#METABOLOMICS WORKBENCH ramkhattri_20221031_122751 DATATRACK_ID:3545 STUDY_ID:ST002426 ANALYSIS_ID:AN003949 PROJECT_ID:PR001513 VERSION 1 CREATED_ON January 4, 2023, 11:58 am #PROJECT PR:PROJECT_TITLE Isolated murine skeletal muscles utilize pyruvate over glucose for PR:PROJECT_TITLE oxidation:Part 2 PR:PROJECT_TYPE Study of the different substrate by isolated skeletal muscle at room temperature PR:PROJECT_TYPE via C-13 isotopomer analysis PR:PROJECT_SUMMARY The goal of this study was to determine the differential utilization of PR:PROJECT_SUMMARY substrates in isolated murine skeletal muscle, and to evalute how isopotomer PR:PROJECT_SUMMARY anlaysis provided insight into skeletal muscle metabolism. PR:INSTITUTE University of Florida PR:DEPARTMENT Applied Physiology and Kinesiology PR:LABORATORY Rm 42 and Rm 43 PR:LAST_NAME Khattri PR:FIRST_NAME Ram PR:ADDRESS 1864 Stadium RD, Gainesville, FL, 32611, USA PR:EMAIL rbk11@ufl.edu PR:PHONE 3307856045 PR:FUNDING_SOURCE This work was supported by grants from the Southeastern Center for Integrated PR:FUNDING_SOURCE Metabolomics (SECIM) (ERB), NIH AR U54 AR052646 (Physiological Assessment Core, PR:FUNDING_SOURCE ERB), and Wellstone Muscular Dystrophy Cooperative Research Center Grant (NIAMS: PR:FUNDING_SOURCE U54AR052646/P50 AR052646). The AMRIS Facility is supported by the National PR:FUNDING_SOURCE Science Foundation Cooperative Agreement No. DMR-1644779 and the State of PR:FUNDING_SOURCE Florida. PR:CONTRIBUTORS Ram B. Khattri, Jason Puglise, Terence E. Ryan, Glenn A. Walter, Matthew E. PR:CONTRIBUTORS Merritt, Elisabeth R. Barton #STUDY ST:STUDY_TITLE Isolated murine skeletal muscles utilize pyruvate over glucose for ST:STUDY_TITLE oxidation:Part 2 ST:STUDY_TYPE Study of the different substrate by isolated skeletal muscle at room temperature ST:STUDY_TYPE via C-13 isotopomer analysis ST:STUDY_SUMMARY Preclinical studies of muscle contractile function often employ ex vivo ST:STUDY_SUMMARY preparations of the soleus and/or extensor digitorum longus (EDL) muscles which ST:STUDY_SUMMARY are relatively easy to prepare and represent slow and fast fiber properties, ST:STUDY_SUMMARY respectively. Therefore, the current study sought to examine the utility of this ST:STUDY_SUMMARY preparation for understanding the metabolic fuel utilization in isolated resting ST:STUDY_SUMMARY mouse muscles at room temperature. 13C-labeling in both muscle types was ST:STUDY_SUMMARY performed using three fuels: glucose, pyruvate, and acetate, followed by ST:STUDY_SUMMARY NMR-based metabolomics analyses. Incubating 13C-labeled substrates in the ST:STUDY_SUMMARY isolated skeletal muscles makes it possible to examine TCA cycle flux and ST:STUDY_SUMMARY substrate selection by these muscles. ST:INSTITUTE University of Florida ST:DEPARTMENT Applied Physiology and Kinesiology ST:LABORATORY Rm 42 and Rm 43 ST:LAST_NAME Khattri ST:FIRST_NAME