Transcriptomic analysis for identification of exercise-induced myokines<\/p>\n
To identify the exercise-induced myokines, transcriptomic data (GSE28422)13<\/a> were analyzed using \u2018GEOquery v2.60.0\u2019 and \u2018LIMMA v3.48.3\u2019 in the R environment (v4.2). We followed the GEOquery-LIMMA pipeline to obtain and normalize the expression data, followed by the identification of differentially expressed genes in age groups between before and after exercise (p-value\u2009\u20091.5). To examine the expression of secretory protein genes, a list of predicted secretory proteins was downloaded from The Human Protein Atlas (2933 genes as of May 2022) at https:\/\/www.proteinatlas.org\/humanproteome\/tissue\/secretome<\/a>, and their expression fold-change values and p-values were extracted from the differential expression analysis result between pre- and post-exercise samples obtained earlier. Volcano plots were generated by the \u2018ggplot2 v3.3.6\u2019 package in R.<\/p>\n Ethics<\/p>\n Human cohorts 1\u20134 were conducted as Basic Experimental Studies involving Humans (BESH), and participants were not enrolled in a clinical trial. All procedures were approved by the relevant institutional review boards and were conducted in accordance with the Declaration of Helsinki. Human cohort 5 consisted of previously collected samples from a published study62<\/a>, which reported the primary outcomes and was not considered a clinical trial. Human studies 1, 3, 4, and 6 were approved by the Institutional Review Board (IRB) at Pusan National University (IRB protocol number PNU_2021_140_HR). Human study 2 was approved by the IRB at Inha University (170621-1AC). Human study 5 was approved by the IRB at Seoul National University (SNUIRB no.1305\/001-009). Written informed consent was obtained from all participants.<\/p>\n Human cohorts<\/p>\n Human study 1 (Young adults_acute resistance exercise): Ten young, active males (age 25.33\u2009\u00b1\u20091.837 years; height 178.4\u2009\u00b1\u20091.913\u2009cm; weight 76.08\u2009\u00b1\u20092.983; BMI 23.84\u2009\u00b1\u20090.5952\u2009kg\/m2) males recruited to partake in this study. Participants were compensated with a monthly gym membership worth 45,000 won. Participants trained at a moderate intensity corresponding to 50\u201369% of their one-repetition maximum (1-RM), which is the heaviest weight they could lift for one repetition in this exercise. The exercise program is described in Table\u00a0S1<\/a>. Participants fasted for at least 8\u2009h prior to exercise. Blood samples were collected before exercise and 1\u2009h post-exercise.<\/p>\n Human study 2 (Young Jiu-Jitsu athletes_High-intensity interval exercise): Participants were selected based on the following criteria: a minimum of 4 years of Jiu-Jitsu training, training at least 4 days per week, a minimum of 8\u2009h of training per week, and a history of placing in national Jiu-Jitsu competitions. Participants were compensated 200,000 won over the course of the study. Blood samples were collected from young Jiu-Jitsu athletes (age: 28.25\u2009\u00b1\u20093.62; height 176.63\u2009\u00b1\u20093.58\u2009cm; weight 76.76\u2009\u00b1\u20098.62\u2009kg; BMI 24.59\u2009\u00b1\u20092.48\u2009kg\/m\u00b2) before and immediately after completing the 4 rounds of the exercise program in a previous study by Park et al.63<\/a>. Briefly, the high-intensity interval resistance exercise program, designed to mimic match conditions and induce exhaustion, consisted of six exercises, each performed for 1\u2009min, followed by a 12-min rest period, and repeated for a total of 4 rounds. The exercises were: 1. Bar Cross Lateral Jump and Sprawl, 2. Bar hanging kimono holding, 3. 