Summary of Study ST003596

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 PR002224. The data can be accessed directly via it's Project DOI: 10.21228/M84Z5P This work is supported by NIH grant, U2C- DK119886.

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Study IDST003596
Study TitleAn Organism-Level Quantitative Flux Model of Energy Metabolism in Mice
Study SummaryMammalian tissues feed on nutrients in the blood circulation. At the organism-level, mammalian energy metabolism comprises of oxidation, storage, interconverting, and releasing of circulating nutrients. Though much is known about the individual processes and nutrients, a holistic and quantitative model describing these processes for all major circulating nutrients is lacking. Here, by integrating isotope tracer infusion, mass spectrometry, and isotope gas analyzer measurement, we developed a framework to systematically quantify fluxes through these metabolic processes for 10 major circulating energy nutrients in mice, resulting in an organism-level quantitative flux model of energy metabolism. This model revealed in wildtype C57BL/6J mice that circulating nutrients have more dominant metabolic cycling fluxes than their oxidation fluxes, with distinct partition between cycling and oxidation flux for individual circulating nutrients. Applications of this framework in obese mouse models showed on a per animal basis extensive elevation of metabolic cycling fluxes in ob/ob mice, but not in diet-induced obese C57BL/6J mice. Thus, our framework describes quantitatively the functioning of energy metabolism at the organism-level, valuable for revealing new features of energy metabolism in physiological and disease conditions.
Institute
Harvard School of Public Health
DepartmentMolecular Metabolism
LaboratorySheng (Tony) Hui
Last NameYuan
First NameBo
Address655 Huntington Avenue
Emailbo_yuan@hsph.harvard.edu
Phone7323189185
Submit Date2024-09-28
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2024-12-30
Release Version1
Bo Yuan Bo Yuan
https://dx.doi.org/10.21228/M84Z5P
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002224
Project DOI:doi: 10.21228/M84Z5P
Project Title:An Organism-Level Quantitative Flux Model of Energy Metabolism in Mice
Project Summary:Mammalian tissues feed on nutrients in the blood circulation. At the organism-level, mammalian energy metabolism comprises of oxidation, storage, interconverting, and releasing of circulating nutrients. Though much is known about the individual processes and nutrients, a holistic and quantitative model describing these processes for all major circulating nutrients is lacking. Here, by integrating isotope tracer infusion, mass spectrometry, and isotope gas analyzer measurement, we developed a framework to systematically quantify fluxes through these metabolic processes for 10 major circulating energy nutrients in mice, resulting in an organism-level quantitative flux model of energy metabolism. This model revealed in wildtype mice that circulating nutrients have more dominant metabolic cycling fluxes than their oxidation fluxes, with distinct partition between cycling and oxidation flux for individual circulating nutrients. Applications of this framework in obese mouse models showed on a per animal basis extensive elevation of metabolic cycling fluxes in ob/ob mice, but not in diet-induced obese mice. Thus, our framework describes quantitatively the functioning of energy metabolism at the organism-level, valuable for revealing new features of energy metabolism in physiological and disease conditions.
Institute:Harvard School of Public Health
Department:Molecular Metabolism
Laboratory:Sheng (Tony) Hui
Last Name:Yuan
First Name:Bo
Address:655 Huntington Avenue
Email:bo_yuan@hsph.harvard.edu
Phone:7323189185
Funding Source:Paul G. Allen Family Foundation 0034665 (S.H.); NIH R00DK117066 (S.H.); NIH R56DK140139 (S.H.); Sabri Ülker Center for Metabolic Research

Subject:

Subject ID:SU003725
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J (control); high fat diet (HFD)-induced obese C57BL/6J mice; ob/ob.
Age Or Age Range:12-24 weeks
Weight Or Weight Range:25-35 g (control); 40-50 g (HFD); 50-65 (ob/ob)
Gender:Male
Animal Animal Supplier:Jackson Laboratory
Animal Light Cycle:7 AM to 7 PM
Animal Feed:chow PicoLab Mouse Diet 5058; Research Diet D12492i, 60% calorie from fat
Species Group:Mammals

Factors:

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

mb_sample_id local_sample_id Genotype Sample source Treatment
SA391905as-H3HFD blood 13C_3-HB
SA391906ar-H1HFD blood 13C_3-HB
SA391907ar-H1 (glycerol anal.)HFD blood 13C_3-HB
SA391908as-H3 (glycerol anal.)HFD blood 13C_3-HB
SA391909as-H4 (glycerol anal.)HFD blood 13C_3-HB
SA391910ba-H1HFD blood 13C_3-HB
SA391911ba-H4HFD blood 13C_3-HB
SA391912ba-H5HFD blood 13C_3-HB
SA391913ba-H6HFD blood 13C_3-HB
SA391914ba-H1 (glycerol anal.)HFD blood 13C_3-HB
SA391915ba-H4 (glycerol anal.)HFD blood 13C_3-HB
SA391916ba-H5 (glycerol anal.)HFD blood 13C_3-HB
SA391917ba-H6 (glycerol anal.)HFD blood 13C_3-HB
SA391918as-H4HFD blood 13C_3-HB
SA391919ca-tail-H102HFD blood 13C_3-HB
SA391920ca-tail-H100HFD blood 13C_3-HB
SA391921bz-tail-H102HFD blood 13C_3-HB
SA391922bz-tail-H101HFD blood 13C_3-HB
SA391923bz-tail-H100HFD blood 13C_3-HB
SA391924ca-tail-H101HFD blood 13C_3-HB
SA391925at-H1HFD blood 13C_Acetate
SA391926at-H4HFD blood 13C_Acetate
SA391927at-H1 (glycerol anal.)HFD blood 13C_Acetate
SA391928at-H4 (glycerol anal.)HFD blood 13C_Acetate
SA391929bi-H7 (glycerol anal.)HFD blood 13C_Alanine
SA391930bi-H52 (glycerol anal.)HFD blood 13C_Alanine
SA391931bi-H51 (glycerol anal.)HFD blood 13C_Alanine
SA391932bi-H5 (glycerol anal.)HFD blood 13C_Alanine
SA391933bc-H18HFD blood 13C_C16 0
SA391934da-tail-H2HFD blood 13C_C16 0
SA391935da-tail-H4HFD blood 13C_C16 0
SA391936bc-H17HFD blood 13C_C16 0
SA391937bc-H15HFD blood 13C_C16 0
SA391938da-tail-H6HFD blood 13C_C16 0
SA391939bc-H15 (glycerol anal.)HFD blood 13C_C16 0
SA391940bc-H17 (glycerol anal.)HFD blood 13C_C16 0
SA391941bc-H18 (glycerol anal.)HFD blood 13C_C16 0
SA391942da-tail-H9HFD blood 13C_C16 0
SA391943ce-H102HFD blood 13C_C18 1
SA391944ce-H106HFD blood 13C_C18 1
SA391945cf-tail-H107HFD blood 13C_C18 1
SA391946ce-H107HFD blood 13C_C18 1
SA391947cf-tail-H102HFD blood 13C_C18 1
SA391948cf-tail-H106HFD blood 13C_C18 1
SA391949bw-H105HFD blood 13C_C18 2
SA391950bw-H104HFD blood 13C_C18 2
SA391951bt-H100HFD blood 13C_C18 2
SA391952bu-H100HFD blood 13C_C18 2
SA391953bu-H101HFD blood 13C_C18 2
SA391954bu-H102HFD blood 13C_C18 2
SA391955bw-H103HFD blood 13C_C18 2
