Summary of Study ST003283

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 PR002035. The data can be accessed directly via it's Project DOI: 10.21228/M8K52N This work is supported by NIH grant, U2C- DK119886.

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Study IDST003283
Study TitleSystematic evaluation of the hepatic de novo lipogenic substrate supply network reveals a hierarchical mitochondrial-cytosolic control structure
Study SummaryHepatic de novo lipogenesis (DNL) is a fundamental physiologic process that becomes pathogenically elevated during metabolic disease. Both cytosolic citrate and acetate supply DNL, but their upstream inputs and pathways to DNL remain unclear. We traced 13C-labeled lactate/pyruvate and acetate into fatty acids in mitochondrial citrate carrier (CiC) and pyruvate carrier (MPC), ATP-citrate lyase (ACLY), acetyl-CoA synthetase 2 (ACSS2) liver-specific knockout (LivKO), and selective double LivKO (DLivKO) mice. We show that MPC and ACLY gate the major mitochondrial-cytosolic acetyl-CoA production pathway that operates in parallel to a cytosolic ACSS2 pathway. Given surprisingly persistent DNL after CiC + ACSS2 DLivKO, we considered a ketone-routed cytosolic acetyl-CoA production pathway as an alternative pathway feeding DNL. CiC LivKO markedly increased acetoacetate- and the ketogenic amino acid leucine-supplied DNL, consistent with ketones functioning as mitochondrial-citrate reciprocal DNL substrates. By delineating a structure for the DNL substrate supply network, these basic findings may inform strategies to therapeutically modulate DNL.
Institute
University of Iowa
DepartmentMolecular Physiology and Biophysics
LaboratoryEric Taylor
Last NameTaylor
First NameEric
Address169 Newton Rd. PBDB 3316
Emaileric-taylor@uiowa.edu
Phone3193357500
Submit Date2024-05-30
Raw Data AvailableYes
Raw Data File Type(s)mzXML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-10-28
Release Version1
Eric Taylor Eric Taylor
https://dx.doi.org/10.21228/M8K52N
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002035
Project DOI:doi: 10.21228/M8K52N
Project Title:Stable Isotope Tracing of Hepatic De Novo Lipogenesis
Project Summary:13C-lactate/pyruvate, 13C-acetate, 13C-acetoacetate, and 13C-leucine traced the hepatic de novo lipogenesis substrate supply network in multiple single and double liver-specific knockout mouse strains. MS data were collected on a ThermoFisher Q-Exactive Orbitrap operating in tSIM mode.
Institute:University of Iowa
Department:Molecular Physiology and Biophysics
Laboratory:Eric Taylor
Last Name:Taylor
First Name:Eric
Address:169 Newton Rd. PBDB 3316
Email:eric-taylor@uiowa.edu
Phone:3193357500
Funding Source:NIDDK

Subject:

