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Skeletal muscle increase FGF21 expression in mitochondrial disorder to compensate for the energy metabolic insufficiency by activating mTOR-YY1-PGC1α pathway Kunqian Ji, Jinfan Zheng, Jingwei Lv, Jingwen Xu, Xinbo Ji, Yue-Bei Luo, Wei Li, Yuying Zhao, Chuanzhu Yan www.elsevier.com/locate/freeradbiomed

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S0891-5849(15)00144-6 http://dx.doi.org/10.1016/j.freeradbiomed.2015.03.020 FRB12360

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Free Radical Biology and Medicine

Received date: 20 August 2014 Revised date: 27 February 2015 Accepted date: 20 March 2015 Cite this article as: Kunqian Ji, Jinfan Zheng, Jingwei Lv, Jingwen Xu, Xinbo Ji, Yue-Bei Luo, Wei Li, Yuying Zhao, Chuanzhu Yan, Skeletal muscle increase FGF21 expression in mitochondrial disorder to compensate for the energy metabolic insufficiency by activating mTOR-YY1-PGC1α pathway, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2015.03.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Skeletal muscle increase FGF21 expression in mitochondrial disorder to compensate for the energy metabolic insufficiency by activating mTOR-YY1-PGC1α pathway Kunqian Ji1#, Jinfan Zheng1#, Jingwei Lv1, Jingwen Xu1, Xinbo Ji1, Yue-Bei Luo1, Wei Li1, Yuying Zhao1, Chuanzhu Yan1, 2, * 1

Laboratory of Neuromuscular Disorders and Department of Neurology, Qilu Hospital, Shandong University, Jinan, 250012, China 2 Department of Neurology, Qilu Hospital of Shandong University, Key Laboratory for Experimental Teratology of the Ministry of Education, Brain Science Research Institute, Shandong University, China. No. 107, West Wenhua Road, Jinan, 250012, China. # These authors contributed equally to this work. *Corresponding author at: Dr C. Yan. Department of Neurology, Qilu Hospital of Shandong University, Key Laboratory for Experimental Teratology of the Ministry of Education, Brain Science Research Institute, Shandong University, China. No. 107, West Wenhua Road, Jinan, 250012, China. Postal codes: 250012；Telephone: 0086-0531-82169217；Fax: 0086-0531-82169217 E-mail address: [email protected]

Abstract Fibroblast growth factor 21 (FGF21) is a growth factor with pleiotropic effects on regulating lipid and glucose metabolism. Its expression is increased in skeletal muscle of mice and human with mitochondrial disorder. However, the effects of FGF21 on skeletal muscle in response to mitochondrial respiratory chain deficiency is largely unknown. Here we demonstrated that the increased expression of FGF21 was a compensatory response to respiratory chain deficiency. The mRNA and protein levels of FGF21 were robustly raised in skeletal muscle from patient with mitochondrial myopathy or MELAS. The mammalian target of rapamycin (mTOR) phosphorylation levels and its downstream targets, Yin Yang 1 (YY1) and Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) were increased by FGF21 treatment in C2C12 myoblasts. Activation of mTOR-YY1-PGC1α pathway by FGF21 in myoblasts regulated energy homeostasis as demonstrated by significant increases in intracellular ATP synthesis, the oxygen consumption rate, the activity of citrate synthase, glycolysis, mitochondrial DNA copy number and induction the expression of key energy metabolic genes. The effects of FGF21 on mitochondrial function required phosphoinositide 3-kinase (PI3K), which activate mTOR. Inhibition of PI3K, mTOR, YY1 and PGC-1α activities attenuated the stimulating effects of FGF21 on intracellular ATP levels and mitochondrial genes expression. Our finding revealed that mitochondrial respiratory chain deficiency elicited a compensatory response in skeletal muscle by increased FGF21 expression levels in muscle, which resulting in enhanced mitochondrial function through an mTOR-YY1-PGC1α dependent pathway in skeletal muscle.

Keywords: Fibroblast growth factor 21; mitochondrial diseases; skeletal muscle; mitochondrial function; oxidative stress Abbreviations: CPEO, chronic progressive external ophthalmoplegia; CS, citrate synthase; cytochrome C oxidase, COX; ECAR, extracellular acidification rate; FGF21, Fibroblast growth factor; MELAS, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; mtDNA, mitochondrial DNA; mTOR, Mammalian target of rapamycin; mTORC1, mTOR complex I; mTORC2, mTOR complex II; PI3K, phosphoinositide 3-kinase; OCR, oxygen consumption rate; PGC-1α, Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha; YY1,Yin Yang 1

