Abnormalities of methyl-CpG binding protein 2 (Mecp2) cause neurological disorders with metabolic dysfunction; however, its role in adipose tissues remains unclear. Here, we report upregulated Mecp2 in white adipose tissues (WAT) of obese humans, as well as in obese mice and during in vitro adipogenesis. Normal chow–fed adipocyte-specific Mecp2 knockout mice (Mecp2Adi KO mice) showed a lean phenotype, with downregulated lipogenic genes and upregulated thermogenic genes that were identified using RNA sequencing. Consistently, the deficiency of Mecp2 in adipocytes protected mice from high-fat diet (HFD)–induced obesity and inhibited in vitro adipogenesis. Furthermore, Mecp2Adi KO mice showed increased browning under different stimuli, including cold treatment. Mechanistically, Mecp2 bound to the promoter of secretory leukocyte protease inhibitor (Slpi) and negatively regulated its expression. Knockdown of Slpi in inguinal WAT of Mecp2Adi KO mice prevented cold-induced browning. Moreover, recombinant SLPI treatment reduced the HFD-induced obesity via enhancing browning. Together, our results suggest a novel non–central nervous system function of Mecp2 in obesity by suppressing browning, at least partially, through regulating adipokine Slpi.

Obesity, which is caused by excessive energy uptake that leads to abnormal accumulation of adipose tissue mass, presents a huge threat to human health. Three major types of adipocytes have been identified so far, including white, brown, and beige adipocytes. Traditionally, white adipose tissue (WAT) is the main site for energy storage, consisting of subcutaneous adipose depots, such as inguinal WAT (iWAT), and visceral adipose depots, such as epididymal WAT (eWAT); whereas brown adipose tissue (BAT) is involved in energy utilization and body temperature maintenance and mainly locates at the interscapular region in mice (1,2). Recently, transdifferentiation between some white adipocytes and beige adipocytes under cold exposure or β3-adrenergic receptor agonist treatment has been revealed, and such a “browning” process promotes weight loss, which makes attractive therapeutic targets for obesity and related diseases (3).

Previously, we reported that under continuous high-fat diet (HFD) stress, offspring developed severe metabolic syndromes, including obesity and insulin resistance, which are associated with the gradual downregulation of DNA methylation on the promoters of inflammatory genes in WAT over generations (4,5). Alterations in DNA methylation have been reported during adipose tissue expansion and adipogenesis (6,7). There are two types of controlling factors for DNA methylation: DNA methylation/demethylation enzymes and methyl-CpG DNA binding proteins. Methyl-CpG binding protein 2 (Mecp2) is an abundant chromatin-associated protein with a high binding affinity to methyl-CpG DNA (8), and it functions as either a transcriptional repressor or an activator (9,10). Neuronal Mecp2 abnormalities (overexpression, deletion, or mutations) are associated with neurological disorders, such as Rett syndrome and autism, and induce unexpected metabolic dysfunctions (11,12). Recent studies on Mecp2 deficiency in peripheral tissues, including heart, liver, and macrophages, have suggested tissue-specific roles of Mecp2 in chronic heart failure, nonalcoholic fatty liver disease (NAFLD), and obesity (1315). However, the exact role that Mecp2 plays in adipose tissue remains to be determined.

Here, we report upregulated Mecp2 in WAT of obese humans and obese mice, as well as during in vitro adipogenesis. Adipocyte-specific Mecp2 knockout mice (Mecp2Adi KO mice) show resistance to HFD-induced obesity and enhanced browning under different stimuli. Our results further suggest negatively regulated secretory leukocyte protease inhibitor (Slpi) by Mecp2, which may contribute to the regulatory role of Mecp2 in browning. Together, we reveal a novel non–central nervous system (CNS) function of Mecp2 in obesity.

Animals, Diets, and Treatments

Mouse strains, loxP-flanked Mecp2flox/y (male), Mecp2flox/flox (female) mice (JAX, No:007177), and adiponectin-Cre (JAX, No:010803) mice were used. Genotyping was performed (primers are listed in Supplementary Table 1), and the male offspring with indicated genotypes was used in the present study. Mice were maintained and handled as previously described (16,17) and as approved by the Committee on Ethics in the Care and Use of Laboratory Animals of College of Life Sciences, Wuhan University. Mice were usually maintained on normal chow (NC). HFD-induced obesity was constructed as previously described (4,5). Food intake (two mice/cage) was monitored for the indicated period of time. For β3-adrenergic receptor agonist treatment, mice were intraperitoneally injected with a single dose of CL316,243 (Sigma) (0.5 mg/kg body weight) (18). For SLPI treatment, mice were fed an HFD for 14 weeks and intraperitoneally injected with recombinant human SLPI (rhSLPI; 0.2 μg/g body weight in saline) (R&D Systems, Minneapolis, MN) once per day. For cold-exposure or thermoneutral experiments, single-housed mice (10 weeks old) were placed at 4°C (3 days) or 30°C (6 weeks), respectively. For adeno-associated virus (AAV) injections, 2.0 × 1011 vg virus-packed mouse Slpi (mSlpi)-shRNA (pAAV-shSlpi) or control (pAAV-Scram) (Vector OBiO, Shanghai, China) (19,20), was injected at multiple sites on the inguinal fat pads. Three weeks after injection, mice were placed at 4°C (cold) for 3 days.

Human Adipose Tissue Samples

WAT tissues (n = 32) were obtained from patients who underwent surgery in Affiliated Tongji Hospital of Tongji Medical College. The study was approved by the ethics committee of Tongji Medical College and was in accordance with the principle of the Declaration of Helsinki. Obese or normal subjects were defined as BMI ≥27 or BMI ≤23 kg/m2, respectively (21,22).

Metabolism Study

Body composition scans were completed with a minispec LF-50 analyzer (Bruker, Rheinstetten, Germany). Metabolic studies were analyzed using CLAMS (Columbus Instruments, Columbus, OH). Oxygen consumption was measured for each mouse over 3 days with 24 h to acclimate to the environment, and the average data of day 2 and 3 was reported.

Glucose and Insulin Tolerance Tests and Measurements of Leptin, Insulin, Triglyceride, Cholesterol, mSlpi, and hSLPI

Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed as previously described (23). Serum levels of leptin, insulin, triglyceride, cholesterol, mSlpi, and human SLPI (hSLPI) were measured using mouse leptin ELISA (Millipore), rat/mouse insulin ELISA (Millipore), triglyceride (Jiancheng, China), total cholesterol (Kehua Bio-engineering, Shanghai, China), and Mouse or human SLPI ELISA (R&D Systems) kits.

Histological Examination, Adipocyte Size Analysis, and Immunohistochemical and Immunofluorescent Staining

Hematoxylin-eosin (H&E) staining and adipocyte analyses were performed as previously described (4,5). For the immunohistochemical study, anti-Mecp2 (1:500) or anti-Ucp1 (1:1,000) was used, and positive staining was visualized with the 3,3′-diaminobenzidine substrate (Vector, Burlingame, CA). For immunofluorescent staining, anti-Slpi (1:100) and anti-perilipin 1 (1:2,000) were used with images taken by a confocal microscope (Leica, Wetzlar, Germany).