4-Step turtle control in the top position, 4. Gi grip jump over and roll back from the top position on the turtle, 5. Jump over the vaulting horse and tackle, 6. Rolling drill between the legs.<\/p>\n Human study 3 (Young adults_acute aerobic exercise): Ten young, active males (age 25.98\u2009\u00b1\u20091.74 years; height 175.9\u2009\u00b1\u20091.53\u2009cm; weight 75.38\u2009\u00b1\u20092.53\u2009kg; BMI 24.44\u2009\u00b1\u20090.98\u2009kg\/m\u00b2) participated in this study. Participants were compensated with a monthly gym membership worth 45,000 won. Participants fasted for a minimum of 8\u2009h prior to exercise and then exercised on a treadmill for 60\u2009min at a moderate intensity (heart rate between 40% and 59% of their maximum). The exercise program is described in Table\u00a0S2<\/a>. Blood was collected at rest before and one hour after exercise to evaluate plasma CLCF1 levels.<\/p>\n Human study 4 (Older adults_Chronic resistance training): Twenty-seven female older participants (age 72.32\u2009\u00b1\u20090.9484 years; height 155.7\u2009\u00b1\u20090.8637\u2009cm; weight 60.04\u2009\u00b1\u20091.257\u2009kg; BMI 24.79\u2009\u00b1\u20090.5447\u2009kg\/m\u00b2) participated in this study (details provided in Table\u00a0S3<\/a>). Participants were compensated with a monthly gym membership worth 45,000 won. Older participants exercised at 30~49% of 1-RM (light intensity) for one hour per day, 5 times per week for 16 weeks. Blood samples were collected from all participants the day after the final exercise session, following a minimum of 8\u2009h of fasting. The exercise program is described in Table\u00a0S4<\/a>.<\/p>\n Human study 5 (Older adults_Chronic resistance band exercise): Blood samples were collected in a project investigating the effect of 12 weeks of elastic band-based high-speed power training on cognition and muscle function in elderly participants (n\u2009=\u200914, age 75.0\u2009\u00b1\u20093.46 years), as described in a study by Yoon et al.62<\/a>. As a reward for their participation, participants were provided with exercise elastic bands and received personalized feedback on their exercise performance and blood test results. Briefly, the intensity was set by adjusting the length of the green elastic band (Hygenic Corporation, Akron, OH, USA). Participants engaged in a 12-week exercise program, completing two weekly 60-min sessions (Tuesday and Thursday). Each session included a 10-min warm-up, 40\u2009min of elastic band training, and a 10-min cool-down period. Blood samples were collected before and after a 12-week exercise program in elderly participants. Following a minimum of 8\u2009h of fasting, blood samples were collected from all participants the day after they completed the final exercise session.<\/p>\n Human study 6 (Plasma levels of CLCF1 in young and elderly subjects): To measure basal CLCF1 levels in young and elderly individuals, we utilized pre-exercise plasma samples from Human studies 1\u20135, along with additional plasma samples from 16 healthy young individuals and 10 healthy elderly individuals (age: 73.36\u2009\u00b1\u20094.69; height 153.57\u2009\u00b1\u20093.14\u2009cm; weight 54.13\u2009\u00b1\u20094.12\u2009kg; BMI 22.72\u2009\u00b1\u20091.92\u2009kg\/m\u00b2).<\/p>\n Animals<\/p>\n All animal experiments conducted in this study were approved by the Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). The mouse strains used in the experiments were of the C57BL\/6J background. Young mice (3 months old), middle-aged mice (8 months old), and aged mice (18\u201324 months old) were obtained from the Laboratory Animal Resource Center (KRIBB). The mice were housed in cages maintained at 22\u201324\u2009\u00b0C under a 12-h light\/12-h dark cycle and were provided with free access to food and water. All mice were euthanized by CO2 inhalation followed by cervical dislocation. Serum and tissue samples were subsequently collected.