#METABOLOMICS WORKBENCH isabelleyao_20230511_141550 DATATRACK_ID:4018 STUDY_ID:ST002710 ANALYSIS_ID:AN004392 PROJECT_ID:PR001680 VERSION 1 CREATED_ON May 19, 2023, 5:57 pm #PROJECT PR:PROJECT_TITLE Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic PR:PROJECT_TITLE GPD is essential to support lipid synthesis PR:PROJECT_SUMMARY The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates PR:PROJECT_SUMMARY reducing equivalents in the cytosol and produces energy in the mitochondria. PR:PROJECT_SUMMARY Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the PR:PROJECT_SUMMARY cytosolic reaction is 4.5 times faster than the mitochondrial reaction. The high PR:PROJECT_SUMMARY flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to PR:PROJECT_SUMMARY maintain redox balance and support lipid synthesis. Interestingly, inhibition of PR:PROJECT_SUMMARY G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial PR:PROJECT_SUMMARY respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a PR:PROJECT_SUMMARY transcriptional level and promotes cancer cell proliferation by increasing PR:PROJECT_SUMMARY glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor PR:PROJECT_SUMMARY can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, PR:PROJECT_SUMMARY our results suggest that G3PS is not required to run as an intact NADH shuttle PR:PROJECT_SUMMARY but is instead truncated to support complex lipid synthesis in kidney cancer. PR:INSTITUTE Harvard Medical School PR:LAST_NAME Yao PR:FIRST_NAME Conghui PR:ADDRESS LHRRB RM301, 240 Longwood Ave, Boston, MA, 02115, USA PR:EMAIL conghui_yao@hms.harvard.edu PR:PHONE 6174326865 #STUDY ST:STUDY_TITLE Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic ST:STUDY_TITLE GPD is essential to support lipid synthesis ST:STUDY_SUMMARY The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates ST:STUDY_SUMMARY reducing equivalents in the cytosol and produces energy in the mitochondria. ST:STUDY_SUMMARY Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the ST:STUDY_SUMMARY cytosolic reaction is 4.5 times faster than the mitochondrial reaction. The high ST:STUDY_SUMMARY flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to ST:STUDY_SUMMARY maintain redox balance and support lipid synthesis. Interestingly, inhibition of ST:STUDY_SUMMARY G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial ST:STUDY_SUMMARY respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a ST:STUDY_SUMMARY transcriptional level and promotes cancer cell proliferation by increasing ST:STUDY_SUMMARY glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor ST:STUDY_SUMMARY can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, ST:STUDY_SUMMARY our results suggest that G3PS is not required to run as an intact NADH shuttle ST:STUDY_SUMMARY but is instead truncated to support complex lipid synthesis in kidney cancer. ST:INSTITUTE Harvard Medical School ST:LAST_NAME Yao ST:FIRST_NAME Conghui ST:ADDRESS LHRRB RM301, 240 Longwood Ave, Boston, MA, 02115, USA ST:EMAIL conghui_yao@hms.harvard.edu ST:PHONE 6174326865 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN balb/c SU:AGE_OR_AGE_RANGE 6 week old SU:GENDER Female #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 - 091622_RPLC_POS_26C_0003 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_26C_0003 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_27C_0010 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_27C_0010 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_29C_0011 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_29C_0011 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_30C_0018 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_30C_0018 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_31C_0019 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_31C_0019 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_32C_0026 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_32C_0026 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_33C_0027 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_33C_0027 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_34C_0034 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_34C_0034 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_35C_0035 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_35C_0035 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_36C_0005 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_36C_0005 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_37C_0008 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_37C_0008 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_3NTC_0032 Treatment:NA RAW_FILE_NAME=091622_RPLC_POS_3NTC_0032 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_40C_0013 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_40C_0013 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_41C_0016 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_41C_0016 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_42C_0021 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_42C_0021 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_43C_0024 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_43C_0024 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_44C_0037 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_44C_0037 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_45C_0029 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_45C_0029 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_26KD_0004 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_26KD_0004 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_27KD_0009 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_27KD_0009 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_28KD_0038 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_28KD_0038 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_29KD_0012 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_29KD_0012 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_30KD_0017 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_30KD_0017 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_31KD_0020 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_31KD_0020 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_32KD_0025 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_32KD_0025 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_33KD_0028 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_33KD_0028 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_34KD_0033 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_34KD_0033 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_35KD_0036 Treatment:PBS RAW_FILE_NAME=091622_RPLC_POS_35KD_0036 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_36KD_0006 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_36KD_0006 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_37KD_0007 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_37KD_0007 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_3NTKD_0031 Treatment:NA RAW_FILE_NAME=091622_RPLC_POS_3NTKD_0031 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_40KD_0014 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_40KD_0014 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_41KD_0015 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_41KD_0015 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_42KD_0022 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_42KD_0022 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_43KD_0023 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_43KD_0023 SUBJECT_SAMPLE_FACTORS - 091622_RPLC_POS_45KD_0030 Treatment:FSG67 RAW_FILE_NAME=091622_RPLC_POS_45KD_0030 #COLLECTION CO:COLLECTION_SUMMARY Tumors were homogenized and extracted with water methanol and chloroform. The CO:COLLECTION_SUMMARY chloroform phase was dried and reconstituted in methanol: chloroform (9:1) CO:SAMPLE_TYPE Renal cancer cells #TREATMENT TR:TREATMENT_SUMMARY Mice were treated with PBS control or 0.1mg FSG67 i.p. daily as the paper TR:TREATMENT_SUMMARY described (DOI:10.1016/j.molcel.2023.03.023). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Tumor samples were quenched and extracted with a mixture of water, methanol and SP:SAMPLEPREP_SUMMARY chloroform. The water-methanol phase containing polar metabolites was dried down SP:SAMPLEPREP_SUMMARY and reconstituted with water/acetonitrile (1:1) with the volume normalized to SP:SAMPLEPREP_SUMMARY tumor wet weight. The chloroform phase containing lipids was dried down and SP:SAMPLEPREP_SUMMARY reconstituted with methanol/ chloroform (9:1). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent QTOF 6546 CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) CH:SOLVENT_A 95% water/5% methanol; 5mM ammonium acetate, pH 6.5 CH:SOLVENT_B 95% isopropanol/5% methanol CH:FLOW_GRADIENT 5% B-100% 5-25min, 100% B 25-30min CH:FLOW_RATE 0.2mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS data processed with XCMS with below settings pw = c(10,60); ppm = 5; bw = 5; MS:MS_COMMENTS method = 'centWave' method = 'obiwarp' MS:MS_RESULTS_FILE ST002710_AN004392_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END