Summary of Study ST003641

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 PR002251. The data can be accessed directly via it's Project DOI: 10.21228/M8NV72 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 IDST003641
Study TitleThe Spatial Transcriptional Activity of Hepatic TCF7L2 Regulates Zonated Metabolic Pathways that Contribute to Liver Fibrosis
Study SummaryBackground and Aims: The molecular mechanisms regulating the zonal distribution of metabolism in liver are incompletely understood. Here we used single nuclei genomics techniques to examine the spatial transcriptional function of the transcription factor 7-like 2 (TCF7L2) in rodent liver. We also determined the consequences of TCF7L2 transcriptional inactivation on the metabolic architecture of the liver, and on the function of key zonated metabolic pathways that influence the development of fibrotic liver diseases. Methods: Multimodal 10X single nuclei RNA- and ATAC-Seq were used to dissect the cell-type specific expression and DNA binding activity of TCF7L2 and transcriptional co-regulators across the liver lobule. The transcriptional activity of TCF7L2 was targeted by excising exon 11 of Tcf7l2, which encodes part of the DNA binding domain (DBD). The consequences of TCF7L2 inactivation on zonation was investigated with a focus on the disruption to zonated metabolic pathways, including those linked to ammonia detoxification and glutamine metabolism, cholesterol homeostasis and bile acid synthesis. The susceptibility of Hep-TCF7L2ΔDBD mice to hepatic fibrosis under dietary stress was investigated using the Gubra Amylin Nash (GAN) and choline-deficient amino acid-defined high fat (CDAHFD) diets. Results: Tcf7l2 mRNA expression was ubiquitous across the liver lobule, but accessibility of the consensus TCF/LEF DNA binding motif was restricted to pericentral (PC) hepatocytes in zone 3. PC hepatocyte-specific gene expression was lost in Hep-TCF7L2ΔDBD mice, which we link to alterations in the transcriptional activity of zonal repressors Tbx3 and Tcf7l1. The absence of a classic zone 3 gene expression program in TCF7L2 mutant mice led to hepatic cholesterol accumulation, impaired bile acid synthesis, and significant disruptions to glutamine and glutamate homeostasis. In human liver biopsy tissue, TCF7L2 expression declined as the severity of fibrosis progressed, and Hep-TCF7L2ΔDBD mice developed more severe hepatic fibrosis when fed the CDAHFD. Conclusion: TCF7L2 is key transcriptional regulator of the PC hepatocyte gene expression program in zone 3, and regulates multiple zonated metabolic pathways that may contribute to the development of fibrotic liver diseases.
Institute
UT Health San Antonio
Last NameNorton
First NameLuke
Address7703 Floyd Curl Drive, San Antonio, TX 78229
Emailnortonl@uthscsa.edu
Phone(210)-567-0739
Submit Date2024-12-23
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2025-01-06
Release Version1
Luke Norton Luke Norton
https://dx.doi.org/10.21228/M8NV72
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002251
Project DOI:doi: 10.21228/M8NV72
Project Title:The Spatial Transcriptional Activity of Hepatic TCF7L2 Regulates Zonated Metabolic Pathways that Contribute to Liver Fibrosis
Project Summary:Study used single nuclei genomics techniques to examine the spatial transcriptional function of the transcription factor 7-like 2 (TCF7L2) in rodent liver. Research aimed to determine the consequences of TCF7L2 transcriptional inactivation on the metabolic architecture of the liver, and on the function of key zonated metabolic pathways that influence the development of fibrotic liver diseases. Dietary stress was investigated using the Gubra Amylin Nash (GAN) and choline-deficient amino acid-defined high fat (CDAHFD) diets to investigate the susceptibility of liver-specific TCF7L2 mutant mice (Hep-TCF7L2ΔDBD) compared to control (TCF7L2LoxP/LoxP) mice in hepatic fibrosis.
Institute:UT Health San Antonio
Last Name:Norton
First Name:Luke
Address:7703 Floyd Curl Drive, San Antonio, TX 78229
Email:nortonl@uthscsa.edu
Phone:(210)-567-0739

Subject:

Subject ID:SU003771
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 Sample source Genotype
SA39776116_BGallbladder Mutant
SA39776215_BGallbladder Mutant
SA39776314_BGallbladder Mutant
SA39776413_BGallbladder Mutant
SA39776512_BGallbladder Mutant
SA39776611_BGallbladder Mutant
SA39776710_BGallbladder Mutant
SA3977689_BGallbladder Mutant
SA3977694_BGallbladder Wild-type
SA3977701_BGallbladder Wild-type
SA3977712_BGallbladder Wild-type
SA3977723_BGallbladder Wild-type
SA3977736_BGallbladder Wild-type
SA3977745_BGallbladder Wild-type
SA3977757_BGallbladder Wild-type
SA3977768_BGallbladder Wild-type
SA39777710_IIntestine Mutant
SA39777816_IIntestine Mutant
SA39777915_IIntestine Mutant
SA39778014_IIntestine Mutant
SA39778113_IIntestine Mutant
SA39778212_IIntestine Mutant
SA39778311_IIntestine Mutant
SA3977849_IIntestine Mutant
SA3977858_IIntestine Wild-type
SA3977867_IIntestine Wild-type
SA3977876_IIntestine Wild-type
SA3977885_IIntestine Wild-type
SA3977894_IIntestine Wild-type
SA3977903_IIntestine Wild-type
SA3977912_IIntestine Wild-type
SA3977921_IIntestine Wild-type
SA39779313_LLiver Mutant
SA3977949_LLiver Mutant
SA39779510_LLiver Mutant
SA39779611_LLiver Mutant
SA39779716_LLiver Mutant
SA39779812_LLiver Mutant
SA39779914_LLiver Mutant
SA39780015_LLiver Mutant
SA3978015_LLiver Wild-type
SA3978023_LLiver Wild-type
SA3978034_LLiver Wild-type
SA3978042_LLiver Wild-type
SA3978056_LLiver Wild-type
SA3978067_LLiver Wild-type
SA3978078_LLiver Wild-type
SA3978081_LLiver Wild-type
Showing results 1 to 48 of 48

