Succinate induces skeletal muscle fiber remodeling via SUCNR1 signaling

Article18 July 2019Open Access Transparent process Succinate induces skeletal muscle fiber remodeling via SUCNR1 signaling Tao Wang Tao Wang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Ya-Qiong Xu Ya-Qiong Xu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Ye-Xian Yuan Ye-Xian Yuan Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Ping-Wen Xu Ping-Wen Xu Division of Endocrinology, Department of Medicine, The University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author Cha Zhang Cha Zhang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Fan Li Fan Li Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Li-Na Wang Li-Na Wang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Cong Yin Cong Yin Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Lin Zhang Lin Zhang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Xing-Cai Cai Xing-Cai Cai Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Can-Jun Zhu Can-Jun Zhu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Jing-Ren Xu Jing-Ren Xu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Bing-Qing Liang Bing-Qing Liang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Sarah Schaul Sarah Schaul Division of Endocrinology, Department of Medicine, The University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author Pei-Pei Xie Pei-Pei Xie Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Dong Yue Dong Yue Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Zheng-Rui Liao Zheng-Rui Liao Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Lu-Lu Yu Lu-Lu Yu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Lv Luo Lv Luo Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Gan Zhou Gan Zhou Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Jin-Ping Yang Jin-Ping Yang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Zhi-Hui He Zhi-Hui He Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Man Du Man Du Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Yu-Ping Zhou Yu-Ping Zhou Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Bai-Chuan Deng Bai-Chuan Deng Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Song-Bo Wang Song-Bo Wang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Ping Gao Ping Gao Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Xiao-Tong Zhu Xiao-Tong Zhu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Qian-Yun Xi Qian-Yun Xi Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Yong-Liang Zhang Yong-Liang Zhang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Gang Shu Corresponding Author Gang Shu [email protected] orcid.org/0000-0002-1321-4396 Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Qing-Yan Jiang Qing-Yan Jiang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Tao Wang Tao Wang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Ya-Qiong Xu Ya-Qiong Xu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Ye-Xian Yuan Ye-Xian Yuan Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Ping-Wen Xu Ping-Wen Xu Division of Endocrinology, Department of Medicine, The University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author Cha Zhang Cha Zhang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Fan Li Fan Li Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Li-Na Wang Li-Na Wang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Cong Yin Cong Yin Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Lin Zhang Lin Zhang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Xing-Cai Cai Xing-Cai Cai Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Can-Jun Zhu Can-Jun Zhu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Jing-Ren Xu Jing-Ren Xu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Bing-Qing Liang Bing-Qing Liang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Sarah Schaul Sarah Schaul Division of Endocrinology, Department of Medicine, The University of Illinois at Chicago, Chicago, IL, USA Search for more papers by this author Pei-Pei Xie Pei-Pei Xie Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Dong Yue Dong Yue Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Zheng-Rui Liao Zheng-Rui Liao Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Lu-Lu Yu Lu-Lu Yu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Lv Luo Lv Luo Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Gan Zhou Gan Zhou Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Jin-Ping Yang Jin-Ping Yang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Zhi-Hui He Zhi-Hui He Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Man Du Man Du Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Yu-Ping Zhou Yu-Ping Zhou Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Bai-Chuan Deng Bai-Chuan Deng Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Song-Bo Wang Song-Bo Wang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Ping Gao Ping