<\/p>\n Generation of CLCF1-CRF1 TG mice and Clcf1-skeletal muscle-specific knockout mice<\/p>\n CLCF1 transgenic C57BL\/6N mice were generated (Macrogen, Seoul, Korea). To facilitate efficient secretion, CRLF1 acted as a chaperone for CLCF140<\/a>. A G4Sx3 linker was used to fuse CLCF1 with CRLF1. The cytomegalovirus (CMV) promoter was utilized to drive the expression of the Clcf1_G4Sx3_Crlf construct. Subsequently, the resulting plasmid, CMV_Clcf1_G4Sx3_Crlf_IRES_EmGFP, underwent digestion using NruI and PsiI enzymes to isolate the transgenic cassette. This expression cassette was purified and delivered through pronuclear injection into mouse zygotes with a C57BL\/6N genetic background.<\/p>\n In order to create mice with skeletal muscle-specific Clcf1 deletion, mice harboring the conditional Clcf1 floxed allele were generated from Clcf1tm1a(EUCOMM)Wtsi ES cells obtained from EUCOMM. The floxed Clcf1 mice were kindly provided by the Korea Mouse Phenotyping Center (KMPC). They were bred with Flp recombinase mice to get homozygous Clcf1 floxed breeders. These breeders were crossed with Mlc-Cre mice to produce a strain of mice that lack Clcf1 specifically in their skeletal muscles.<\/p>\n Plasma sample assays ELISA<\/p>\n Blood samples were collected into tubes containing the anticoagulant EDTA (Greiner Bio-One, 450542) to prevent coagulation. The collected blood was then centrifuged at 1000\u2009\u00d7\u2009g for 10\u2009min at 4\u2009\u00b0C to separate the plasma. After centrifugation, the plasma was carefully collected into new tubes to avoid contamination with cellular components and stored at \u221280\u2009\u00b0C until further analysis. The concentration of CLCF1 in the plasma was measured using an enzyme-linked immunosorbent assay (ELISA) kit (USCN, SEC389Hu; Mybiosource, MBS762838) according to the manufacturer\u2019s instructions. Mouse and human plasma samples were diluted 5-fold prior to performing the ELISA experiments. Samples were incubated at 37\u2009\u00b0C for 1\u2009h, followed by three washes with PBST (1 \u00d7 PBS\u2009containing 0.05% Tween 20). The secondary antibody was diluted at 1:1000 in dilution buffer and incubated for 1\u2009h. The plates were then washed three times with PBST, followed by incubation with TMB substrate solution at 37\u2009\u00b0C for 20\u2009min. The reaction was stopped by adding the Stop Solution. All samples and standards were assayed in duplicate, and absorbance was measured using a microplate reader.<\/p>\n Cell culture<\/p>\n The C2C12 mouse muscle cell line (ATCC, #CRL-1772) was maintained in a humidified incubator at 37\u2009\u00b0C with 5% CO2 and cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin\/streptomycin (100\u2009U\/mL, 100\u2009\u03bcg\/mL). To induce differentiation, C2C12 cells were grown in DMEM containing 2% FBS and 1% penicillin\/streptomycin (Pen\/Strep) until they formed myotubes. 3T3-L1 cells were differentiated in DMEM supplemented with 10% FBS, 1% Pen\/Strep, and MDI (5\u2009mM isobutylmethylxanthine (IBMX), 5\u2009\u03bcg\/mL insulin, and 1\u2009\u03bcM dexamethasone) for 2 days following cell confluence. The cells were then maintained in medium containing 5\u2009\u03bcg\/mL insulin for an additional 2 days.