Mitochondrial glycerol 3‐phosphate dehydrogenase promotes skeletal muscle regeneration
2018; Springer Nature; Volume: 10; Issue: 12 Linguagem: Inglês
10.15252/emmm.201809390
ISSN1757-4684
AutoresXiufei Liu, Hua Qu, Yi Zheng, Qian Liao, Linlin Zhang, Xiaoyu Liao, Xin Xiong, Yuren Wang, Rui Zhang, Hui Wang, Qiang Tong, Zhenqi Liu, Hui Dong, Gangyi Yang, Zhiming Zhu, Jing Xu, Hongting Zheng,
Tópico(s)Pancreatic function and diabetes
ResumoResearch Article2 November 2018Open Access Source DataTransparent process Mitochondrial glycerol 3-phosphate dehydrogenase promotes skeletal muscle regeneration Xiufei Liu Xiufei Liu Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Hua Qu Hua Qu Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Yi Zheng Yi Zheng Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Qian Liao Qian Liao Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Linlin Zhang Linlin Zhang Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Xiaoyu Liao Xiaoyu Liao Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Xin Xiong Xin Xiong Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Yuren Wang Yuren Wang Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Rui Zhang Rui Zhang Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Hui Wang Hui Wang Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Qiang Tong Qiang Tong Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Zhenqi Liu Zhenqi Liu Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health System, Charlottesville, VA, USA Search for more papers by this author Hui Dong Hui Dong Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Gangyi Yang Gangyi Yang orcid.org/0000-0002-6458-6747 Department of Endocrinology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China Search for more papers by this author Zhiming Zhu Zhiming Zhu Department of Hypertension and Endocrinology, Daping Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Jing Xu Jing Xu Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Hongting Zheng Corresponding Author Hongting Zheng [email protected] orcid.org/0000-0002-6930-0103 Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Xiufei Liu Xiufei Liu Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Hua Qu Hua Qu Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Yi Zheng Yi Zheng Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Qian Liao Qian Liao Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Linlin Zhang Linlin Zhang Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Xiaoyu Liao Xiaoyu Liao Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Xin Xiong Xin Xiong Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Yuren Wang Yuren Wang Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Rui Zhang Rui Zhang Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Hui Wang Hui Wang Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Qiang Tong Qiang Tong Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Zhenqi Liu Zhenqi Liu Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health System, Charlottesville, VA, USA Search for more papers by this author Hui Dong Hui Dong Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Gangyi Yang Gangyi Yang orcid.org/0000-0002-6458-6747 Department of Endocrinology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China Search for more papers by this author Zhiming Zhu Zhiming Zhu Department of Hypertension and Endocrinology, Daping Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Jing Xu Jing Xu Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Hongting Zheng Corresponding Author Hongting Zheng [email protected] orcid.org/0000-0002-6930-0103 Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China Search for more papers by this author Author Information Xiufei Liu1,‡, Hua Qu1,‡, Yi Zheng1,‡, Qian Liao1,‡, Linlin Zhang1, Xiaoyu Liao1, Xin Xiong1, Yuren Wang1, Rui Zhang1, Hui Wang1, Qiang Tong1, Zhenqi Liu2, Hui Dong3, Gangyi Yang4, Zhiming Zhu5, Jing Xu1 and Hongting Zheng *,1 1Translational Research Key Laboratory for Diabetes, Department of Endocrinology, Xinqiao Hospital, Third Military Medical University, Chongqing, China 2Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health System, Charlottesville, VA, USA 3Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, China 4Department of Endocrinology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China 5Department of Hypertension and Endocrinology, Daping Hospital, Third Military Medical University, Chongqing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +8602368755709; Fax: +8602368755707; E-mail: [email protected] EMBO Mol Med (2018)10:e9390https://doi.org/10.15252/emmm.201809390 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 While adult mammalian skeletal muscle is stable due to its post-mitotic nature, muscle regeneration is still essential throughout life for maintaining functional fitness. During certain diseases, such as the modern pandemics of obesity and diabetes, the regeneration process becomes impaired, which leads to the loss of muscle function and contributes to the global burden of these diseases. However, the underlying mechanisms of the impairment are not well defined. Here, we identify mGPDH as a critical regulator of skeletal muscle regeneration. Specifically, it regulates myogenic markers and myoblast differentiation by controlling mitochondrial biogenesis via CaMKKβ/AMPK. mGPDH−/− attenuated skeletal muscle regeneration in vitro and in vivo, while mGPDH overexpression ameliorated dystrophic pathology in mdx mice. Moreover, in patients and animal models of obesity and diabetes, mGPDH expression in skeletal muscle was reduced, further suggesting a direct correlation between its abundance and muscular regeneration capability. Rescuing mGPDH expression in obese and diabetic mice led to a significant improvement in their muscle regeneration. Our study provides a potential therapeutic target for skeletal muscle regeneration impairment during obesity and diabetes. Synopsis mGPDH is here identified as an important regulator of muscle differentiation and regeneration. Activation of the mGPDH/AMPK/mitochondrial biogenesis pathway provides a new strategy for improving muscle frailty. Direct targeting of mGPDH may also have therapeutic benefits for obesity and diabetes. mGPDH improves skeletal muscle regeneration by promoting myoblast differentiation. mGPDH promotes myoblast differentiation via CaMKKβ/AMPK control of mitochondrial biogenesis. mGPDH expression was reduced in skeletal muscle obtained from patients and animal models of obesity and diabetes. Rescuing mGPDH deficiency improves skeletal muscle regeneration during these pathological processes. Introduction Adult mammalian skeletal muscle is a stable tissue that is post-mitotic; however, it actively undergoes regeneration following injury (Charge & Rudnicki, 2004). In certain diseases, such as the modern pandemics of obesity and diabetes, skeletal muscle regeneration becomes impaired, which leads to the loss of muscle function and contributes to the global burden of these diseases (Fu et al, 2016; Benoit et al, 2017). However, the mechanisms that underlie the regeneration impairment are poorly understood. mGPDH is an integral component of the mitochondrial respiratory chain and functions as the rate-limiting step in the glycerophosphate (GP) shuttle (Eto et al, 1999b). Due to a different structure and cell localization, the function and regulation of this enzyme are distinct from those of cytoplasmic glycerol 3-phosphate dehydrogenase (cGPDH, also referred to as GPD1; Mracek et al, 2013). At the same time, despite the relatively simple structure of mGPDH, its functions remain largely unknown. Recently, mGPDH has been reported to be involved in hepatic glucose metabolism (Baur & Birnbaum, 2014; Madiraju et al, 2014). To gain a more complete understanding of mGPDH functions, we examined the role of mGPDH in skeletal muscle, which is a major insulin-sensitive tissue that plays an essential role in glucose metabolism. Our results showed that although mGPDH is vital in regulating hepatic glucose metabolism, it did not significantly affect the glucose uptake and insulin signaling within skeletal muscle (differentiated C2C12 myoblasts). Interestingly, however, the mGPDH expression significantly increased over the course of C2C12 myocyte differentiation, with an expression profile similar to that of myogenic markers (myogenin and MyHC). These