Introduction Since its discovery in 2005, fibroblast growth factor (FGF21) has attracted high interest due to its wide range of beneficial effect on metabolic homeostasis[1]. These effects include regulating glucose and lipid metabolism[2], and enhancing insulin sensitivity[3], mitochondrial oxidative function and thermogenesis[4]. Unlike most of the members from FGFs family, which require a heparin domain for efficient binding to the FGF receptors (FGFRs), FGF21, as a member of the endocrine FGF19 family, is secreted into the circulation, and can travel to sites distal from its origin and acts predominantly via endocrine mechanism[5]. Human FGF21 is a 187 amino acid protein which is predominantly secreted by liver and other tissues involved in glucose and lipid metabolism such as adipose, pancrease and muscle[6]. Studies in mouse indicate that the major site for FGF21 production is liver. Nevertheless, both the liver and serum levels of FGF21 are low under normal physiological condition. However, both hepatic and serum levels of FGF21 are dramatically elevated during fasting and ketogenic diet, and rapidly suppressed by refeeding[7]. Apart from the liver, the adipocytes also express and secrete FGF21 at time of thermogenic activation. However, the major source of the serum levels of FGF21 under normal physiological condition has yet to be discovered and some debate exists as to the relevance of basal serum levels of FGF21 to its regulation of physiology. Apart from starvation and obesity, there are studies on animals and human indicating that FGF21 is induced in individual tissues in response to specific diseases such as type 2 diabetes, coronary heart disease, liver injury, chemical insult and hepatic regenerative response[8-10]. Interestingly, several recent studies demonstrate that FGF21 is upregulated in patients with mitochondrial disorders[11], mice with mitochondrial respiratory chain deficiency[12] and mice defective in muscular autophagy/mitophagy[13]. Despite the importance of FGF21 as a sensitive biomarker of muscle-manifesting mitochondrial diseases, little is known about the role of FGF21 in skeletal muscle tissue. Additionally, the mechanisms by which FGF21 regulates mitochondrial oxidative function remain unclear. The mammalian target of rapamycin (mTOR) signaling pathway is a master regulator of cell metabolism and energy homeostasis[14]. The mTOR protein is a 289-kDa serine-threonine kinase and nucleates two distinct multi-protein complex, mTOR complex I (mTORC1) and mTOR complex II (mTORC2). Several signals has been demonstrated to activate mTORC1, such as Ras signaling, PI3K-AKT pathway and Wnt signaling[15]. Recently, mTORC1 has been to shown to play an

important role in mitochondrial metabolism and biogenesis. Here we demonstrate that FGF21 is induced in skeletal muscle tissue of patients with mitochondrial oxidative phosphorylation deficiency and compensates for energy metabolism deficiency by modulating mTOR activities via PI3K-AKT pathway in skeletal muscle cell. FGF21 increases the expression levels of Yin Yang 1 (YY1) and Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α). The activation of these key metabolic regulators result in enhancing mitochondrial oxidative function, accounting for the compensatory beneficial effect of FGF21 on mitochondrial diseases.

Materials and Methods Patients and muscle biopsy Muscle biopsy specimens from 18 patients with mitochondrial diseases were evaluated. The cohort included 13 patients with mitochondrial encephalomyopathy with lactic acidosis and stroke like episodes (MELAS) and confirmed mtDNA A3243G mutation. 5 patients with mitochondrial myopathy, including 2 patients with large-scale mtDNA deletion and 3 patients with cytochrome C oxidase (COX) deficiency. Fourteen muscle biopsies from subjects who were ultimately deemed to be free of neuromuscular diseases were selected as non-disease controls. Muscle biopsies were performed for diagnostic purpose after written informed consent and this study was approved by the local ethical board. Cell culture, treatments and retroviral transduction C2C12 myoblasts were grown at 37°C in a humidified 5%CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 10% (V/V) fetal calf serum, 1 mM sodium pyruvate, 200 U/ml Penicillin G, 200 mg/ml streptomycin, and 4 mM glutamine. Differentiation was induced by incubating the cells with DMEM containing 2% horse serum and penicillin-streptomycin for 6 days. Recombinant mouse FGF21 purchased from USCN Life Science (China) and was diluted to various concentrations in culture media ranging from 25ng/ml to 100ng/ml. Cells were treated for either 2, 12, 24, 36, or 48h. For rapamycin experiments, cells were incubated in growth medium containing 250nM rapamycin (Sigma) for 36h. For knockdown experiments, C2C12 myoblasts were infected with retrovirus expressing shRNA constructs against YY1 and PGC-1α. YY1 and PGC-1α shRNA lentivirus were purchased from GENECHEM (China) and ORIGENE respectively. Blocking PI3K-AKT pathway The C2C12 myoblasts were pretreated with 30µM LY294002 (Sigma) for 2 h and then treated with FGF21 (50ng/ml). After 24 h, the medium and cells were harvested for the detection of mitochondrial genes. The ATP levels were measured after 36h treatment with FGF21. RNA preparation, reverse transcription, Real-time Quantitative PCR (qPCR) and statistical analysis RNA from muscle biopsies and cells pellets was prepared using TRIzol reagent (sigma). RNA

