Leptin, an anorexigenic hormone in the hypothalamus, suppresses food intake and increases energy expenditure. Failure to respond to leptin will lead to obesity. Here, we discovered that nuclear receptor Nur77 expression is lower in the hypothalamus of obese mice compared with normal mice. Injection of leptin results in significant reduction in body weight in wild-type mice but not in Nur77 knockout (KO) littermates or mice with specific Nur77 knockdown in the hypothalamus. Hypothalamic Nur77 not only participates in leptin central control of food intake but also expands leptin’s reach to liver and adipose tissues to regulate lipid metabolism. Nur77 facilitates signal transducer and activator of transcription 3 (STAT3) acetylation by recruiting acetylase p300 and disassociating deacetylase histone deacetylase 1 (HDAC1) to enhance the transcriptional activity of STAT3 and consequently modulates the expression of downstream gene Pomc in the hypothalamus. Nur77 deficiency compromises response to leptin in mice fed a high-fat diet. Severe leptin resistance in Nur77 KO mice with increased appetite, lower energy expenditure, and hyperleptinemia contributes to aging-induced obesity. Our study opens a new avenue for regulating metabolism with Nur77 as the positive modulator in the leptin-driven antiobesity in the hypothalamus.
Introduction
The hypothalamus is the major center for regulating energy homeostasis by sensing and responding to input from hormonal and nutrient-related signals (1). Among various signals sensed by the hypothalamus, leptin is an important hormone derived from adipocytes with a key role in representing nutritional status to the hypothalamus for the regulation of energy balance (2). This regulation is mostly mediated by several well-structured nuclei in the hypothalamus, especially in arcuate nucleus (ARC) (3). Two distinct populations of neurons in ARC, proopiomelanocortin (Pomc) neurons and agouti-related protein (AgRp) neurons, are regulated by leptin in completely different fashions. Leptin suppresses the expression of orexigenic neuropeptide AgRp and neuropeptide Y (NPY) in AgRp neurons but upregulates the expression of anorexigenic neuropeptide Pomc in Pomc neurons (3). Leptin’s function in these neurons is critical to normal food intake and body weight, and its dysregulation would break energy homeostasis. Leptin signaling ablation from the mutations of db or ob genes in mice would result in severe obesity and diabetes (4). Administration of leptin greatly reduces body weight and food intake in normal mice but not in obese mice. This phenomenon, together with higher circulating leptin level in the obese, is the so-called leptin resistance.
Signal transducer and activator of transcription 3 (STAT3) is an important downstream component in leptin signaling on energy balance for the regulation of body weight and food intake (5). Mouse models with neural deletion of STAT3 develop hyperphagia and obesity, as well as severe diabetes (6). As Tyr1138 of OB-Rb (the long form of leptin receptor) is specific for STAT3 binding, mice with this point mutation show a phenotype similar to that of db/db mice by failing to activate the leptin-STAT3 pathway (7). Constitutively expressing active STAT3 in AgRp neurons elevates locomotor activity of mice and protects mice from diet-induced obesity (8). Upon leptin binding to its receptor OB-Rb, Janus kinase 2 is activated to phosphorylate Tyr705 of STAT3, which subsequently dimerizes and translocates into the nucleus to regulate the transcription and expression of downstream genes. Besides phosphorylation, STAT3 acetylation also regulates the physiological function of STAT3 and is critical to its transcriptional activity (9,10). Therefore, impairment of STAT3 modification may lead to different biological functions.
Orphan nuclear receptor Nur77 (also called TR3) is a member of the nuclear hormone receptor 4A subgroup and has emerged as an important regulator of metabolism in different tissues (11). Hepatic overexpression of Nur77 modulates the lipid content through downregulating expression of SREBP1c, which then controls the downstream genes in lipid and cholesterol metabolism (12). In skeletal muscle, Nur77 promotes lipolysis and modulates expression of genes in lipid, carbohydrate, and energy metabolism (13). To date, however, the direct Nur77 role in hypothalamus-controlled weight balance has not been established. This study demonstrated that hypothalamic Nur77 participates in the leptin-associated reduction in body weight and food intake. Nur77 enhances STAT3 transcriptional activity via acetylation to regulate the downstream gene expression in the hypothalamus. Furthermore, Nur77 expands leptin regulatory function to lipid metabolism in liver and adipose tissues. It implicates Nur77 as a positive regulator in the leptin-induced STAT3 signaling pathway and provides a novel target for combating leptin-resistant obesity.