differentiation-associated increases in mGPDH expression and activity were also clearly visible in mitochondrial fractions, which indicate that mGPDH might be involved in myogenic differentiation. In the current study, we identify a novel characteristic of mGPDH in regulating myogenic differentiation and a potential therapeutic target for ameliorating muscle regeneration impairment and muscle pathology. In addition, the activation of the mGPDH/AMPK/mitochondrial biogenesis pathway of skeletal muscle might represent a new mechanism for treatment during obesity and diabetes. Results mGPDH regulates myoblast differentiation Our preliminary observations showed that mGPDH did not significantly influence glucose uptake or insulin signaling under both non-insulin- and insulin-treated conditions (Appendix Fig S1A and B), but its expression was augmented during the course of C2C12 myocyte differentiation (Fig 1A–D). To further explore the possibility of mGPDH involvement in myogenic differentiation, we regulated mGPDH expression by overexpression (plasmid pPR-mGPDH) or inhibition (specific siRNA si-mGPDH) in C2C12. Less cell fusion and multinuclear myotube formation events were observed in the si-mGPDH group than those in the control, and striking increases in these events accompanied overexpression (Fig 1E–G). Furthermore, the protein expression of myogenin and MyHC was also reduced by si-mGPDH during the course of differentiation (Fig 1H–K). Consistent changes in the corresponding mRNA levels following mGPDH expression perturbation were also identified (Fig 1L and M). These findings indicate that mGPDH is essential for myoblast differentiation. A major function of mGPDH is to form the GP shuttle with cGPDH. The expression of cGPDH was not significantly changed during the course of C2C12 myocyte differentiation, and the knockdown of cGPDH by siRNA did not show significant effects on C2C12 myocyte differentiation (Fig EV1A–F). Figure 1. mGPDH regulates myoblast differentiation A, B. qRT–PCR (A) and immunoblot (B) of mGPDH, myogenin, and myosin heavy chain (MyHC) levels during C2C12 myocyte differentiation. Quantification represents the levels of the indicated protein normalized to β-actin. C. Immunoblot of mGPDH, voltage-dependent anion channel (VDAC), and cytochrome c (Cyt C) levels in mitochondrial lysate during C2C12 myocyte differentiation. Quantification represents the levels of the indicated protein normalized to COX IV. D. Activity assay of mGPDH at days 0 and 7 after C2C12 myocyte differentiation. E–G. Representative images of MyHC immunofluorescence (E) of C2C12 myocyte transfected with the siRNA or the overexpression plasmid for mGPDH; the fusion index (F) and the distribution of nuclei per myotube (G) were calculated at day 5 after differentiation. H–K. Immunoblot of mGPDH, myogenin, and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH. Quantification (I–K) represents the levels of the indicated protein normalized to β-actin at the indicated day after differentiation. L, M. qRT–PCR analysis of mGPDH, myogenin, and MyHC in C2C12 myocytes transfected with the siRNA or the overexpression plasmid for mGPDH at day 4 after differentiation. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 50 μm in panel (E). In panels (A–D) and (H–M), n = 3; in panels (E–G), n = 15. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired t-test was used for all analyses except in panel (G), where Kolmogorov–Smirnov test was used. Source data are available online for this figure. Source Data for Figure 1 [emmm201809390-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Effect of cGPDH on myoblast differentiation A. cGPDH expression during C2C12 myocyte differentiation. B–D. Representative images of MyHC immunofluorescence (B) of C2C12 myocytes transfected with siRNA targeting cGPDH; the fusion index (C) and the distribution of nuclei per myotube (D) were calculated. E, F. qRT–PCR (E) and Western blot analysis (F) of myogenin and MyHC in C2C12 myocytes transfected with siRNA targeting cGPDH. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 50 μm in panel (B). In panels (A–F), n = 3. *P < 0.05. Unpaired t-test was used for all analyses except in panel (D), where the Kolmogorov–Smirnov test was used. Download figure Download PowerPoint mGPDH is essential in skeletal muscle regeneration Myoblast differentiation occurs during muscle development and also during adulthood for muscle mass maintenance and muscle regeneration (Charge & Rudnicki, 2004). Here, we aim to identify the role of mGPDH in both stages. First, we examined the mGPDH distribution among different skeletal muscles and found that it was abundantly expressed in the gastrocnemius (GA) and quadriceps femoris (QUA), particularly in the GA (Fig EV2A); it seems that mGPDH does not match with the muscle fiber type. To further observe this issue, we costained MHC IIb (the most abundant fiber type in GA muscle) with mGPDH. The results showed the fibers were stained as three colors (Fig EV2B), which indicates that mGPDH did not match with the fiber type in GA muscle. Moreover, the expressions of MHC isoforms (MHC I, IIa, and IIb) were not significantly changed in mGPDH-depleted skeletal muscle (Fig EV2C). During muscle development, the mGPDH expression increased after birth, but only for the first few postnatal days (Fig EV2D). In the mGPDH knockout (mGPDH−/−) mice (Fig EV2E), there were no significant differences in the body and muscle weight compared with the wild-type (WT) mice during development (Fig EV2F and G). Histological analysis also showed no differences in the muscle appearance or myofiber size between these two genotypes (Fig EV2H and I), which suggests mGPDH is not essential for muscle development. Click here to expand this figure. Figure EV2. mGPDH is not essential to muscle development A. Immunoblot of mGPDH in the quadriceps (QUA), gastrocnemius (GA), soleus (SOL), extensor digitorum longus (EDL), and tibialis anterior (TA) muscles of 8-week-old C57BL/6J mice. B. Immunofluorescence showing localization of mGPDH with fiber type marker MHC IIb on cryosections from uninjured GA muscle of 8-week-old C57BL/6J mice. C. qRT–PCR analyses of the indicated fiber type markers (MHC I, IIa, and IIb) in the uninjured GA muscles of 8-week-old WT and mGPDH−/− mice. D. Immunoblot of mGPDH in C57BL/6J mouse skeletal muscle at postnatal days 1, 5, and 10 and 8 weeks. E. Immunoblot of mGPDH in the GA muscle of 8-week WT and mGPDH−/− mice. F. Body weight of WT and mGPDH−/− mice at the indicated week of age. G. Muscle weight of the indicated 8-week-old mGPDH−/− mice normalized to WT. H, I. Hematoxylin–eosin (H&E) staining (H) and average myofiber cross-sectional area (CSA) (I) in the GA muscle of 8-week-old WT and mGPDH−/− mice. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 200 μm in panel (B) and 100 μm in panel (H). In panels (A and D), n = 3 mice per group; in panels (B and C), n = 6 mice per group; in panels (E–I), n = 4 mice per group; in panels (H and I), three sections were obtained per mouse. n.s., not significant. Unpaired t-test was used for all analyses except in panel (I), where the Kolmogorov–Smirnov test was used. Download figure Download PowerPoint Thus, we subsequently assessed the role of mGPDH in muscle regeneration post-injury. Both the mGPDH expression and activity were increased in GA muscle after cardiotoxin (CTX) injury and paralleled the changes of myogenic markers and developmental myosin heavy chain (Fig 2A–C), which is consistent with our observation in vitro (Fig 1A–D). In addition, compared with the basal expression of mGPDH in normal fibers with peripheral nuclei, the injury-induced higher expression of mGPDH was mainly localized in regenerating fibers with central nuclei (Appendix Fig S2), which indicates the injury-induced mGPDH expression predominately presented in newly formed myofibers. Although both the mGPDH−/− and WT mice exhibited extensive muscle damage at day 3 post-injury, the mGPDH−/− mice showed a delay in the disappearance of necrotic fibers and inflammatory cells and had fewer and more unevenly distributed newly formed myofibers with multiple centrally located nuclei at day 7 (Fig 2D–F). The immunofluorescence of desmin, an intermediate filament protein in newly generated myofibers (Liu et al, 2012), further confirmed the impaired muscle regeneration in mGPDH−/− mice (Fig 2G). At day 14, the muscle weight was decreased (Fig 2H), while the collagen