RNA Sequencing

Total RNA was isolated, and RNA sequencing and data analyses were performed by Novogene Bioinformatics Technology (Beijing, China) as previously reported (24).

Isolation of Mouse Stromal Vascular Fraction and Mature Adipocytes

Stromal vascular fraction (SVF) and mature adipocytes were isolated as reported (25). Briefly, minced iWAT was digested at 37°C in digestion buffer containing collagenase D and dispase II (Sigma), and then filtered. Mature adipocytes and SVF cells were separated by centrifugation.

Cell Culture, Transfection, In Vitro Adipocyte Differentiation, and Oil Red O Staining

Culture and differentiation of 3T3-L1 cells were performed as previously reported (26). To knockdown Mecp2 in 3T3-L1 cells, cells were transfected with pLKO.1 vector containing scrambled shRNA or shRNAs targeting Mecp2 (Supplementary Table 1). Stable knockdown cell lines were established as previously described (27). To overexpress Mecp2 in 3T3-L1 cells, cells were transfected with plasmid pCMV6-mMecp2-Flag (Origene).

Mouse SVFs were grown in DMEM/F12 media (Hyclone) supplemented with 10% FBS (Gibco). Differentiation of primary white or beige adipocytes was done as described (25). In some experiments, Mecp2 knockout in SVF cells was achieved by infecting SVF cells isolated from Mecp2flox/y mice with lentivirus expressing Cre-GFP. To overexpress Mecp2, mouse Mecp2 (phage-Mecp2-Flag) was packed into lentivirus expressing system to infect beige differentiated SVF cells. For SLPI treatment, cells were treated with rhSLPI (0.2 µg/mL) for 24 h. Differentiated cells were stained with Oil Red O (26).

Chromatin Immunoprecipitation and Coimmunoprecipitation

Chromatin immunoprecipitation (ChIP) experiments were performed as previously described (28) (Supplementary Table 1). The 293T cells were grown in DMEM media supplemented with 10% FBS, and the 3T3-L1 cells at day 8 or SVF cells at day 7 after white adipocyte induction, or 293T cells, or iWAT tissues, were lysed. Coimmunoprecipitation (Co-IP) was performed as previously described (27).

Reporter Assays

Slpi promoter from −3,000 to transcription start site was cloned into pGL3-enhancer (Promega). The 293T cells were transfected with pGL3-enhancer-Slpi, pRL-TK, and different dosages of Mecp2 plasmid. Luciferase assays were performed and analyzed as previously described (27).

Quantitative Real-time PCR and Western Blots

Quantitative real-time PCR (qPCR) and Western blots were performed as previously described (29). The primers and antibodies used are provided (Supplementary Tables 1 and 2).

Statistical Analysis

Results were expressed as mean ± SD. Data were analyzed using the Student t test except the metabolic chamber studies for which ANCOVA was used. Differences were considered statistically significant at P < 0.05.

Data and Resource Availability

RNA sequencing data are available at Gene Expression Omnibus (GEO) (code GSE117517).

Mecp2 Is Upregulated in WAT of Obese Humans and Mice

After 6 months of HFD stress, the transcription levels and the protein levels of Mecp2 were increased in WAT of wildtype (WT) mice (Fig. 1A–C). To determine the clinical relevance of adipocyte Mecp2 in obesity, subcutaneous fat depots from humans with different BMIs were obtained during surgery and analyzed. The mRNA levels of MECP2 were significantly increased in obese subjects and positively correlated with BMI (Fig. 1D and E). Consistently, upregulated MECP2 in subcutaneous WAT (sWAT) and adipocytes of obese subjects was observed by Western blots and immunohistochemical staining, respectively (Fig. 1F and G).

Figure 1

Mecp2 is upregulated in WAT of obese humans/mice and during in vivo adipogenesis. AC: qPCR analysis (A), representative images (B), and Western blots (left) with quantitative results (right) (C) of Mecp2 in eWAT of NC-fed or 6-month HFD-fed male mice (n = 5–6 per mouse group). D: qPCR analysis of MECP2 in sWAT of human subjects (normal human subjects, n = 17, and obese human subjects, n = 15). E: Correlation between MECP2 and BMI. F and G: Western blots (F) (normal human subjects, n = 5, and obese human subjects, n = 7) and representative images (G) (normal human subjects, n = 10, and obese human subjects, n = 12) of MECP2 in sWAT of human subjects. HK: Increased Mecp2 levels during white adipocyte differentiation in SVF and 3T3-L1 cells. The representative mRNA levels of indicated genes (H and J) and representative Western blots (left) with quantitative results (right) of Pparγ1/2, Fabp4, and Mecp2 (I and K) during SVF and 3T3-L1 white differentiation, respectively. *P < 0.05; **P < 0.01.

Figure 1

Mecp2 is upregulated in WAT of obese humans/mice and during in vivo adipogenesis. AC: qPCR analysis (A), representative images (B), and Western blots (left) with quantitative results (right) (C) of Mecp2 in eWAT of NC-fed or 6-month HFD-fed male mice (n = 5–6 per mouse group). D: qPCR analysis of MECP2 in sWAT of human subjects (normal human subjects, n = 17, and obese human subjects, n = 15). E: Correlation between MECP2 and BMI. F and G: Western blots (F) (normal human subjects, n = 5, and obese human subjects, n = 7) and representative images (G) (normal human subjects, n = 10, and obese human subjects, n = 12) of MECP2 in sWAT of human subjects. HK: Increased Mecp2 levels during white adipocyte differentiation in SVF and 3T3-L1 cells. The representative mRNA levels of indicated genes (H and J) and representative Western blots (left) with quantitative results (right) of Pparγ1/2, Fabp4, and Mecp2 (I and K) during SVF and 3T3-L1 white differentiation, respectively. *P < 0.05; **P < 0.01.

Next, we examined whether Mecp2 expression levels are altered during adipocyte differentiation. Successful differentiation was demonstrated by gradually elevated mRNA levels of Cebpa, Pparg1/2, Fabp4, Leptin, and Adipoq as well as elevated protein levels of Pparγ1/2 and Fabp4 in SVF and 3T3-L1 cells after differentiation to white adipocytes (Fig. 1H–K). During this adipogenesis process, gradually upregulated mRNA and protein levels of Mecp2 were also observed (Fig. 1H–K). However, the mRNA levels of other DNA methylation binding proteins, such as Mbd2, Mbd3, and Mbd4, were unchanged (Fig. 1I and K).

NC-Fed Adipocyte-Specific Mecp2 Knockout Mice Show a Lean Phenotype

To study the role that Mecp2 plays in obesity, we crossed adiponectin-cre male mice (expressing Cre recombinase under the control of adiponectin promoter) with the loxP-flanked Mecp2flox/+ females. Adiponectin is a mature adipocyte marker expressed at late phase of adipocyte differentiation (30,31). Since Mecp2 is X-linked (32), males genotyped as Mecp2flox/y (WT) and Mecp2flox/yadiponectin-Cre (Mecp2Adi KO) were used (Supplementary Fig. 1). Significantly reduced mRNA and protein levels of Mecp2 in WAT and BAT but not in liver and skeletal muscle were found in Mecp2Adi KO mice (Fig. 2A–D). Compared with WT mice, NC-fed Mecp2Adi KO mice showed lower body weight (Fig. 2E) with reduced fat mass but not lean mass (Fig. 2F). Moreover, reduced eWAT/iWAT/BAT and liver weights, as well as decreased iWAT/eWAT adipocyte size, were observed (Fig. 2G–L). Elevated oxygen consumption was observed in Mecp2Adi KO mice (Fig. 2M). However, WT and Mecp2Adi KO mice showed a similar amount of food intake and serum levels of leptin, insulin, triglyceride, cholesterol, fasting blood glucose (FBG), and glucose tolerance (Fig. 2N and O and Supplementary Table 3).