Subject ID:SU003403
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Sample source Tracer Sex Diet
SA35528401047b_248ACLY-LivKO Liver 13C-acetate Male NCD
SA35528501047b_265ACLY-LivKO Liver 13C-acetate Male NCD
SA35528601047b_257ACLY-LivKO Liver 13C-acetate Male NCD
SA35528701047b_249ACLY-LivKO Liver 13C-acetate Male NCD
SA35528801047b_252ACLY-LivKO Liver 13C-acetate Male NCD
SA35528901047b_253ACLY-LivKO Liver 13C-acetate Male NCD
SA35529001047b_254ACLY-LivKO Liver 13C-acetate Male NCD
SA35527700763_16ACLY-LivKO Liver 13C-Acetate Male NCD
SA35527800763_5ACLY-LivKO Liver 13C-Acetate Male NCD
SA35527900763_8ACLY-LivKO Liver 13C-Acetate Male NCD
SA35528000763_9ACLY-LivKO Liver 13C-Acetate Male NCD
SA35528100763_12ACLY-LivKO Liver 13C-Acetate Male NCD
SA35528200763_14ACLY-LivKO Liver 13C-Acetate Male NCD
SA35528300763_13ACLY-LivKO Liver 13C-Acetate Male NCD
SA35529100835_11ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35529200835_9ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35529300835_6ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35529400835_5ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35529500835_2ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35529600835_1ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35529700763_2ACLY-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35529800763_23ACLY-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35529900763_21ACLY-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35530000763_19ACLY-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35530100763_17ACLY-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35530200763_3ACLY-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35530301047_183ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35530401047_167ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35530501047_168ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35530601047_171ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35530701047_172ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35530801047_176ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35530901047_179ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531001047_181ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531100835_23ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531201047_185ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531300835_17ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531400835_15ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531501047_191ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531600835_13ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531700835_18ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531801047_190ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35531901047_186ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35532000835_22ACLY-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35532801047b_236ACSS2-LivKO Liver 13C-acetate Male NCD
SA35532901047b_238ACSS2-LivKO Liver 13C-acetate Male NCD
SA35533001047b_237ACSS2-LivKO Liver 13C-acetate Male NCD
SA35533101047b_231ACSS2-LivKO Liver 13C-acetate Male NCD
SA35533201047b_232ACSS2-LivKO Liver 13C-acetate Male NCD
SA35533301047b_229ACSS2-LivKO Liver 13C-acetate Male NCD
SA35533401047b_227ACSS2-LivKO Liver 13C-acetate Male NCD
SA35532100622_20ACSS2-LivKO Liver 13C-Acetate Male NCD
SA35532200622_24ACSS2-LivKO Liver 13C-Acetate Male NCD
SA35532300622_25ACSS2-LivKO Liver 13C-Acetate Male NCD
SA35532400622_26ACSS2-LivKO Liver 13C-Acetate Male NCD
SA35532500622_17ACSS2-LivKO Liver 13C-Acetate Male NCD
SA35532600622_15ACSS2-LivKO Liver 13C-Acetate Male NCD
SA35532700622_19ACSS2-LivKO Liver 13C-Acetate Male NCD
SA35533501047_86ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35533601047_87ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35533701047_97ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35533801047_98ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35533901047_84ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35534001047_109ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35534100510d_84ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35534200510d_87ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35534300510d_97ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35534400510d_98ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35534500510d_102ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35534600510d_109ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35534701047_102ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35534800510d_86ACSS2-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35536300622_13Acss2-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35536400622_11Acss2-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35536500622_9Acss2-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35536600622_8Acss2-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35536700622_7Acss2-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35536800622_2Acss2-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35536900622_3Acss2-LivKO Liver 13C-lactate/13C-pyruvate Male NCD
SA35534900510d_101ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535001047_101ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535100510d_92ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535201047_89ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535301047_92ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535401047_93ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535501047_96ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535600510d_96ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535700510d_105ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535800510d_104ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35535900510d_89ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35536001047_104ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35536101047_105ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35536200510d_93ACSS2-LivKO Liver Lactate/pyruvate+13C-acetate Male HSD
SA35537001047_138CiC+ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35537101047_157CiC+ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35537201047_148CiC+ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35537301047_147CiC+ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35537401047_146CiC+ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35537500912_25CiC+ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
SA35537600912_12CiC+ACLY-LivKO Liver 13C-lactate/13C-pyruvate+acetate Male HSD
Showing page 1 of 9     Results:    1  2  3  4  5  Next  Last     Showing results 1 to 100 of 872

Collection:

Collection ID:CO003396
Collection Summary:Mice were anesthetized by isoflurane inhalation for 2.5 minutes. Liver tissue was dissected from breathing mice and rapidly frozen using a freeze-clamp.
Sample Type:Liver
Storage Conditions:-80℃

Treatment:

Treatment ID:TR003412
Treatment Summary:Mice fasted from around the start of the light cycle (~zeitgeber time 0, ZT0) for 4 hours (~ZT4), received an intraperitoneal bolus injection of a 13C-labeled tracer: 13C-lactate/13C-pyruvate - an 11% w/v solution of U13C-lactate/U13C-pyruvate (10:1) was administered by intraperitoneal (IP) injection at a dosage of 2.2 g/kg. 30 minutes later, livers tissue was harvested under freeze-clamped conditions. 13C-acetate - a 5% w/v solution of U13C-acetate was administered by IP injection at a dosage of 0.75 g/kg. 40 minute later, livers tissue was harvested under freeze-clamped conditions. 13C-lactate/13C-pyruvate + acetate and lactate/pyruvate + 13C-acetate - a solution of 11% w/v of U13C-lactate/U13C-pyruvate (10:1) + 2.5% w/v acetate or 11% w/v or lactate:pyruvate + 2.5% w/v U13C-acetate was administered by IP injection at a dosage of 2.2 g/kg lactate:pyruvate and 0.5 g/kg acetate. A second injection equivalent in all ways to the first was administered 45 minutes later. 75 minutes, livers tissue was harvested under freeze-clamped conditions. 13C-ccetoacetate + lactate/pyruvate - a solution containing 5% w/v U13C4-acetoactate + 11% w/v + lactate/pyruvate (10:1) + was administered over the course of two IP injection spaced 15 minutes apart at a dosage of 1.0 g/kg acetoacetate and 2.2 g/kg lactate:pyruvate. 30-minutes after the second injection (45-minutes after the first injection), livers tissue was harvested under freeze-clamped conditions. 13C-leucine and 13C-leucine + lactate/pyruvate - a solution containig 2.5% w/v of U13C6-Leucine or 2.5% w/v of U13C6-Leucine + 11% w/v of lactate/pyruvate (10:1) was administered over the course of two IP injection spaced 15 minutes apart at a dosage of 0.5 g/kg leucine and 2.2 g/kg lactate:pyruvate. 30-minutes after the second injection (45-minutes after the first injection), livers tissue was harvested under freeze-clamped conditions.

