#METABOLOMICS WORKBENCH oilkay_20241205_115113 DATATRACK_ID:5430 STUDY_ID:ST003647 ANALYSIS_ID:AN005991 PROJECT_ID:PR002257 VERSION 1 CREATED_ON 01-03-2025 #PROJECT PR:PROJECT_TITLE Integration of metabolomic and transcriptomic analyses reveals novel regulatory PR:PROJECT_TITLE functions of the ChREBP transcription factor in energy metabolism. PR:PROJECT_SUMMARY In this study, we have integrated transcriptomic and metabolomic analyses to PR:PROJECT_SUMMARY better understand the role of the metabolic regulatory transcription factor PR:PROJECT_SUMMARY ChREBP in coordinated regulation of key pathways of intermediary metabolism in PR:PROJECT_SUMMARY the liver. We have uncovered regulatory effects of ChREBP on metabolic PR:PROJECT_SUMMARY homeostasis beyond itâs historical role in control of core glucose and lipid PR:PROJECT_SUMMARY metabolic pathways, to now include effects on co-factors, transporters for amino PR:PROJECT_SUMMARY acids and other small molecules, nucleotide metabolism, and control of PR:PROJECT_SUMMARY mitochondrial substrate supply. PR:INSTITUTE Duke University PR:LAST_NAME Ilkayeva PR:FIRST_NAME Olga PR:ADDRESS 300 N Duke St, Durham, NC, 27701, USA PR:EMAIL olga.ilkayeva@duke.edu PR:PHONE 919-479-2370 PR:DOI http://dx.doi.org/10.21228/M8WC2H #STUDY ST:STUDY_TITLE Targeted mass spec-based metabolomic and clinical analyte analyses of liver and ST:STUDY_TITLE plasma samples from rats with and without hepatic knockdown of ChREBP ST:STUDY_TITLE expression. ST:STUDY_SUMMARY The transcription factor Carbohydrate Response Element-Binding Protein (ChREBP) ST:STUDY_SUMMARY activates genes of glucose, fructose and lipid metabolism in response to ST:STUDY_SUMMARY carbohydrate feeding. Integrated transcriptomic and metabolomic analyses in rats ST:STUDY_SUMMARY with GalNac-siRNA-mediated suppression of ChREBP expression (GalNac-siChREBP ST:STUDY_SUMMARY treatment) in liver revealed novel ChREBP functions relative to rats treated ST:STUDY_SUMMARY with a GalNac vector expressing a non-targeting siRNA (GalNac-siCtrl treatment). ST:STUDY_SUMMARY GalNac-siChREBP treatment reduced expression of genes involved in coenzyme A ST:STUDY_SUMMARY (CoA) biosynthesis, and lowered CoA and short chain acyl CoA levels. Despite ST:STUDY_SUMMARY suppression of pyruvate kinase, pyruvate levels were maintained, possibly via ST:STUDY_SUMMARY increased expression of pyruvate and amino acid transporters. In addition, ST:STUDY_SUMMARY expression of multiple anaplerotic enzymes was decreased by GalNac-siChREBP ST:STUDY_SUMMARY treatment, affecting TCA cycle intermediates. Finally, GalNAc-siChREBP treatment ST:STUDY_SUMMARY suppressed late steps in purine and NAD synthesis, with increases in precursors ST:STUDY_SUMMARY and lowering of end products in both pathways. In sum, our studies have revealed ST:STUDY_SUMMARY functions of ChREBP beyond its canonical roles in carbohydrate and lipid ST:STUDY_SUMMARY metabolism to include regulation of substrate transport, mitochondrial function, ST:STUDY_SUMMARY and cellular energy balance. ST:INSTITUTE Duke University ST:LAST_NAME Ilkayeva ST:FIRST_NAME Olga ST:ADDRESS 300 N Duke St, Durham, NC, 27701, USA ST:EMAIL olga.ilkayeva@duke.edu ST:PHONE 919-479-2370 ST:SUBMIT_DATE 2024-12-05 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS R01E4-1 1 Treatment:GalNAc-siChrebp | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a032; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a032; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a020; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a032; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a028; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a012; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a018 SUBJECT_SAMPLE_FACTORS R01E4-19 19 Treatment:GalNAc-siChrebp | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a050; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a050; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a038; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a050; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a023; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a030; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a036 