Figure 2

NC-fed Mecp2Adi KO mice show a lean phenotype. A and B: qPCR (A) and Western blot (B) analysis of Mecp2 levels in eWAT, iWAT, and BAT of male WT or Mecp2Adi KO mice. C and D: qPCR (C) and Western blot (D) of Mecp2 levels in the livers and muscle of NC-fed WT or Mecp2Adi KO mice. EI: Growth curves (E), fat mass (F), abdominal view (G), and representative images (H) of different adipose tissues and their weights (I) for NC-fed WT or Mecp2Adi KO mice. J: Tissue weights of NC-fed WT or Mecp2Adi KO mice. K and L: Representative images of H&E staining (left), adipocyte size distribution (middle), and average cell area (right) in iWAT (K) and eWAT (L) of NC-fed WT or Mecp2Adi KO mice. M: Oxygen consumption of NC-fed WT or Mecp2Adi KO mice. N: Food intake (monitored from ages 14–16 weeks for both groups; two mice per cage) of NC-fed WT or Mecp2Adi KO mice. O: GTT of NC-fed WT or Mecp2Adi KO mice. n = 5–6 per mouse group. *P < 0.05; **P < 0.01.

Figure 2

NC-fed Mecp2Adi KO mice show a lean phenotype. A and B: qPCR (A) and Western blot (B) analysis of Mecp2 levels in eWAT, iWAT, and BAT of male WT or Mecp2Adi KO mice. C and D: qPCR (C) and Western blot (D) of Mecp2 levels in the livers and muscle of NC-fed WT or Mecp2Adi KO mice. EI: Growth curves (E), fat mass (F), abdominal view (G), and representative images (H) of different adipose tissues and their weights (I) for NC-fed WT or Mecp2Adi KO mice. J: Tissue weights of NC-fed WT or Mecp2Adi KO mice. K and L: Representative images of H&E staining (left), adipocyte size distribution (middle), and average cell area (right) in iWAT (K) and eWAT (L) of NC-fed WT or Mecp2Adi KO mice. M: Oxygen consumption of NC-fed WT or Mecp2Adi KO mice. N: Food intake (monitored from ages 14–16 weeks for both groups; two mice per cage) of NC-fed WT or Mecp2Adi KO mice. O: GTT of NC-fed WT or Mecp2Adi KO mice. n = 5–6 per mouse group. *P < 0.05; **P < 0.01.

Differentially Expressed Genes in Adipose Tissues of Mecp2Adi KO Mice

To perform an unbiased study of signaling pathways altered by Mecp2 knockout in adipose tissues, the global gene expression profiles of iWAT and BAT from NC-fed WT and Mecp2Adi KO mice were analyzed by RNA sequencing. A deficiency of Mecp2 in iWAT and BAT led to 1,375 and 1,030 differentially expressed genes, and among them, 218 were seen in both tissues (Fig. 3A). Interestingly, in both iWAT and BAT of Mecp2Adi KO mice, genes involved in thermogenesis, such as Ucp1, Elovl3, Cox7a1, Dio2, Cox8b, and Cidea were upregulated, while genes related to adipogenesis and lipogenesis, such as Leptin, Sncg, Nnat, Srebp1, and Fasn were downregulated (Fig. 3A).

Figure 3

Increased thermogenic genes and decreased lipogenic genes in NC-fed Mecp2Adi KO mice. A: RNA sequencing revealed differentially expressed genes in iWAT and BAT of Mecp2Adi KO mice compared with those of WT mice. BE: qPCR analysis for indicated genes in iWAT (B and D) or BAT (C and E) of NC-fed WT or Mecp2Adi KO mice. F: Representative Western blots (left) with quantitative results (right) of mSrebp1, Fasn, Cidea, and Ucp1 in iWAT of NC-fed WT or Mecp2Adi KO mice. G: Representative images of Ucp1 staining in iWAT of NC-fed WT or Mecp2Adi KO mice. n = 3–6. *P < 0.05; **P < 0.01.

Figure 3

Increased thermogenic genes and decreased lipogenic genes in NC-fed Mecp2Adi KO mice. A: RNA sequencing revealed differentially expressed genes in iWAT and BAT of Mecp2Adi KO mice compared with those of WT mice. BE: qPCR analysis for indicated genes in iWAT (B and D) or BAT (C and E) of NC-fed WT or Mecp2Adi KO mice. F: Representative Western blots (left) with quantitative results (right) of mSrebp1, Fasn, Cidea, and Ucp1 in iWAT of NC-fed WT or Mecp2Adi KO mice. G: Representative images of Ucp1 staining in iWAT of NC-fed WT or Mecp2Adi KO mice. n = 3–6. *P < 0.05; **P < 0.01.

Consistent with RNA-sequencing results, qPCR verified these changes in iWAT and BAT of Mecp2Adi KO mice (Fig. 3B–E). Moreover, significantly decreased protein levels of mature Srebp1 and Fasn, as well as significantly upregulated Ucp1 and Cidea, were seen in iWAT of NC-fed Mecp2Adi KO mice (Fig. 3F). In addition, upregulated Ucp1 was further verified by immunostaining (Fig. 3G). Together, these results indicate suppressed lipogenesis and enhanced thermogenesis in Mecp2-deficient adipose tissues.

Adipocyte-Specific Mecp2 Knockout Prevents HFD-Induced Obesity in Mice

Altered mRNA and protein levels of lipogenic and thermogenic factors in Mecp2Adi KO mice enable us to hypothesize that Mecp2 deficiency in adipocytes may attenuate obesity. To test this possibility, we fed WT and Mecp2Adi KO mice with an HFD for 12 weeks. Compared with HFD-fed WT mice, HFD-fed Mecp2Adi KO mice showed significantly lower body weights, reduced fat but not lean mass, and reduced WAT and BAT weights (Fig. 4A–E). Moreover, HFD-fed Mecp2Adi KO mice had significantly decreased liver and kidney weights (Supplementary Fig. 2A) as well as decreased adipocyte size of iWAT and eWAT (Fig. 4F and G and Supplementary Fig. 2B). Furthermore, HFD-fed Mecp2Adi KO mice showed significantly decreased FBG, serum leptin, insulin, and triglyceride levels (Supplementary Table 3) as well as significantly improved glucose tolerance and insulin tolerance (Fig. 4H and I and Supplementary Fig. 2C). A similar amount of food intake was observed in two groups (Supplementary Fig. 2D). In HFD-fed Mecp2Adi KO mice, dramatically downregulated lipogenic genes, together with significantly upregulated thermogenic genes, were found in iWAT (Fig. 4J). Significantly increased Ucp1 and Cidea levels were also found in iWAT (Fig. 4K) and in BAT of HFD-fed Mecp2Adi KO mice (Fig. 4L and M). Moreover, HFD-fed Mecp2Adi KO mice showed elevated oxygen consumption (Fig. 4N), which was more obvious during the light cycle possibly because of increased motor activities (data not shown); and attenuated hepatic steatosis as demonstrated by liver appearance, Oil Red O staining and pathological examination (Fig. 4O and P).

