Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity methods
Aim. Evidence-backed execution summary for Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity methods from Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Intestine-specific FXR disruption reduces obesity.
reagent used in the protocol.
- Use
- To further explore the role of intestinal FXR in lipid and glucose metabolism, male wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice were fed a HFD, revealing that the body weights of Fxr ΔIE mice are lower than Fxr fl/fl mice after 3 weeks of treatment ( ). NMR measurements con...
Intestine-specific FXR disruption reduces obesity.
reagent used in the protocol.
- Use
- Earlier studies revealed that C14, C18, C18:1, C20:4 sphingomyelins (SMs) and ceramide to sphingosine-1-phosphate ratios were positively correlated with insulin resistance,. Serum lipidomics using tandem mass spectrometry and retention time comparisons with ion identification and quantification with authentic stan...
Tempol improves obesity via inhibition of intestinal FXR.
reagent used in the protocol.
- Use
- To further investigate the role of FXR in the tempol-improved metabolic phenotype of HFD-fed mice, wild-type ( Fxr +/+ ) mice and whole-body Fxr -null ( Fxr -/- ) mice, wild-type ( Fxr fl/fl ) mice and intestine-specific Fxr -null ( Fxr ΔIE ) mice on a HFD were administered tempol in the drinking wa...
Methods
reagent used in the protocol.
- Use
- Tempol was purchased from Sigma (St. Louis, MO). Bile acids were ordered from Steraloid, Inc. (Newport, RI) and Sigma, and TCA-d5 sodium salt was from Toronto Research Chemicals Inc. (Toronto, Ontario). SMs and ceramides were obtained from Avanti Polar Lipids. The S1P ELISA kit was purchased from Echelon Biosciences...
Preparation and culture of primary hepatocytes.
reagent used in the protocol.
- Use
- Hepatocytes were prepared by the general perfusion method. Primary hepatocytes from 6-week-old C57BL/6N mice were obtained by collagenase 1 (Invitrogen, Carlsbad, CA) perfusion. Thecells were purified by 45% percoll (Sigma-Aldrich, St Louis, MO) density centrifugation and cultured in DMEM (Invitrogen) with 10% feta...
RNA analysis.
reagent used in the protocol.
- Use
- The mucosa of intestine was gently scraped and flash frozen in liquid nitrogen and stored at - 80 °C until RNA was prepared. RNA was extracted from frozen intestine using TRIzol reagent (Invitrogen). Complementary DNA was synthesized from 1 µg of total RNA using Superscript II reverse transcriptase (...
Luciferase assays.
reagent used in the protocol.
- Use
- Grace Guo (Rutgers University) provided the PGL4-Shp-TK firefly luciferase construct and human Fxr expression plasmid. Paul A. Dawson (Wake Forest University School of Medicine) provided human Asbt expression plasmid. The plasmids were transfected into Caco2 (ATCC HTB-37) cellsusing the X-tremeGENE HP DNA Transfecti...
Metabolic assays.
reagent used in the protocol.
- Use
- For GTT, mice were fasted for 16 h, blood was drawn and mice were injected intraperitoneally with 1 g kg -1 glucose. For ITT, mice were fasted for 4 h, blood was drawn and then injected intraperitoneally with insulin (Eli Lilly, Washington, DC) 1 U kg -1 bodyweight. Blood samples were taken from the tail...
Tempol affects gut microbiota and BSH activity.
Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gut,. These observations suggested that tempol might affect gut microbial metabolism or the composition of the gut microbiome. Indeed, signi...
- Use
- Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gut,. These observations suggested that tempol might affect gut microbial metabolism or the composition of the gut microbiome. Indeed, signi...
Animal studies.
The wild-type ( Fxr +/+ ) mice and whole-body Fxr -null Fxr( -/- ) mice, and, wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice are all on a C57BL/6N genetic background,. Fxr +/+ and Fxr -/- mice were backcrossed with C57BL/6N mice for over four generations....
- Use
- The wild-type ( Fxr +/+ ) mice and whole-body Fxr -null Fxr( -/- ) mice, and, wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice are all on a C57BL/6N genetic background,. Fxr +/+ and Fxr -/- mice were backcrossed with C57BL/6N mice for over four generations....