deposition was increased (Fig 2I) in the mGPDH−/− mice. These results suggested that mGPDH loss attenuates muscle regeneration. At the same time, expressions of the satellite cell marker paired box protein 7 (PAX7; Zhang et al, 2016; Bi et al, 2017) and the satellite cell activation marker myoblast determination protein (MyoD; Zhang et al, 2016; Bi et al, 2017) were not different between the mGPDH−/− and WT mice (Appendix Fig S3A–F), which suggests that mGPDH has no significant effects on myoblast quantity and activation. However, the differentiation markers myogenin and myh3 (Park et al, 2016) were reduced in the mGPDH−/− mice (Fig 2J and K), which is consistent with our in vitro data and indicates that mGPDH deletion inhibits skeletal muscle regeneration by diminishing myoblast differentiation. Figure 2. mGPDH is essential to skeletal muscle regeneration A, B. qRT–PCR (A) and immunoblot (B) of mGPDH, myogenin, and developmental myosin heavy chain (myh8, myl4, and myh3) in gastrocnemius (GA) muscle from C57BL/6J mice at the indicated day after CTX intramuscular injection. C. Activity assay of mGPDH in GA muscle from C57BL/6J mice at days 0 and 7 after CTX injection. D–G. Representative images of the H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (D), distribution of the fiber cross-sectional area (CSA) (E), percentage of myofibers with central nuclei (F), and immunofluorescence staining of desmin (green) (G) in GA muscle from WT and mGPDH−/− mice at day 7 post-CTX injection. H, I. Muscle weight (H) and trichrome staining (I) in GA muscle from WT and mGPDH−/− mice at day 14 post-CTX injection. Quantification represents the fibrotic areas. J, K. qRT–PCR (J) and immunoblot (K) for mGPDH, myogenin, and myh3 in GA muscle from WT and mGPDH−/− mice at day 7 post-CTX injection. L–Q. qRT–PCR for mGPDH, myogenin, and myh3 (L), H&E staining (M), distribution of the fibers CSA (N), qRT–PCR (O), and immunofluorescence staining (P) for utrophin and trichrome staining (Q) in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injection. R. Exercise capacity of mdx mice 6 weeks after AAV-mGPDH tail vein injection. Data information: Data are presented as the mean ± s.e.m. Scale bars represent 100 μm (25 μm for magnification insets) in panels (D, I, M, and Q) and 50 μm in panels (G, P). In panels (A–C), n = 3; in panels (D–R), n = 6 mice per group; in panels (D–F, M, and N), three sections were obtained per mouse. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired t-test was used for all analyses except in panels (E, N), where the Kolmogorov–Smirnov test was used. Source data are available online for this figure. Source Data for Figure 2 [emmm201809390-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Next, we activated mGPDH via AAV in mdx mice, which represent a model of Duchenne muscular dystrophy, in which there is a persistent damage and loss of myofibers induced by the Dmd gene mutation (Barton et al, 2002; Duddy et al, 2015; Novak et al, 2017). The basal expression levels of mGPDH and myogenin were increased in the mdx mice compared to the normal mice, which indicated an activated regeneration process that was insufficient to compensate (Appendix Fig S4A and B). The overexpression of mGPDH via intramuscular injection of AAV into the GA muscle induced a further increase in myogenin and myh3 expression (Fig 2L); in line with this finding, the number of small regenerating fibers and the variability in the myofiber size were decreased (Fig 2M), and the distribution of the cross-sectional area (CSA) shifted to the right (Fig 2N). Moreover, the mRNA and protein levels of utrophin, an indicator of regeneration in mdx mice (Durko et al, 2010), were increased (Fig 2O and P), while muscle fibrosis decreased (Fig 2Q). Furthermore, systematically up-regulating mGPDH via tail vein injection of AAV improved the exercise capacity of the mdx mice (Appendix Fig S5 and Fig 2R). Taken together, these in vivo data of mGPDH deletion and overexpression suggest that mGPDH plays a pivotal role in regulating myoblast differentiation and muscle regeneration. mGPDH effects occur via the CaMKKβ/AMPK control of mitochondrial biogenesis To gain further insights into the underlying