Gao Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Xiao-Tong Zhu Xiao-Tong Zhu Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Qian-Yun Xi Qian-Yun Xi Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Yong-Liang Zhang Yong-Liang Zhang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Gang Shu Corresponding Author Gang Shu [email protected] orcid.org/0000-0002-1321-4396 Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Qing-Yan Jiang Qing-Yan Jiang Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China Search for more papers by this author Author Information Tao Wang1,‡, Ya-Qiong Xu1,‡, Ye-Xian Yuan1,‡, Ping-Wen Xu2, Cha Zhang1, Fan Li1, Li-Na Wang1, Cong Yin1, Lin Zhang1, Xing-Cai Cai1, Can-Jun Zhu1, Jing-Ren Xu1, Bing-Qing Liang1, Sarah Schaul2, Pei-Pei Xie1, Dong Yue1, Zheng-Rui Liao1, Lu-Lu Yu1, Lv Luo1, Gan Zhou1, Jin-Ping Yang1, Zhi-Hui He1, Man Du1, Yu-Ping Zhou1, Bai-Chuan Deng1, Song-Bo Wang1, Ping Gao1, Xiao-Tong Zhu1, Qian-Yun Xi1, Yong-Liang Zhang1, Gang Shu *,1,3 and Qing-Yan Jiang1,3 1Guangdong Province Key Laboratory of Animal Nutritional Regulation, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China 2Division of Endocrinology, Department of Medicine, The University of Illinois at Chicago, Chicago, IL, USA 3National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, China ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: 86 020 85284901; E-mail: [email protected] EMBO Reports (2019)20:e47892https://doi.org/10.15252/embr.201947892 Correction(s) for this article Succinate induces skeletal muscle fiber remodeling via SUCNR1 signaling07 June 2021 Succinate induces skeletal muscle fiber remodeling via SUCNR1 signaling06 May 2020 [Correction added on 5 September 2019, after first online publication: the article title has been corrected.] [Correction added on 25 April 2019, after first online publication: the abbreviation for succinate receptor 1 has been corrected to SUCNR1.] Correction added on 4 June 2021, after first online publication: the phrase "dose-dependently" was removed. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The conversion of skeletal muscle fiber from fast twitch to slow-twitch is important for sustained and tonic contractile events, maintenance of energy homeostasis, and the alleviation of fatigue. Skeletal muscle remodeling is effectively induced by endurance or aerobic exercise, which also generates several tricarboxylic acid (TCA) cycle intermediates, including succinate. However, whether succinate regulates muscle fiber-type transitions remains unclear. Here, we found that dietary succinate supplementation increased endurance exercise ability, myosin heavy chain I expression, aerobic enzyme activity, oxygen consumption, and mitochondrial biogenesis in mouse skeletal muscle. By contrast, succinate decreased lactate dehydrogenase activity, lactate production, and myosin heavy chain IIb expression. Further, by using pharmacological or genetic loss-of-function models generated by phospholipase Cβ antagonists, SUCNR1 global knockout, or SUCNR1 gastrocnemius-specific knockdown, we found that the effects of succinate on skeletal muscle fiber-type remodeling are mediated by SUCNR1 and its downstream calcium/NFAT signaling pathway. In summary, our results demonstrate succinate induces transition of skeletal muscle fiber via SUCNR1 signaling pathway. These findings suggest the potential beneficial use of succinate-based compounds in both athletic and sedentary populations. Synopsis Aerobic exercise leads to skeletal muscle remodelling. This study reveals that dietary succinate is sufficient to elicit muscle remodelling and increased endurance in sedentary mice. Dietary succinate increases endurance exercise ability in mice. Dietary succinate induces skeletal muscle fiber transition from fast-twitch to slow-twitch. SUCNR1 signaling pathway is required for the succinate induced skeletal muscle remodeling. Introduction In mammals, skeletal muscle comprises about 55% of the individual body mass 12. Skeletal muscle is heterogeneous and composed of slow- and fast-twitch fiber types, which differ in contractile-protein composition, oxidative capacity, and substrate preference for ATP production 3. Slow-twitch fibers have more myoglobin, more mitochondria 4, a higher level of intracellular calcium concentrations 5, and higher activity of oxidative metabolic enzymes than fast-twitch fibers. Therefore, the switching of skeletal muscle fiber from fast twitch to slow twitch is important for sustained and tonic contractile events 67, maintenance of energy homeostasis 8, and alleviation of fatigue. Endurance or aerobic exercise is crucial to muscle fiber-type remodeling by increasing the mechanical and metabolic demand on skeletal muscle 9. Previous study showed endurance training increases intracellular calcium concentration ([Ca2+]i) 1011, which activates the calcineurin/nuclear factor of activated T