Ram ST:ADDRESS 1864 Stadium RD, Gainesville, FL, 32611, USA ST:EMAIL rbk11@ufl.edu ST:PHONE 3307856045 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 18 ST:NUM_MALES NA ST:NUM_FEMALES NA ST:PUBLICATIONS Metabolomics journal (submitted) #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 16±3 weeks SU:GENDER Not applicable SU:ANIMAL_ANIMAL_SUPPLIER Jackson Labs (Stock # 000664) SU:ANIMAL_HOUSING Housed in a temperature of 22 oC SU:ANIMAL_LIGHT_CYCLE 12-hour light/12-hour dark SU:ANIMAL_FEED Ad libitum chow diet food SU:ANIMAL_WATER free access to food and water (3-5 animals per cage). #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 - Glucose_EDL_13CNMR-1 Group:Glucose_EDL RAW_FILE_NAME=Glucose_EDL_13CNMR-1 SUBJECT_SAMPLE_FACTORS - Glucose_EDL_13CNMR-2 Group:Glucose_EDL RAW_FILE_NAME=Glucose_EDL_13CNMR-2 SUBJECT_SAMPLE_FACTORS - Glucose_EDL_13CNMR-3 Group:Glucose_EDL RAW_FILE_NAME=Glucose_EDL_13CNMR-3 SUBJECT_SAMPLE_FACTORS - Glucose_EDL_13CNMR-4 Group:Glucose_EDL RAW_FILE_NAME=Glucose_EDL_13CNMR-4 SUBJECT_SAMPLE_FACTORS - Glucose_Soleus_13CNMR-1 Group:Glucose_Soleus RAW_FILE_NAME=Glucose_Soleus_13CNMR-1 SUBJECT_SAMPLE_FACTORS - Glucose_Soleus_13CNMR-2 Group:Glucose_Soleus RAW_FILE_NAME=Glucose_Soleus_13CNMR-2 SUBJECT_SAMPLE_FACTORS - Glucose_Soleus_13CNMR-3 Group:Glucose_Soleus RAW_FILE_NAME=Glucose_Soleus_13CNMR-3 SUBJECT_SAMPLE_FACTORS - Glucose_Soleus_13CNMR-4 Group:Glucose_Soleus RAW_FILE_NAME=Glucose_Soleus_13CNMR-4 SUBJECT_SAMPLE_FACTORS - Pyruvate_EDL_13CNMR-1 Group:Pyruvate_EDL RAW_FILE_NAME=Pyruvate_EDL_13CNMR-1 SUBJECT_SAMPLE_FACTORS - Pyruvate_EDL_13CNMR-2 Group:Pyruvate_EDL RAW_FILE_NAME=Pyruvate_EDL_13CNMR-2 SUBJECT_SAMPLE_FACTORS - Pyruvate_EDL_13CNMR-3 Group:Pyruvate_EDL RAW_FILE_NAME=Pyruvate_EDL_13CNMR-3 SUBJECT_SAMPLE_FACTORS - Pyruvate_Soleus_13CNMR-1 Group:Pyruvate_Soleus RAW_FILE_NAME=Pyruvate_Soleus_13CNMR-1 SUBJECT_SAMPLE_FACTORS - Pyruvate_Soleus_13CNMR-2 Group:Pyruvate_Soleus RAW_FILE_NAME=Pyruvate_Soleus_13CNMR-2 SUBJECT_SAMPLE_FACTORS - Pyruvate_Soleus_13CNMR-3 Group:Pyruvate_Soleus RAW_FILE_NAME=Pyruvate_Soleus_13CNMR-3 #COLLECTION CO:COLLECTION_SUMMARY Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine CO:COLLECTION_SUMMARY (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) CO:COLLECTION_SUMMARY muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution CO:COLLECTION_SUMMARY gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a CO:COLLECTION_SUMMARY perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. CO:COLLECTION_SUMMARY These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] CO:COLLECTION_SUMMARY pyruvate, or 16.5 mM [13C2] labeled Na-acetate. Following incubation, muscles CO:COLLECTION_SUMMARY were quickly removed, blotted, and then rapidly frozen in liquid nitrogen for CO:COLLECTION_SUMMARY subsequent NMR analysis. N=4 muscles were pooled into a single biological CO:COLLECTION_SUMMARY