Figure 4

Mecp2Adi KO mice are resistant to HFD stress–induced obesity. AC: Growth curves (A), body mass (B), and abdominal view (C) of HFD-fed WT or Mecp2Adi KO mice. DF: Representative pictures (D), weights (E), and H&E staining (F) of different adipose tissues in HFD-fed WT or Mecp2Adi KO mice. G: Adipocyte size distribution (left) and average cell area (right) in iWAT of HFD-fed WT or Mecp2Adi KO mice. H and I: GTT (H) and ITT (I) of HFD-fed WT or Mecp2Adi KO mice. J and K: qPCR analysis of indicated genes (J) and representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea in iWAT (K). L and M: Representative pictures of Ucp1 staining (M) and representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea (L) in BAT of HFD-fed WT or Mecp2Adi KO mice. NP: Oxygen consumption (N), representative gross livers (O), H&E and Oil Red O staining (P, left) and steatosis score (P, right) for the liver of HFD-fed WT and Mecp2Adi KO mice. n = 6 per group. *P < 0.05; **P < 0.01.

Figure 4

Mecp2Adi KO mice are resistant to HFD stress–induced obesity. AC: Growth curves (A), body mass (B), and abdominal view (C) of HFD-fed WT or Mecp2Adi KO mice. DF: Representative pictures (D), weights (E), and H&E staining (F) of different adipose tissues in HFD-fed WT or Mecp2Adi KO mice. G: Adipocyte size distribution (left) and average cell area (right) in iWAT of HFD-fed WT or Mecp2Adi KO mice. H and I: GTT (H) and ITT (I) of HFD-fed WT or Mecp2Adi KO mice. J and K: qPCR analysis of indicated genes (J) and representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea in iWAT (K). L and M: Representative pictures of Ucp1 staining (M) and representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea (L) in BAT of HFD-fed WT or Mecp2Adi KO mice. NP: Oxygen consumption (N), representative gross livers (O), H&E and Oil Red O staining (P, left) and steatosis score (P, right) for the liver of HFD-fed WT and Mecp2Adi KO mice. n = 6 per group. *P < 0.05; **P < 0.01.

Chronic inflammation in adipose tissue contributes to the development of obesity (33), the transcriptional levels of inflammatory factors Tnf, Ccl2, Il1b, and Il8 were evaluated. Comparable levels of these inflammatory genes were found in NC- or HFD-fed Mecp2Adi KO and WT mice, except for downregulated Tnf in HFD-fed Mecp2Adi KO mice (Supplementary Fig. 3A and B). Alterations in bioenergenesis may contribute to the development of obesity (25); however, similar mRNA and protein levels of Pgc1α, a key bioenergetic factor (34), were found in NC- or HFD-fed WT mice and Mecp2Adi KO mice (Supplementary Fig. 3).

Mecp2 Regulates In Vitro Adipogenesis

To investigate whether Mecp2 regulates adipogenesis in vitro, knockdown of Mecp2 in SVF cells were achieved by infecting lentivirus packed Cre-recombinase into SVF cells isolated from Mecp2flox/y mice (Supplementary Fig. 4A and B). Ablation of Mecp2 in SVF cells resulted in pronounced defects in white adipocyte differentiation as demonstrated by Oil Red O staining (Supplementary Fig. 4C). Furthermore, drastic adipogenic defects were observed in two stable Mecp2 knockdown 3T3-L1 cell lines (Supplementary Fig. 4D–F). To investigate how Mecp2 regulates white adipocyte differentiation, we examined the mRNA levels of several key adipogenesis activators or inhibitors in these Mecp2 knockdown cell lines. Significantly downregulated Pparg2 and Fabp4 before induction, as well as significantly downregulated adipogenic genes (Cebpa, Cebpb, Pparg1/2, and Fabp4) and lipogenic genes (Srebp1, Chrebp, Fasn, Scd1, Acly, and Acaca) after induction, were identified (Supplementary Fig. 4G–J). To explore whether Mecp2-deficiency–caused adipogenesis defects can be rescued, Mecp2 was transfected into the stable Mcep2 knockdown 3T3-L1 cells, and restoration of Mecp2 fully rescued adipogenesis (Supplementary Fig. 4K–M).

Increased Browning in Mecp2Adi KO Mice Under Different Stimuli

Dramatically upregulated thermogenic genes in iWAT of Mecp2Adi KO mice (Figs. 2 and 3) inspired us to challenge Mecp2Adi KO mice with a β-norepinephrine (β-NE, CL316,243), cold or thermoneutral stimuli. Under the β-NE challenge, dramatically upregulated thermogenic genes in iWAT of WT mice were found, such as Ucp1, Elovl3, Cox7a1, Dio2, and Cidea (data not shown), which were further significantly upregulated in iWAT of β-NE–challenged Mecp2Adi KO mice (Fig. 5A). Moreover, compared with WT mice, significantly upregulated browning, increased Ucp1 staining and increased Ucp1/Cidea levels were also found in iWAT of Mecp2Adi KO mice under a β-NE challenge (Fig. 5B and C). Similar results were found in iWAT of Mecp2Adi KO mice under cold or thermoneutral conditions (Fig. 5D–G). Comparable mRNA levels of Pgc1a were found in WAT of WT and Mecp2Adi KO mice under a β-NE challenge and cold stress (Supplementary Fig. 5).

Figure 5

Mecp2Adi KO mice show enhanced browning under β-adrenergic agonist or cold exposure. AC: qPCR analysis for indicated genes (A), representative images of H&E and Ucp1 staining (B), representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea in iWAT (C) after Cl316,243 injection. D: qPCR analysis for indicated genes in iWAT under 4°C for 3 days (top) or 30°C for 6 weeks (bottom). E: Representative images of H&E and Ucp1 staining in iWAT under RT, 4°C for 3 days or 30°C for 6 weeks. F and G: Representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea in iWAT under 4°C for 3 days (F) or 30°C for 6 weeks (G). n = 5–6 per group. RT, room temperature. *P < 0.05; **P < 0.01.

Figure 5

Mecp2Adi KO mice show enhanced browning under β-adrenergic agonist or cold exposure. AC: qPCR analysis for indicated genes (A), representative images of H&E and Ucp1 staining (B), representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea in iWAT (C) after Cl316,243 injection. D: qPCR analysis for indicated genes in iWAT under 4°C for 3 days (top) or 30°C for 6 weeks (bottom). E: Representative images of H&E and Ucp1 staining in iWAT under RT, 4°C for 3 days or 30°C for 6 weeks. F and G: Representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea in iWAT under 4°C for 3 days (F) or 30°C for 6 weeks (G). n = 5–6 per group. RT, room temperature. *P < 0.05; **P < 0.01.