RNA analysis.
The mucosa of intestine was gently scraped and flash frozen in liquid nitrogen and stored at - 80 °C until RNA was prepared. RNA was extracted from frozen intestine using TRIzol reagent (Invitrogen). Complementary DNA was synthesized from 1 µg of total RNA using Superscript II reverse transcriptase (...
- Use
- The mucosa of intestine was gently scraped and flash frozen in liquid nitrogen and stored at - 80 °C until RNA was prepared. RNA was extracted from frozen intestine using TRIzol reagent (Invitrogen). Complementary DNA was synthesized from 1 µg of total RNA using Superscript II reverse transcriptase (...
16S rRNA gene sequencing of the intestinal microbiome.
The bacteria in feces and caecum content were extracted using PowerSoil DNA Isolation Kit (Mo Bio laboratory, Inc., Carlsbad, CA). The PCR products (~ 1,000 bp) were purified using the AgencourtAMPure technology (Beckman Coulter, Brea, CA) as described in454 Technical Bulletin number 2011-002, Short Fragment R...
- Use
- The bacteria in feces and caecum content were extracted using PowerSoil DNA Isolation Kit (Mo Bio laboratory, Inc., Carlsbad, CA). The PCR products (~ 1,000 bp) were purified using the AgencourtAMPure technology (Beckman Coulter, Brea, CA) as described in454 Technical Bulletin number 2011-002, Short Fragment R...
Metagenomic data analysis.
The experimental set-up consisted of 14 samples distributed as 6 vehicle replicates and 8 tempol gavage replicates. After quality filtering and deduplication, each sample contains on average 11,000 reads. The mothur software package was used to preprocess the sequencing data andthe Ribosomal Database Project multi-c...
- Use
- The experimental set-up consisted of 14 samples distributed as 6 vehicle replicates and 8 tempol gavage replicates. After quality filtering and deduplication, each sample contains on average 11,000 reads. The mothur software package was used to preprocess the sequencing data andthe Ribosomal Database Project multi-c...
Metagenomic data analysis.
Study: study 1, study 2 (re-sequencing of study 1)
- Use
- Study: study 1, study 2 (re-sequencing of study 1)
UPLC-ESI-QTOFMS analysis.
UPLC-ESI-QTOFMS analysis was performed in positive and negative mode, which was operated in full-scan mode at m/z 100-1,000 ( ). The liquid chromatography system was an ACQUITY UPLC (Waters Corp., Milford, MA) consisting of a reverse-phase 2.1 × 50 mm ACQUITY UPLC BEH C18 1.7 µm column (Waters Corp.)...
- Use
- UPLC-ESI-QTOFMS analysis was performed in positive and negative mode, which was operated in full-scan mode at m/z 100-1,000 ( ). The liquid chromatography system was an ACQUITY UPLC (Waters Corp., Milford, MA) consisting of a reverse-phase 2.1 × 50 mm ACQUITY UPLC BEH C18 1.7 µm column (Waters Corp.)...
Data processing and multivariate data analysis.
Raw data were aligned using MarkerLynx software (Waters Corp.) to generate a data matrix consisting of peak areas corresponding to a unique m/z and retention time without normalization. Metabolomics analysis including principal component analysis and PLS-DA models was performed with SIMCA-P + 12 (Umetrics, Kinnelon,...
- Use
- Raw data were aligned using MarkerLynx software (Waters Corp.) to generate a data matrix consisting of peak areas corresponding to a unique m/z and retention time without normalization. Metabolomics analysis including principal component analysis and PLS-DA models was performed with SIMCA-P + 12 (Umetrics, Kinnelon,...
Metagenomic data analysis.
Software used for acquisition, scoring, statistics, or reporting.
- Use
- The experimental set-up consisted of 14 samples distributed as 6 vehicle replicates and 8 tempol gavage replicates. After quality filtering and deduplication, each sample contains on average 11,000 reads. The mothur software package was used to preprocess the sequencing data andthe Ribosomal Database Project multi-c...
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Tempol affects gut microbiota and BSH activity.
Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gut,. These observations suggested that tempol might affect gut microbial metabolism or the composition of the gut microbiome. Indeed, significant phylum-level shifts from Firmicutes to Bacteroidetes in the gut microbiome composition are observed in mouse caecum following 5 days of tempol administered by gavage (250 mg kg -1 per day) to mice on a normal chow diet ( ). Heat map diagrams of 16S rRNA sequencing results indicate that tempol treatment dramatically decreases the family Lactobacillaceae ( ) and robustly reduces the genus Lactobacillus ( ). Similar to the results of acute treatment via gavage, quantitative real-time PCR (qPCR) analysis of suspected caecum microbes obtained from mice on a HFD reve...
Tempol enhances the intestinal taurine-conjugated bile acids.
Previous studies demonstrated that alteration of the gut micro-biome affects the levels of bile acids in the liver, heart and kidney. To determine whether the composition of bile acids were changed in the tempol group, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-based metabolomics analysis was used to examine the bile-acid composition and their levels in the feces and intestinal tissue. Partial least-squares discriminant analysis (PLS-DA) modelling of the UPLC-ESI-QTOFMS-negative mode data from mouse feces and intestine reveal distinct clustering of the control and tempol groups ( ). Levels of the four ions (m/z, 453.2842 at 3.14min; m/z, 391.2841 at 4.11min; m/z, 407.2782 at 3.33 min; and m/z, 391.2838 at 4.06 min) are significantly reduced in the feces by tempol ( ), whereas four ions...
Intestine-specific FXR disruption reduces obesity.
To further explore the role of intestinal FXR in lipid and glucose metabolism, male wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice were fed a HFD, revealing that the body weights of Fxr ΔIE mice are lower than Fxr fl/fl mice after 3 weeks of treatment ( ). NMR measurements confirm that the fat mass, the ratio of fat and body mass and the ratio of fat and lean mass of Fxr fl/fl mice on a HFD are higher than Fxr ΔIE mice (, ). Food intake measurements are comparable between Fxr fl/fl and Fxr ΔIE mice ( ). GTT reveals that Fxr ΔIE mice have improved glucose intolerance compared with Fxr fl/fl mice ( ) and ITT demonstrates that the insulin sensitivity in Fxr ΔIE mice is significantly increased ( ). Moreover, fasted serum insulin levels and the homoeostasis model assessment index are significantly reduced in Fxr ΔIE mice ( )...
Animal studies.
The wild-type ( Fxr +/+ ) mice and whole-body Fxr -null Fxr( -/- ) mice, and, wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice are all on a C57BL/6N genetic background,. Fxr +/+ and Fxr -/- mice were backcrossed with C57BL/6N mice for over four generations. Fxr fl/fl and Fxr ΔIE mice were backcrossed with C57BL/6N mice for over 10 generations. The mice were housed in temperature- and light-controlled rooms and given water and pelleted NIH-31 chow ad libitum. All animal studies were performed in accordance with Institute of Laboratory Animal Resources guidelines and approved by the National Cancer Institute Animal Care and Use Committee. In the tempol study, male mice were fed a HFD (Bio-Serv, Frenchtown, NJ; 60kcal% fat) from 6 weeks of age and administered 0.064% (w/v) tempol in the drinking water. For the chow diet stu...
Metabolic assays.
For GTT, mice were fasted for 16 h, blood was drawn and mice were injected intraperitoneally with 1 g kg -1 glucose. For ITT, mice were fasted for 4 h, blood was drawn and then injected intraperitoneally with insulin (Eli Lilly, Washington, DC) 1 U kg -1 bodyweight. Blood samples were taken from the tail at 15, 30, 60 and 90 min after injection, and glucose was measured using a glucometer (Bayer, Pittsburgh, PA). Fasted serum insulin was measured using an enzyme-linked immunosorbent assay kit (Crystal Chem Inc., Downers Grove, IL).
16S rRNA gene sequencing of the intestinal microbiome.
