#METABOLOMICS WORKBENCH qiuxu_20250101_180456 DATATRACK_ID:5501 STUDY_ID:ST003650 ANALYSIS_ID:AN005996 PROJECT_ID:PR002260 VERSION 1 CREATED_ON January 2, 2025, 10:06 pm #PROJECT PR:PROJECT_TITLE 3-Hydroxybutyrate promotes myoblast proliferation and differentiation PR:PROJECT_SUMMARY Muscle wasting is a major clinical challenge in various diseases and PR:PROJECT_SUMMARY physiological states, as the loss of skeletal muscle mass adversely affects PR:PROJECT_SUMMARY patient outcomes. This study elucidates the role of the endogenously supplied PR:PROJECT_SUMMARY metabolite 3-hydroxybutyrate (3-HB) in promoting proliferation and PR:PROJECT_SUMMARY differentiation of C2C12 myoblasts through nuclear magnetic resonance PR:PROJECT_SUMMARY (NMR)-based metabolomic analysis. PR:INSTITUTE Xiamen University PR:LAST_NAME Qiu PR:FIRST_NAME Xu PR:ADDRESS Xiamen University PR:EMAIL qiuxu@stu.xmu.edu.cn PR:PHONE 13395036603 #STUDY ST:STUDY_TITLE Investigation of the effects of 3-hydroxybutyrate on myoblast proliferation and ST:STUDY_TITLE differentiation using NMR-based metabolomics ST:STUDY_SUMMARY This study aims to further elucidate the molecular mechanisms by which 3-HB ST:STUDY_SUMMARY promotes the proliferation and differentiation of C2C12 myoblasts. By performing ST:STUDY_SUMMARY NMR-based metabolomic analysis on myoblasts, we explored the dual roles of 3-HB ST:STUDY_SUMMARY functioning as a metabolic substrate and a signaling molecule in myoblasts, and ST:STUDY_SUMMARY uncovered the underlying molecular mechanisms. Our results may significantly ST:STUDY_SUMMARY contribute to the development of 3-HB as a potential therapeutic agent against ST:STUDY_SUMMARY skeletal muscle atrophy. ST:INSTITUTE Xiamen University ST:LAST_NAME Qiu ST:FIRST_NAME Xu ST:ADDRESS Xiamen University ST:EMAIL qiuxu@stu.xmu.edu.cn ST:PHONE 13395036603 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - C1 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=1 SUBJECT_SAMPLE_FACTORS - C2 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=2 SUBJECT_SAMPLE_FACTORS - C3 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=3 SUBJECT_SAMPLE_FACTORS - C4 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=4 SUBJECT_SAMPLE_FACTORS - C5 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=5 SUBJECT_SAMPLE_FACTORS - C6 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=6 SUBJECT_SAMPLE_FACTORS - C7 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=7 SUBJECT_SAMPLE_FACTORS - C8 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=8 SUBJECT_SAMPLE_FACTORS - C9 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=9 SUBJECT_SAMPLE_FACTORS - C10 Sample source:C2C12 Myoblast cells | Factors:Treatment Control RAW_FILE_NAME(Fid ID)=10 SUBJECT_SAMPLE_FACTORS - CK1 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=11 SUBJECT_SAMPLE_FACTORS - CK2 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=12 SUBJECT_SAMPLE_FACTORS - CK3 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=13 SUBJECT_SAMPLE_FACTORS - CK4 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=14 SUBJECT_SAMPLE_FACTORS - CK5 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=15 SUBJECT_SAMPLE_FACTORS - CK6 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=16 SUBJECT_SAMPLE_FACTORS - CK7 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=17 SUBJECT_SAMPLE_FACTORS - CK8 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=18 SUBJECT_SAMPLE_FACTORS - CK9 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=19 SUBJECT_SAMPLE_FACTORS - CK10 Sample source:C2C12 Myoblast cells | Factors:Treatment 5 mM-3HB RAW_FILE_NAME(Fid ID)=20 SUBJECT_SAMPLE_FACTORS - L1 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=21 SUBJECT_SAMPLE_FACTORS - L2 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=22 SUBJECT_SAMPLE_FACTORS - L3 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=23 SUBJECT_SAMPLE_FACTORS - L4 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=24 SUBJECT_SAMPLE_FACTORS - L5 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=25 