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Research ArticleEndocrinology
Open Access | 
10.1172/JCI178303
1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
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1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
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1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
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1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
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1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
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1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
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1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
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1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
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1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
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1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
Find articles by Wu, W. in: JCI | PubMed | Google Scholar
1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
Find articles by Xu, K. in: JCI | PubMed | Google Scholar
1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
Find articles by Chen, Y. in: JCI | PubMed | Google Scholar
1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
Find articles by Zhang, H. in: JCI | PubMed | Google Scholar
1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
Find articles by Liu, T. in: JCI | PubMed | Google Scholar
1State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai, China.
2Department of Sports Medicine and Arthroscopy Surgery, Huashan Hospital, Fudan University, Shanghai, China.
3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China.
4Shanghai Medical College, Fudan University, Shanghai, China.
5Department of Endocrinology, Jinshan Hospital, Fudan University, Shanghai, China.
6Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai, China.
7School of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China..
Address correspondence to: Xingxing Kong, State Key Laboratory of Genetic Engineering and School of Life Sciences, Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Human Phenome Institute, Fudan University, Shanghai 200438, China. Phone: 86.21.31246753; Email: kongxingxing@fudan.edu.cn.
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
Find articles by Kong, X. in: JCI | PubMed | Google Scholar
Authorship note: HW, SG, HG, JD, and HL contributed equally to this work.
Published June 18, 2024 - More info
Published in Volume 134, Issue 16 on August 15, 2024
AbstractMyostatin (MSTN) has long been recognized as a critical regulator of muscle mass. Recently, there has been increasing interest in its role in metabolism. In our study, we specifically knocked out MSTN in brown adipose tissue (BAT) from mice (MSTNΔUCP1) and found that the mice gained more weight than did controls when fed a high-fat diet, with progressive hepatosteatosis and impaired skeletal muscle activity. RNA-Seq analysis indicated signatures of mitochondrial dysfunction and inflammation in the MSTN-ablated BAT. Further studies demonstrated that Kruppel-like factor 4 (KLF4) was responsible for the metabolic phenotypes observed, whereas fibroblast growth factor 21 (FGF21) contributed to the microenvironment communication between adipocytes and macrophages induced by the loss of MSTN. Moreover, the MSTN/SMAD2/3-p38 signaling pathway mediated the expression of KLF4 and FGF21 in adipocytes. In summary, our findings suggest that brown adipocyte–derived MSTN regulated BAT thermogenesis via autocrine and paracrine effects on adipocytes or macrophages, ultimately regulating systemic energy homeostasis.
IntroductionBrown adipose tissue (BAT) plays a crucial role in whole-body energy balance and fuel metabolism, mediating nonshivering thermogenesis in mammals exposed to subthermoneutral temperatures (1). The abundance of mitochondria and expression of uncoupling protein 1 (UCP1) in thermogenic adipocytes equip brown fat with a unique thermogenic capacity (2). Furthermore, BAT is now recognized as a dynamic endocrine organ, secreting adipokines, gaseous messengers, and microvesicles that can target distant tissues such as white adipose tissue (WAT), liver, pancreas, heart, and bone (3, 4). Experimental studies involving BAT transplantation and activation have demonstrated notable improvements in metabolism and cardiac protection through the release of endocrine factors such as insulin-like growth factor I, IL-6, and fibroblast growth factors (FGFs) (5). In a previous study, we demonstrated that KO of IFN regulatory factor 4 in brown fat cells could reduce the secretion of myostatin (MSTN, also known as growth differentiation factor 8), impairing the exercise capacity of mice (6). However, the role of MSTN in brown fat cells remains unclear.