Research Design and Methods
Cell Culture and Transient Transfection
Human embryonic kidney (HEK) 293T cell lines were obtained from the American Type Culture Collection. Mouse pituitary tumor cell line AtT-20 was purchased from the Institute of Cell Biology (China). GT1-7 cells were a gift from Professor Xiaoying Li (Institute of Endocrinology and Metabolism, Shanghai, China). Cells were cultured in DMEM medium (for HEK293T and GT1-7 cells) and RPMI 1640 medium (for AtT-20 cells) with 10% FBS (GIBCO), 100 units/mL penicillin, and 10 mg/mL streptomycin. Transfection was performed by using a TurboFect kit (Thermo Scientific). Before leptin treatment, AtT-20 and GT1-7 cells were serum starved overnight, and GT1-7 cells were further subjected to 1 mmol/L glucose DMEM for 6 h.
Plasmid Constructions
The mouse pGL3-Pomc construct was purchased from Addgene. Different mutants of STAT3 and pGL3-Pomc were generated by using the QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA), and the primers used are indicated in Supplementary Table 1.
Mouse Models and Leptin Treatment
All animal experiments were approved by the Animal Ethics Committee of Xiamen University (accepted no. XMULAC20120030). Wild-type and Nur77 knockout (KO) mice with C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). Leptin receptor–mutated db/db mice, leptin-deficient ob/ob mice, and their corresponding normal mice with C57BL/6 background were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China).
In leptin sensitivity detection, by intraperitoneal injection, 12-week-old male mice with similar body weight were injected with saline for three consecutive days and subsequently with leptin (0.5 μg/g; R&D Systems), twice daily at fixed times (9:00 a.m. and 5:00 p.m.), for seven consecutive days. By intracerebroventricular injection, lateral ventricle cannulations were first done. Mice were anesthetized with 1% pentobarbital and a sterile guide cannula stereotaxically inserted to the right lateral ventricle (0.2 mm posterior, 1 mm lateral relative to bregma, and 2 mm depth) and then embedded in dental cement. Mice were housed individually for 4–5 days’ recovery and injected with leptin (1 μg) daily at 9:00 a.m. for 6 days.
Indirect Calorimetry and Locomotor Activity Measurements
Mice were single housed and acclimatized to the respiratory chambers for 48 h. Indirect calorimetry and locomotor activity were measured using a TSE LabMaster system for 1 week. The indirect calorimetry was normalized to body weight raised by allometric scaling exponent of 0.75 (14).
Recombinant Adenovirus and Lentivirus Hypothalamic Injection
Adenovirus vector pAdEasy (GFP) was used to generate recombinant adenoviruses. Fourteen days after linearized adenovirus DNA transfection, HEK293 cells were collected and subjected to four freeze-thaw-vortex cycles. Finally, recombinant adenovirus was stepwise amplified and purified by cesium chloride gradient centrifugation and concentrated to 1012 viral particles/mL.
The lentivirus system was generated as previously described (15). The knockdown efficiency was determined by Western blot or real-time PCR. The oligonucleotide sequences for short hairpin RNA (shRNA) are provided in Supplementary Table 2.
The adenoviruses or lentivirus was separately injected bilaterally into the mediobasal hypothalamus (1.4 mm posterior, 0.5 mm lateral to bregma, and 5.5 mm depth). After adenovirus injection, mice were recovered for 7 days, and then a long-time intraperitoneal leptin injection was performed. After lentivirus injection, body weight and food intake of mice were recorded.
Sequential ChIP
Sequential ChIP (SeqChIP) was performed as previously describe (16). IgG antibody was used as a negative control. Both first and second eluate was purified by a DNA purification kit (Axygen, Inc., Shanghai, China) and subjected to PCR. Primers are provided in Supplementary Table 2.
Measure of Metabolic Factors
Serum leptin level was estimated by a mouse Leptin ELISA kit (Crystal Chem Inc.). Serum total cholesterol and triglyceride levels were measured by total cholesterol kits and triglyceride reagent (Biosino Bio-Technology & Science Inc., Beijing, China). Nonesterified fatty acid (NEFA) was tested with a Wako LabAssay NEFA kit (Wako, Osaka, Japan).
