can specify alternative programs of lipid storage or thermogenesis (Koppen & Kalkhoven, 2010;Peirce, Carobbio & Vidal-Puig, 2014;Villanueva et al., 2013), it can be envisioned that preponderance of one type of cofactor in an aging tissue may drive PPARc to activate only select gene targets. Future studies will determine whether the preferential binding of PPARc to brown fat gene promoters we observed in 12month-old mice is driven by differential amounts of brown versus white cofactors present in subcutaneous adipose tissues in aging mice.
It is also plausible that posttranslational modifications in PPARc occurring specifically in aging could modify PPARc target gene promoter binding choices by potentially altering PPARc affinity for specific cofactors, given that it has been previously demonstrated F I G U R E 5 PPARc deficiency in iWAT of young and aging mice leads to distinct adiposity phenotypes. (a) Illustration of the strategy used to reduce PPARc levels in the iWAT of young and aging mice via adenoviral delivery. (b) PPARc mRNA levels; (c) weight of iWAT; (d) representative H&E images of iWAT and (e) adipocyte area in 2-month-old and 12month-old mice injected with shRNA control (shCon) or shPPARc (shPPARc) adenoviruses. Data are presented as mean AE SEM and *, p < .05; **, p < .01 that the recruitment of brown fat coactivators can be modulated by the PPARc acetylation status in young mice (Qiang et al., 2012) and that phosphorylation of PPARc promotes the interaction with specific coregulators (Choi et al., 2014). Here, we show for the first time that the phospho-status of PPARc is modified during the aging process; whether phosphorylation at serine 273 in PPARc may direct the selective expression of target genes in an age-specific manner remains to be determined.

| Adenoviral delivery into inguinal fat
PPARc flox/flox mice were purchased from Jax laboratory. Adenoviruses expressing control (CMV-GFP), Cre (Cre-CMV-GFP), control shRNA (U6-shRNA-CMV-GFP), and shPPARc (U6-shPPARc-CMV-GFP) were constructed, amplified, and purified by Vector BioLabs, Malvern, PA. 50 ll of each adenovirus diluted in saline was injected F I G U R E 6 PPARc preferentially regulates brown gene programs in inguinal fat of aging mice. (a) Heat map of white and brown gene transcripts in iWAT of 2-month-old (young) and 12-month-old (aging) mice with knockdown of PPARc (shPPARc) compared to control (shCon) (b, c) White and brown PPARc gene targets in iWAT of 2-month-old (b) and 12-month-old (c) mice after PPARc knockdown. (d) Chromatin IP at the aP2 promoter and at the Ucp1 enhancer in iWAT of 2-month-old (Young) and 12-month-old (Aging) mice. The b-globin promoter was used as a negative control. Data are presented as mean AE SEM and *, p < .05; **, p < .01. n = 7-8 per group unilaterally (5 9 10 9 pfu, for acute purposes) or bilaterally (2 9 10 9 pfu, for chronic purposes) into the inguinal fat pads of mice (Ma, Xu, Gavrilova & Mueller, 2014;Ma et al., 2015;Xu, Ma, Bagattin & Mueller, 2016). For acute analysis, mice were euthanized on the fourth day after viral delivery and for long-term studies, mice were injected once a week for up to 6 weeks.

| Isolation of adipose macrophage (ATM) from inguinal fat
Inguinal fat from mice was excised under sterile conditions and fractionated to obtain stromal vascular cells (SVF), as previously described (Ma et al., 2015). Briefly, inguinal fat was minced and Plasma glucose levels were measured from tail blood before or 15, 30, 60, 90, and 120 min after insulin or glucose injections via automatic reader (Bayer, Leverkusen, Germany). AUC (area under the curve) was calculated with GraphPad software, as previously described .

| Real-time PCR and PCR array
Total RNA was extracted from tissues with TRIzol (Invitrogen, Waltham, MA, USA) or RNeasy (Qiagen), and 1 lg total RNA was reverse-transcribed to cDNA with First Strand cDNA Synthesis Kit (Roche). Quantitative real-time PCR was performed with the ABI PRISM 7900HT sequence detection system (Applied Biosystems, Waltham, MA, USA) using SYBR green (Roche). Gene expression levels were determined by the delta-delta Ct method, after normalization to 36B4 expression. Primer sequences are listed in Table S2.

| In vivo chromatin immunoprecipitation assays
For in vivo ChIP analysis, inguinal fat was first processed as previously reported (Haim, Tarnovscki, Bashari & Rudich, 2013). Briefly, inguinal fat tissues were freshly dissected, minced in small pieces, and cross-linked with 1.5% formaldehyde and subsequently treated with 0.125 M glycine. After incubation, samples were centrifuged at room temperature at 2,500 rpm for 5 min and placed on ice. The upper phase including lipid-rich tissue pieces and fat was washed twice with ice-cold PBS supplemented with protease inhibitors (Roche) followed by centrifugation (5 min, 2,500 rpm, 4°C). After removal of the liquid phase, small adipose tissue pieces were resuspended in adipocyte lysis buffer containing 500 mM PIPES, 80 mM KCl, and 1% Igepal (Sigma) supplemented with protease inhibitors, homogenized using a Dounce homogenizer (Thomas Scientific, Swedesboro, NJ, USA) and incubated on ice for 15 min by vortexing.
Larger particles were removed using a 250-lm mesh. Then, samples were centrifuged (5 min, 2,500 rpm, 4°C) and the pellet of nuclei was resuspended in 500 ll of SDS lysis buffer supplemented with protease inhibitors and incubated on ice for 20 min prior to sonication. The following steps were performed according to the standard protocols described in the manuals accompanying the ChIP assay kit

| Statistical analysis
Student's t test was used for comparison between two groups using GraphPad software. Paired t test was used to compare iWAT weights with unilateral adenoviral delivery of control or shPPARc in iWAT of 2-and 12-month-old mice by SPSS software. p < .05 was considered as statistically significant. Results are shown as mean AE SEM.

ACKNOWLEDGMENTS
We are thankful to Pasha Sarraf for discussions throughout the project. We also thank Gregory G. Germino and Cheng-Chao Lin for kindly sharing their MidiMACS separator and Danielle Springer, Michele Allen, and Audrey Noguchi at the NHLBI murine core facility for technical support in metabolic phenotyping. This project was supported by funding from Shanghai Pujiang Program (17PJ1402700 to L.X and 17PJ1402600 to X.M), National Natural Science Founda-

CONF LICT OF I NTEREST
The authors have declared no conflict of interest