Summary of study ST000380

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 PR000298. The data can be accessed directly via it's Project DOI: 10.21228/M8HW28 This work is supported by NIH grant, U2C- DK119886.

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Study IDST000380
Study TitleTemporal metabolomic responses of cultured HepG2 liver cells to high fructose and high glucose exposures (part II)
Study SummaryHigh fructose consumption has been implicated with deleterious effects on human health, including hyperlipidemia elicited through de novo lipogenesis. However, more global effects of fructose on cellular metabolism have not been elucidated. In order to explore the metabolic impact of fructose-containing nutrients, we applied both GC-TOF and HILIC-QTOF mass spectrometry metabolomic strategies using extracts from cultured HepG2 cells exposed to fructose, glucose, or fructose + glucose. Cellular responses were analyzed in a time-dependent manner, incubated in media containing 5.5 mM glucose + 5.0 mM fructose in comparison to controls incubated in media containing either 5.5 mM glucose or 10.5 mM glucose. Mass spectrometry identified 156 unique known metabolites and a large number of unknown compounds, which revealed metabolite changes due to both utilization of fructose and high-carbohydrate loads independent of hexose structure. Fructose was shown to be partially converted to sorbitol, and generated higher levels of fructose-1-phosphate as a precursor for glycolytic intermediates. Differentially regulated ratios of 3-phosphoglycerate to serine pathway intermediates in high fructose media indicated a diversion of carbon backbones away from energy metabolism. Additionally, high fructoseconditions changed levels of complex lipids toward phosphatidylethanolamines. Patterns of acylcarnitines in response to high hexose exposure (10.5 mM glucose or glucose/fructose combination) suggested a reduction in mitochondrial beta-oxidation.
Institute
University of California, Davis
DepartmentGenome and Biomedical Sciences Facility
LaboratoryWCMC Metabolomics Core
Last NameFiehn
First NameOliver
Address1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616
Emailofiehn@ucdavis.edu
Phone(530) 754-8258
Submit Date2016-04-14
Raw Data AvailableYes
Raw Data File Type(s).peg
Analysis Type DetailLC-MS
Release Date2016-04-25
Release Version1
Oliver Fiehn Oliver Fiehn
https://dx.doi.org/10.21228/M8HW28
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000298
Project DOI:doi: 10.21228/M8HW28
Project Title:Temporal metabolomic responses of cultured HepG2 liver cells to high fructose and high glucose exposures
Project Type:Time-course Study
Project Summary:High fructose consumption has been implicated with deleterious effects on human health, including hyperlipidemia elicited through de novo lipogenesis. However, more global effects of fructose on cellular metabolism have not been elucidated. In order to explore the metabolic impact of fructose-containing nutrients, we applied both GC-TOF and HILIC-QTOF mass spectrometry metabolomic strategies using extracts from cultured HepG2 cells exposed to fructose, glucose, or fructose + glucose. Cellular responses were analyzed in a time-dependent manner, incubated in media containing 5.5 mM glucose + 5.0 mM fructose in comparison to controls incubated in media containing either 5.5 mM glucose or 10.5 mM glucose. Mass spectrometry identified 156 unique known metabolites and a large number of unknown compounds, which revealed metabolite changes due to both utilization of fructose and high-carbohydrate loads independent of hexose structure. Fructose was shown to be partially converted to sorbitol, and generated higher levels of fructose-1-phosphate as a precursor for glycolytic intermediates. Differentially regulated ratios of 3-phosphoglycerate to serine pathway intermediates in high fructose media indicated a diversion of carbon backbones away from energy metabolism. Additionally, high fructoseconditions changed levels of complex lipids toward phosphatidylethanolamines. Patterns of acylcarnitines in response to high hexose exposure (10.5 mM glucose or glucose/fructose combination) suggested a reduction in mitochondrial beta-oxidation.
Institute:University of California, Davis
Department:Genome and Biomedical Sciences Facility
Laboratory:WCMC Metabolomics Core
Last Name:Fiehn
First Name:Oliver
Address:1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616
Email:ofiehn@ucdavis.edu
Phone:(530) 754-8258
Funding Source:NIH U24DK097154

Subject:

Subject ID:SU000401
Subject Type:Cells
Subject Species:Homo sapiens
Species Group:Human

Factors:

Subject type: Cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatment Time
SA01715710Glucose10minReplicate3_0022.d10.5mM Glu 10min
SA01715810Glucose10minReplicate1_0042.d10.5mM Glu 10min
SA01715910Glucose10minReplicate4_0008.d10.5mM Glu 10min
SA01716010Glucose10minReplicate5_0094.d10.5mM Glu 10min
SA01716110Glucose10minReplicate6_0024.d10.5mM Glu 10min
SA01716210Glucose10minReplicate2_0077.d10.5mM Glu 10min
SA01716310Glucose1hrReplicate2_0034.d10.5mM Glu 1hr
SA01716410Glucose1hrReplicate5_0092.d10.5mM Glu 1hr
SA01716510Glucose1hrReplicate4_0037.d10.5mM Glu 1hr
SA01716610Glucose1hrReplicate3_0083.d10.5mM Glu 1hr
SA01716710Glucose1hrReplicate1_0005.d10.5mM Glu 1hr
SA01716810Glucose1hrReplicate6_0030.d10.5mM Glu 1hr
SA01716910Glucose24hrReplicate3_0044.d10.5mM Glu 24hr
SA01717010Glucose24hrReplicate4_0061.d10.5mM Glu 24hr
SA01717110Glucose24hrReplicate5_0062.d10.5mM Glu 24hr
SA01717210Glucose24hrReplicate6_0053.d10.5mM Glu 24hr
SA01717310Glucose24hrReplicate2_0090.d10.5mM Glu 24hr
SA01717410Glucose24hrReplicate1_0043.d10.5mM Glu 24hr
SA01717510Glucose6hrReplicate1_0071.d10.5mM Glu 6hr
SA01717610Glucose6hrReplicate2_0070.d10.5mM Glu 6hr
SA01717710Glucose6hrReplicate5_0046.d10.5mM Glu 6hr
SA01717810Glucose6hrReplicate6_0031.d10.5mM Glu 6hr
SA01717910Glucose6hrReplicate3_0006.d10.5mM Glu 6hr
SA01718010Glucose6hrReplicate4_0009.d10.5mM Glu 6hr
SA0171815Glucose5Fructose10minReplicate1_0036.d5.5mM Glu 5mM Fru 10min
SA0171825Glucose5Fructose10minReplicate3_0013.d5.5mM Glu 5mM Fru 10min
SA0171835Glucose5Fructose10minReplicate4_0023.d5.5mM Glu 5mM Fru 10min
SA0171845Glucose5Fructose10minReplicate6_0082.d5.5mM Glu 5mM Fru 10min
SA0171855Glucose5Fructose10minReplicate2_0065.d5.5mM Glu 5mM Fru 10min
SA0171865Glucose5Fructose10minReplicate5_0029.d5.5mM Glu 5mM Fru 10min
SA0171875Glucose5Fructose1hrReplicate6_0017.d5.5mM Glu 5mM Fru 1hr
SA0171885Glucose5Fructose1hrReplicate2_0081.d5.5mM Glu 5mM Fru 1hr
SA0171895Glucose5Fructose1hrReplicate1_0038.d5.5mM Glu 5mM Fru 1hr
SA0171905Glucose5Fructose1hrReplicate5_0059.d5.5mM Glu 5mM Fru 1hr
SA0171915Glucose5Fructose1hrReplicate4_0048.d5.5mM Glu 5mM Fru 1hr
SA0171925Glucose5Fructose1hrReplicate3_0067.d5.5mM Glu 5mM Fru 1hr
SA0171935Glucose5Fructose24hrReplicate2_0054.d5.5mM Glu 5mM Fru 24hr
SA0171945Glucose5Fructose24hrReplicate1_0056.d5.5mM Glu 5mM Fru 24hr
SA0171955Glucose5Fructose24hrReplicate3_0033.d5.5mM Glu 5mM Fru 24hr
SA0171965Glucose5Fructose24hrReplicate4_0007.d5.5mM Glu 5mM Fru 24hr
SA0171975Glucose5Fructose24hrReplicate6_0035.d5.5mM Glu 5mM Fru 24hr
SA0171985Glucose5Fructose24hrReplicate5_0074.d5.5mM Glu 5mM Fru 24hr
SA0171995Glucose5Fructose6hrReplicate6_0045.d5.5mM Glu 5mM Fru 6hr
SA0172005Glucose5Fructose6hrReplicate4_0041.d5.5mM Glu 5mM Fru 6hr
SA0172015Glucose5Fructose6hrReplicate3_0018.d5.5mM Glu 5mM Fru 6hr
SA0172025Glucose5Fructose6hrReplicate1_0014.d5.5mM Glu 5mM Fru 6hr
SA0172035Glucose5Fructose6hrReplicate2_0012.d5.5mM Glu 5mM Fru 6hr
SA0172045Glucose10minReplicate6_0019.d5.5mM Glu 10min
SA0172055Glucose10minReplicate5_0089.d5.5mM Glu 10min
SA0172065Glucose10minReplicate2_0020.d5.5mM Glu 10min
SA0172075Glucose10minReplicate1_0068.d5.5mM Glu 10min
SA0172085Glucose10minReplicate3_0086.d5.5mM Glu 10min
SA0172095Glucose10minReplicate4_0032.d5.5mM Glu 10min
SA0172105Glucose1hrReplicate3_0057.d5.5mM Glu 1hr
SA0172115Glucose1hrReplicate4_0095.d5.5mM Glu 1hr
SA0172125Glucose1hrReplicate5_0080.d5.5mM Glu 1hr
SA0172135Glucose1hrReplicate6_0085.d5.5mM Glu 1hr
SA0172145Glucose1hrReplicate1_0069.d5.5mM Glu 1hr
SA0172155Glucose1hrReplicate2_0060.d5.5mM Glu 1hr
SA0172165Glucose24hrReplicate1_0050.d5.5mM Glu 24hr
SA0172175Glucose24hrReplicate5_0025.d5.5mM Glu 24hr
SA0172185Glucose24hrReplicate4_0084.d5.5mM Glu 24hr
SA0172195Glucose24hrReplicate3_0078.d5.5mM Glu 24hr
SA0172205Glucose24hrReplicate2_0066.d5.5mM Glu 24hr
SA0172215Glucose6hrReplicate5_0072.d5.5mM Glu 6hr
SA0172225Glucose6hrReplicate6_0055.d5.5mM Glu 6hr
SA0172235Glucose6hrReplicate4_0011.d5.5mM Glu 6hr
SA0172245Glucose6hrReplicate1_0010.d5.5mM Glu 6hr
SA0172255Glucose6hrReplicate3_0079.d5.5mM Glu 6hr
SA0172265Glucose6hrReplicate2_0049.d5.5mM Glu 6hr
SA01722710Glucosamine10minReplicate4_0073.dGlucosamine 10min
SA01722810Glucosamine10minReplicate2_0093.dGlucosamine 10min
SA01722910Glucosamine10minReplicate6_0091.dGlucosamine 10min
SA01723010Glucosamine10minReplicate1_0047.dGlucosamine 10min
SA01723110Glucosamine10minReplicate3_0026.dGlucosamine 10min
SA01723210Glucosamine10minReplicate5_0021.dGlucosamine 10min
Showing results 1 to 76 of 76