Luciferase Assay
Cells were transfected with required plasmids and then treated with leptin (0.5 μg/mL, 6 h). Luciferase assay was performed as described previously (17). The reporter gene APRE, a response element of STAT3, was used to assess the STAT3 transcriptional activity. The reporter gene pGL3-Pomc carrying the Pomc promoter (−646 to 65) was used to test the Pomc transcriptional activity.
Real-Time PCR
Real-time PCR was performed as previously described (18). The different primers are indicated in Supplementary Table 3.
Coimmunoprecipitation and Western Blot Analysis
Coimmunoprecipitation (Co-IP) and Western blot analysis were performed as previously described (19). The different primary antibodies are indicated in Supplementary Table 4.
Histological Analysis
Mice were perfused with PBS, followed by 4% paraformaldehyde. Tissues were isolated and fixed overnight at 4°C, treated with 30% sucrose for cryoprotection, and embedded in optimum cutting temperature compound. Sections of liver were stained with Oil Red O for the determination of lipid content. For immunofluorescent staining, sections were incubated with anti-Nur77 rabbit antibody (LifeSpan Biosciences) overnight at 4°C followed by Alexa 488-conjugated donkey anti-rabbit antibody (Invitrogen) for 1 h at room temperature. The section was stained with DAPI (50 μg/mL) for 5 min. For in situ hybridization, the anti-sense riboprobe was transcribed from Pomc (base 331–1017) into digoxigenin-labeled RNA, using the DIG RNA Labeling Kit (Roche). Hybridization was carried out with 2 ng/μL digoxigenin-labeled Pomc riboprobe. The mRNA of Pomc was visualized by using BCIP and NBT.
Statistical Analyses
Data are expressed as means with SEM. Statistical significance was determined with a two-tailed Student t test or one-way ANOVA followed by Tukey post hoc test. P < 0.05 is considered significant.
Results
Nur77 Is Linked to Leptin-Controlled Obesity and Lipid Metabolism
Despite the fact that Nur77 has been reported to play important roles in metabolism, there has been no in-depth investigation on its regulatory function for body weight and energy homeostasis in hypothalamus to date. Analysis of samples from hypothalamus showed a significant decrease of Nur77 expression in both mRNA and protein level in obese db/db and ob/ob mice compared with corresponding normal mice (Fig. 1A). Immunofluorescent staining also revealed a distinct reduction of Nur77 expression in ARC of ob/ob mice (Fig. 1B). Since leptin is the predominant factor in controlling energy balance in hypothalamus (20), there could be a direct link between hypothalamic Nur77 and leptin-controlled energy homeostasis. To this end, 12-week-old WT and Nur77 KO mice with similar body weight before the onset of obesity were administrated with leptin. The body weight and food intake were significantly reduced after leptin administration for the WT mice, but such reduction was significantly attenuated for the KO littermates (Fig. 1C). These results suggest that Nur77, presumably in the hypothalamus, might take part in the leptin-controlled body weight.
The body weight loss was correlated to the significant reduction in weight and size of the white adipose tissue (WAT) in WT mice compared with Nur77 KO mice with leptin administration (Fig. 1D). Leptin treatment led to smaller adipocytes for WT mice but not for KO mice (Fig. 1E). Detection of a series of relevant gene products supports the role of Nur77 on lipid metabolism. In WAT, the gene expressions for lipid synthesis were decreased in leptin-treated WT mice (such as SREBP1c, FASN, ACC, and SCD1); in contrast, there was no obvious change in KO mice. Conversely, the mRNA expressions in lipid oxidation and lipolysis (such as PGC1α, CPT1, and HSL) were significantly increased in leptin-treated WT mice compared with the Nur77 KO mice (Fig. 1F). These results indicate the association of Nur77 with leptin-induced inhibition of lipid synthesis as well as enhancement of lipid oxidation and lipolysis in adipose tissue.
Although no obvious difference was detected in liver weight between WT and Nur77 KO mice, there is a significant decrease in lipid content in WT mice compared with the KO mice with the leptin treatment (Fig. 1G). Similarly, leptin also significantly affected the gene expression of those key metabolic enzymes in the liver of WT mice but not in that of KO mice (Fig. 1F). Clearly, the leptin-controlled lipid metabolism in liver is also impaired with Nur77 deficiency.