cells (NFAT) 1213 and myocyte enhancer factor-2 (MEF2) 14. These two transcription factors play a dominant role in muscle formation and fiber remodeling. In addition to transient elevation of [Ca2+]i, endurance exercise also increases several specific TCA cycle intermediates, among which succinate increases the most 1516. However, whether these intermediates mediate endurance exercise-induced muscle fiber transition is rarely investigated. Succinate has been reported to induce cardiomyocyte hypertrophy 17 and osteoclastogenesis 18. It also plays an important role in energy 19 and glucose 20 homeostasis by regulating mitochondrial oxygen consumption 21 and heat production from brown adipose tissue (BAT) 22. Therefore, we hypothesize that succinate regulates skeletal muscle fiber remodeling. To test this hypothesis, we first examined the effects of succinate on skeletal muscle fiber composition, metabolism, and exercise tolerance. By combining pharmacological and siRNA-mediated knockdown model both in vitro and in vivo, we demonstrated that succinate induces skeletal muscle transition from fast twitch to slow twitch through the SUCNR1 signaling pathway. Our results indicate potential use of succinate as a dietary supplement to improve physical fitness and counteract fatigue. Results The dietary supplement of succinate shifts skeletal muscle fiber size distribution To determine the effects of succinate on skeletal muscle growth, we fed male C57BL/6J mice with chow diet supplemented with 0, 0.5%, or 1% succinic acid disodium salt for 8 weeks. We found that succinate-supplemented diet increased serum SUA level (Fig 1A) but had no effects on the body weight gain (Fig 1B), food intake (Fig EV1A), fat mass (Fig 1C), lean mass (Fig 1D), gastrocnemius muscle index (Fig 1E), or liver index (Fig EV1B). Additionally, consistent with our previous report 23, we found that succinate activated Akt/mTOR cascade and inhibited FoxO3a (Fig EV1C and D). Interestingly, we also found that 1% succinate increased the proportion of small muscle fiber (200–400 μm2), while decreased the proportion of large muscle fiber (600–800 μm2; Fig 1F and G). This shift of muscle fiber size distribution indicates that succinate may affect skeletal muscle contraction properties. Figure 1. Effects of succinate on growth performance and serum concentration in miceMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5, and 1% SUC for 8 weeks. A–E. (A) Serum SUA level, (B) body weight gain, (C) fat and (D) lean mass and (E) gastrocnemius index. F, G. (F) Gastrointestinal muscle fiber immunofluorescent laminin staining and (G) frequency histogram of fiber cross-sectional area. Scale bar in (F) represents 100 μm. Data information: Results are presented as mean ± SEM (n = 6–8). Different letters between bars mean P ≤ 0.05 in one-way ANOVA analyses followed by post hoc Tukey's tests. *: significant difference (P ≤ 0.05) between 0.5% SUC and control group by non-paired Student's t-test. #: significant difference (P ≤ 0.05) between 1% SUC and control group by non-paired Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Effects of succinate on growth performance and muscle fiber composition in mice (related to Figs 1 and 3)Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, or 1% SUC for 8 weeks. A, B. (A) Cumulative food intake and (B) liver index of mice treated with SUC for 8 weeks (n = 8). C, D. Immunoblots and quantification of p-mTOR, mTOR, p-FoxO3a, FoxO3a, p-AKT, and AKT protein in gastrocnemius (n = 3). E–H. Representative images and quantification of laminin (green), MyHC I (red), and MyHC IIb (red) immunofluorescent staining in the (E, F) soleus and (G, H) extensor digitorum longus muscle. The graphs show the MyHC I and MyHC IIb fiber ratios (n = 6). Scale bar in (E, G) represents 100 μm. I–N. The percentage of SDH positive in the (I, J) gastrocnemius, (K, L) soleus, and (M, N) extensor digitorum longus muscle is shown by SDH enzyme staining. Only darkly stained SDH fibers are treated as SDH-positive fibers. The graphs show the SDH-positive fiber ratios (n = 4–6). Scale bar in I, K, and M represents 100 μm. Data information: Results are presented as mean ± SEM. Different letters between bars mean P ≤ 0.05 in one-way ANOVA analyses followed by post hoc Tukey's tests. Download figure Download PowerPoint Succinate enhances endurance exercise capacity and reduces muscle fatigue To further investigate the effects of succinate on skeletal muscle contraction properties, we first tested the exercise capacity of mice. We found that succinate dose-dependently increased muscle grip strength (Fig 2A), low-speed running time (Fig 2B), and decreased falling time in four-limb handing test (Fig 2C). However, high-speed running time was unchanged by succinate supplementation (Fig 2D), which indicates succinate may specifically improve endurance exercise performance, but not explosive exercise