quality was verified using Bioanalyzer and was reverse-transcribed into cDNA. Real-time Quantitative PCR was performed using SYBR®Green (TOYOBO) according to the manufacturer’s instructions and performed in triplicate on an ABI Prism 7900HT (Applied Biosystems) and were normalized to GAPDH or ACTIN proteins. The primer sequences are list in Supplementary Material, Table S1. Relative transcript abundance was calculated using the 2-ΔΔCt values and analyzed by paired t-tests to evaluate statistical significance relative to controls. Correct qPCR product size was verified by agarose gel electrophoresis, and melting curve measurements were made after every experiment. Western blot analysis Cells and muscle biopsies were lysed in cell lysis buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors. Lysates were sonicated for 1 min and centrifuged at 12, 000×g for 10 min at 4 °C and then 30-50 mg protein were run on a 8-12% SDS–PAGE gel at 100 V, 2-3h. Proteins were transferred to NC (Bio-Rad) for 1-3 h at 100 V, 4°C. After protein transfer, the membrane was incubated with various primary antibodies. All the total and phosphorylated antibodies, such as mTOR, Akt and p70s6, were obtained from Cell Signaling Technology. PGC-1α antibody was purchased from Santa Cruz Biotechnology. YY1 antibody was purchased from Abcam. Real-time Quantitative PCR for mtDNA copy number Total genomic DNA were extracted from the C2C12 myoblasts with a genomic DNA extraction kit (Tiangen, China). For the determination of mtDNA copy number relative to nDNA in cells, ND1 gene was used as a marker of mtDNA and ACTB gene for nDNA. Quantitative PCR was performed on a real-time PCR detection system using SYBR®Green, as described above. Measurement of oxygen consumption rate The oxygen consumption rate (OCR) measurements were performed using a Seahorse Bioscience XFe24 instruments (Seahorse Bioscience, North Billerica, MA). The C2C12 myoblasts were seeded into 24-wells Seahorse XF24e plates at a density of 50X103 cells/well in 200µL of DMEM. For the basal measurement of OCR, the C2C12 myoblasts were measured in the absence of uncoupler for three measurement cycles. The mitochondrial inhibitors oligomycin (1µM final) and FCCP (0.7µM final) were injected into the wells, followed by measurement cycles for OCR. The oxygen consumption rates were calculated from the continuous average slope the O2 decreases using a compartmentalization model. Basal OCR is [OCR with substrates – OCR with rotenone and antimycin A]. Maximal OCR is [OCR with FCCP – OCR with rotenone and antimycin A]. Assay for glycolysis, glycolytic capacity in C2C12 cells using the XFe-24 Flux Analyzer C2C12 cells were seeded using DMEM at 50X103 cells/well of cell plate (Seahorse Bioscience, North Billerica, MA) 24 hours before the assay. On the day of the assay, the media was changed to DMEM (without serum, glucose or bicarbonate, but with 2mM glutamine), and incubated for 1 hour before the assay in a non-CO2 incubator at 37°C. Injection of glucose (10mM final), oligomycin (1µM final) and 2-DG (100mM final) were diluted in the assay media and loaded onto port A, B and C respectively. The analyzer was calibrated and the assay was performed using glycolysis stress test assay protocol as suggested by the manufacturer (Seahorse Bioscience,

North Billerica, MA). The rate of glycolysis is the extracellular acidification rate (ECAR) after the addition of glucose. The glycolytic capacity is the rate of increase in ECAR after the injection of oligomycin following glucose. Citrate synthase activity and ATP concentration Citrate synthase (CS) activity was determined spectrophotometrically, as described previously[16]. Briefly, cells pellets were lysed in 50 mM Tris, 1 mM EDTA (pH 7.4), and 0.1% Triton X-100 and centrifuged at 12,000g for 10 min at 4°C. The supernatant was used to determine the protein content and CS activity levels and then loaded into one well of a 96-well plate. 190 µL of reaction buffer [100 mM Tris, 1 mM MgCl2, 1 mM EDTA (pH 8.2), and 0.1M DTNB] and 25 of acetyl-CoA (3.6 mM) were added. All analyses were completed in triplicate. To start the reaction, 50 of oxaloacetate (3 mM) was added, and the absorbance change at 412 nm was measured for 10 min at 37°C. The CS activity was calculated from the slope of the linear portion of the absorbance curve. ATP concentrations were determined using the luciferase-based ATP Assay purchased from Invitrogen according to the instructions of the manufacturer. The results of this assay were expressed as nanomoles ATP per milligram cell protein. Flow cytometry and confocal microscopy Cells were seeded in 6-well plates at a density of 1 X 105 cells/well and incubated as previously described. The cells were treated with 50ng/ml FGF21 for 48h and the media were changed very 24h. Following treatment, the media was removed and the cells were re-suspended in pre-warmed media with 200 nM Mitotracker red (Invitrogen, USA) incubated in a humidified 5% CO2 atmosphere at 37°C for 30 min. The cells were pelleted, the media with Mitotracker was removed and the cells were rinsed and suspended in pre-warmed media. Group mean fluorescence was measured using Facscalibur filtering 579nm. For confocal microscopy experiment, cells are seeded in Chamber-slides and after FGF21 treatment for 48h, cells were stained with Mitotracker red as described above. Cells were imaged by confocal microscope (Carl Zeiss LSM 780). Statistical analyses Quantitative date are presented as mean±SEM. Data were analyzed by the one-way ANOVA and Student’s t-test, using the SPSS 13.0 software. p