MSTN belongs to the TGF-β superfamily and serves as a critical regulator of skeletal muscle mass (7). Inhibitors targeting the MSTN signaling pathway have been developed for the treatment of sarcopenia and muscular dystrophy (8). However, the response to MSTN inhibitors in terms of functional improvements has been inconsistent. Although increased muscle mass has been observed in most clinical trials, this often does not translate into clinically meaningful enhancements in strength (9, 10). However, targeting the MSTN signaling pathway consistently reduces fat mass (11–13). These observations align with findings from mouse studies, in which MSTN global-KO mice exhibit increased muscle mass, reduced fat deposition, improved insulin sensitivity, enhanced fatty acid oxidation, and resistance to obesity (14, 15). Subsequent studies in mice treated with MSTN inhibitors have further elucidated the role of MSTN in metabolic regulation (16, 17). Notably, clinical observations have increasingly associated variations in MSTN expression with metabolic conditions. For instance, elevated MSTN levels have been observed in individuals with obesity and insulin resistance, implicating it in the pathophysiology of metabolic syndrome (18). Conversely, reduced MSTN activity is linked to increased muscle mass and improved metabolic profiles, suggesting a protective role against metabolic dysfunction (8). Conversely, reduced MSTN activity is linked to increased muscle mass and improved metabolic profiles, suggesting a protective role against metabolic dysfunction (8). Additionally, MSTN deletion has been found to prevent age-related increases in adipose tissue mass and to partially improve obesity diabetes in mice (19). Furthermore, specific overexpression of MSTN in adipose tissue has been demonstrated to increase the metabolic rate and resistance to diet-induced obesity (DIO) (20). The dual role of MSTN in muscle and adipose tissue underscores its potential as a therapeutic target. Clinical studies have explored MSTN inhibitors in muscle-wasting diseases, noting improvements in muscle mass and preliminary indications of metabolic benefits. These observations raise compelling questions about the broader implications of MSTN modulation in metabolic health, particularly through its effects on adipose tissues.
Kruppel-like factor 4 (KLF4) is a member of a large family of zinc-finger proteins that are critical for various development processes, including differentiation, proliferation, and inflammation. KLF4 serves as an essential early regulator of adipogenesis by regulating C/EBPβ (21). Moreover, cells deficient in KLF4 exhibit mitochondrial dysfunction and impaired mitophagy (22). Specifically, in KLF4-null cells, there is a reduction in the expression of the mitophagy-associated protein Bnip3 and the antioxidant protein GSTα4 (22). Despite substantial contextual evidence of the role of KLF4 in development, the specific molecular mechanisms in metabolism, especially in BAT, are unclear.
FGF21, a member of the endocrine FGF subfamily, has pleiotropic effects on energy homeostasis. Emerging clinical evidence demonstrates that elevated circulating FGF21 can be used as a biomarker of metabolic diseases such as metabolic dysfunction–associated steatohepatitis (MASH) and type 2 diabetes (23, 24). Notably, several FGF21 analogs and mimetics have progressed to early phases of clinical trials involving patients with obesity, type 2 diabetes mellitus, or MASH (25). Global deletion of FGF21 in mice leads to impairments in cold-induced browning of inguinal white adipose tissue (iWAT), whereas administration of recombinant FGF21 increases browning and total energy expenditure in mice (26). Huang et al. reported that adipocyte-derived FGF21 exerts autocrine effects, inducing CCL11 production in adipocytes to promote recruitment of eosinophils, thereby stimulating M2 macrophage activity (27). However, it is currently unclear what regulates FGF21 in adipocytes.
The present study found that mice with brown adipocyte–specific deletion of MSTN exhibited diet-induced insulin resistance, glucose intolerance, and hepatosteatosis, contrary to the phenotypes of MSTN global-KO mice. Furthermore, BAT-specific KO of MSTN led to a marked reduction in browning and adaptive thermogenesis. Mechanistic studies revealed that MSTN regulated the expression of KLF4 and FGF21 via the SMAD2/3 and p38 signaling pathways in adipocytes. The decreased levels of KLF4 and FGF21 contributed to MSTN deficiency–induced mitochondrial dysfunction and inflammation, respectively. These findings provide critical insights into the function of MSTN in BAT and its potential as a modulator of metabolic health, paving the way for novel interventions targeting BAT function to ameliorate obesity and metabolic diseases.