performance. Figure 2. Succinate enhances the endurance exercise capacity of skeletal muscle in miceMale C57BL/6J mice fed with chow diet supplemented with 0, 0.5, and 1% SUC for 8 weeks. A–D. (A) The muscle grip strength, (B) running time in low speed, (C) four-limb handing time, and (D) running time in high speed. E, F. (E) Serum concentration of RBC and (F) HGB in whole blood. G–L. (G–I) Ex vivo gastrocnemius muscle force, (J) fatigability, (K) glucose consumption, and (L) lactate production were tested. Data information: Results are presented as mean ± SEM (n = 5–8). Different letters between bars mean P ≤ 0.05 in one-way ANOVA analyses followed by post hoc Tukey's tests. Download figure Download PowerPoint It is well-known that endurance exercise performance is determined by oxygen supply and muscle fiber type 19. We first tested if the oxygen-carrying capacity of muscle was enhanced by succinate. We found although succinate slightly increased the number of red blood cells (RBC; Fig 2E) and the hemoglobin (HGB) level (Fig 2F), the extent of these increases is not comparable to the dramatic improvement of endurance exercise capacity. In order to further characterize other parameters related to endurance exercise capability, we used an ex vivo strategy to evaluate isolated muscle contraction properties (Fig 2G). We found that dietary supplementation of succinate did not affect the maximum contractile force (Fig 2I), but significantly improved fatigue resistance of muscle (Fig 2H and J), with less glucose consumption (Fig 2K), and lactate production (Fig 2L) during contraction. Taken together, our data indicate that succinate can increase oxygen-carrying capacity and reduce muscle fatigue. Succinate induces skeletal muscle fiber-type transition in vivo There are four types of skeletal muscle fiber, including I, IIa, IIx, and IIb. Each of them expresses different myosin heavy chain and troponin isoforms. Here, we studied the effects of succinate on muscle fiber-type transaction in three different muscles, including soleus, extensor digitorum longus (EDL), and gastrocnemius. Soleus is known as a typical slow-twitch muscle (slow/slow), whereas EDL is a typical fast-twitch muscle (fast/fast). Gastrocnemius usually has a lot of fast-twitch muscle fibers, or an equal number of fast and slow-twitch fibers (fast/slow mixed). In mixed gastrocnemius muscle, we found that succinate upregulated slow-twitch fiber-associated genes MyHC I, MyHC IIa, PGC-1α, myoglobin, and TnnT1, whereas it downregulated fast-twitch fiber-associated genes, including MyHC IIb and TnnT3 (Fig 3A). Further, both Western blot (Fig 3B) and immunofluorescence (Fig 3C and D) demonstrated that succinate increased MyHC I/IIa protein expression and slow-twitch fiber percentage, while decreased MyHC IIb protein and fast-twitch fiber percentage. These results indicate that succinate induces a fast twitch to slow-twitch transition in skeletal muscle. Figure 3. Effects of succinate on MyHC expression in miceMale C57BL/6J mice were fed with chow diet supplemented with 0, 0.5, and 1% SUC for 8 weeks. A. The mRNA expression of MyHC I, MyHC IIa, PGC-1α, myoglobin, TnnT1 MyHC IIb, MyHC IIx, and TnnT3 in the gastrocnemius muscle (n = 5–6). B. Immunoblots and quantification of MyHC I, MyHC IIa, and MyHC IIb protein expression in gastrocnemius (n = 3–4). C, D. Representative images and quantification of laminin (green), MyHC I, and MyHC IIb immunofluorescent staining (red) in gastrocnemius (n = 3). Scale bar in (C) represents 100 μm. Data information: Results are presented as mean ± SEM. Different letters between bars mean P ≤ 0.05 in one-way ANOVA analyses followed by post hoc Tukey's tests. Download figure Download PowerPoint Consistently, we found that succinate increased‡ MyHC I but not MyHC IIb protein expression in soleus, suggesting an increased proportion of slow-twitch fiber (Fig EV1E and F). On the other hand, succinate failed to affect the muscle fiber composition of EDL muscle (Fig EV1G and H). Oxidative capacity of three muscles was also evaluated by the staining of succinate dehydrogenase (SDH), a marker of oxidative capacity of skeletal muscle at the fiber level. We found that succinate dose-dependently increased the percentage of SDH-positive fibers in SOL, EDL, and gastrocnemius muscles (Fig EV1I–N), suggesting succinate is sufficient to improve mitochondrial content and oxidative capacity of mixed (gastrocnemius), slow/slow (SOL), or fast/fast (EDL) muscles. Succinate increases aerobic oxidation and mitochondrial biogenesis in skeletal muscle A high number of mitochondrial and metabolic adaptation are generally accompanied with endurance exercise and skeletal muscle type transition 24. Here, we tested the effects of succinate on metabolism and mitochondrial properties. We found that succinate increased whole-body oxygen consumption (Fig 4A and B) and decreased whole-body respiratory exchang