<\/p>\n Primary osteoblast precursor cells were isolated from neonatal mouse calvariae using enzymatic digestion. The first set of cells following an initial treatment of calvariae for 10\u2009min at 37\u2009\u00b0C with 0.1% collagenase (Life Technologies, Carlsbad, CA) and 0.2% dispase II (Roche Diagnostics GmbH, Mannheim, Germany) were discarded. Primary osteoblast precursor cells were isolated from clavariae by sequential enzymatic digestion with collagenase for four 10-min periods. Osteoblast differentiation was then induced using osteogenic medium (OGM) containing 100\u2009ng\/mL BMP2, 50\u2009\u03bcg\/mL ascorbic acid, and 100\u2009mM \u03b2-glycerophosphate for 3 or 6 days. ALP activity analysis was performed on day 3 of osteogenic differentiation. The following lysis of cells using an osteoblast lysis buffer [50\u2009mM Tris-HCl (pH 7.4), 1% Triton X-100, 150\u2009mM NaCl, and 1\u2009mM EDTA], cell lysates were incubated with p-nitrophenyl phosphate substrate (Sigma-Aldrich, St Louis, MO) and ALP activity was determined by measuring the absorbance at 405\u2009nm using a spectrophotometer. The alizarin red staining was performed after 6 days of osteogenic differentiation. The cultured cells were fixed with 70% ethanol and treated with a 40\u2009mM alizarin red (pH 4.2) for 10\u2009min. The stained culture was visualized with a CanoScan 9900F (Canon Inc., Japan), and then alizarin red was extracted with 10% acetic acid to quantify the substrate calcification. The concentration of alizarin red was determined by measuring the absorbance at 405\u2009nm using a spectrophotometer.<\/p>\n Bone marrow cells were isolated from the long bones of ICR mice by flushing with \u03b1-MEM and incubated with a Red Blood Cell Lysis Buffer (Sigma-Aldrich, St. Louis, MO, USA). Bone marrow cells were cultured in \u03b1-MEM containing 10% FBS and antibiotics in the presence of M-CSF (30\u2009ng\/mL) for 3 days. Adherent cells (osteoclast precursors) were cultured in the presence of M-CSF and RANKL for 3 days. The cultured cells were fixed with 3.7% formalin and treated with TRAP staining solution (Sigma-Aldrich) for 10\u2009min. The stained cells were visualized with a ProgRes CFscan camera (Jenoptik, Jena, Germany) and ProgRes Capture Pro software (Jenoptik). TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclasts.<\/p>\n Electrical pulse stimulation (EPS)<\/p>\n C2C12 myoblasts were seeded in six-well plates and differentiated into myotubes by culturing in differentiation medium (DMEM supplemented with 2% FBS) for 3 days. Following the differentiation period, the myotubes were subjected to electrical pulse stimulation (EPS) using the C-PACE EP culture pacer system (IonOptix, Dublin, Ireland) in a humidified incubator maintained at 37\u2009\u00b0C with 5% CO2. The EPS protocol consisted of monophasic square wave pulses with a pulse duration of 2 milliseconds (ms) delivered at a frequency of 1\u2009Hz. The voltage applied was 11.5 volts (V). Prior to the initiation of stimulation, cells were allowed to equilibrate in the stimulation setup for 1\u2009h to recover from handling stress. Electrical stimulation was then applied continuously for 24\u2009h under the specified conditions.<\/p>\n All stimulation parameters, including frequency, pulse width, and voltage, were optimized based on previous studies to mimic in vivo muscle contraction and ensure viability of C2C12 myotubes during prolonged stimulation.<\/p>\n Treadmill exercise<\/p>\n Mice underwent a gradual acclimatization process over two days, starting with a low incline and slow speed to familiarize them with the exercise equipment. Over the next two days, the incline was moderately increased while progressively extending the session duration. From day 5 onwards, the incline was further increased, and speed was slightly adjusted based on the mice\u2019s tolerance. The objective was to push both young and aged mice near exhaustion without causing complete physical collapse. After exercise adaptation, all exercise bouts were performed at 70% of maximal speed on an incline greater than 5\u00b0. While young mice could easily maintain a speed of 24\u2009m\/min on a 10\u00b0 incline, aged mice could only manage a speed of 15\u201318\u2009m\/min on a 5\u00b0 incline. Each exercise bout was maintained for a total duration of 1\u2009h. High-resistance running was defined as maintaining a speed of 24\u2009m\/min on a 10\u00b0 incline. The chronic training protocol involved daily exercise sessions conducted over 30 consecutive days. Blood samples were collected from the mice before the exercise and immediately after the final exercise session.