ResultsMice with BAT-specific MSTN KO are prone to DIO. Previous studies have suggested that BAT-derived MSTN may play a role in energy metabolism (6, 28). To further investigate the role of MSTN in regulating BAT homeostasis, we examined the expression of MSTN in response to varying nutrient states. Our findings revealed a reduction in MSTN expression in the DIO mouse model (Figure 1, A and B).
Figure 1Mice with BAT-specific MSTN KO are prone to DIO. (A) Western blot analysis of the expression of MSTN in BAT from DIO mice (n = 3). (B) mRNA expression of Mstn in BAT from DIO mice (n = 5). Con, control. (C) Western blot analysis of the expression of MSTN in BAT from male BKO and Flox mice on a 12-week HFD (n = 3). (D) mRNA expression of Mstn in BAT, GAS, and iWAT from male BKO and Flox mice on a 12-week HFD (n = 7). (E) Body weight of male BKO and Flox mice on a HFD (n = 7–10). (F) Body composition of male BKO and Flox mice on a HFD (n = 7). (G) Weight of BAT, iWAT, epididymal WAT (eWAT), and GAS tissue from male BKO and Flox mice on a 12-week HFD (n = 7). (H) Images showing the morphology of BAT, iWAT, and eWAT. (I) H&E staining of BAT, iWAT of male BKO and Flox mice on a 12-week HFD. Scale bars: 20 μm. (J–M) GTTs and ITTs for male BKO and Flox mice (n = 6–7). (N–Q) The OCR (VO2), carbon dioxide production (VCO2), energy expenditure, and RER of male BKO and Flox mice on a 12-week HFD (n = 6–7). (R) Body temperature of male BKO and Flox mice during cold challenges (n = 7). All results are shown as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test.
Subsequently, we generated MSTNfl/fl (referred to hereafter as Flox) mice and crossed them with CAG-Cre (MSTNΔCAG) mice to mimic the effects observed in MSTN global-KO mice (7). Notably, the MSTNΔCAG mice were noticeably more muscular than the control mice when fed a chow diet (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI178303DS1). Additionally, the heterozygous MSTNΔCAG mice showed resistance to DIO, with reduced fat mass but increased lean mass compared with the controls (Supplemental Figure 1, C and D). Adipocyte size was also smaller in adipose tissue from heterozygous MSTNΔCAG mice on a high-fat diet (HFD) (Supplemental Figure 1E).
We then crossed the Flox mice with UCP1-Cre mice to study the thermogenic function of MSTN. The protein and mRNA levels of MSTN were markedly decreased in BAT but remained normal in other tissues (Figure 1, C and D). The protein levels of MSTN in plasma were not altered in MSTNΔUCP1 mice compared with Flox mice (Supplemental Figure 2A), indicating that MSTN deletion in BAT did not affect the circulating levels of MSTN. Compared with the Flox mouse group, BAT-specific MSTN-KO male mice (referred to hereafter as MSTNΔUCP1) showed no defective developmental or metabolic phenotypes in body weight and body composition when fed a normal chow (NC) diet (Supplemental Figure 2, B–I). Surprisingly, unlike the MSTNΔCAG mice, the MSTNΔUCP1 mice exhibited a more pronounced increase in body weight and adiposity, without marked changes in their lean mass when fed a HFD (Figure 1, E–H). The increased body weight and adiposity were also observed in female mice (Supplemental Figure 2, J and K). To address developmental concerns, we crossed Rosa26CAG-LSL-Cas9-tdTomato mice with UCP1-Cre–transgenic mice and obtained UCP1-Cre Cas9 mice. We performed in situ injection of adeno-associated virus–sgMstn (AAV-sgMstn) into BAT to specifically knock out MSTN in the BAT of UCP1-Cre mice. We found that MSTN protein levels were markedly decreased in BAT (Supplemental Figure 2L), whereas the phenotypes of AAV8-sgMstn mice were consistent with those of brown adipose tissue knockout (BKO) mice (Supplemental Figure 2, M and N). Furthermore, the MSTNΔUCP1 mice displayed a more deteriorative adipose tissue phenotype characterized by larger adipocytes (Figure 1I), a finding opposite to the adipocytes observed in MSTN global-KO mice (29). Additionally, the MSTNΔUCP1 mice showed insulin resistance (Figure 1, J–M). To determine whether brown adipocyte MSTN deficiency affects energy balance, we placed the mice in metabolic cages. The oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory exchange rate (RER), and energy expenditure were lower in MSTNΔUCP1 mice than in the control littermates (Figure 1, N–Q). To confirm the role of MSTN in adaptive thermogenesis, the mice were subjected to cold stress. As expected, MSTNΔUCP1 mice were cold intolerant (Figure 1R). In summary, loss of MSTN in brown adipocytes resulted in impaired energy expenditure, which was different from the phenotypes observed among MSTN global-KO mice.