For better evaluation of the regulation of hypothalamic Nur77 on leptin signaling for body weight and lipid metabolism in liver and WAT, intracerebroventricular injection of leptin was carried out to complement the intraperitoneal injection study. Long-term intracerebroventricular administration of leptin triggered more pronounced body weight loss and food intake reduction in WT mice (Supplementary Fig. 1A). Although both mouse strains exhibited similar liver weight after leptin treatment, the attenuation of WAT weight loss in Nur77 KO mice was still observed (Supplementary Fig. 1B). Consistently, the modulation of lipid- and liver metabolism–related genes by leptin was also compromised by the deletion of Nur77 (Supplementary Fig. 1C). A series of results further implicates Nur77 as a crucial factor in leptin control of body weight and lipid metabolism in peripheral tissues.
Knockdown of Nur77 in Hypothalamus Directly Affects Leptin Function
To establish a direct link between Nur77 in hypothalamus and leptin functions on body weight loss and lipid metabolism of WAT and liver, we specifically knocked down Nur77 in hypothalamus using adenovirus-based RNA interference technique. Bilateral injection of adenovirus-packed Nur77 shRNA to the mediobasal hypothalamus resulted in an obvious decrease in Nur77 expression in hypothalamus but not in any other tissues, including WAT, muscle, and liver (Supplementary Fig. 2A). After leptin treatment, control mice injected with adenovirus-packed scramble shRNA (termed Ctrl) were of less body weight and food intake than the Nur77 knockdown mice (termed KD) (Fig. 2A), which suggests a decreased hypothalamic response to the leptin sensitivity in KD mice. Similar to the Nur77 KO mice, the leptin-induced reduction of WAT mass was alleviated in the KD mice with similar liver mass (Fig. 2B). Moreover, the expression of genes involved in lipid metabolism followed a similar trend in WAT and liver of the control and KD mice (Fig. 2C) compared with WT and KO mice under leptin treatment (Fig. 1F). It is the hypothalamic Nur77 that participates in the regulatory pathway of leptin to modulate the body weight and lipid metabolism.
Nur77 Negatively Regulates High-Fat Diet–Induced Leptin Resistance
Feeding mice with a high-fat diet (HFD) would result in chronic leptin resistance (21). WT and Nur77 KO mice were challenged with 60% HFD for 2 months, which resulted in a significant increase in the body weight of KO mice (Fig. 3A) accompanied by the markedly higher serum leptin level in KO mice (Fig. 3B). These results indicate an enhanced susceptibility to diet-induced obesity and severe leptin resistance due to Nur77 deficiency. Injection of leptin to HFD mice failed to influence the body weight of Nur77 KO mice, indicating that these mice had become insensitive to leptin (Fig. 3C). In contrast, the body weight of HFD WT mice decreased with leptin treatment with considerably diminished WAT (Fig. 3D and E). In addition, the lipid content in the liver was reduced in WT mice but not in Nur77 KO mice, despite the fact that the liver weight was not significantly changed (Supplementary Fig. 3A). The data that leptin attenuated lipid synthesis and promoted lipid oxidation and lipolysis in WAT and liver of HFD WT mice but not Nur77 KO mice (Fig. 3F) further corroborate the notion that the inhibition of Nur77 would lead to leptin resistance and obesity.
Nur77 Activates STAT3 Transcriptional Activity Through an Acetylation Pathway
STAT3 is a critical component for leptin-activated signal pathways in the hypothalamus (22). Not surprisingly, STAT3 gene expression was much higher in the hypothalamus of normal mice than that in db/db and ob/ob mice (Fig. 4A). STAT3 gene expression tendency in the hypothalamus of different mouse models was similar to Nur77 expression (Fig. 1A), suggesting a correlation between STAT3 and Nur77. Nur77 could interact with STAT3 in HEK293T cells that were cotransfected with Myc-Nur77 and Flag-STAT3 (Supplementary Fig. 4A), and in the hypothalamus of WT mice in which the in vivo Nur77-STAT3 interaction was enhanced by leptin (Supplementary Fig. 4A). Leptin also induced the interaction between Nur77 and STAT3 in GT1-7 cells, an immortalized neuronal cell line derived from the mouse hypothalamus (Supplementary Fig. 4A). These results suggest a leptin-associated regulation of Nur77-STAT3 interaction.