<\/p>\n Mitochondrial DNA contents<\/p>\n Genomic DNA was isolated from differentiated C2C12 myotubes using the Quick-DNA Miniprep Plus Kit (Zymo Research, D4068) according to the manufacturer\u2019s instructions. Mitochondrial DNA (mtDNA) content was assessed by measuring the relative abundance of the mitochondrial gene ND1 to the nuclear gene HK2 using quantitative real-time PCR (qRT-PCR). The relative mtDNA content was calculated using the \u0394Ct method, where \u0394Ct\u2009=\u2009Ct(HK2)\u2009\u2212\u2009Ct(ND1). Lower \u0394Ct values indicate a higher mitochondrial-to-nuclear DNA ratio. Primer sequences used for ND1 and HK2 are provided in Supplementary Table\u00a0S6<\/a>.<\/p>\n Glucose uptake and free fatty acid uptake assay<\/p>\n Glucose uptake was measured in vitro using differentiated C2C12 myotubes and 3T3-L1 adipocytes. After full differentiation, cells were serum-starved for 4\u2009h in glucose-free DMEM prior to treatment. For stimulation of glucose uptake, cells were incubated with 1\u2009\u03bcM insulin for 20\u2009min at 37\u2009\u00b0C. Following treatment, cells were washed with PBS, lysed, and analyzed using the Glucose Uptake Assay Kit (Abcam, ab136955) according to the manufacturer\u2019s instructions. Absorbance was measured using a microplate reader at the recommended wavelength, and relative glucose uptake was quantified by interpolation from a standard curve. To measure free fatty acid (FFA) uptake, differentiated C2C12 myotubes and 3T3-L1 adipocytes were cultured in 96-well plates and treated with recombinant CLCF1 (100\u2009ng\/mL) for 24\u2009h. FFA uptake was then assessed using the Free Fatty Acid Uptake Assay Kit (Abcam, ab176768), according to the manufacturer\u2019s instructions. Briefly, after CLCF1 treatment, cells were incubated with a fluorescent fatty acid substrate provided in the kit, and uptake was measured using a fluorescence microplate reader at Ex\/Em\u2009=\u2009485\/535\u2009nm. Background signal was subtracted using control wells containing no cells, and relative fluorescence units (RFU) were normalized to total protein concentration per well. All experiments were performed in at least five independent biological replicates.<\/p>\n Glucose tolerance and insulin tolerance test<\/p>\n For glucose tolerance tests (GTT), mice were fasted overnight (10\u201312\u2009h) before receiving an intraperitoneal injection of glucose at a dose of 2\u2009g\/kg body weight. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120\u2009min using the ACCU-CHEK Performa test strip. Similarly, for insulin tolerance tests (ITT), mice were fasted for 4\u2009h before receiving an intraperitoneal injection of insulin at a dose of 0.75\u2009U\/kg body weight. Blood glucose levels were measured at the same time points (0, 15, 30, 60, 90, and 120\u2009min) using ACCU-CHEK Performa test strips. For both tests, blood samples were collected from the tail vein at the indicated time points.