Collection:

Collection ID:CO000395
Collection Summary:HepG2 cells (ATCC HB-8065) were cultured in MEM containing 10 % (v:v) FBS, 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA), 1 x MEM non-essential amino acids, and 5.5 mM glucose at 37 °C in a 5 % CO2 environment. Cells were grown for four to six passages in 10 cm tissue culture dishes with 14 mL MEM, and transferred to MULTIWELL™ 12 well culture dishes for incubation in 2 mL of the treatment medium. Upon reaching 80 % confluency, the cell culture medium was changed to media representative of experimental condition: 5.5 mM glucose MEM, 5.5 mM glucose MEM + 5 mM glucose, or 5.5 mM glucose MEM + 5 mM fructose. Media was replenished after 24 and 48 h. Cell material was collected at 10 min, 1, 6, and 24 h time points following the 48 h media replacement. Culture dishes were placed on ice and each well was washed twice with 1 mL ice-cold PBS. 4 mL ice-cold 3:1 methanol/H2O extraction solvent was added to each well. Cell material was manually scraped from each well, and the extraction solvent cell material suspension was transferred to collection tubes and frozen at −80 °C prior to further processing.
Collection Protocol Filename:metabolomic_responses_of_cultured_HepG2_liver_cells.pdf
Sample Type:Cell
Collection Location:Invitrogen, Carlsbad, CA
Collection Frequency:Cell material was collected at 10 min, 1, 6, and 24 h time points following the 48 h media replacement.
Tissue Cell Identification:HepG2