Although the expression levels of STAT3 protein were about the same in hypothalamus of both WT and Nur77 KO mice, leptin administration significantly increased STAT3 acetylation only in the hypothalamus of WT mice (Fig. 4B). The level of elevated phosphorylated STAT3 was about the same in the hypothalamus of both WT and KO mice after leptin treatment. Leptin treatment also elevated STAT3 acetylation in hypothalamus of WT mice fed with HFD (Fig. 4B). Consistent with the observation in hypothalamus, both GT1-7 cells and Pomc-producing pituitary-derived AtT-20 cells displayed pronounced leptin-induced STAT3 acetylation when Nur77 was overexpressed (Supplementary Fig. 4B). In contrast, leptin lost its ability to enhance STAT3 acetylation with the knockdown of endogenous Nur77 (termed Nur77-KD) in cell lines by lentivirus-based RNA interference (Supplementary Fig. 4B). Furthermore, luciferase assays showed that overexpression of Nur77 significantly strengthened leptin-induced APRE (a response element of STAT3) reporter activity in both GT1-7 and AtT-20 cells, while STAT3 transcriptional activity was obviously decreased in these two Nur77-KD cell lines even in the presence of leptin (Fig. 4C). It is likely that Nur77 is a novel factor to enhance STAT3 activity through the regulation of STAT3 acetylation.
Since Lys49, Lys87, and Lys685 of STAT3 are critical for its acetylation (9,10), these sites are mutated for investigation. 2KR is a mutant with double point mutation at Lys49 and Lys87, and nonacetylation mutant (3KR) is of triple point mutation at Lys49, Lys87, and Lys685. These mutations did not interfere with the STAT3 interactions with Nur77 (Fig. 4D). However, 3KR completely and 2KR mutant partially abolished the Nur77’s ability to activate APRE reporter activity induced by leptin in both AtT-20 and GT1-7 cells (Fig. 4E). Thus, these Lys sites are all critical for Nur77 function in elevating leptin-induced STAT3 transcriptional activity. For verification that Nur77-regulated acetylation of these sites in STAT3 would lead to mice resistance to obesity, 3KR mutant and acetylation-mimicking mutant (3KQ) of STAT3 were separately introduced into hypothalamus using lentivirus (Fig. 4F). Although at the first day the body weight and food intake were pronounced decreased in all strains of mice, due to the surgery, mice expressing 3KQ maintained lower body weight and food intake than those expressing 3KR afterward (Fig. 4F). Clearly, STAT3 acetylation contributes to control of body weight and food intake in mice. Since these three acetylation-related residues are responsible for Nur77 regulation of STAT3 function, the enhancement of leptin-induced STAT3 acetylation by Nur77 might contribute to the leptin’s effect on body weight control.
Leptin signaling begins with leptin binding to its receptor OB-Rb, followed by STAT3 activation (2). We tested whether Nur77-elevated and leptin-induced STAT3 acetylation is associated with OB-Rb expression. In comparison of WT and Nur77 KO mice, OB-Rb expression in hypothalamus was not changed (Supplementary Fig. 4C), indicating that Nur77 does not influence OB-Rb expression. The role of Nur77-associated STAT3 acetylation on recruitment of cofactors was further investigated. In GT1-7 and AtT-20 cells, p300 (acetylase) positively and HDAC1 (deacytalase) negatively modulates the STAT3 transcriptional activity under leptin stimulation (Supplementary Fig. 4D). Knockdown of Nur77 abolished p300-induced APRE reporter activity, whereas overexpression of Nur77 diminished HDAC1-inhibited APRE reporter activity even in the presence of leptin (Supplementary Fig. 4E). These results suggest that both p300 and HDAC1 engage in Nur77 regulation of STAT3 transcriptional activity. Moreover, overexpression of Nur77 enhanced the p300-STAT3 interaction but decreased the HDAC1-STAT3 interaction (Fig. 4G). Therefore, Nur77 enhanced STAT3 acetylation through recruiting acetylase p300 to and disassociating deacetylase HDAC1 from STAT3.