<\/p>\n Western blotting<\/p>\n Cells and tissues were lysed with RIPA buffer containing 0.1\u2009mM Na3VO4, 1\u2009mM NaF, 1\u2009mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), and 5\u2009mg\/ml aprotinin (Sigma-Aldrich). Total proteins were quantified using the BCA (bicinchoninic acid) assay kit and Bradford assay (Thermo Fisher, 23227). Protein samples were loaded on SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes and nitrocellulose (NC) membranes. The membranes were blocked for 1\u2009h at RT with 5% skim milk or 2% BSA in Tris-buffered saline (TBS). The membrane was incubated with the respective primary antibodies at 4\u2009\u00b0C overnight. All listed primary antibodies were used 1:1000. After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000\u20131:10,000) for 1\u2009h at RT. The image was detected using the ChemiDoc (Thermo Fisher, iBright CL1500). All primary antibodies used in this study are listed in Supplementary Table\u00a0S5<\/a>, along with their sources and catalog numbers.<\/p>\n Retroviral gene transduction<\/p>\n Retroviral vectors were transfected into the packaging cell line, Plat E, using FuGENE 6 (Promega, Madison, WI) according to the manufacturer\u2019s instructions. Target cells were plated on 48-well or 6-well plates 24\u2009h prior to infection. Viral supernatants, collected from the culture medium 48\u2009h after transfection, were then incubated with the target cells for 6\u2009h in the presence of 10\u2009\u03bcg\/mL polybrene (Sigma-Aldrich, St Louis, MO, USA).<\/p>\n Immunocytochemistry<\/p>\n C2C12 myoblasts were seeded on 24-well plates and differentiated into myotubes. Myotubes were rinsed in PBS, fixed in 4% paraformaldehyde for 15\u2009min at room temperature, and washed three times with PBS and permeabilized in 0.25% Triton X-100 in PBS for 10\u2009min. The myotubes were blocked with 1% BSA in PBS containing 0.1% Tween 20 for 30\u2009min and incubated with anti-MYH (sc-376157) in blocking buffer overnight at 4\u2009\u00b0C, followed by Alexa Fluor 488 (#A-21121, Thermo Fisher) with 1% BSA in PBS for 1\u2009h at room temperature. And the myotubes were incubated with Vectashield with DAPI mounting medium (VECTOR Laboratories) for 1\u2009min in the dark. Images were captured using a Nikon Eclipse Ti-U inverted microscope and Nikon DS-Ri2 camera using NIS-Elements software. The short-axis diameters of myotubes were analyzed by Image J software.<\/p>\n Immunohistology<\/p>\n Muscles were mounted in optimal cutting temperature medium (O.C.T, Tissue-Tek) at resting length and frozen in dry ice and stored at \u221280\u2009\u00b0C. Muscle sections (7\u2009\u03bcm) were cut from the mid at \u221220\u2009\u00b0C on a cryostat (Leica, CM1950) and collected on Micro Slides (MUTO, Japan) and stored at \u221280\u2009\u00b0C. For immunofluorescence staining, serial sections were air-dried for 30\u2009min and washed three times with PBS. Sections were blocked and permeabilized in PBS and 0.4% Triton X-100 for 30\u2009min before being incubated for 2\u2009h at room temperature in a primary antibody solution containing laminin (L9393, Sigma-Aldrich) and 10% goat serum (ab7481, Abcam) followed by Alexa555 (A32732, Invitrogen), Alexa555 (A28180, Invitrogen) in PBS containing 10% goat serum. Sections were washed three times in PBS and mounted with Vectashield with DAPI mounting medium (VECTOR Laboratories). Images were captured using a Nikon Eclipse Ti-U inverted microscope and Nikon DS-Ri2 camera using NIS-Elements software.<\/p>\n Quantitative RT-PCR (qPCR)<\/p>\n Total RNA was isolated from tissues and cells using RiboEx reagent (GeneAll Biotechnology Co., South Korea). cDNA synthesis was performed using the iScript cDNA synthesis kit (Bio-Rad). Quantitative RT-PCR analysis was performed using StepOnePlus (Applied Biosystems) with a 20\u2009\u03bcl reaction volume containing cDNA, primers, and SYBR Master Mix (Applied Biosystems). The sequences of the primers used in this study are provided in Supporting Information, Table\u00a0S6<\/a>.<\/p>\n CLCF1 supplementation<\/p>\n 3-month-old and 20-month-old C57BL6 mice were injected intraperitoneally with either recombinant CLCF1 (Abcam, ab50021 or Enzynomics, C045) or PBS (vehicle control) at a dose of 0.1 or 0.5\u2009mg\/kg body weight per day for 14 consecutive days.