molecular mechanisms, we subsequently assessed a number of the common factors related to myoblast differentiation, such as the cell cycle, apoptosis, autophagy, insulin-like growth factor-1 (IGF-1), and mitochondrial biogenesis (Musaro et al, 2001; Kim et al, 2010; Hochreiter-Hufford et al, 2013; Zhang et al, 2014; Garcia-Prat et al, 2016). mGPDH had no significant effects on the cell cycle, apoptosis, autophagy, and IGF-1 receptor expression (Appendix Fig S6A–D), but obviously changed the mitochondrial content of C2C12 myocytes (Fig 3A). Moreover, it regulated the expression of nuclear-encoded oxidative phosphorylation (OXPHOS) subunits (NDUFS8, SDHb, Uqcrc1, COX5, and ATP5a; Fig 3B), despite no substantial impact on the mitochondrial genomes (ND1, Cytb, COX1, and ATP6; Appendix Fig S7A and B). The mitochondrial respiration rate further confirmed these links to mitochondrial biogenesis (Fig 3C). AMP-activated protein kinase (AMPK) is a key regulator of nuclear-encoded OXPHOS subunits and mitochondrial function (Xiao et al, 2011; Lin et al, 2012; Gomes et al, 2013; Mottillo et al, 2016), and it has also been reported to influence myoblast differentiation (Mounier et al, 2013). Therefore, we assessed whether AMPK is involved in mGPDH effects. As shown in Fig 3D, mGPDH expression significantly activated AMPK and its downstream acetyl-CoA carboxylase (ACC), as well as the mitochondrial biogenesis marker peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α; Fig 3D–F). Strikingly, the activated mitochondrial biogenesis caused by mGPDH overexpression, including increased PGC1α, mitochondrial content, and nuclear-encoded OXPHOS, was abrogated when the AMPK inhibitor compound C was applied (Fig 3G–I), which indicates that the effects of mGPDH on nuclear-encoded OXPHOS subunits and mitochondrial biogenesis are AMPK-dependent. Previous studies have reported that to a large extent, AMPK regulated mitochondrial biogenesis mainly through the modulation of PGC1α activity by the NAD+/NADH ratio (Iwabu et al, 2010; Meng et al, 2013; Woldt et al, 2013). Our results showed that mGPDH loss- and gain-of-function manipulations affected the NAD+/NADH ratio (Fig 3J). Moreover, PGC1α acetylation was altered when mGPDH expression changed (Fig 3K). Mitochondrial biogenesis is critical for skeletal muscle differentiation. It activates myoblast differentiation markers by suppressing c-myc expression, which represses myoblast differentiation through direct binding to the promoters or enhancers of myogenin (Miner & Wold, 1991; Seyer et al, 2010; Ravel-Chapuis et al, 2014). In our observation, mGPDH overexpression repressed c-myc expression and increased myogenin expression and myoblast differentiation, and these effects were abolished by the AMPK inhibitor compound C (Fig 3L–P). Figure 3. mGPDH effect occurs via the CaMKKβ/AMPK control of mitochondrial biogenesis A–F. Mitochondrial DNA (A), nuclear-encoded OXPHOS genes (B), respirometry analysis (C), and immunoblots of mGPDH, phospho-Thr172 AMPK (p-AMPK), total AMPK (AMPK), phospho-Ser79-ACC (p-ACC), total ACC and PGC1α, and corresponding quantifications represent mGPDH, p-AMPK, p-ACC, and PGC1α protein levels (D–F) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation. G–I. Immunoblots of p-AMPK, p-ACC, and PGC1α and corresponding quantifications represent p-AMPK, p-ACC, and PGC1α protein levels (G), mitochondrial DNA (H), and nuclear-encoded OXPHOS genes combined by NDUFS8, SDHb, Uqcrc1, COX5b, and ATP5a1 (I) in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiation. J, K. NAD+/NADH ratio (J) and immunoprecipitation analysis for PGC1α acetyl-lysine (Ac-Lys) level (K) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation. L–P. Immunoblot of c-myc and myogenin (L) and corresponding quantifications represent c-myc and myogenin protein levels (M), representative images of MyHC immunofluorescence (N), fusion index (O), and the distribution of nuclei per myotube (P) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L, M) or 72 h (N–P) after differentiation. Q. Immunoblots of p-AMPK, p-ACC, PGC1α, and myogenin in C2C12 myocytes transfected with mGPDH plasmid with the CaMKKβ inhib
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