Nur77 and STAT3 Cooperatively Stimulate the Activity of Pomc Promoter
Leptin-stimulated reduction of body weight and food intake has been reported to be through enhancing Pomc and suppressing AgRp and NPY gene expressions (20). In the hypothalamus, these leptin-regulated gene expressions can be shown to be Nur77 dependent. After treatment of the fasting mice with leptin, the expression of the Pomc gene was elevated, while the expression of AgRp and NPY genes were repressed in WT mice but not in Nur77 KO mice (Fig. 5A and Supplementary Fig. 5A). An induction of leptin on Pomc gene expression in the ARC of hypothalamus, detected by in situ hybridization (ISH) assay, was also observed in WT mice compared with the KO mice (Fig. 5A). Moreover, overexpression of Nur77 significantly enhanced leptin-induced Pomc promoter activity (Fig. 5B), whereas leptin-elevated Pomc promoter activity was decreased when Nur77 was knocked down (Fig. 5B). When STAT3 was present, leptin-stimulated Pomc promoter activity could be detected regardless of whether Nur77 was transfected or knocked down (Fig. 5B). However, once STAT3 was knocked down, leptin lost the ability to activate Pomc even with the transfection of Nur77 (Fig. 5B). These results not only demonstrate STAT3’s dominant role in response to leptin induction but also support that Nur77 is a cofactor for STAT3 in a leptin-stimulated signaling pathway. That the Nur77-associated activation of leptin-induced Pomc promoter was blocked only by 3KR and not any other mutants (Fig. 5C) is another indication that this Nur77 activity is through the STAT3 acetylation pathway.
Two typical Nur77 binding sites are located separately in a proximal NGFI-B response element (NBRE) site (−70 to −63 for monomeric Nur77 binding) and a distal Nur response element (NurRE) site (−378 to −357 for homodimeric/heterodimeric Nur77 binding) (23) (Fig. 5D). For determination of the mechanism of Nur77-enhanced leptin activation of the Pomc promoter, the NBRE and NurRE were separately mutated (termed NBRE mut and NurRE mut). The mutation of NurRE not only compromised the Nur77-associated Pomc promoter activity (Fig. 5D) but also attenuated the STAT3 activation of the Pomc promoter activity (Fig. 5D). In contrast, a similar phenomenon was not observed with NBRE mut. However, a mutation at the STAT3 binding site (termed STAT3 response element [STRE] mut), which overlaps with NurRE, could impair neither STAT3- nor Nur77-stimulated Pomc promoter activity (Fig. 5D). It can also be demonstrated that the specificity protein 1 (SP-1) binding site (SP-1 response element [SPRE]) to which STAT3 is recruited was critical for response to leptin function, as mutation at SPRE (termed SPRE mut) completely blocked leptin-induced Pomc promoter activity even with the overexpression of STAT3 or Nur77 (Fig. 5D). Together, it is likely that 1) STRE is not required for leptin-induced and STAT3-associated Pomc promoter activity, while SPRE is responsible for a leptin effect, and 2) Nur77-STAT3 may both bind to NurRE to promote Pomc promoter activity.
The above results suggest that Nur77 may form a heterodimer with STAT3 to bind to NurRE in response to leptin. For corroboration of this hypothesis, SeqChIP assays were carried out. In the first round of SeqChIP analysis, leptin dramatically induced the association of STAT3 with both the proximal promoter (containing SPRE) and distal promoter (containing both STRE and NurRE). However, in the second round of SeqChIP, the immunoprecipitates of Nur77 were present only in the distal Pomc promoter (Fig. 5E). Conversely, Nur77 were recruited to the distal Pomc promoter with immunoprecipitation of Nur77 in the first step. When STAT3 was immunoprecipitated in the second round, it was still associated with the distal but not the proximal Pomc promoter (Fig. 5E). Clearly, besides SPRE, which STAT3 alone binds to, the co-occupation of Nur77 and STAT3 on the distal NurRE site of Pomc promoter contributes to the mediation of leptin function in Pomc promoter activity.