SUBJECT_SAMPLE_FACTORS - L6 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=26 SUBJECT_SAMPLE_FACTORS - L7 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=27 SUBJECT_SAMPLE_FACTORS - L8 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=28 SUBJECT_SAMPLE_FACTORS - L9 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=29 SUBJECT_SAMPLE_FACTORS - L10 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose RAW_FILE_NAME(Fid ID)=30 SUBJECT_SAMPLE_FACTORS - LK1 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=31 SUBJECT_SAMPLE_FACTORS - LK2 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=32 SUBJECT_SAMPLE_FACTORS - LK3 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=33 SUBJECT_SAMPLE_FACTORS - LK4 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=34 SUBJECT_SAMPLE_FACTORS - LK5 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=35 SUBJECT_SAMPLE_FACTORS - LK6 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=36 SUBJECT_SAMPLE_FACTORS - LK7 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=37 SUBJECT_SAMPLE_FACTORS - LK8 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=38 SUBJECT_SAMPLE_FACTORS - LK9 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=39 SUBJECT_SAMPLE_FACTORS - LK10 Sample source:C2C12 Myoblast cells | Factors:Treatment Low glucose+3HB RAW_FILE_NAME(Fid ID)=40 #COLLECTION CO:COLLECTION_SUMMARY Cells were rinsed three times with precooled phosphate-buffered saline (PBS) to CO:COLLECTION_SUMMARY remove residual medium. To stop metabolic activity, 3 mL of pre-cooled (-80°C) CO:COLLECTION_SUMMARY methanol was added to the cells. The cells were then scraped with a cell scraper CO:COLLECTION_SUMMARY and collected in 15 mL centrifuge tubes. A two-phase extraction was performed CO:COLLECTION_SUMMARY using a methanol: chloroform: water mixture in a ratio of 4:4:2.85 (v/v) to CO:COLLECTION_SUMMARY extract water-soluble metabolites from the cells. The aqueous phase was CO:COLLECTION_SUMMARY collected and lyophilized. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY C2C12 myoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM, TR:TREATMENT_SUMMARY Corning, USA) supplemented with 10% fetal bovine serum (FBS, Corning, USA) and TR:TREATMENT_SUMMARY 1% penicillin-streptomycin (PS, Hyclone, USA) for cell growth and maintenance. TR:TREATMENT_SUMMARY The medium was changed periodically to ensure optimal cell health. To prepare TR:TREATMENT_SUMMARY low-glucose DMEM (0.5 g/L D-glucose, without sodium pyruvate), we mixed standard TR:TREATMENT_SUMMARY DMEM (4.5 g/L D-glucose, without sodium pyruvate, Corning, USA) with sugar-free TR:TREATMENT_SUMMARY DMEM (without sodium pyruvate, Corning, USA) in a ratio of 1:8. This was TR:TREATMENT_SUMMARY followed by the addition of 10% FBS to create the conditions necessary to model TR:TREATMENT_SUMMARY myoblasts and myotubes, respectively, under low-glucose conditions. Sodium TR:TREATMENT_SUMMARY (R)-3-hydroxybutyrate (Sigma-Aldrich, China) was added to the medium. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The resulting metabolite powder was dissolved in 550 μL of NMR buffer (prepared SP:SAMPLEPREP_SUMMARY in D2O, 50 mM PO₄³-, 0.05 mM TSP, pH 7.4) and transferred to a 5 mm NMR tube SP:SAMPLEPREP_SUMMARY for acquiring the NMR spectrum. #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker Avance III HD 850 MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:STANDARD_CONCENTRATION 0.05 mM TSP NM:SPECTROMETER_FREQUENCY 850 MHz NM:NMR_SOLVENT H2O+D2O NM:NMR_TUBE_SIZE 5 mm NM:PULSE_SEQUENCE noesygppr1d [(RD)-90°-t1-90°-τm-90°-ACQ] NM:RECEIVER_GAIN 144 NM:OFFSET_FREQUENCY 15.02 ppm NM:TEMPERATURE 25°C NM:NUMBER_OF_SCANS 32 NM:DUMMY_SCANS 4 NM:ACQUISITION_TIME 2 s NM:SPECTRAL_WIDTH 20 ppm NM:NUM_DATA_POINTS_ACQUIRED 64 K NM:LINE_BROADENING 0.3 Hz NM:BASELINE_CORRECTION_METHOD Auto-baseline correction of integral by abs NM:CHEMICAL_SHIFT_REF_STD TSP (0.000 ppm) NM:NMR_RESULTS_FILE ST003650_AN005996_Results.txt UNITS:area under the curve #END