Treatment:

Treatment ID:TR000415
Treatment Summary:3 Treatments: A) Control group of HepG2 cells incubated in media with 5.5 mM glucose (Glc5) B) HepG2 cells incubated in media containing 5.5 mM glucose + 5.0 mM fructose (Glc5Fru5) C) HepG2 cells incubated in media containing 10.5 mM glucose (Glc10)
Treatment Protocol Filename:metabolomic_responses_of_cultured_HepG2_liver_cells.pdf
Treatment Protocol Comments:HepG2 cells incubated in media containing 10.5 mM glucose (Glc10) was included to enable differentiation of metabolic effects caused by fructose from metabolic effects caused by increased hexose resources
Cell Media:Cells were acclimated to their respective media condition for 48 h prior to collection of sample material to obtain a metabolite profile representative of continued exposure instead of response to a sudden change in carbohydrate resources. Media was replenished after 24 and 48 h

Sample Preparation:

Sampleprep ID:SP000408
Sampleprep Summary:Sample material was thawed on ice, vortexed for 20 s, sonicated for 5 min with a VWR 50HT Ultrasonic Bath (VWR International Inc., Bridgeport, NJ), and separated into 500 μL aliquots. Each aliquot was centrifuged for 5 min @ 14,000 rcf, and supernatant was collected and lyophilized to dryness. Samples was kept on ice and removed only for sonication, centrifugation, and lyophilization steps. Lyophilized material was used for HILIC-QTOF metabolite profiling without additional clean-up steps. Lyophilized material for GC-TOF analysis was redissolved in 1:1 acetonitrile/H2O, vortexed for 10 s, and centrifuged for 5 min @ 14,000 rcf. Supernatant was collected and lyophilized to dryness.
Sampleprep Protocol Filename:metabolomic_responses_of_cultured_HepG2_liver_cells.pdf
Processing Method:Lysophilization

Combined analysis:

Analysis ID AN000614
Analysis type MS
Chromatography type HILIC
Chromatography system Agilent 6530
Column Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
MS Type ESI
MS instrument type QTOF
MS instrument name Agilent 6530 QTOF
Ion Mode POSITIVE
Units counts

Chromatography:

Chromatography ID:CH000440
Methods Filename:Data_Dictionary_Fiehn_laboratory_HILIC_QTOF_MS.pdf
Instrument Name:Agilent 6530
Column Name:Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
Column Pressure:200-700 bar
Column Temperature:45 C
Flow Gradient:100% B to 30% B
Flow Rate:0.4 mL/min
Injection Temperature:4 C
Internal Standard:See data dictionary
Retention Time:See data dictionary
Sample Injection:3uL
Solvent A:Ultrapure water with 10 mM ammonium formiate + 0.125% formic acid, pH 3
Solvent B:Ultrapure water with 10 mM ammonium formiate + 0.125% formic acid, pH 3
Analytical Time:14 min
Capillary Voltage:4500 V
Time Program:16.75 min
Weak Wash Solvent Name:1:1 ACN:H2O
Strong Wash Solvent Name:1:1 ACN:H2O
Target Sample Temperature:Autosampler temp 4 C
Randomization Order:Excel generated
Chromatography Type:HILIC

MS:

MS ID:MS000547
Analysis ID:AN000614
Instrument Name:Agilent 6530 QTOF
Instrument Type:QTOF
MS Type:ESI
Ion Mode:POSITIVE
Capillary Voltage:3500
Collision Gas:Nitrogen
Dry Gas Flow:8 L/min
Dry Gas Temp:325 C
Fragment Voltage:120 V
Fragmentation Method:Auto MS/MS
Ion Source Temperature:325
Ion Spray Voltage:1000
Ionization:Pos
Precursor Type:Intact Molecule
Reagent Gas:Nitrogen
Source Temperature:325 C
Dataformat:.d
Desolvation Gas Flow:11 L/min
Desolvation Temperature:350 C
Nebulizer:35 psig
Octpole Voltage:750
Resolution Setting:extended dynamic range
Scan Range Moverz:60-1700 Da
Scanning Cycle:2 Hz
Scanning Range:60-1700 Da
Skimmer Voltage:1850 V
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