Nur77 Deficiency Promotes Leptin Resistance in Age-Induced Obesity
Leptin resistance and associated obesity between WT and Nur77 KO mice were evaluated under a normal physiological condition. In WT mice fed a normal diet, body weight was indistinguishable from the corresponding KO littermates in the first 15 weeks of age. The body weight of KO mice started to overtake that of the WT mice in the 16th week and became significantly higher in the 40th week (Fig. 6A). Comparing the ratio of organ-to-body weight showed that the mass of WAT or brown adipose tissue (BAT) was much higher in KO mice at the age of 40 weeks but not 12 weeks, though there was no difference in liver at both ages (Fig. 6B). Furthermore, 40-week-old KO mice had elevated total cholesterol and triglyceride levels compared with the age-matched WT mice, while the NEFA in plasma was higher at both 12 and 40 weeks old (Fig. 6C). This suggests that Nur77 KO mice develop more severe obesity than WT mice in older age.
The aging-induced obesity is accompanied by leptin resistance (24). There is a significant difference in food intake between WT and Nur77 KO mice at the age of 32 and 40 weeks old but not 16 weeks old (Fig. 6A), indicating an attenuated control of appetite by leptin with Nur77 deficiency. Compared with the age-matched WT mice, the KO mice at 40 weeks old had significantly lower energy expenditure, with metabolically active mass-corrected O2 consumption and CO2 production and lower body temperature and locomotor activity (Fig. 6D–F). This difference in energy expenditure was negligible for mice at the age of 12 weeks. In a cold environment, the capacity of WT mice to maintain body temperature was stronger than KO littermates at both 12 and 40 weeks old (Supplementary Fig. 6A). Along with the larger adiposity, KO mice had much higher serum leptin levels at the age of 40 weeks (Supplementary Fig. 6B), which is an indication of leptin resistance (2). In addition, hypothalamic Pomc gene expression was also decreased in 40-week-old KO mice (Supplementary Fig. 6C). Together, this demonstrated that severe leptin resistance in Nur77 KO mice would contribute to the aging-dependent obesity and corroborated the notion that Nur77 plays an important regulatory role in leptin-controlled obesity.
It could also be seen that the weight of BAT increased (Fig. 6B) with the decrease in energy expenditure (Fig. 6D) in KO mice. Histological analysis further showed that brown adipocytes became larger and numbers of multicocular adipocytes were decreased in KO mice (Supplementary Fig. 6D), which suggests that the capacity of BAT to burn lipid is attenuated in KO mice, as multicocular adipocytes are a functional indication of BAT (25). Consistent with the impaired energy expenditure in KO mice, the expressions of thermogenic genes, such as UCP1, UCP2, and UCP3, were all decreased with Nur77 deficiency (Supplementary Fig. 6D). It is likely that the attenuated capacity of BAT to burn lipid in Nur77 KO mice may lead to lipid accumulation and cell enlargement in brown adipocytes.
Discussion
Obesity is one of the most serious health problems and associated with various diseases. Leptin, a key hormone secreted from adipocytes, regulates body weight and energy expenditure to maintain metabolic homeostasis. Obese individuals are typically associated with higher serum leptin levels and leptin resistance. Thus, it is imperative to unravel the mechanism of leptin signaling to combat obesity. In this study, Nur77 is demonstrated to be a positive factor to regulate hypothalamus/leptin-controlled obesity. In hypothalamus, Nur77 elevates STAT3 activity through enhancing its acetylation, thereby regulating the expression of Pomc, a gene downstream of STAT3. These activating events in hypothalamus greatly augment the mouse’s response to leptin, resulting in the increase of lipid oxidation and lipolysis and the decrease of lipid synthesis in adipose and liver, finally controlling body weight (Fig. 6G).
Much evidence supports Nur77’s role in regulating metabolism in a context-dependent manner. Although the roles of Nur77 in controlling the metabolisms of liver (26), muscle (27), and WAT (28) have been revealed, Nur77’s role in the hypothalamus is unknown. Expression level of hypothalamic neuron-derived orphan receptor 1, another family member of Nur77, is reported to be downregulated in obese mice compared with WT mice, which inhibits the levels of AgRp and NPY to impair the feeding behavior and energy balance in mice (29,30). The current study demonstrates that Nur77 is reduced in hypothalamus in both ob/ob and db/db obese mice, suggesting that the hypothalamus may be a new organ for Nur77 to regulate obesity. Knockout or specific knockdown of Nur77 in hypothalamus confirmed this finding, in which impaired leptin-controlled food intake and lipid metabolism were evident. Therefore, Nur77 is a factor in the regulation of obesity by different signaling pathways in different tissues, implicating a fresh therapeutic target to combat obesity.