MSTNΔUCP1 mice exhibit progressive fatty liver disease. To evaluate whether BAT MSTN affects systemic metabolism, we performed targeted metabolomics, encompassing 600 metabolites. Principal component analysis (PCA) revealed a clear distinction between the BKO and Flox groups (Figure 2A). Elevated levels of triglycerides (TGs) and ceramides were detected in plasma from MSTNΔUCP1 mice (Figure 2B). In addition to TG levels, total cholesterol (TC) was also increased in MSTN-KO mice (Figure 2C). Given that hepatic steatosis is closely associated with obesity and insulin resistance, we next assessed the effects of MSTN deletion on hepatic lipid deposition under HFD conditions. Elevated levels of TGs and TC were observed in the livers of the MSTNΔUCP1 mice (Figure 2D). The liver mass of MSTNΔUCP1 mice was heavier than that of control mice after HFD feeding (Figure 2E). MSTNΔUCP1 mice showed more lipid accumulation in the liver than did controls (Figure 2F). The mRNA levels of fatty acid synthesis genes, such as fatty acid synthase (Fas) and sterol regulatory element–binding protein 1c (Srebp1c) were markedly increased in livers from BKO mice compared with those from control mice (Figure 2G). Conversely, the expression of lipolysis genes, including Pnpla2 and Lipe, was decreased compared with expression in controls (Figure 2G). We observed similar impairments in lipid metabolism in AAV8-sgMstn mice (Figure 2H). Thus, BAT-specific MSTN deficiency aggravated hepatic steatosis.
Figure 2MSTN ablation in BAT shows progressive fatty liver. (A) PCA plot of metabolomics from the BKO and Flox groups. expl var, explained variables. (B) Heatmap of metabolites with significantly differential (P < 0.05) expression in the BKO versus the control group. (C) Plasma TG and TC levels in BKO and Flox mice on a 12-week HFD (n = 6–7). (D) Liver TG and TC levels in BKO and Flox mice on a 12-week HFD (n = 6–7). (E) Liver weight/body weight ratio in BKO and Flox mice on a 12-week HFD (n = 6–7). (F) H&E and Oil Red O staining of liver from BKO and Flox mice on a 12-week HFD. Scale bars: 50 μm. (G) Relative mRNA expression of lipid metabolism–related genes in the liver of BKO and Flox mice on a 12-week HFD (n = 7). (H) Relative mRNA expression of lipid metabolism–related genes in the liver of AAV8-sgCon and AAV8-sgMstn mice on a 12-week HFD (n = 3). All results are shown as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test.
Ablation of MSTN in BAT impairs skeletal muscle function. Despite the comparable lean mass of MSTNΔUCP1 mice compared with control mice, unlike the extremely muscular phenotype observed in MSTN global mutant animals (7), we found that skeletal muscle function was impaired. Grip strength and exercise capacity were both lower in MSTNΔUCP1 mice compared with Flox mice (Figure 3, A and B). Additionally, the latency of muscle contraction was prolonged in MSTNΔUCP1 mice (Figure 3C). Consistent with these findings, the oxygen consumption rate (OCR) was decreased in muscle from the MSTNΔUCP1 mice (Figure 3D). Different muscle fiber types were reported to contribute to muscle strength (30). Interestingly, the MSTNΔUCP1 mice exhibited a decrease in the proportion of type IIa muscle fibers, but there was no significant difference in cross-sectional area (CSA) of the fibers (Figure 3, E–G). Skeletal muscle injuries are common occurrences in daily life and exercise, and the capacity for regeneration is critical for muscle repair and functional maintenance. We injected cardiotoxin (CTX), which can induce a transient and reproducible acute injury without affecting the vasculature or nerves (31), into the tibialis anterior (TA) muscle. The MSTNΔUCP1 mice exhibited delayed muscle regeneration compared with the control mice (Figure 3H).