The bacteria in feces and caecum content were extracted using PowerSoil DNA Isolation Kit (Mo Bio laboratory, Inc., Carlsbad, CA). The PCR products (~ 1,000 bp) were purified using the AgencourtAMPure technology (Beckman Coulter, Brea, CA) as described in454 Technical Bulletin number 2011-002, Short Fragment Removal Procedure. After purification, the products were quantified by both Qubit (Lifetech, Carlsbad, CA) and qPCR, using the KAPA Biosystems Library Quantification Kit (Kapa-Biosystems, Woburn, MA). Products were pooled based on molar amounts, run ona 1% agarose gel and extracted. After clean-up with a QIAquick PCR Purificationkit (Qiagen, Valencia, CA), quality and quantity were assessed using a DNA 7500LabChip on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Qubit quantification. The sequencing was performed using a quarter Picotiter Plate on a...
Metagenomic data analysis.
Full model: (1) Y i j k = u + Dose i + Study j + Error i j k with i = 1, 2, 3; j = 1,2; k = 1, 2, 3, 4
Metagenomic data analysis.
Reduced model: (2) Y i k = u + Dose i + Error i k with i = 1, 2, 3; k = 1, 2, 3, 4
Measurement outputs
What raw and processed outputs should exist?
Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gu...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Previous studies demonstrated that alteration of the gut micro-biome affects the levels of bile acids in the liver, heart and kidney. To determine whether the composition of bi...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Bile-acid metabolism is tightly associated with the FXR signalling pathway, and HFD can induce FXR signalling in the intestine. Tempol treatment inhibits expression of the FXR...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
To further explore the role of intestinal FXR in lipid and glucose metabolism, male wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice were fed a...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gut,.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gu...; Previous studies demonstrated that alteration of the gut micro-biome affects the levels of bile acids in the liver, heart and kidney. To determine whether the composition of bi...; Bile-acid metabolism is tightly associated with the FXR signalling pathway, and HFD can induce FXR signalling in the intestine. Tempol treatment inhibits expression of the FXR...; To further explore the role of intestinal FXR in lipid and glucose metabolism, male wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice were fed a....
from paperStatistical comparison
Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gu...; To further investigate the role of FXR in the tempol-improved metabolic phenotype of HFD-fed mice, wild-type ( Fxr +/+ ) mice and whole-body Fxr -null ( Fxr -/- ) mi...; The experimental set-up consisted of 14 samples distributed as 6 vehicle replicates and 8 tempol gavage replicates. After quality filtering and deduplication, each sample contai...; ANOVA analysis with factorial treatment design was introduced to detect bacteria that significantly changed in numbers.
from paperReporting output
Report representative outputs alongside summary comparisons for Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gu..., Previous studies demonstrated that alteration of the gut micro-biome affects the levels of bile acids in the liver, heart and kidney. To determine whether the composition of bi..., Bile-acid metabolism is tightly associated with the FXR signalling pathway, and HFD can induce FXR signalling in the intestine. Tempol treatment inhibits expression of the FXR..., To further explore the role of intestinal FXR in lipid and glucose metabolism, male wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice were fed a....
inferred from protocolStructured statistical methods
Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gu...; To further investigate the role of FXR in the tempol-improved metabolic phenotype of HFD-fed mice, wild-type ( Fxr +/+ ) mice and whole-body Fxr -null ( Fxr -/- ) mi...; The experimental set-up consisted of 14 samples distributed as 6 vehicle replicates and 8 tempol gavage replicates. After quality filtering and deduplication, each sample contai...; ANOVA analysis with factorial treatment design was introduced to detect bacteria that significantly changed in numbers.
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gut,. These observations suggested that tempol might affect gut microbial metabolism or the composition of the gut microbiome. Indeed, significant phylum-level shifts from Firmicutes to Bacteroidetes in the gut microbiome composition are observed in mouse caecum following 5 days of tempol administered by gavage (250 mg kg -1 per day) to mice on a normal chow diet ( ). Heat map diagrams of 16S rRNA sequencing results indicate that tempol treatment dramatically decreases the family Lactobacillaceae ( ) and robustly reduces the genus Lactobacillus ( ). Similar to the results of acute treatment via gavage, quantitative real-time PCR (qPCR) analysis of suspected caecum microbes obtained from mice on a HFD reveals that tempol treatment causes a shift from Firmicutes to Bacteroidetes ( ). These results indicate that the effects of tempol on the gut microbiome are independent of diet, delivery method, and obesity. Further, the genus Lactobacillus of the Lactobacillaceae is decreased, coincident with a signi...