SUBJECT_SAMPLE_FACTORS R01E4-21 21 Treatment:GalNAc-siChrebp | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a052; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a052; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a040; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a052; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a038; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a032; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a038 SUBJECT_SAMPLE_FACTORS R01E4-3 3 Treatment:GalNAc-siChrebp | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a034; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a034; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a022; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a034; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a033; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a014; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a020 SUBJECT_SAMPLE_FACTORS R01E4-6 6 Treatment:GalNAc-siChrebp | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a037; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a037; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a025; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a037; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a031; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a017; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a023 SUBJECT_SAMPLE_FACTORS R01E4-7 7 Treatment:GalNAc-siChrebp | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a038; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a038; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a026; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a038; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a042; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a018; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a024 SUBJECT_SAMPLE_FACTORS R01E4-15 15 Treatment:GalNAc-siCtrl | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a046; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a046; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a034; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a046; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a029; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a026; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a032 SUBJECT_SAMPLE_FACTORS R01E4-17 17 Treatment:GalNAc-siCtrl | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a048; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a048; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a036; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a048; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a021; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a028; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a034 SUBJECT_SAMPLE_FACTORS R01E4-18 18 Treatment:GalNAc-siCtrl | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a049; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a049; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a037; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a049; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a045; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a029; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a035 SUBJECT_SAMPLE_FACTORS R01E4-2 2 Treatment:GalNAc-siCtrl | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a033; RAW_FILE_NAME(AA raw file name)=AnJie_Newgard_AA_013123_a033; RAW_FILE_NAME(OA raw file name)=AnJie_Newgard_27Liver_OA_052924_a021; RAW_FILE_NAME(BCKA raw file name)=AnJie_Newgard_KA_012723_a033; RAW_FILE_NAME(Nucleotides raw file name)=AnJie_Newgard_Liver_NucleoTides_061024_a026; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=AnJie_Newgard_27_Liver_061124_a013; RAW_FILE_NAME(SC Acyl CoAs raw file name)=LC_SC_CoA_032123_a019 SUBJECT_SAMPLE_FACTORS R01E4-10 10 Treatment:Sal | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a041; RAW_FILE_NAME(AA raw file name)=-; RAW_FILE_NAME(OA raw file name)=-; RAW_FILE_NAME(BCKA raw file name)=-; RAW_FILE_NAME(Nucleotides raw file