replicate of 30-50 mg tissue to afford detectable levels of substrates in the CO:COLLECTION_SUMMARY NMR analysis. CO:SAMPLE_TYPE Muscle CO:COLLECTION_LOCATION University of Florida, Applied Physiology and Kinesiology, MBI, 1149 Newell Dr, CO:COLLECTION_LOCATION Gainesville, FL 32610 CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS cryovials CO:STORAGE_VIALS cryovials #TREATMENT TR:TREATMENT_SUMMARY Mice were anesthetized (using a combination of xylazine (80mg/kg) and ketamine TR:TREATMENT_SUMMARY (10mg/kg)) to allow removal of soleus and extensor digitorum longus (EDL) TR:TREATMENT_SUMMARY muscles. Upon removal, muscles were incubated at 22 oC in Ringer/MEM solution TR:TREATMENT_SUMMARY gas equilibrated with 95/5% O2/CO2 with appropriate 13C labeled substrates in a TR:TREATMENT_SUMMARY perfusion chamber routinely used for isolated muscle mechanics for 30 minutes. TR:TREATMENT_SUMMARY These included the following: 5.5 mM [U-13C6] glucose; 5.5 mM [U-13C3] TR:TREATMENT_SUMMARY pyruvate, or 16.5 mM [13C2] labeled Na-acetate. Following incubation, muscles TR:TREATMENT_SUMMARY were quickly removed, blotted, and then rapidly frozen in liquid nitrogen for TR:TREATMENT_SUMMARY subsequent NMR analysis. N=4 muscles were pooled into a single biological TR:TREATMENT_SUMMARY replicate of 30-50 mg tissue to afford detectable levels of substrates in the TR:TREATMENT_SUMMARY NMR analysis. TR:ANIMAL_VET_TREATMENTS none TR:ANIMAL_ANESTHESIA a combination of xylazine (80mg/kg) and ketamine (10mg/kg)) TR:ANIMAL_FASTING non-fasted TR:ANIMAL_ENDP_EUTHANASIA Euthanasia was carried out by thoracotomy followed by cervical dislocation. TR:ANIMAL_ENDP_TISSUE_COLL_LIST Skeletal muscle (soleus and EDL) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Perchloric acid (PCA) or acetonitrile:isopropanol:water (3:3:2) extractions were SP:SAMPLEPREP_SUMMARY performed for all samples to isolate metabolites. The latter method was more SP:SAMPLEPREP_SUMMARY efficient in sample recovery due to the reduced number of steps in the procedure SP:SAMPLEPREP_SUMMARY but did not affect the proportion of metabolites. For PCA extraction, isolated SP:SAMPLEPREP_SUMMARY muscle samples were homogenized with a FASTPREP-24 (MP Biomedicals, Solon, Ohio, SP:SAMPLEPREP_SUMMARY USA) with 6% (v/v) ice cold PCA and centrifuged with 13.2 K rpm at 4 oC. The SP:SAMPLEPREP_SUMMARY solid muscle portion was washed again with the 6% (v/v) ice cold PCA followed by SP:SAMPLEPREP_SUMMARY centrifugation (13.2 K rpm) at 4 oC. The supernatant (combined) obtained was SP:SAMPLEPREP_SUMMARY further neutralized with 5M potassium hydroxide and centrifuged again SP:SAMPLEPREP_SUMMARY maintaining 13.2 K rpm speed at 4 oC. The resulting supernatants were then SP:SAMPLEPREP_SUMMARY lyophilized (Thermo-Scientific, Dallas, USA). The pH of the dried powder was SP:SAMPLEPREP_SUMMARY adjusted to 7.2 after dissolving it in 