SA391956bt-H101HFD blood 13C_C18 2
SA391957bt-H102HFD blood 13C_C18 2
SA391958cg-tail-H110HFD blood 13C_Glucose
SA391959cg-tail-H109HFD blood 13C_Glucose
SA391960cg-tail-H108HFD blood 13C_Glucose
SA391961az-H9HFD blood 13C_Glucose
SA391962az-H8HFD blood 13C_Glucose
SA391963az-H7HFD blood 13C_Glucose
SA391964az-H10HFD blood 13C_Glucose
SA391965az-H11HFD blood 13C_Glucose
SA391966az-H8 (glycerol anal.)HFD blood 13C_Glucose
SA391967az-H9 (glycerol anal.)HFD blood 13C_Glucose
SA391968az-H7 (glycerol anal.)HFD blood 13C_Glucose
SA391969az-H10 (glycerol anal.)HFD blood 13C_Glucose
SA391970az-H11 (glycerol anal.)HFD blood 13C_Glucose
SA391971bh-H5 (glycerol anal.)HFD blood 13C_Glutamine
SA391972bh-H14 (glycerol anal.)HFD blood 13C_Glutamine
SA391973bh-H7HFD blood 13C_Glutamine
SA391974bh-H5HFD blood 13C_Glutamine
SA391975bh-H7 (glycerol anal.)HFD blood 13C_Glutamine
SA391976bh-H14HFD blood 13C_Glutamine
SA391977bh-H19 (glycerol anal.)HFD blood 13C_Glutamine
SA391978bb-H10 (glycerol anal.)HFD blood 13C_Glycerol
SA391979bb-H10HFD blood 13C_Glycerol
SA391980bb-H13 (glycerol anal.)HFD blood 13C_Glycerol
SA391981bb-H11HFD blood 13C_Glycerol
SA391982bb-H13HFD blood 13C_Glycerol
SA391983bb-H14HFD blood 13C_Glycerol
SA391984bb-H7HFD blood 13C_Glycerol
SA391985bb-H11 (glycerol anal.)HFD blood 13C_Glycerol
SA391986bb-H9 (glycerol anal.)HFD blood 13C_Glycerol
SA391987bb-H14 (glycerol anal.)HFD blood 13C_Glycerol
SA391988bb-H7 (glycerol anal.)HFD blood 13C_Glycerol
SA391989bb-H8 (glycerol anal.)HFD blood 13C_Glycerol
SA391990bb-H9HFD blood 13C_Glycerol
SA391991bb-H8HFD blood 13C_Glycerol
SA391992bg-H19HFD blood 13C_Lactate
SA391993bg-H14 (glycerol anal.)HFD blood 13C_Lactate
SA391994bg-H19 (glycerol anal.)HFD blood 13C_Lactate
SA391995bg-H7HFD blood 13C_Lactate
SA391996bg-H5HFD blood 13C_Lactate
SA391997bg-H7 (glycerol anal.)HFD blood 13C_Lactate
SA391998bg-H14HFD blood 13C_Lactate
SA391999bd-H10HFD blood 13C_Valine
SA392000bd-H7HFD blood 13C_Valine
SA392001be-H14HFD blood 13C_Valine
SA392002be-H22HFD blood 13C_Valine
SA392003bd-H10 (glycerol anal.)HFD blood 13C_Valine
SA392004bd-H7 (glycerol anal.)HFD blood 13C_Valine
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Collection:

Collection ID:CO003718
Collection Summary:Blood was collected by tail snip, or from implanted carotid artery catheter.
Sample Type:Blood (serum)
Storage Conditions:-80℃

Treatment:

Treatment ID:TR003734
Treatment Summary:Treatment #1 Infusion of Isotope Tracers with serum metabolites labeling measurement. On the day of tracer infusion, the mouse was transferred to a new cage without food at 9 AM, and fasted for 4 hours before infusion. Right before the start of infusion, the lock solution was drawn and removed from the in-dwelling jugular vein catheter, and the catheter was flushed with 50 µL saline. The vascular access button was then connected to the infusion tether, which was in turn connected with a swivel on a counter-balance arm. This allowed the mouse to move freely around in the cage. The infusion of U-13C-tracer at a small non-perturbing dose started at 2 PM, and lasted for 30 min for fatty acids and 2.5-3 h for all other tracers. The tracer concentration and infusion rates can be found in the Study Design section. At the end of infusion, 40 µL blood was collected by tail snip. For infusion of 13C-lactate, alanine, and glutamine, given their significant labeling difference in the arterial and venous blood, 40 µL arterial blood was sampled from the carotid artery via the pre-implanted catheter, and used for analysis. The blood was collected into a Microvette tube with clotting activator, and was kept on ice during collection. After infusion, the catheter was flushed with 50 µL saline, and then sealed with 10 ~ 20 µL lock solution. The blood was centrifuged at 5,000 ×g for 10 min. 4 µL serum was aliquoted in separate tubes, and stored at -80°C. --- Treatment #2 Hyperinsulinemia Clamp. C57BL/6J mouse (26.5 ± 1.8 g) was transferred to a new cage without food at 9 AM. After fasting for 4 hours, blood was collected via tail snip for basal glycemia and insulin measurement (noted as time point 0, or T0). A mixture of U-13C-glucose (200 mM), unlabeled glucose (2361 mM) and insulin (66.6 mU/mL) was infused at 3 µL/min/mouse. After 2.5 h, blood was collected for serum glucose labeling and insulin measurement (noted as time point T1). Then U-13C palmitate (8 mM) was infused at 6 µL/min/animal for another 30 min (delivered by a second pump), simultaneously with the glucose and insulin infusion (in the Study Design section, it is noted as "13C-glucose and 13C-C16" with "200 and 8" for concentration and "3 and 6" for infusion rate). Blood was collected at the end for serum glucose and palmitate labeling and insulin measurement (time point T2). Insulin was measured by ELISA Kit following the vendor’s protocol. ---- Treatment #3 Measurement of Triglyceride Circulatory Turnover Flux. The serum triglyceride turnover was measured with a pulse-chase strategy. After fasting for 4 hours, BL/6J mouse was infused with 8 mM U-13C-palmitate at 0.25 µL/min/g for 1-2 hours to label serum triglyceride. The infusion was stopped, and blood was collected by tail snip every 15 minutes for one hour to monitor the decay of M+16 labeling L in palmitoyl-containing triglyceride. An exponential curve was fitted with L=Ae^(-kt) for data points from 15 to 60 min. 0-15 min was excluded from modeling for clearance of labeling in free fatty acids. The triglyceride turnover flux R_a was calculated as R_a=k⋅C⋅V, where C is the total triglyceride concentration in serum, and V the serum volume of 49 µL/g (Clemons, 1992).

Sample Preparation:

Sampleprep ID:SP003732
Sampleprep Summary:For general metabolomics analysis with HILIC-MS method #1 (see MS section), 4 µL serum was mixed with 60 µL -20°C methanol: acetonitrile: water at 40: 40: 20. The sample was vigorously vortexed, and centrifuged at 4°C at 13,000 g for 10 min. 30 µL supernatant was transferred to the HPLC vial from which 5 µL was injected to the LC-MS. For serum glycerol analysis with HILIC-MS method #2, serum glycerol was first enzymatically derivatized into glycerol-3-phosphate before LC-MS analysis. 4 µL serum was mixed with 90 µL freshly made enzyme solution containing 2 U/mL glycerol kinase, 25 mM Tris-HCl (pH 8.0), 50 mM sodium chloride, 10 mM magnesium chloride, and 1.5 mM ATP, and incubated at room temperature for 15 min. The reaction was stopped by the addition of 400 µL methanol. The sample was vortexed, and centrifuged at 16,000 g for 10 min. The supernatant was transferred to a new tube, and dried using SpeedVac at 45 °C for 90 min. 60 µL of methanol:acetonitrile:water at 40:40:20 was added to the residue, vortexed, and centrifuged at 16,000 g for 10 min. 30 µL supernatant was transferred to the HPLC vial, from which 5 µL was injected to the LC-MS. For serum triglyceride analysis with RP-MS method #3, 4 µL serum was mixed with 50 µL extraction buffer of butanol:methanol at 1:1 volume ratio containing 5 mM ammonium formate. The sample was vigorously vortexed, and centrifuged at 4°C at 13,000 g for 10 min. 30 µL supernatant was carefully taken and transferred to glass insert of LC/MS vials for LC/MS analysis. SPLASH® LIPIDOMIX internal standard with serial dilution was spiked in serum for calibration and triglyceride quantification. In the Data(results) section, "0" means non-detectable labeling, and NA refers to missing measurement (we did not measure certain metabolites in certain samples).
Processing Storage Conditions:On ice

Combined analysis:

Analysis ID AN005905 AN005906 AN005907
Analysis type MS MS MS
Chromatography type HILIC HILIC Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish Thermo Dionex Ultimate 3000 RS
Column Waters XBridge BEH Amide XP (150 x 2.1mm, 2.5um) Waters XBridge BEH Amide (100 x 2.1mm,2.5um) Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
MS Type ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode NEGATIVE NEGATIVE POSITIVE
Units average 13C labeling fraction M+16 normalized labeling 13C labeling fraction

Chromatography:

Chromatography ID:CH004483
Chromatography Summary:HILIC method to detect a wide range of serum metabolomics
Instrument Name:Thermo Vanquish
Column Name:Waters XBridge BEH Amide XP (150 x 2.1mm, 2.5um)
Column Temperature:25
Flow Gradient:0 - 3 min, 100% B; 3.2 - 6.2 min, 90% B; 6.5. - 10.5 min, 80% B; 10.7 - 13.5 min, 70% B; 13.7 - 16 min, 45% B; and 16.5 - 22 min, 100% B
Flow Rate:300 uL/min
Solvent A:95% water/5% acetonitrile; 10 mM ammonium acetate; 10 mM ammonium hydroxide
Solvent B:20% water/80% acetonitrile; 10 mM ammonium acetate; 10 mM ammonium hydroxide
Chromatography Type:HILIC
  
Chromatography ID:CH004484
Chromatography Summary:HILIC, similar to HILIC method 1, but focuses on glycerol analysis after serum glycerol is enzymatically derivatized to glycerol-3-phosphate (G3P). Only eluent 1 min around the retention time of G3P is directed to MS.
Instrument Name:Thermo Vanquish
Column Name:Waters XBridge BEH Amide (100 x 2.1mm,2.5um)
Column Temperature:25
Flow Gradient:0 - 3 min, 100% B; 3.2 - 6.2 min, 90% B; 6.5. - 10.5 min, 80% B; 10.7 - 13.5 min, 70% B; 13.7 - 16 min, 45% B; and 16.5 - 22 min, 100% B
Flow Rate:300 uL/min
Solvent A:95% water/5% acetonitrile; 10 mM ammonium acetate; 10 mM ammonium hydroxide
Solvent B:20% water/80% acetonitrile; 10 mM ammonium acetate; 10 mM ammonium hydroxide
Chromatography Type:HILIC
  
Chromatography ID:CH004485
Chromatography Summary:Reverse phase for lipidomics
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:50
Flow Gradient:20% B from 0 to 3 min, 55% B at 7 min, 100% B at 24 min and held until 26 min, 20% B at 28 min and held until 30 min
Flow Rate:350 uL/min
Solvent A:40% water/60% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Solvent B:90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS005623
Analysis ID:AN005905
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The mass spectrometry used was Q Exactive HF (Thermo Fisher Scientific, San Jose, CA), and scanned from 70 to 1000 m/z with switching polarity. The resolution was 120,000. Metabolites were identified based on accurate mass and retention time using an in-house library, and the isotopic labeling was analyzed by EI-Maven.
Ion Mode:NEGATIVE
  
MS ID:MS005624
Analysis ID:AN005906
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The mass spectrometry condition used was the same as HILIC-MS method (1), except that only eluent 1 min around the retention time of gycerol-3-phosphate is directed to MS, otherwise to waste.
Ion Mode:NEGATIVE
  
MS ID:MS005625
Analysis ID:AN005907
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The mass spectrometry used as Thermo Q Exactive. For lipid identification, the scan mode was data dependent (dd)-MS2, covering 100-1200 m/z in both positive and negative polarities. The resolving power was 70,000 for precursor ion scan, and 17,500 for product ion scan. The isolation width was 1.0 m/z, and stepped normalized collision energy (NCE) was 10, 20, and 40 eV. Serum triglycerides were identified in LipidSearch as ammonium-adduct cations. For quantitative and isotopic labeling analysis, the scan range was 700 to 1,000 m/z under positive mode with resolution 70,000 for faster scan speed. The isotopic labeling was further analyzed in EI-Maven.
Ion Mode:POSITIVE
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