Mecp2 Negatively Regulates SLPI

To identify downstream factors that may be responsible for the increased browning phenotype in Mecp2Adi KO mice, a highly upregulated gene in iWAT of Mecp2Adi KO mice, Slpi, which encodes the SLPI, caught our attention (Fig. 6A). Immunofluroscent staining suggested that SLPI can locate on mature adipocytes of mouse iWAT and human sWAT (Supplementary Fig. 6A and B). Under NC-fed conditions and at room temperature, compared with WT, significantly increased Slpi levels in iWAT, eWAT, BAT and mature adipocytes of iWAT, of the Mecp2Adi KO mice were observed (Fig. 6B and C and Supplementary Fig. 6C). Upregulated Slpi on adipocytes in iWAT of the Mecp2Adi KO mice were also found (Supplementary Fig. 6D). Moreover, upregulated Slpi in iWAT of the Mecp2Adi KO mice under HFD stress, cold or thermoneutral stimuli, and β-NE treatment was observed (Fig. 6D). Furthermore, reintroducing Mecp2 into the Mecp2 knockdown 3T3-L1 cells inhibited the Mecp2 deficiency–induced Slpi upregulation (Fig. 6E), and an in vitro luciferase reporter assay confirmed the binding and dose dependently negative regulation of Mecp2 on Slpi (Supplementary Fig. 6E). These data indicate a negative regulation of Slpi by Mecp2.

Figure 6

Mecp2 negatively regulates Slpi level. A: Volcanic map of several highly upregulated genes in iWAT of Mecp2Adi KO mice. B and C: Slpi level in iWAT (B) and SVF or mature adipocytes of iWAT (C) of WT or Mecp2Adi KO mice at normal conditions. D: Slpi level in iWAT of WT or Mecp2Adi KO mice at indicated conditions (4°C for 3 days; 30°C for 6 weeks; HFD for 6 months; CL316,243 injection for 7 days); n = 5–6 per group. E: Slpi level in indicated 3T3-L1 cells. FI: Slpi level in iWAT (F) and Slpi level in serum (G) of indicated conditions of NC-fed WT mice, and Slpi level in WAT of obese humans (H) or obese mice (I); n = 5–6 per group for mice; n = 15–17 per group for humans. J and K: Mecp2 binding affinity on the promoter of Slpi (J) (four different regions with the exact location of primer shown at the top of J) and on the promoter of Ucp1 (K) (two different regions with the exact location of each primer shown at the top of K) in WT and Mecp2Adi KO mice (left) and in WT mice under cold stimulation or β-NE challenge (right); n = 4 per group. TSS, transcription start site. *P < 0.05; **P < 0.01.

Figure 6

Mecp2 negatively regulates Slpi level. A: Volcanic map of several highly upregulated genes in iWAT of Mecp2Adi KO mice. B and C: Slpi level in iWAT (B) and SVF or mature adipocytes of iWAT (C) of WT or Mecp2Adi KO mice at normal conditions. D: Slpi level in iWAT of WT or Mecp2Adi KO mice at indicated conditions (4°C for 3 days; 30°C for 6 weeks; HFD for 6 months; CL316,243 injection for 7 days); n = 5–6 per group. E: Slpi level in indicated 3T3-L1 cells. FI: Slpi level in iWAT (F) and Slpi level in serum (G) of indicated conditions of NC-fed WT mice, and Slpi level in WAT of obese humans (H) or obese mice (I); n = 5–6 per group for mice; n = 15–17 per group for humans. J and K: Mecp2 binding affinity on the promoter of Slpi (J) (four different regions with the exact location of primer shown at the top of J) and on the promoter of Ucp1 (K) (two different regions with the exact location of each primer shown at the top of K) in WT and Mecp2Adi KO mice (left) and in WT mice under cold stimulation or β-NE challenge (right); n = 4 per group. TSS, transcription start site. *P < 0.05; **P < 0.01.

Moreover, the Slpi level was markedly increased in iWAT of cold or β-NE challenged WT mice (Fig. 6F). Upregulated serum Slpi were observed in Mecp2Adi KO mice and cold-treated WT mice (Fig. 6G). Dramatically downregulated mRNA and protein levels of SLPI in obese mice or humans were observed (Fig. 6H and I and Supplementary Fig. 6A and B). These data suggest that the Slpi level is positively correlated with browning but negatively correlated with obesity.

It has been reported that Mecp2 binds histone deacetylase 3 (HDAC3) and cooperates together to regulate gene expression (35). We performed Co-IP experiments using 293T cells, white adipocyte–differentiated SVF cells or 3T3-L1 cells, and iWAT. Consistent with previous reports that Mecp2 associates with transcriptional repression regulatory complexes (36), binding of Mecp2 to nuclear receptor corepressor (NcoR) was found; however, no binding of Mecp2 with HDAC3 was detected (Supplementary Fig. 7). The ChIP analysis suggested that an adipocyte deficiency of Mecp2 abolished the Mecp2 affinity to the promoter of Slpi in iWAT of the Mecp2Adi KO mice (Fig. 6J). Moreover, cold or β-NE treatment inhibited Mecp2 binding to the promoter of Slpi in iWAT of WT mice (Fig. 6J). Similar results were also found on the promoter of Ucp1 (Fig. 6K).

SLPI Inhibits Obesity by Increasing Browning

To investigate whether upregulated Slpi is responsible for adipocyte Mecp2 deficiency-induced browning enhancement, we first knockdown Slpi in the left iWAT pad by in situ injection of AAV-shSlpi, with the AAV-shScram–injected right iWAT pad as the controls, then cold challenged for 3 days at 3 weeks after injection (Fig. 7A). A significantly downregulated mRNA level of Slpi, without affecting inflammatory genes Mecp2 and Pgc1a, and iWAT weights, was found in AAV-shSlpi–injected iWAT (Fig. 7B and C and Supplementary Fig. 8A). Furthermore, AAV-shSlpi significantly decreased the adipocyte-Mecp2-deficiency–induced browning enhancement, Ucp1/Cidea and thermogenic gene upregulation, but not Mecp2 or Pgc1a, in iWAT of Mecp2Adi KO mice upon cold stimuli (Fig. 7D–F and Supplementary Fig. 8B). Next, we knockdown Slpi in iWAT pads of both flanks, and a mild but significant downregulation of serum Slpi levels was found (Fig. 7G).

Figure 7

Mecp2 regulates browning by Slpi. A: Experimental design and iWAT AAV injection. B: mRNA level of Slpi in iWAT injected with AAV-shSlpi or AAV-Scram. C: Weights of iWAT. D and F: Representative images of H&E/Ucp1 staining (D) and representative Western blots (left) with quantitative results (right) of Ucp1 (F) in iWAT of WT or Mecp2Adi KO mice after 4°C stress. E: mRNA levels of thermogenic genes in iWAT of WT or Mecp2Adi KO mice after 4°C stress. G: Serum Slpi level after iWAT pads of both flanks injected with AAV-shSlpi or AAV-Scram. n = 3–7 per group. *P < 0.05; **P < 0.01.