name)=-; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=-; RAW_FILE_NAME(SC Acyl CoAs raw file name)=- SUBJECT_SAMPLE_FACTORS R01E4-11 11 Treatment:Sal | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a042; RAW_FILE_NAME(AA raw file name)=-; RAW_FILE_NAME(OA raw file name)=-; RAW_FILE_NAME(BCKA raw file name)=-; RAW_FILE_NAME(Nucleotides raw file name)=-; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=-; RAW_FILE_NAME(SC Acyl CoAs raw file name)=- SUBJECT_SAMPLE_FACTORS R01E4-13 13 Treatment:Sal | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a044; RAW_FILE_NAME(AA raw file name)=-; RAW_FILE_NAME(OA raw file name)=-; RAW_FILE_NAME(BCKA raw file name)=-; RAW_FILE_NAME(Nucleotides raw file name)=-; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=-; RAW_FILE_NAME(SC Acyl CoAs raw file name)=- SUBJECT_SAMPLE_FACTORS R01E4-4 4 Treatment:Sal | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a035; RAW_FILE_NAME(AA raw file name)=-; RAW_FILE_NAME(OA raw file name)=-; RAW_FILE_NAME(BCKA raw file name)=-; RAW_FILE_NAME(Nucleotides raw file name)=-; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=-; RAW_FILE_NAME(SC Acyl CoAs raw file name)=- SUBJECT_SAMPLE_FACTORS R01E4-9 9 Treatment:Sal | Sample source:liver RAW_FILE_NAME(AC raw file name)=AnJie_Newgard_AC_013123_a040; RAW_FILE_NAME(AA raw file name)=-; RAW_FILE_NAME(OA raw file name)=-; RAW_FILE_NAME(BCKA raw file name)=-; RAW_FILE_NAME(Nucleotides raw file name)=-; RAW_FILE_NAME(Creatine/Phosphocreatine raw file name)=-; RAW_FILE_NAME(SC Acyl CoAs raw file name)=- #COLLECTION CO:COLLECTION_SUMMARY On day 28 between 8 AM-noon, animals were anesthetized and sacrificed for CO:COLLECTION_SUMMARY collection of plasma and tissue samples. To minimize tissue harvest time, a team CO:COLLECTION_SUMMARY of 3 scientists collected tissues from the animals via the following procedure. CO:COLLECTION_SUMMARY Animals were anesthetized with 250 mg/kg Nembutal, and the abdomen and diaphragm CO:COLLECTION_SUMMARY were surgically opened. Five ml of blood was drawn from the heart, and one CO:COLLECTION_SUMMARY operator centrifuged the sample, collected the plasma, and transferred it to CO:COLLECTION_SUMMARY Eppendorf tubes for rapid freezing by submersion in liquid nitrogen. The other CO:COLLECTION_SUMMARY two team members proceeded to immediate surgical excision of the heart and CO:COLLECTION_SUMMARY liver, which were briefly rinsed with ice-cold PBS, quickly wrapped in aluminum CO:COLLECTION_SUMMARY foil, and then frozen by submersion in liquid nitrogen. The time elapsed between CO:COLLECTION_SUMMARY beginning of surgery to removal and freezing of the heart and liver was less CO:COLLECTION_SUMMARY than 2 minutes. Other tissues (skeletal muscle, adipose, kidney) were collected CO:COLLECTION_SUMMARY after excision of the heart and liver and rapidly frozen in liquid nitrogen. All CO:COLLECTION_SUMMARY tissues were stored at -80C until processing for metabolomic, transcriptomic, CO:COLLECTION_SUMMARY and proteomic analyses. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY All procedures were approved by Duke University Institutional Animal Care and TR:TREATMENT_SUMMARY Use Committee and performed according to the regulations of the committee. TR:TREATMENT_SUMMARY Breeding pairs of Obese Prone CD (OP/CD) Sprague Dawley rats were gifts from Dr. TR:TREATMENT_SUMMARY Warren Grill and Dr. Eric Gonzalez, Duke University, and a colony was TR:TREATMENT_SUMMARY established and maintained by Duke Laboratory Animal Resources (DLAR). Starting TR:TREATMENT_SUMMARY at 4 weeks of age, male OP/CD rats were single-housed with a light cycle of 7 AM TR:TREATMENT_SUMMARY on/7 PM off, and fed ad libitum with a high-fat/high-sucrose (HF/HS) diet TR:TREATMENT_SUMMARY (D12451i, Research Diets) containing 47% fat (kcal) and 17% sucrose (kcal). Body TR:TREATMENT_SUMMARY weight and food intake were monitored weekly. After 9 weeks of feeding of the TR:TREATMENT_SUMMARY HF/HS diet, plasma samples were collected via saphenous vein bleeding. One week TR:TREATMENT_SUMMARY later, animals received an initial subcutaneous injection of one