Collection:

Collection ID:CO003764
Collection Summary:Tissues were collected from control (TCF7L2LoxP/LoxP) and liver-specific TCF7L2 mutant mice (Hep-TCF7L2ΔDBD) then stored at -80’C until further analysis was performed.
Sample Type:Liver, Intestine, Gallbladder

Treatment:

Treatment ID:TR003780
Treatment Summary:Wild-type control (TCF7L2LoxP/LoxP) and liver-specific TCF7L2 mutant mice (Hep-TCF7L2ΔDBD) were fed methionine and choline deficient, 60% HFD diet (CDAHFD, Research Diets, Cat#: A06071302) for 8-weeks.

Sample Preparation:

Sampleprep ID:SP003778
Sampleprep Summary:Gallbladder, liver and intestine were isolated from Wild-type control (TCF7L2LoxP/LoxP) and liver-specific TCF7L2 mutant mice (Hep-TCF7L2ΔDBD). Bile acids were extracted from liver and intestine in Optima Methanol containing recovery bile acid spikes (G-UDCA, G-CDCA) overnight at room temp. The following day, samples were centrifuged at 4000rpm for 10min. Prior to analysis, bile was diluted 1:1000 into Optima Methanol, then further diluted 1:2 into autosampler vials. Liver supernatant was vortexed then filtered through a 0.45 µm syringe-driven filter unit directly into autosampler vials. Intestine supernatant was thoroughly vortexed, then an aliquot centrifuged at 15,000xg for 5min. Intestine samples were then diluted 1:20 directly into autosampler vials. For bile acid analysis, bile, intestine (2uL) and liver (5uL) were injected via an Ultimate3000 autosampler module. For C4 analysis, 10uL of liver sample was injected.

Combined analysis:

Analysis ID AN005979 AN005980
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column ACE Excel C18-PFP (150 x 2.1mm, 2um, 100Å) ACE Excel SuperC18 (150 x 2.1mm, 2um, 90Å)
MS Type ESI ESI
MS instrument type Triple quadrupole Triple quadrupole
MS instrument name Thermo Quantiva Thermo Quantiva
Ion Mode NEGATIVE POSITIVE
Units nmol/g dry weight (liver, intestine), nmol/uL (gallbladder) nmol/g dry weight of liver

Chromatography:

Chromatography ID:CH004541
Chromatography Summary:Separation and detection of bile acids using a UHPLC gradient method. Duration = 33min. Please see ETV-Lab-Bile-Acids-Methods.pdf for details.
Methods Filename:ETV-Lab-Bile-Acids-Methods.pdf
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:ACE Excel C18-PFP (150 x 2.1mm, 2um, 100Å)
Column Temperature:50
Flow Gradient:0.325 ml/min: 0min: 26% B, 74% A, 5min: 27.5% B, 72.5% A, 14min: 40% B, 60% A, 22min: 85% B, 15% A, 22.1min: 98% B, 2% A, 25.1min 98%, 2% A; 0.2 ml/min: 26min: 100% C, 27.9min: 100% C; 0.3 ml/min: 29min: 28.5% B, 71.5% A; 0.325 ml/min: 30-33min: 28.5% B, 71.5% A
Flow Rate:0.325 mL/min
Injection Temperature:5
Solvent A:Water 100%; 0.1% formic acid; 10 mM Ammonium acetate
Solvent B:25% Methanol/75% Acetonitrile; 0.1% formic acid; Ammonium acetate (10mM)
Chromatography Type:Reversed phase
Solvent C:100% Methanol
  
Chromatography ID:CH004542
Chromatography Summary:Detection and separation of 7α-hydroxy-4-cholesten-3-one (C4) using a UHPLC gradient method. Duration = 15min. Please see ETV-Lab-C4-Methods.pdf for details.
Methods Filename:ETV-Lab-C4-Methods.pdf
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:ACE Excel SuperC18 (150 x 2.1mm, 2um, 90Å)
Column Temperature:50
Flow Gradient:0.3 ml/min: 0min: 60% C, 40% A, 0.5min: 60% C, 40% A, 2min: 90% C, 10% A, 5min: 100% C, 9min: 100% C, 10-15min: 60% C, 40% A
Flow Rate:0.3 mL/min
Injection Temperature:5
Solvent A:Water 80%/20% methanol; 0.1% formic acid;
Solvent B:-
Chromatography Type:Reversed phase
Solvent C:100% Methanol

MS:

MS ID:MS005692
Analysis ID:AN005979
Instrument Name:Thermo Quantiva
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Bile acid concentrations (nM) were quantified in Skyline-daily software (MacCross Lab Software) from standard curves of each individual bile acid generated from a mix of purified bile acid standards, with the exception of T-a-MCA, T-b-MCA, and T-w-MCA where T-CA was used. Total bile acid levels (nmol) per tissue were determined per intestine (g), whole liver weight (g), and gallbladder volume (uL).
Ion Mode:NEGATIVE
  
MS ID:MS005693
Analysis ID:AN005980
Instrument Name:Thermo Quantiva
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:C4 levels (uM) were quantified in Skyline-daily software (MacCross Lab Software) from a standard curve generated using C4 purified standards. Total C4 levels were determined per whole liver weight.
Ion Mode:POSITIVE
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