Sample Preparation:

Sampleprep ID:SP003410
Sampleprep Summary:Freeze-clamped liver tissue (40 ± 5 mg) was weighed without thawing and lyophilized overnight. Lyophilized tissue was homogenized in 18 volumes of ice-cold 2:2:1 acetonitrile:methanol:water extraction solvent per milligram of wet tissue weight using a BeadRupter bead mill homogenizer (Omni International) operated for 30 seconds at 6.45 MHz. Immediately afterwards, homogenization tubes were rotated for 60 minutes at -20°C. Samples were centrifuged at 21,000 x g for 10 minutes, after which the supernatant (metabolite extract) was transferred to a new 1.7 mL microcentrifuge tube and pulse vortexed to ensure uniform mixing. 300 μL of the metabolite extract was transferred to a new 1.7 ml microcentrifuge tube and dried using a Speedvac Vacuum concentrator (Thermo) for 2 hours without heating at a vacuum ramp = 4 (~50 Torr/min). Samples were rehydrated in 30 uL of 1:1 acetonitrile:water and allowed to reconstitute overnight at -20C. The following day, samples were centrifuged 21,000 x g for 10 minutes and supernatants were transferred to autosampler vials for analysis. For determination of acetate enrichment, metabolite extracts were analyzed direction following being centrifuged 21,000 x g for 10 minutes.

Combined analysis:

Analysis ID AN005378 AN005379
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Vanquish Thermo Vanquish
Column SeQuant ZIC-pHILIC (150 x 2.1mm, 5um) SeQuant ZIC-pHILIC (50 x 2.1mm, 5um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode NEGATIVE NEGATIVE
Units Intensity Intensity

Chromatography:

Chromatography ID:CH004077
Chromatography Summary:For each prepared sample, 2 µL was separated using a Millipore SeQuant ZIC-pHILIC (2.1 X 150 mm, 5 µm particle size, Millipore Sigma #150460) column with a ZIC-pHILIC guard column (20 x 2.1 mm, Millipore Sigma #150437) attached to a Thermo Vanquish Flex UHPLC. The mobile phase comprised Buffer A [20 mM (NH4)2CO3, 0.1% NH4OH (v/v)] and Buffer B [acetonitrile]. The chromatographic gradient was run at a flow rate of 0.150 mL/min as follows: 0–21 min-linear gradient from 80 to 20% Buffer B; 21-21.5 min-linear gradient from 20 to 80% Buffer B; and 21.5–28 min-hold at 80% Buffer B.
Instrument Name:Thermo Vanquish
Column Name:SeQuant ZIC-pHILIC (150 x 2.1mm, 5um)
Column Temperature:25
Flow Gradient:0–21 min-linear gradient from 80 to 20% B; 21-21.5 min-linear gradient from 20 to 80% B; and 21.5–28 min-hold at 80% B
Flow Rate:150 ul/min
Solvent A:100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide
Solvent B:100% acetonitrile
Chromatography Type:HILIC
  
Chromatography ID:CH004078
Chromatography Summary:For each prepared sample, 6 µL was separated using a Millipore SeQuant ZIC-pHILIC (2.1 X 50 mm, 5 µm particle size, Millipore Sigma #150459) column with a ZIC-pHILIC guard column (20 x 2.1 mm, Millipore Sigma #150437) attached to a Thermo Vanquish Flex UHPLC. The mobile phase comprised Buffer A [20 mM (NH4)2CO3, 0.1% NH4OH (v/v)] and Buffer B [acetonitrile]. The chromatographic gradient was run at a flow rate of 0.300 mL/min as follows: 0–3.33 min-linear gradient from 80 to 20% Buffer B; 3.33-3.42 min-linear gradient from 20 to 80% Buffer B; and 3.42-6 min-hold at 80% Buffer B.
Instrument Name:Thermo Vanquish
Column Name:SeQuant ZIC-pHILIC (50 x 2.1mm, 5um)
Column Temperature:25
Flow Gradient:0–3.33 min-linear gradient from 80 to 20% B; 3.33-3.42 min-linear gradient from 20 to 80% B; and 3.42-6 min-hold at 80% B
Flow Rate:300 ul/min
Solvent A:100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS005107
Analysis ID:AN005378
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Data was acquired using a Thermo Q Exactive MS with a spray voltage set to 3.0 kV, the heated capillary held at 275°C, and the HESI probe held at 350°C. The sheath gas flow was set to 40 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 1 unit. MS data resolution was set at 70,000, the AGC target at 10e6, and the maximum injection time at 200 ms. For 13C tracing experiments, the MS was operated in negative polarity tSIM mode.
Ion Mode:NEGATIVE
  
MS ID:MS005108
Analysis ID:AN005379
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Data was acquired using a Thermo Q Exactive MS with a spray voltage set to 3.0 kV, the heated capillary held at 275°C, and the HESI probe held at 350°C. The sheath gas flow was set to 40 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 1 unit. MS data resolution was set at 70,000, the AGC target at 10e6, and the maximum injection time at 200 ms. For 13C tracing experiments, the MS was operated in negative polarity tSIM mode.
Ion Mode:NEGATIVE
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