A direct link between Nur77 deficiency and leptin resistance is demonstrated in our work. Nur77 knockout or a specific knockdown of Nur77 in hypothalamus reduced the leptin sensitivity compared with the corresponding control. Leptin-enhanced STAT3 acetylation was attenuated by the knockdown of Nur77 in either mice or immortalized neuronal cells. Compromised regulation of leptin function by Nur77 in HFD mice, a chronic leptin resistance model, also leads to obesity. Moreover, Nur77-deficient mice develop age-dependent obesity when fed a normal diet. The higher body fat composition, increased serum lipid content, and attenuated energy expenditure were obvious in Nur77 KO mice at the age of 40 weeks but not at the age of 12 weeks. It is an indication of leptin resistance with the higher appetite and serum leptin levels yet reduced hypothalamic Pomc gene expression in those Nur77 KO mice aged 40 weeks. Consequently, these results associate Nur77 with leptin resistance, and it can be argued that Nur77 regulation of leptin sensitivity is one of the important factors in aging-induced obesity.
The binding site that STAT3 is recruited to is dependent on various stimulators (31,32). In contrast to the recruitment of STAT3 to the STRE site under leukemia inhibitory factor stimulation, leptin promotes STAT3 activating Pomc transcription by binding to SPRE (31). Here, STAT3 is not only recruited to SPRE in specific response to the leptin stimulation but also interacts with Nur77 to bind to NurRE on the Pomc promoter, possibly as a heterodimer, to enhance Pomc gene expression. Although there is another STAT3 response element, STRE, which overlaps with NurRE in the Pomc promoter (33), it does not seem to be required for STAT3 binding, as STAT3 can still activate the Pomc activity when STRE is mutated. However, when NurRE in the Pomc promoter is mutated, both Nur77 and STAT3 lose their ability to bind to NurRE and to activate downstream gene transcription activity. Therefore, Nur77, as a necessary cofactor in STAT3 binding to the promoter of the downstream gene, may associate with another mechanism in a leptin-stimulated signaling pathway.
Posttranslational modification is particularly important for STAT3 function. Loss of STAT3 methylation prolongs its phosphorylation and enhances the leptin sensitivity, thus leading to reduced adiposity and obesity resistance (34). Acetylation contributes to the STAT3-suppressed expression of genes relating to gluconeogenesis and peroxisome proliferator–activated receptor γ coactivator 1α, leading to the repression of hepatic glucose production (35). Here, a potential strategy for antiobesity with Nur77 as the positive regulator in leptin-induced STAT3 acetylation to strengthen leptin sensitivity is first demonstrated. With this strategy, the regulatory mechanism of Nur77 for STAT3 acetylation has been revealed with Nur77-enhanced STAT3 acetylation via the recruitment of acetylase p300 and dissociation of deacetylase HDAC1.
Article Information
Acknowledgments. The authors are grateful to Professor Xiaoying Li (Institute of Endocrinology and Metabolism, Ruijin Hospital, Shanghai Jiao Tong University, Shanghai, China) for the GT1-7 cell line. The expressing vector encoding the mouse OB-Rb was kindly provided by Professor Christian Bjorbaek (Harvard Medical School).
Funding. This work was supported by grants from the 973 Program of the Ministry of Science and Technology and the National Natural Science Foundation of China (2011CB910802, 2014CB910602, U1405224, 31221065, and 31230019); the Open Research Fund of State Key Laboratory of Cellular Stress Biology, Xiamen University; and the National Natural Science Foundation of China for Fostering Talents in Basic Research (J1310027).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. Y.C., R.W., H.-z.C., Q.X., W.-j.W., J.-p.H., X.-x.L., and L.L. were responsible for the experiments on molecular cellular biology and mouse detections. X.-w.Y. was responsible for setting up ISH assay. P.W., X.-c.W., and X.-h.T. were responsible for setting up the mouse model of hypothalamus adenovirus injection. S.-j.L. and X.Y. were responsible for setting up the mouse model of intracerebroventricular injection. Q.W. designed the experiments and wrote the manuscript. Q.W. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.