<\/p>\n eCNTFR production and supplementation<\/p>\n We used an engineered CNTFR (eCNTFR)18<\/a> to achieve effective blockade of CLCF1. This blockade requires both increased binding of the decoy to CLCF1 and decreased binding of CLCF1 to gp130 and LIFR. The eCNTFR-Fc recombinant protein, containing eight consensus mutations (R110Q, T174P, S237F, I287F, Y177H, K178N, T268A, D269A), was synthesized by Y-Biologics (Y-Biologics, Deajeon, South Korea). Mice were treated with eCNTFR-Fc (10\u2009mg\/kg) or PBS (vehicle) by i.p injection for 1 month, three times per week.<\/p>\n Production of CLCF1\/CRLF1 protein complex<\/p>\n The human Cardiotrophin-like cytokine factor 1 (hCLCF1, Uniprot Q9UBD9) and human Cytokine receptor-like factor 1 (hCRLF1, Uniprot O75462) genes were amplified by PCR (Sogent, SPX16-R250). Amplicons were digested with BamHI (New England Biolabs, R0136) \/ XhoI (New England Biolabs, R0146) and inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, A14697). A polyhistidine tag was placed at the C-terminus of CLCF1 for affinity chromatography. For transient expression of CLCF1\/CRLF1 protein complex, Expi293FTM cells (Thermo Fisher Scientific, A14527) were cultured in Expi293TM expression medium (Thermo Fisher Scientific, A1435102). The cells were maintained in a humidified chamber at 37\u2009\u00b0C with 8% CO2. ExpiFectamineTM 293 transfection kit (Thermo Fisher Scientific, A14525) was used following the manufacturer\u2019s instructions for efficient transfection of Expi293FTM cells with plasmids. Briefly, the cells were diluted at a density of 1.7\u2009\u00d7\u2009106 cells\/ml in 300\u2009ml of fresh media in 1\u2009L Erlenmeyer shake flask (Corning, 7513). On the following day, DNA plasmids encoding hCLCF1 and hCRLF1 genes and ExpiFectamineTM 293 were separately mixed in Opti-MEMTM (Thermo Fisher Scientific, 31985070) and incubated at room temperature for 5\u2009min. Then, the ExpiFectamineTM 293 and DNA mixtures were combined and incubated for another 20\u2009min at room temperature. Finally, the mixture was added to flasks containing the cells and incubated in a humidified chamber at 37\u2009\u00b0C with 8% CO2. After 18\u2009h, a cocktail of Enhancer I and II was added to the flask. The cells were then incubated for 72~96\u2009h, and the culture media were collected. Recombinant CLCF1\/CRLF1 protein complex was purified by affinity and size exclusion chromatography using a fast protein liquid chromatography machine (FPCL) (Cytiva, \u00c4KTA Go system). The flited culture media was applied at a flow rate of 5\u2009ml\/min to the HisTrap Excel column (Cytiva, 17371205) previously equilibrated with 20\u2009mM imidazole, 500\u2009mM NaCl, and 20\u2009mM Na2HPO4 at pH 7.4. Unbound proteins were washed away with 50\u2009ml equilibration buffer. Bound fractions were eluted with 75\u2009mL elution buffer containing a 20-to-500\u2009mM imidazole gradient in 20\u2009mM Na2HPO4 and 500\u2009mM NaCl at pH 7.4 for 15\u2009min. The protein complex was collected, pooled, and analyzed by SDS-PAGE. The eluted proteins were polished by SEC on an FPLC machine using a HiLoad 16\/600 Superdex 200 pg (Cytiva, 28989335) previously equilibrated with PBS (pH 7.4) at a flow rate of 0.5\u2009ml\/min. Finally, Fractions containing CLCF1\/CRLF protein complex were collected, pooled, and analyzed by SDS-PAGE and immunoblot analysis.