Figure 3MSTN ablation in BAT impairs the function of skeletal muscle. (A) Grip strength of male Flox and BKO mice on a HFD (n = 7–11). (B) Total distance achieved by male Flox and BKO mice on a HFD in the exhaustion test (n = 6–8). (C) Latency (Lat) of compound muscle action potentials in GAS muscles of mice (n = 4–6). (D) OCR in GAS muscles from BKO and Flox mice on a HFD (n = 6). ADP, adenosine diphosphate); PMG, pyruvate, malate, glutamine; CYC, cytochrome C; SUC, succinate; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; ROT, rotenone; AMA, antimycin A. (E) Immunofluorescence analysis of fiber type composition in GAS. The different myosin heavy chain isoforms are stained in blue (MyHC-I), green (MyHC-IIA), or red (MyHC-IIB). Scale bars: 50 μm. (F) Representative H&E staining of GAS from BKO and Flox mice on a HFD. Scale bars: 50 μm. (G) Fiber CSA distribution and median CSA of GAS. (H) Representative H&E staining of TA tissue, 3 days, 5 days, 7 days, and 28 days after CTX injury. Scale bars: 50 μm. (I) TG levels in GAS from BKO and Flox mice on a 12-week HFD (n = 6–8). dpi, days post injection. (J) Representative Oil Red O staining of GAS muscle from BKO and Flox mice on a HFD. Scale bars: 50 μm. (K) Representative electron micrographs of lipid droplets in muscle from male mice. Scale bars: 1 μm. (L) Heatmap of 323 DEGs of GAS from BKO and Flox mice on a HFD. up, upregulated; down, downregulated. (M and N) KEGG analysis and GSEA based on downregulated genes. (O) Relative mRNA expression of lipid metabolism–related genes in GAS of BKO and Flox mice on a 12-week HFD (n = 5). (P) Relative mRNA expression of lipid metabolism related genes in GAS of AAV8-sgCon and AAV8-sgMstn mice on a 12-week HFD (n = 3). All results are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-tailed Student’s t test.
Lipid accumulation in skeletal muscles is implicated in insulin resistance and type 2 diabetes (32). Therefore, we measured the TG levels in muscle, revealing an increase in TG levels in muscles from the MSTNΔUCP1 mice (Figure 3I). The MSTNΔUCP1 mice exhibited greater lipid accumulation in the gastrocnemius (GAS) than did control mice (Figure 3J). Electron microscopy images showed an elevated number of lipid droplets in muscle from MSTNΔUCP1 mice compared with controls (Figure 3K). RNA-Seq analysis revealed 227 downregulated genes and 96 upregulated genes in muscle from the MSTNΔUCP1 mice compared with Flox mice (Figure 3L). Pathway analysis suggested attenuation of lipid catabolism (Figure 3, M and N). Furthermore, quantitative PCR (qPCR) data revealed that expression levels of fatty acid oxidation genes were decreased in muscle from MSTNΔUCP1 mice (Figure 3O). Impaired lipid metabolism was also observed in GAS muscle from AAV8-sgMstn mice, which showed lower expression levels of lipid metabolism–related genes (Figure 3P). Collectively, these findings demonstrated impaired lipid metabolism in muscle obtained from MSTNΔUCP1 mice.
Loss of MSTN attenuates mitochondrial biogenesis and mitophagy. MSTN has been reported to influence adipogenesis in vitro (20). To further explore this, we induced overexpression of MSTN in stromal vascular fractions (SVFs) and then induced their differentiation into
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