Summary of Study ST003647

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002257. The data can be accessed directly via it's Project DOI: 10.21228/M8WC2H This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003647
Study TitleTargeted mass spec-based metabolomic and clinical analyte analyses of liver and plasma samples from rats with and without hepatic knockdown of ChREBP expression.
Study SummaryThe transcription factor Carbohydrate Response Element-Binding Protein (ChREBP) activates genes of glucose, fructose and lipid metabolism in response to carbohydrate feeding. Integrated transcriptomic and metabolomic analyses in rats with GalNac-siRNA-mediated suppression of ChREBP expression (GalNac-siChREBP treatment) in liver revealed novel ChREBP functions relative to rats treated with a GalNac vector expressing a non-targeting siRNA (GalNac-siCtrl treatment). GalNac-siChREBP treatment reduced expression of genes involved in coenzyme A (CoA) biosynthesis, and lowered CoA and short chain acyl CoA levels. Despite suppression of pyruvate kinase, pyruvate levels were maintained, possibly via increased expression of pyruvate and amino acid transporters. In addition, expression of multiple anaplerotic enzymes was decreased by GalNac-siChREBP treatment, affecting TCA cycle intermediates. Finally, GalNAc-siChREBP treatment suppressed late steps in purine and NAD synthesis, with increases in precursors and lowering of end products in both pathways. In sum, our studies have revealed functions of ChREBP beyond its canonical roles in carbohydrate and lipid metabolism to include regulation of substrate transport, mitochondrial function, and cellular energy balance.
Institute
Duke University
Last NameIlkayeva
First NameOlga
Address300 N Duke St, Durham, NC, 27701, USA
Emailolga.ilkayeva@duke.edu
Phone919-479-2370
Submit Date2024-12-05
Raw Data AvailableYes
Raw Data File Type(s)cdf
Analysis Type DetailLC-MS/Direct infusion
Release Date2025-01-12
Release Version1
Olga Ilkayeva Olga Ilkayeva
https://dx.doi.org/10.21228/M8WC2H
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002257
Project DOI:doi: 10.21228/M8WC2H
Project Title:Integration of metabolomic and transcriptomic analyses reveals novel regulatory functions of the ChREBP transcription factor in energy metabolism.
Project Summary:In this study, we have integrated transcriptomic and metabolomic analyses to better understand the role of the metabolic regulatory transcription factor ChREBP in coordinated regulation of key pathways of intermediary metabolism in the liver. We have uncovered regulatory effects of ChREBP on metabolic homeostasis beyond it’s historical role in control of core glucose and lipid metabolic pathways, to now include effects on co-factors, transporters for amino acids and other small molecules, nucleotide metabolism, and control of mitochondrial substrate supply.
Institute:Duke University
Last Name:Ilkayeva
First Name:Olga
Address:300 N Duke St, Durham, NC, 27701, USA
Email:olga.ilkayeva@duke.edu
Phone:919-479-2370

Subject:

Subject ID:SU003777
Subject Type:Mammal
Subject Species:Rattus norvegicus
Taxonomy ID:10116

Factors:

Subject type: Mammal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment Sample source
SA3982711GalNAc-siChrebp liver
SA3982723GalNAc-siChrebp liver
SA3982736GalNAc-siChrebp liver
SA3982747GalNAc-siChrebp liver
SA39827519GalNAc-siChrebp liver
SA39827621GalNAc-siChrebp liver
SA3982772GalNAc-siCtrl liver
SA39827815GalNAc-siCtrl liver
SA39827917GalNAc-siCtrl liver
SA39828018GalNAc-siCtrl liver
SA3982814Sal liver
SA3982829Sal liver
SA39828310Sal liver
SA39828411Sal liver
SA39828513Sal liver
Showing results 1 to 15 of 15

Collection:

Collection ID:CO003770
Collection Summary:On day 28 between 8 AM-noon, animals were anesthetized and sacrificed for collection of plasma and tissue samples. To minimize tissue harvest time, a team of 3 scientists collected tissues from the animals via the following procedure. Animals were anesthetized with 250 mg/kg Nembutal, and the abdomen and diaphragm were surgically opened. Five ml of blood was drawn from the heart, and one operator centrifuged the sample, collected the plasma, and transferred it to Eppendorf tubes for rapid freezing by submersion in liquid nitrogen. The other two team members proceeded to immediate surgical excision of the heart and liver, which were briefly rinsed with ice-cold PBS, quickly wrapped in aluminum foil, and then frozen by submersion in liquid nitrogen. The time elapsed between beginning of surgery to removal and freezing of the heart and liver was less than 2 minutes. Other tissues (skeletal muscle, adipose, kidney) were collected after excision of the heart and liver and rapidly frozen in liquid nitrogen. All tissues were stored at -80C until processing for metabolomic, transcriptomic, and proteomic analyses.
Sample Type:Liver

Treatment:

Treatment ID:TR003786
Treatment Summary:All procedures were approved by Duke University Institutional Animal Care and Use Committee and performed according to the regulations of the committee. Breeding pairs of Obese Prone CD (OP/CD) Sprague Dawley rats were gifts from Dr. Warren Grill and Dr. Eric Gonzalez, Duke University, and a colony was established and maintained by Duke Laboratory Animal Resources (DLAR). Starting at 4 weeks of age, male OP/CD rats were single-housed with a light cycle of 7 AM on/7 PM off, and fed ad libitum with a high-fat/high-sucrose (HF/HS) diet (D12451i, Research Diets) containing 47% fat (kcal) and 17% sucrose (kcal). Body weight and food intake were monitored weekly. After 9 weeks of feeding of the HF/HS diet, plasma samples were collected via saphenous vein bleeding. One week later, animals received an initial subcutaneous injection of one of two GalNAc-siRNA constructs at a dose of 9 mg/kg body weight, or an equal volume of the diluent (PBS), (see below for description of the two GalNAc-siRNA reagents). Additional doses of each GalNAc-siRNA construct were injected at 10, 18 and 25 days after the first injection. Animals were fasted overnight one day after the third injection (day 19), and subjected to an intraperitoneal glucose tolerance test (IPGTT) on the following day. Animals were weighed and a glucose solution (1g/kg body weight) was administered via intraperitoneal injection. Tail blood samples were obtained and glucose levels measured with a blood glucose meter (CVSHealth) immediately before and at 30,60, 90, 120, and 180 minutes after bolus injection of glucose. One day after the fourth GalNAc-siRNA or saline injection on day 25, plasma samples were collected via saphenous vein bleeding. A bolus of deuterium oxide (D2O, 10 ml/kg body weight, Sigma Aldrich) was then given by intraperitoneal injection and followed by free access to drinking water supplemented with 4% D2O for the rest of the experimental period. Saphenous plasma samples were collected again one day after the bolus delivery of D2O (day 27).

Sample Preparation:

Sampleprep ID:SP003784
Sampleprep Summary:To prepare samples for analyses, frozen livers were pulverized under liquid nitrogen, and weighed aliquots of the powder (50 mg powdered tissue/aliquot) were transferred into pre-frozen Eppendorf tubes for further homogenization at 50 mg of wet tissue per 1 ml of homogenate using the following buffers: 50% acetonitrile/0.3% formic acid for the analysis of amino acids, acylcarnitines, organic acids, creatine, and phosphocreatine; 3M perchloric acid for the analysis of branched-chain keto acids; 0.3M perchloric acid for the analysis of short-chain acyl CoAs; 100% methanol for the analysis of nucleotides.