of two TR:TREATMENT_SUMMARY GalNAc-siRNA constructs at a dose of 9 mg/kg body weight, or an equal volume of TR:TREATMENT_SUMMARY the diluent (PBS), (see below for description of the two GalNAc-siRNA reagents). TR:TREATMENT_SUMMARY Additional doses of each GalNAc-siRNA construct were injected at 10, 18 and 25 TR:TREATMENT_SUMMARY days after the first injection. Animals were fasted overnight one day after the TR:TREATMENT_SUMMARY third injection (day 19), and subjected to an intraperitoneal glucose tolerance TR:TREATMENT_SUMMARY test (IPGTT) on the following day. Animals were weighed and a glucose solution TR:TREATMENT_SUMMARY (1g/kg body weight) was administered via intraperitoneal injection. Tail blood TR:TREATMENT_SUMMARY samples were obtained and glucose levels measured with a blood glucose meter TR:TREATMENT_SUMMARY (CVSHealth) immediately before and at 30,60, 90, 120, and 180 minutes after TR:TREATMENT_SUMMARY bolus injection of glucose. One day after the fourth GalNAc-siRNA or saline TR:TREATMENT_SUMMARY injection on day 25, plasma samples were collected via saphenous vein bleeding. TR:TREATMENT_SUMMARY A bolus of deuterium oxide (D2O, 10 ml/kg body weight, Sigma Aldrich) was then TR:TREATMENT_SUMMARY given by intraperitoneal injection and followed by free access to drinking water TR:TREATMENT_SUMMARY supplemented with 4% D2O for the rest of the experimental period. Saphenous TR:TREATMENT_SUMMARY plasma samples were collected again one day after the bolus delivery of D2O (day TR:TREATMENT_SUMMARY 27). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To prepare samples for analyses, frozen livers were pulverized under liquid SP:SAMPLEPREP_SUMMARY nitrogen, and weighed aliquots of the powder (50 mg powdered tissue/aliquot) SP:SAMPLEPREP_SUMMARY were transferred into pre-frozen Eppendorf tubes for further homogenization at SP:SAMPLEPREP_SUMMARY 50 mg of wet tissue per 1 ml of homogenate using the following buffers: 50% SP:SAMPLEPREP_SUMMARY acetonitrile/0.3% formic acid for the analysis of amino acids, acylcarnitines, SP:SAMPLEPREP_SUMMARY organic acids, creatine, and phosphocreatine; 3M perchloric acid for the SP:SAMPLEPREP_SUMMARY analysis of branched-chain keto acids; 0.3M perchloric acid for the analysis of SP:SAMPLEPREP_SUMMARY short-chain acyl CoAs; 100% methanol for the analysis of nucleotides. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY LC-MS/MS analysis of creatine and phosphocreatine CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Thermo Hypercarb (100 x 2.1mm,3um) CH:COLUMN_TEMPERATURE 30 CH:FLOW_GRADIENT t=0, B=0%; t=4 min., B=60%; t=4.1 min., B=90% followed by 1 min wash and 2 min CH:FLOW_GRADIENT re-equilibration at the initial conditions. CH:FLOW_RATE 0.4 ml/min CH:SOLVENT_A 20 mM ammonium bicarbonate, pH=10 CH:SOLVENT_B acetonitrile CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo TQ-XS MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS Creatine and phosphocreatine were detected in the negative MRM mode based on a MS:MS_COMMENTS characteristic fragmentation reaction: creatine 130/88, creatine-d3 133/91, MS:MS_COMMENTS phosphocreatine 210/79. Metabolite concentrations were computed using a ratio to MS:MS_COMMENTS creatinine-d3. TargetLynx was used for data processing. MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS µM MS_METABOLITE_DATA_START Samples 1 19 21 3 6 7 15 17 18 2 Factors Treatment:GalNAc-siChrebp | Sample source:liver Treatment:GalNAc-siChrebp | Sample source:liver Treatment:GalNAc-siChrebp | Sample source:liver Treatment:GalNAc-siChrebp | Sample source:liver Treatment:GalNAc-siChrebp | Sample source:liver Treatment:GalNAc-siChrebp | Sample source:liver Treatment:GalNAc-siCtrl | Sample source:liver Treatment:GalNAc-siCtrl | Sample source:liver Treatment:GalNAc-siCtrl | Sample source:liver Treatment:GalNAc-siCtrl | Sample source:liver creatine 14.9507 6.2131 9.3114 7.4083 7.4624 7.5548 9.7310 11.4744 13.9289 13.2387 phosphocreatine 3.7423 2.3899 2.7960 2.2669 1.9981 2.0984 1.2078 1.0474 1.2990 1.8318 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant creatine phosphocreatine METABOLITES_END #END