200 μL of ultra-pure water using 1M SP:SAMPLEPREP_SUMMARY sodium hydroxide and 1 M hydrochloric acid. The pH-adjusted solution was further SP:SAMPLEPREP_SUMMARY centrifuged, the resulting supernatant was dried and the powder was used to SP:SAMPLEPREP_SUMMARY prepare the NMR sample. For acetonitrile:isopropanol:water extraction, SP:SAMPLEPREP_SUMMARY homogenization of isolated muscle samples was carried out in 1 mL SP:SAMPLEPREP_SUMMARY acetonitrile:isopropanol:water (3:3:2, v:v:v) ice cold mixture with a SP:SAMPLEPREP_SUMMARY FASTPREP-24 (MP Biomedicals, Solon, Ohio, USA) and centrifuged at 4 oC in SP:SAMPLEPREP_SUMMARY separate vials. Resultant supernatants were further lyophilized till dryness SP:SAMPLEPREP_SUMMARY (Thermo-Scientific, Dallas, USA). The dried powder was further dissolved in 1 mL SP:SAMPLEPREP_SUMMARY of Acetonitrile:Water (1:1, v:v) mixture, vortexed well for ~5 minutes. The SP:SAMPLEPREP_SUMMARY resultant solution was further centrifuged, the supernatant obtained was further SP:SAMPLEPREP_SUMMARY dried and the powder was used to prepare the NMR sample. The centrifugation SP:SAMPLEPREP_SUMMARY speed for each step used was 13.2K rpm. Each NMR sample consisted of 50 mM SP:SAMPLEPREP_SUMMARY phosphate buffer (pH 7), 2 mM EDTA, 0.02% of NaN3 with 0.5 mM of DSS as a SP:SAMPLEPREP_SUMMARY standard internal reference in deuterated environment. 1H NMR spectra were taken SP:SAMPLEPREP_SUMMARY at 25oC using a 600 MHz Bruker Avance II Console equipped with a TCI CryoProbe SP:SAMPLEPREP_SUMMARY that utilized Bruker Topspin 4 software (Bruker BioSpin Corporation, Billerica, SP:SAMPLEPREP_SUMMARY MA, USA). The first slice of a NOESY pulse sequence (noesypr1d) was used to SP:SAMPLEPREP_SUMMARY acquire proton NMR. Fractional enrichment for glutamate, lactate and alanine SP:SAMPLEPREP_SUMMARY were determined using 13C decoupling ON/OFF 1H proton spectra as well as 1D SP:SAMPLEPREP_SUMMARY NOESY spectra. To determine enrichements, a standard zgig pulse sequence was SP:SAMPLEPREP_SUMMARY adapted to allow 13C decoupling during the acquistion period (1.36 s) to remove SP:SAMPLEPREP_SUMMARY the satellites. Total enrichment was measured by taking a ratio of the SP:SAMPLEPREP_SUMMARY metabolite peak heights in the decoupling on/off experiments. NOESY spectra were SP:SAMPLEPREP_SUMMARY collected with a 1 s relaxation delay (d1), and a 4 s acqusition time (at), in SP:SAMPLEPREP_SUMMARY accordance with Chenomx recommendations for producing quantitative estimates of SP:SAMPLEPREP_SUMMARY concentration. Using the Chenomx quantification and the fractional enrichments, SP:SAMPLEPREP_SUMMARY a final concentration of the metabolites was calculated. Conventional 1H SP:SAMPLEPREP_SUMMARY decoupled 13C spectra were acquired using a 600 MHz Agilent with a specially SP:SAMPLEPREP_SUMMARY designed 1.5 mm superconducting (HTS) probe at 30oC. SP:SAMPLEPREP_PROTOCOL_FILENAME