<\/p>\n Glycolysis measurement and oxygen consumption rate<\/p>\n Oxygen consumption rates of C2C12 myotube cells, seeded at a density of X cells\/well in a 96-well cell culture plate, were recorded on an XF96 Extracellular Flux Analyzer using the XF Cell Mito Stress Test Kit (Seahorse Bioscience, North Billerica, MA) according to the manufacturer\u2019s protocol. Following seeding, cells were washed and pre-incubated in XF DMEM media (pH 7.4) for 1\u2009h prior to the assay. The XF Cell Mito Stress Test was then performed according to the manufacturer\u2019s protocol. Oligomycin (1\u2009\u03bcM), FCCP (2\u2009\u03bcM), and rotenone & antimycin A (0.5\u2009\u03bcM each Rot\/Anti) were added as indicated. All measured values were normalized to total protein content, which was quantified using the BCA protein assay.<\/p>\n siRNA transfection<\/p>\n C2C12 cells were seeded in 24-well plates with 1\u2009\u00d7\u2009105 cells per well and then differentiated into myotubes for 4 days using a differentiation medium (2% FBS DMEM). At 3 days during differentiation, myotube was transiently transfected with siRNA control (Dhamacon, ON-TARGET plus siRNA) or siRNA Cntfr, Il6st (gp130), and Lifr using Lipofectamin RNAiMAX reagent (Thermo Fisher Scientific, USA) according to the manufacturer\u2019s protocols. Primary osteoblast precursor cells or osteoclast precursor cells were transfected with siRNA control, siRNA Stat1, or siRNA Stat3 (Dhamacon, ON-TARGET plus siRNA) using Lipofectamine RNAiMAX (Thermo Fisher Scientific, USA), according to the manufacturer\u2019s instructions.<\/p>\n Skeletal muscle function explorationTime and distance to exhaustion<\/p>\n After five consecutive days of acclimation, running performance was tested in mice by measuring their ability to run until exhaustion. Mice were exercised on a treadmill at 10\u2009m\/min for 10\u2009min, and the speed was increased every 2\u2009min by 2\u2009m\/min until 24\u2009m\/min. Mice were considered to be exhausted when mice remained on the electric shock grid for more than 10\u2009s. Time and distance were recorded and calculated.<\/p>\n Strength by grip test<\/p>\n Mice were allowed to grasp an angled grid with all four limbs. They were then gently pulled backwards until they lost their grip, for a total of three attempts. The force (in Newtons) applied to the grid during each attempt was recorded. The average of these three measurements was then normalized to the body weight of each mouse.<\/p>\n Inverted grid test<\/p>\n Mice were placed in the center of a wire grid system, which was then inverted and held steadily 40\u2009cm above a padded surface. The latency to fall, measured by the time the mice spent hanging onto the grid system, was then recorded.<\/p>\n Ex vivo isometric force and fatigue measurements<\/p>\n Intact TA muscles were isolated from the hindlimb of euthanized mice and mounted vertically between a force transducer (Model FT03, Glass Instruments, USA) in an organ bath with platinum electrodes and continuous perfusion with 95% O2\u2009+\u20095% CO2-saturated Krebs\u2013Ringer solution (118\u2009mM NaCl, 4.75\u2009mM KCl, 24.8\u2009mM NaHCO3, 1.18\u2009mM KH2PO4, 2.5\u2009mM CaCl2\u2009\u2219\u20092H20, 1.18\u2009mM MgSO4, and 10\u2009mM glucose). Optimal muscle stretch was determined by applying a single twitch at a supramaximal voltage of 100\u2009V for 1\u2009ms, and set at the length that generated maximal twitch force after 10\u2009min of equilibration, TA muscles were tested with a range of stimulation frequencies (tetanic with increasing frequencies from 30 to 200\u2009Hz every 500\u2009ms, with 2-min recovery intervals between each frequency). The fatigue properties of the TA muscles were assessed by repeated stimulation at 1\u2009Hz and 100\u2009V for 10\u2009min. All experiments were conducted at 25\u2009\u00b0C. Data acquisition and analysis were performed using LabChart Pro Software (Version 8; AD Instruments, Pty Ltd.). At the end of each experiment, muscle length, diameter, and wet weight were measured.<\/p>\n RANKL-induced bone loss and ectopic bone formation<\/p>\n