Figure 7

Mecp2 regulates browning by Slpi. A: Experimental design and iWAT AAV injection. B: mRNA level of Slpi in iWAT injected with AAV-shSlpi or AAV-Scram. C: Weights of iWAT. D and F: Representative images of H&E/Ucp1 staining (D) and representative Western blots (left) with quantitative results (right) of Ucp1 (F) in iWAT of WT or Mecp2Adi KO mice after 4°C stress. E: mRNA levels of thermogenic genes in iWAT of WT or Mecp2Adi KO mice after 4°C stress. G: Serum Slpi level after iWAT pads of both flanks injected with AAV-shSlpi or AAV-Scram. n = 3–7 per group. *P < 0.05; **P < 0.01.

To test whether Slpi plays a role in obesity, rhSLPI was injected once per day for 5 days to WT mice fed with an HFD for 14 weeks (Fig. 8A). Dramatically increased serum hSLPI levels were detected after injection (Fig. 8B). Compared with the control mice, rhSLPI treatment significantly decreased body weights, reduced fat mass but not lean mass, and reduced iWAT, eWAT, and liver weights in HFD-fed mice (Fig. 8C–F). Notably, a similar amount of food intake was observed among HFD-fed mice with or without the rhSLPI treatment (Fig. 8G). Moreover, rhSLPI injection significantly upregulated thermogenic genes and increased Ucp1 staining in iWAT, eWAT, and BAT, as well as increased Ucp1/Cidea protein levels without affecting inflammatory genes, Pgc1a, mRNA, and protein levels of Mecp2 in iWAT of HFD-fed mice (Fig. 8H–J and Supplementary Fig. 9). Injection of rhSLPI also significantly attenuated HFD-induced hepatic steatosis (Fig. 8K). Furthermore, in vitro experiments using beige differentiated SVF cells demonstrated that overexpression of Mecp2 in mature beige adipocytes inhibited the transcription of Ucp1 and Cidea, which was prevented by rhSLPI treatment (Supplementary Fig. 10).

Figure 8

rhSLPI inhibits diet-induced obesity by increasing browning. A: Experimental design for rhSLPI treatment. B: Serum level of hSLPI. CF: Changed body weight (C), fat mass (D), weights of different tissues (E), and representative H&E images of different adipose tissues (F) of HFD-fed WT mice with or without rhSLPI treatment. G: Food intake (monitored from 14 to 15 weeks upon HFD stress; age for mice: 18–19 weeks; two mice per cage). H: qPCR analysis of indicated genes in eWAT (left), iWAT (middle), and BAT (right) of HFD-fed WT mice with or without rhSLPI treatment. IK: Ucp1 staining in different fat pads (I), representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea in iWAT (J), and hepatic H&E staining (left) and steatosis score (right) of HFD-fed WT mice with or without rhSLPI treatment (K). n = 5–7 per group. *P < 0.05; **P < 0.01.

Figure 8

rhSLPI inhibits diet-induced obesity by increasing browning. A: Experimental design for rhSLPI treatment. B: Serum level of hSLPI. CF: Changed body weight (C), fat mass (D), weights of different tissues (E), and representative H&E images of different adipose tissues (F) of HFD-fed WT mice with or without rhSLPI treatment. G: Food intake (monitored from 14 to 15 weeks upon HFD stress; age for mice: 18–19 weeks; two mice per cage). H: qPCR analysis of indicated genes in eWAT (left), iWAT (middle), and BAT (right) of HFD-fed WT mice with or without rhSLPI treatment. IK: Ucp1 staining in different fat pads (I), representative Western blots (top) with quantitative results (bottom) of Ucp1 and Cidea in iWAT (J), and hepatic H&E staining (left) and steatosis score (right) of HFD-fed WT mice with or without rhSLPI treatment (K). n = 5–7 per group. *P < 0.05; **P < 0.01.

Mecp2 is involved in neurological and nonneurological disorders. In humans, mutations or deletion of Mecp2 causes Rett syndrome (11) and autism (37), suggesting its critical role in neuron functions. Most Rett syndrome patients have lipid and cholesterol metabolic dysfunctions (38,39). Contradictory results on weight gain have been reported in NC-fed whole body Mecp2 KO mice (4042), which may be because of different stain backgrounds (B6 vs. B6/129 vs. BALB/c) and deletion of different exons (exons 3 and 4 vs. exon 3 only). Neuron-specific, including whole brain, forebrain, hypothalamus and amygdala, and hypothalamic pro-opiomelanocortin neurons, deletion of Mecp2 in mouse results in increased body weights (41,43,44). In addition, liver-specific Mecp2 KO male mice show spontaneous fatty liver diseases without affecting body weights (14). Upregulated lipogenic genes have been found in the livers of whole body or liver-specific Mecp2 KO mice, which may be because of less enrichment of HDAC3 on the promoters of lipogenic genes caused by impaired binding of Mecp2 with HDAC3 (14). Here, we found a lean phenotype, reduced fat mass and adipocyte size, and less hepatic steatosis in Mecp2Adi KO mice under either NC or HFD conditions (Figs. 2 and 4 and Supplementary Fig. 2). Direct binding between Mecp2 and NcoR, but not HDAC3, was found in examined cells or iWAT (Supplementary Fig. 7), indicating, at least in the present setting, that HDAC3 may not be responsible for the phenotypes observed.

Our results clearly demonstrated that adipocyte-specific deletion of Mecp2 enhanced iWAT browning in Mecp2Adi KO mice under HFD, β-NE, or cold stress. Mecp2Adi KO mice are sensitive to stimuli-induced browning possibly for at least two reasons. Dramatically reduced Fasn in the adipose tissues of Mecp2Adi KO mice (Fig. 3) is consistent with a report that mice with Fasn depletion in mature adipose tissue show increased energy expenditure, beige-like adipocytes in iWAT, and resistance to diet-induced obesity (45). Another possibility is the reduced accumulation of Mecp2 on the Ucp1 promoter and upregulated Ucp1, a master regulator of browning, in iWAT of Mecp2Adi KO mice (Figs. 35 and 6K).

Our results suggested that Mecp2 also binds to the Slpi promoter and downregulates its level (Fig. 6 and Supplementary Fig. 6). The regulatory role of adipocyte Mecp2 on cold-induced browning is, at least partially, dependent on Slpi since knockdown of Slpi drastically reversed the cold-induced browning enhancement in Mecp2Adi KO mice and rhSLPI treatment inhibited the Mecp2-induced downregulation of thermogenic genes in beige differentiated SVF cells (Fig. 7 and Supplementary Fig. 10). SLPI is a small, nonglycosylated protein which can suppress monocyte/macrophage proinflammatory responses through inhibition of NF-κB signaling, thus exerts immune-modulatory activities in inflammatory diseases, such as sepsis, asthma, and cancer (46). However, in the current study, neither manipulating Slpi level nor knockout Mecp2 in mouse iWAT showed significant effects on inflammatory genes (Supplementary Figs. 3, 8, and 9). Such a difference may be due to the subclinical, chronic inflammation of obese adipose tissues in this study, compared with the acute inflammatory models that most SLPI studies have investigated (46). Notably, our observations are that rhSLPI treatment reduces obesity and NAFLD and enhances browning in an HFD-fed model (Fig. 8), suggesting its potential as a clinical intervention target for obesity and NAFLD.