Combined analysis:

Analysis ID AN005988 AN005989 AN005990 AN005991 AN005992 AN005993
Analysis type MS MS MS MS MS MS
Chromatography type Reversed phase Reversed phase Reversed phase Reversed phase Reversed phase None (Direct infusion)
Chromatography system Waters Acquity I-Class Waters Acquity I-Class Waters Acquity I-Class Waters Acquity I-Class Waters Acquity I-Class Waters Acquity I-Class
Column Waters ACQUITY UPLC BEH C18 (50 x 2.1mm,1.7um) Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) EMD Millipore Chromolith FastGradient RP-18e 50-2mm Thermo Hypercarb (100 x 2.1mm,3um) Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) N/A
MS Type ESI ESI ESI ESI ESI ESI
MS instrument type Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole
MS instrument name Waters Xevo TQ-XS Waters Xevo TQ-XS Waters Xevo TQ-XS Waters Xevo TQ-XS Waters Xevo TQ-XS Waters Xevo-TQ
Ion Mode POSITIVE POSITIVE NEGATIVE NEGATIVE POSITIVE POSITIVE
Units µM µM µM µM µM µM

Chromatography:

Chromatography ID:CH004548
Chromatography Summary:LC-MS/MS analysis of branched-chain keto acids
Instrument Name:Waters Acquity I-Class
Column Name:Waters ACQUITY UPLC BEH C18 (50 x 2.1mm,1.7um)
Column Temperature:30
Flow Gradient:t=0, B=55%; t=2 min., B=55%; t=2.5 min., B=95% followed by 1 min wash and 1 min re-equilibration at the initial conditions.
Flow Rate:0.4 ml/min
Solvent A:5 mM ammonium acetate in water
Solvent B:methanol
Chromatography Type:Reversed phase
  
Chromatography ID:CH004549
Chromatography Summary:LC-MS/MS analysis of organic acids
Instrument Name:Waters Acquity I-Class
Column Name:Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
Column Temperature:50
Flow Gradient:t=0, B=25%; t=6 min., B=33%; t=9.5 min., B=95% followed by 1 min wash and 1 min re-equilibration at the initial conditions.
Flow Rate:0.4 ml/min
Solvent A:0.1% formic acid in water
Solvent B:acetonitrile
Chromatography Type:Reversed phase
  
Chromatography ID:CH004550
Chromatography Summary:LC-MS/MS analysis of nucleotides
Instrument Name:Waters Acquity I-Class
Column Name:EMD Millipore Chromolith FastGradient RP-18e 50-2mm
Column Temperature:40
Flow Gradient:t=0, B=0%; t=22 min., B=40%; t=22.5 min., B=95% followed by 1 min wash and 2 min re-equilibration at the initial conditions.
Flow Rate:0.3 ml/min
Solvent A:95% water, 5% methanol and 5 mM dimethylhexylamine adjusted to pH 7.5 with acetic acid
Solvent B:20% water, 80% methanol and 10 mM dimethylhexylamine
Chromatography Type:Reversed phase
  
Chromatography ID:CH004551
Chromatography Summary:LC-MS/MS analysis of creatine and phosphocreatine
Instrument Name:Waters Acquity I-Class
Column Name:Thermo Hypercarb (100 x 2.1mm,3um)
Column Temperature:30
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 re-equilibration at the initial conditions.
Flow Rate:0.4 ml/min
Solvent A:20 mM ammonium bicarbonate, pH=10
Solvent B:acetonitrile
Chromatography Type:Reversed phase
  
Chromatography ID:CH004552
Chromatography Summary:LC-MS/MS analysis of short-chain acyl CoAs
Instrument Name:Waters Acquity I-Class
Column Name:Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
Column Temperature:30
Flow Gradient:t=0, B=5%; t=10 min., B=25%; t=10.5 min., B=90% followed by 1 min wash and 1 min re-equilibration at the initial conditions.
Flow Rate:0.4 ml/min
Solvent A:5 mM ammonium acetate/5 mM DMBA, pH 7
Solvent B:acetonitrile
Chromatography Type:Reversed phase
  