Isolated_muscle_Procedures.docx SP:PROCESSING_METHOD Lyophilization and Homogenization SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Perchloric acid (PCA) or acetonitrile:isopropanol:water (3:3:2) extractions SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION In 35 microliter of 50 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 0.5 mM DSS SP:SAMPLE_RESUSPENSION and 0.2% sodium azide for aqueous phase samples. SP:SAMPLE_SPIKING 0.5 mM DSS #ANALYSIS AN:ANALYSIS_TYPE NMR AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME Agilent 600 MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-13C NM:FIELD_FREQUENCY_LOCK 13C NM:STANDARD_CONCENTRATION 0.5mM DSS NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_PROBE HTS-1A NM:NMR_SOLVENT Phosphate buffer (pH 7.2) + 2 mM EDTA + 0.5 mM DSS + 0.2% of sodium azide in NM:NMR_SOLVENT deuterated environment NM:NMR_TUBE_SIZE 1.5 mm NMR tube NM:SHIMMING_METHOD Varian NM:PULSE_SEQUENCE s2pul NM:WATER_SUPPRESSION WALTZ-16 NM:PULSE_WIDTH 45 degree NM:RECEIVER_GAIN 60 NM:CHEMICAL_SHIFT_REF_CPD DSS NM:TEMPERATURE 30oC NM:NUMBER_OF_SCANS 6000 to 30000 scans NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 1.5 s NM:RELAXATION_DELAY 1.5 s NM:SPECTRAL_WIDTH 36764.7 Hz NM:NUM_DATA_POINTS_ACQUIRED 55147 NM:REAL_DATA_POINTS 65536 NM:LINE_BROADENING 0.5 Hz NM:ZERO_FILLING 65,536 points NM:APODIZATION Exponential NM:BASELINE_CORRECTION_METHOD Whittaker Smoother NM:CHEMICAL_SHIFT_REF_STD 0 ppm for DSS #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS For Fc3, % unit NMR_METABOLITE_DATA_START Samples Glucose_EDL_13CNMR-1 Glucose_EDL_13CNMR-2 Glucose_EDL_13CNMR-3 Glucose_EDL_13CNMR-4 Glucose_Soleus_13CNMR-1 Glucose_Soleus_13CNMR-2 Glucose_Soleus_13CNMR-3 Glucose_Soleus_13CNMR-4 Pyruvate_EDL_13CNMR-1 Pyruvate_EDL_13CNMR-2 Pyruvate_EDL_13CNMR-3 Pyruvate_Soleus_13CNMR-1 Pyruvate_Soleus_13CNMR-2 Pyruvate_Soleus_13CNMR-3 Factors Group:Glucose_EDL Group:Glucose_EDL Group:Glucose_EDL Group:Glucose_EDL Group:Glucose_Soleus Group:Glucose_Soleus Group:Glucose_Soleus Group:Glucose_Soleus Group:Pyruvate_EDL Group:Pyruvate_EDL Group:Pyruvate_EDL Group:Pyruvate_Soleus Group:Pyruvate_Soleus Group:Pyruvate_Soleus Fc3(%) N/A N/A N/A N/A N/A N/A N/A N/A 0.76 0.284 0.329 0.44 0.42 0.365 C4/C3 1.176 1.641 1.463 1.55 2.492 2.618 2.686 2.12 1.04 0.811 0.846 0.97 1.599 1.124 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Glucose_EDL_13CNMR-1 Glucose_EDL_13CNMR-2 Glucose_EDL_13CNMR-3 Glucose_EDL_13CNMR-4 Glucose_Soleus_13CNMR-1 Glucose_Soleus_13CNMR-2 Glucose_Soleus_13CNMR-3 Glucose_Soleus_13CNMR-4 Pyruvate_EDL_13CNMR-1 Pyruvate_EDL_13CNMR-2 Pyruvate_EDL_13CNMR-3 Pyruvate_Soleus_13CNMR-1 Pyruvate_Soleus_13CNMR-2 Pyruvate_Soleus_13CNMR-3 Fc3(%) N/A N/A N/A N/A N/A N/A N/A N/A 0.76 0.284 0.329 0.44 0.42 0.365 C4/C3 1.176 1.641 1.463 1.55 2.492 2.618 2.686 2.12 1.04 0.811 0.846 0.97 1.599 1.124 METABOLITES_END #END