SLPI has also been predicted in the secretome of human adipose tissues (47). Upregulated serum Slpi levels after cold or rhSLPI treatment, as well as downregulated serum Slpi levels after knockdown Slpi in iWAT (Figs. 6G, 7G, and 8B), indicate that Slpi can be released to serum by adipose tissues. The results that rhSLPI regulates browning in the HFD-fed mice and thermogenic genes in differentiated beige adipocytes (Fig. 8 and Supplementary Fig. 10) suggest that Slpi may act through autocrine and endocrine manners.

Consistent with a previous study (48), we demonstrated that at normal conditions, the SVF part of adipose tissue has a higher Slpi level compared with that of mature adipocytes (Fig. 6C). However, a dramatically increased Slpi level was found in mature adipocytes of Mecp2Adi KO mice, which was also significantly higher than that of the SVF parts isolated from WT or Mecp2Adi KO mice. An increased protein level of Slpi was also found in adipocytes of Mecp2Adi KO mice (Fig. 6C and Supplementary Fig. 6D). Moreover, the mRNA and protein levels of Slpi were downregulated in the iWAT of obese mice or obese humans in the current study (Fig. 6H and I and Supplementary Fig. 6A and B). These differences may be due to different durations of HFD feeding (6 months vs. 12 weeks) and different fat pads studied (iWAT vs. eWAT).

Enhanced mitochondrial biogenesis is an important component of adaptive thermogenesis, especially in brown fat and skeletal muscle, and contributes to the development of obesity (25). PGC1α connects environmental stimuli, mitochondrial biogenesis, and respiration upon altered energy or thermogenic requirements (34). Knockout Mecp2 in adipose tissue or a knockdown Slpi level in iWAT did not significantly affect Pgc1a levels in the examined conditions (Supplementary Figs. 3, 5, 8, and 9). Additional bioenergenesis factors may be investigated in future studies.

Although Mecp2 negatively regulated the Slpi level as demonstrated in this study, Slpi did not affect the Mecp2 level, at least in iWAT (Supplementary Figs. 8 and 9). However, under cold stress, knockdown Slpi downregulated both mRNA and protein levels of Ucp1 in WT mice and prevented browning in Mecp2Adi KO mice (Fig. 7). Future studies, such as using adipocyte-specific knockout Slpi and RNA sequencing, may reveal the overlapping and unique biological functions between Slpi and Mecp2 in browning. In summary, we report a novel non-CNS function of Mecp2 in adipocytes by suppressing browning, at least partially, through regulating adipokine Slpi, thus suggesting the novel roles that Mecp2 and Slpi play in obesity. Their therapeutic potentials for obesity and metabolic syndromes are suggested.

Acknowledgments. The authors thank Dr. Bo Zhong (Wuhan University) for providing lentivirus expressing Cre-GFP.

Funding. The work is technically supported by the Analytical and Testing Core of College of Life Sciences of Wuhan University and by the Analytical and Testing Center of Huazhong University of Science and Technology (HUST). This work is supported by the National Key R&D Program of China (2018YFA0800700), the National Natural Science Foundation of China (31671195, 31871381, 31971066, and 31871411), the Natural Science Foundation of Hubei Province (2016CFA012), the Front Youth Program of HUST, and the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, HUST.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. C.L., J.W., Y.W., W.Z., M.G., Y.Y., Y.C., Y.S., H.C., Y.Z., M.X., and Y.L. performed the experiments and analyzed the data. C.L., L.Z., and K.H. wrote the manuscript. L.Z. and K.H. designed the study and analyzed the data. L.Z. and K.H. are the guarantors of this work with full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