Chromatography ID:CH004553
Chromatography Summary:MS/MS analysis of amino acid and acylcarnitines
Instrument Name:Waters Acquity I-Class
Column Name:N/A
Column Temperature:N/A
Flow Gradient:isocratic
Flow Rate:0.1 ml/min
Solvent A:80% methanol
Solvent B:80% methanol
Chromatography Type:None (Direct infusion)

MS:

MS ID:MS005701
Analysis ID:AN005988
Instrument Name:Waters Xevo TQ-XS
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Mass transitions of m/z 203/161 (KIC), 206/161 (KIC-d3), 189/174 (KIV), 194/178 (KIV-5C13), 203/174 (KMV), and 211/177 (KMV-d8) were monitored in the positive ion mode. Metabolite concentrations were computed using a ratio to heavy isotope-labeled internal standards. TargetLynx was used for data processing.
Ion Mode:POSITIVE
  
MS ID:MS005702
Analysis ID:AN005989
Instrument Name:Waters Xevo TQ-XS
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:The following mass transitions were monitored in the positive ion mode: Lactate 196/91; Pyruvate 299/91; Succinate 329/91; Fumarate 327/91; Malate 345/222; α-Ketoglutarate 462/91; Citrate, Isocitrate 508/91; Oxaloacetate 448/91; Lactate-d3 199/91; Pyruvate-13C3 302/91; Succininate-13C4 333/91; Fumarate-d2 329/91; Malate-d3 348/225; α-Ketoglutarate-13C4 466/91; Citrate-d4 512/91. Metabolite concentrations were computed using a ratio to heavy isotope-labeled internal standards. TargetLynx was used for data processing.
Ion Mode:POSITIVE
  
MS ID:MS005703
Analysis ID:AN005990
Instrument Name:Waters Xevo TQ-XS
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:All nucleotides were detected in the negative ion MRM mode based on a characteristic fragmentation reaction as descibed in Gooding JR, Jensen MV, Dai X, Wenner BR, Lu D, Arumugam R, Ferdaoussi M, MacDonald PE, Newgard CB. Cell Rep. 2015 Oct 6;13(1):157-167. Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism. PMID: 26411681. PMCID: PMC4598307. TargetLynx was used for data processing.
Ion Mode:NEGATIVE
  
MS ID:MS005704
Analysis ID:AN005991
Instrument Name:Waters Xevo TQ-XS
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Creatine and phosphocreatine were detected in the negative MRM mode based on a characteristic fragmentation reaction: creatine 130/88, creatine-d3 133/91, phosphocreatine 210/79. Metabolite concentrations were computed using a ratio to creatinine-d3. TargetLynx was used for data processing.
Ion Mode:NEGATIVE
  
MS ID:MS005705
Analysis ID:AN005992
Instrument Name:Waters Xevo TQ-XS
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:The following mass transitions were monitored in the positive ion mode: Free CoA 768.2/261.1; Acetyl-CoA 810.2/303.1; Propionyl-CoA 824.2/317.1; Butyryl-CoA 838.2/331.1; Malonyl-CoA 854.2/347.1; HMG-CoA 912.2/405.2; Acetyl CoA-13C2 812.2/305.1. Metabolite concentrations were computed using a ratio to Acetyl-CoA-13C2. TargetLynx was used for data processing.
Ion Mode:POSITIVE
  
MS ID:MS005706
Analysis ID:AN005993
Instrument Name:Waters Xevo-TQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Mass spectra for acylcarnitine and amino acid esters were obtained using precursor ion (parents of m/z = 99) and neutral loss (NTL 102 amu for acidic and neutral amino acids; NTL 119 amu for basic amino acids; and NTL 161 amu for arginine) scanning methods, respectively. The spectra were acquired in a multi-channel analyzer (MCA) mode to improve signal-to-noise. Metabolite concentrations were computed using a ratio to heavy isotope-labeled internal standards. NeoLynx was used for data processing.
Ion Mode:POSITIVE
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