Fat-Specific Knockout of Mecp2 Upregulates Slpi to Reduce Obesity by Enhancing Browning

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 (13–15). 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 (Mecp2^Adi 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.

Mouse strains, loxP-flanked Mecp2^flox/y (male), Mecp2^flox/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.

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).

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 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.

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).

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

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.

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 Mecp2^flox/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 (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).

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

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.

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

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).

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).

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 Mecp2^flox/+ 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 Mecp2^flox/y (WT) and Mecp2^flox/yadiponectin-Cre (Mecp2^Adi 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 Mecp2^Adi KO mice (Fig. 2A–D). Compared with WT mice, NC-fed Mecp2^Adi 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 Mecp2^Adi KO mice (Fig. 2M). However, WT and Mecp2^Adi 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).

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 Mecp2^Adi 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 Mecp2^Adi 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).

Consistent with RNA-sequencing results, qPCR verified these changes in iWAT and BAT of Mecp2^Adi 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 Mecp2^Adi 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.

Altered mRNA and protein levels of lipogenic and thermogenic factors in Mecp2^Adi KO mice enable us to hypothesize that Mecp2 deficiency in adipocytes may attenuate obesity. To test this possibility, we fed WT and Mecp2^Adi KO mice with an HFD for 12 weeks. Compared with HFD-fed WT mice, HFD-fed Mecp2^Adi 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 Mecp2^Adi 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 Mecp2^Adi 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 Mecp2^Adi 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 Mecp2^Adi KO mice (Fig. 4L and M). Moreover, HFD-fed Mecp2^Adi 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).

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 Mecp2^Adi KO and WT mice, except for downregulated Tnf in HFD-fed Mecp2^Adi 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 Mecp2^Adi KO mice (Supplementary Fig. 3).

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 Mecp2^flox/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).

Dramatically upregulated thermogenic genes in iWAT of Mecp2^Adi KO mice (Figs. 2 and 3) inspired us to challenge Mecp2^Adi 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 Mecp2^Adi 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 Mecp2^Adi KO mice under a β-NE challenge (Fig. 5B and C). Similar results were found in iWAT of Mecp2^Adi KO mice under cold or thermoneutral conditions (Fig. 5D–G). Comparable mRNA levels of Pgc1a were found in WAT of WT and Mecp2^Adi KO mice under a β-NE challenge and cold stress (Supplementary Fig. 5).

To identify downstream factors that may be responsible for the increased browning phenotype in Mecp2^Adi KO mice, a highly upregulated gene in iWAT of Mecp2^Adi 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 Mecp2^Adi KO mice were observed (Fig. 6B and C and Supplementary Fig. 6C). Upregulated Slpi on adipocytes in iWAT of the Mecp2^Adi KO mice were also found (Supplementary Fig. 6D). Moreover, upregulated Slpi in iWAT of the Mecp2^Adi 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.

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 Mecp2^Adi 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 Mecp2^Adi 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).

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 Mecp2^Adi 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).

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).

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 (40–42), 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 Mecp2^Adi 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 Mecp2^Adi KO mice under HFD, β-NE, or cold stress. Mecp2^Adi KO mice are sensitive to stimuli-induced browning possibly for at least two reasons. Dramatically reduced Fasn in the adipose tissues of Mecp2^Adi 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 Mecp2^Adi KO mice (Figs. 3–5 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 Mecp2^Adi 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 Mecp2^Adi KO mice, which was also significantly higher than that of the SVF parts isolated from WT or Mecp2^Adi KO mice. An increased protein level of Slpi was also found in adipocytes of Mecp2^Adi 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 Mecp2^Adi 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.