1.
Zwick
RK
,
Guerrero-Juarez
CF
,
Horsley
V
,
Plikus
MV
.
Anatomical, physiological, and functional diversity of adipose tissue
.
Cell Metab
2018
;
27
:
68
83
2.
Harms
M
,
Seale
P
.
Brown and beige fat: development, function and therapeutic potential
.
Nat Med
2013
;
19
:
1252
1263
3.
Tseng
YH
,
Cypess
AM
,
Kahn
CR
.
Cellular bioenergetics as a target for obesity therapy
.
Nat Rev Drug Discov
2010
;
9
:
465
482
4.
Li
J
,
Huang
J
,
Li
JS
,
Chen
H
,
Huang
K
,
Zheng
L
.
Accumulation of endoplasmic reticulum stress and lipogenesis in the liver through generational effects of high fat diets
.
J Hepatol
2012
;
56
:
900
907
5.
Ding
Y
,
Li
J
,
Liu
S
, et al
.
DNA hypomethylation of inflammation-associated genes in adipose tissue of female mice after multigenerational high fat diet feeding
.
Int J Obes
2014
;
38
:
198
204
6.
Fujiki
K
,
Kano
F
,
Shiota
K
,
Murata
M
.
Expression of the peroxisome proliferator activated receptor gamma gene is repressed by DNA methylation in visceral adipose tissue of mouse models of diabetes
.
BMC Biol
2009
;
7
:
38
7.
Kamei
Y
,
Suganami
T
,
Ehara
T
, et al
.
Increased expression of DNA methyltransferase 3a in obese adipose tissue: studies with transgenic mice
.
Obesity (Silver Spring)
2010
;
18
:
314
321
8.
Meehan
RR
,
Lewis
JD
,
Bird
AP
.
Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA
.
Nucleic Acids Res
1992
;
20
:
5085
5092
9.
Bird
A
.
DNA methylation patterns and epigenetic memory
.
Genes Dev
2002
;
16
:
6
21
10.
Chahrour
M
,
Jung
SY
,
Shaw
C
, et al
.
MeCP2, a key contributor to neurological disease, activates and represses transcription
.
Science
2008
;
320
:
1224
1229
11.
Amir
RE
,
Van den Veyver
IB
,
Wan
M
,
Tran
CQ
,
Francke
U
,
Zoghbi
HY
.
Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2
.
Nat Genet
1999
;
23
:
185
188
12.
Lyst
MJ
,
Bird
A
.
Rett syndrome: a complex disorder with simple roots
.
Nat Rev Genet
2015
;
16
:
261
275
13.
Wolf
Y
,
Boura-Halfon
S
,
Cortese
N
, et al
.
Brown-adipose-tissue macrophages control tissue innervation and homeostatic energy expenditure
.
Nat Immunol
2017
;
18
:
665
674
14.
Kyle
SM
,
Saha
PK
,
Brown
HM
,
Chan
LC
,
Justice
MJ
.
MeCP2 co-ordinates liver lipid metabolism with the NCoR1/HDAC3 corepressor complex
.
Hum Mol Genet
2016
;
25
:
3029
3041
15.
Mayer
SC
,
Gilsbach
R
,
Preissl
S
, et al
.
Adrenergic repression of the epigenetic reader MeCP2 facilitates cardiac adaptation in chronic heart failure
.
Circ Res
2015
;
117
:
622
633
16.
Chen
H
,
Wan
D
,
Wang
L
, et al
.
Apelin protects against acute renal injury by inhibiting TGF-β1
.
Biochim Biophys Acta
2015
;
1852
:
1278
1287
17.
Chen
H
,
Huang
Y
,
Zhu
X
, et al
.
Histone demethylase UTX is a therapeutic target for diabetic kidney disease
.
J Physiol
2019
;
597
:
1643
1660
18.
Ohno
H
,
Shinoda
K
,
Ohyama
K
,
Sharp
LZ
,
Kajimura
S
.
EHMT1 controls brown adipose cell fate and thermogenesis through the PRDM16 complex
.
Nature
2013
;
504
:
163
167
19.
Liu
X
,
Zhou
Y
,
Liu
X
, et al
.
MPHOSPH1: a potential therapeutic target for hepatocellular carcinoma
.
Cancer Res
2014
;
74
:
6623
6634
20.
Wang
W
,
Wang
Q
,
Wan
D
, et al
.
Histone HIST1H1C/H1.2 regulates autophagy in the development of diabetic retinopathy
.
Autophagy
2017
;
13
:
941
954
21.
Hsu
WC
,
Araneta
MR
,
Kanaya
AM
,
Chiang
JL
,
Fujimoto
W
.
BMI cut points to identify at-risk Asian Americans for type 2 diabetes screening
.
Diabetes Care
2015
;
38
:
150
158
22.
WHO Expert Consultation
.
Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies
[published correction appears in Lancet 2004;363:902]
.
Lancet
2004
;
363
:
157
163
23.
Xue
W
,
Huang
J
,
Chen
H
, et al
.
Histone methyltransferase G9a modulates hepatic insulin signaling via regulating HMGA1
.
Biochim Biophys Acta Mol Basis Dis
2018
;
1864
:
338
346
24.
Zhang
Y
,
Xue
W
,
Zhang
W
, et al
.
Histone methyltransferase G9a protects against acute liver injury through GSTP1
.
Cell Death Differ
.
12 September 2019 [Epub ahead of print]. DOI: 10.1038/s41418-019-0412-8
.
25.
Yao
L
,
Cui
X
,
Chen
Q
, et al
.
Cold-inducible SIRT6 regulates thermogenesis of brown and beige fat
.
Cell Rep
2017
;
20
:
641
654
26.
Wan
D
,
Liu
C
,
Sun
Y
,
Wang
W
,
Huang
K
,
Zheng
L
.
MacroH2A1.1 cooperates with EZH2 to promote adipogenesis by regulating Wnt signaling
.
J Mol Cell Biol
2017
;
9
:
325
337
27.
Zhang
Y
,
Guo
X
,
Yan
W
, et al
.
ANGPTL8 negatively regulates NF-κB activation by facilitating selective autophagic degradation of IKKγ
.
Nat Commun
2017
;
8
:
2164
28.
Chen
H
,
Li
J
,
Jiao
L
, et al
.
Apelin inhibits the development of diabetic nephropathy by regulating histone acetylation in Akita mouse
.
J Physiol
2014
;
592
:
505
521
29.
Cai
R
,
Xue
W
,
Liu
S
,
Petersen
RB
,
Huang
K
,
Zheng
L
.
Overexpression of glyceraldehyde 3-phosphate dehydrogenase prevents neurovascular degeneration after retinal injury
.
FASEB J
2015
;
29
:
2749
2758
30.
Cristancho
AG
,
Lazar
MA
.
Forming functional fat: a growing understanding of adipocyte differentiation
.
Nat Rev Mol Cell Biol
2011
;
12
:
722
734
31.
Lee
KY
,
Russell
SJ
,
Ussar
S
, et al
.
Lessons on conditional gene targeting in mouse adipose tissue
.
Diabetes
2013
;
62
:
864
874
32.
Lewis
JD
,
Meehan
RR
,
Henzel
WJ
, et al
.
Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA
.
Cell
1992
;
69
:
905
914
33.
McNelis
JC
,
Olefsky
JM
.
Macrophages, immunity, and metabolic disease
.
Immunity
2014
;
41
:
36
48
34.
Finck
BN
,
Kelly
DP
.
PGC-1 coactivators: inducible regulators of energy metabolism in health and disease
.
J Clin Invest
2006
;
116
:
615
622
35.
Nott
A
,
Cheng
J
,
Gao
F
, et al
.
Histone deacetylase 3 associates with MeCP2 to regulate FOXO and social behavior
.
Nat Neurosci
2016
;
19
:
1497
1505
36.
Bienvenu
T
,
Chelly
J
.
Molecular genetics of Rett syndrome: when DNA methylation goes unrecognized
.
Nat Rev Genet
2006
;
7
:
415
426
37.
Carney
RM
,
Wolpert
CM
,
Ravan
SA
, et al
.
Identification of MeCP2 mutations in a series of females with autistic disorder
.
Pediatr Neurol
2003
;
28
:
205
211
38.
Justice
MJ
,
Buchovecky
CM
,
Kyle
SM
,
Djukic
A
.
A role for metabolism in Rett syndrome pathogenesis: new clinical findings and potential treatment targets
.
Rare Dis
2013
;
1
:
e27265
39.
Buchovecky
CM
,
Turley
SD
,
Brown
HM
, et al
.
A suppressor screen in Mecp2 mutant mice implicates cholesterol metabolism in Rett syndrome
.
Nat Genet
2013
;
45
:
1013
1020
40.
Guy
J
,
Hendrich
B
,
Holmes
M
,
Martin
JE
,
Bird
A
.
A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome
.
Nat Genet
2001
;
27
:
322
326
41.
Chen
RZ
,
Akbarian
S
,
Tudor
M
,
Jaenisch
R
.
Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice
.
Nat Genet
2001
;
27
:
327
331
42.
Baker
SA
,
Chen
L
,
Wilkins
AD
,
Yu
P
,
Lichtarge
O
,
Zoghbi
HY
.
An AT-hook domain in MeCP2 determines the clinical course of Rett syndrome and related disorders
.
Cell
2013
;
152
:
984
996
43.
Fyffe
SL
,
Neul
JL
,
Samaco
RC
, et al
.
Deletion of Mecp2 in Sim1-expressing neurons reveals a critical role for MeCP2 in feeding behavior, aggression, and the response to stress
.
Neuron
2008
;
59
:
947
958
44.
Wang
X
,
Lacza
Z
,
Sun
YE
,
Han
W
.
Leptin resistance and obesity in mice with deletion of methyl-CpG-binding protein 2 (MeCP2) in hypothalamic pro-opiomelanocortin (POMC) neurons
.
Diabetologia
2014
;
57
:
236
245
45.
Lodhi
IJ
,
Yin
L
,
Jensen-Urstad
AP
, et al
.
Inhibiting adipose tissue lipogenesis reprograms thermogenesis and PPARγ activation to decrease diet-induced obesity
.
Cell Metab
2012
;
16
:
189
201
46.
Majchrzak-Gorecka
M
,
Majewski
P
,
Grygier
B
,
Murzyn
K
,
Cichy
J
.
Secretory leukocyte protease inhibitor (SLPI), a multifunctional protein in the host defense response
.
Cytokine Growth Factor Rev
2016
;
28
:
79
93
47.
Hoggard
N
,
Cruickshank
M
,
Moar
KM
,
Bashir
S
,
Mayer
CD
.
Using gene expression to predict differences in the secretome of human omental vs. subcutaneous adipose tissue
.
Obesity (Silver Spring)
2012
;
20
:
1158
1167
48.
Adapala
VJ
,
Buhman
KK
,
Ajuwon
KM
.
Novel anti-inflammatory role of SLPI in adipose tissue and its regulation by high fat diet
.
J Inflamm (Lond)
2011
;
8
:
5
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Supplementary data