Previous studies demonstrated that alteration of the gut micro-biome affects the levels of bile acids in the liver, heart and kidney. To determine whether the composition of bile acids were changed in the tempol group, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-based metabolomics analysis was used to examine the bile-acid composition and their levels in the feces and intestinal tissue. Partial least-squares discriminant analysis (PLS-DA) modelling of the UPLC-ESI-QTOFMS-negative mode data from mouse feces and intestine reveal distinct clustering of the control and tempol groups ( ). Levels of the four ions (m/z, 453.2842 at 3.14min; m/z, 391.2841 at 4.11min; m/z, 407.2782 at 3.33 min; and m/z, 391.2838 at 4.06 min) are significantly reduced in the feces by tempol ( ), whereas four ions (m/z, 514.2864 at 2.53 min; m/z, 514.2862 at 2.91 min; m/z, 498.2910 at 2.85 min; and m/z, 498.2891 at 3.22 min) are markedly enriched in the intestine of tempol-treated mice ( ). Ion identification was performed by tandem mass spectrometry and retention time comparisons with authentic standard...
To further explore the role of intestinal FXR in lipid and glucose metabolism, male wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice were fed a HFD, revealing that the body weights of Fxr ΔIE mice are lower than Fxr fl/fl mice after 3 weeks of treatment ( ). NMR measurements confirm that the fat mass, the ratio of fat and body mass and the ratio of fat and lean mass of Fxr fl/fl mice on a HFD are higher than Fxr ΔIE mice (, ). Food intake measurements are comparable between Fxr fl/fl and Fxr ΔIE mice ( ). GTT reveals that Fxr ΔIE mice have improved glucose intolerance compared with Fxr fl/fl mice ( ) and ITT demonstrates that the insulin sensitivity in Fxr ΔIE mice is significantly increased ( ). Moreover, fasted serum insulin levels and the homoeostasis model assessment index are significantly reduced in Fxr ΔIE mice ( ). The expression of Fxr mRNA is nearly absent in the intestinal mucosa of Fxr ΔIE mice and, accordingly, expression of the FXR target genes Fgf15, organic solute transporter-α and organic solute transporter-β are significantly lower ( ). In particular, the fatty-acid trafficking-rela...
The wild-type ( Fxr +/+ ) mice and whole-body Fxr -null Fxr( -/- ) mice, and, wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice are all on a C57BL/6N genetic background,. Fxr +/+ and Fxr -/- mice were backcrossed with C57BL/6N mice for over four generations. Fxr fl/fl and Fxr ΔIE mice were backcrossed with C57BL/6N mice for over 10 generations. The mice were housed in temperature- and light-controlled rooms and given water and pelleted NIH-31 chow ad libitum. All animal studies were performed in accordance with Institute of Laboratory Animal Resources guidelines and approved by the National Cancer Institute Animal Care and Use Committee. In the tempol study, male mice were fed a HFD (Bio-Serv, Frenchtown, NJ; 60kcal% fat) from 6 weeks of age and administered 0.064% (w/v) tempol in the drinking water. For the chow diet study, tempol (dissolved in 0.9% normal saline) was orally administered to C57BL/6N mice for 5 days. When food and water intakes were measured, the mice were removed from their home cage and transferred to a metabolic cage and housed individually. The mice were adapted to the metabolic cages for 1 day...
For GTT, mice were fasted for 16 h, blood was drawn and mice were injected intraperitoneally with 1 g kg -1 glucose. For ITT, mice were fasted for 4 h, blood was drawn and then injected intraperitoneally with insulin (Eli Lilly, Washington, DC) 1 U kg -1 bodyweight. Blood samples were taken from the tail at 15, 30, 60 and 90 min after injection, and glucose was measured using a glucometer (Bayer, Pittsburgh, PA). Fasted serum insulin was measured using an enzyme-linked immunosorbent assay kit (Crystal Chem Inc., Downers Grove, IL).
The bacteria in feces and caecum content were extracted using PowerSoil DNA Isolation Kit (Mo Bio laboratory, Inc., Carlsbad, CA). The PCR products (~ 1,000 bp) were purified using the AgencourtAMPure technology (Beckman Coulter, Brea, CA) as described in454 Technical Bulletin number 2011-002, Short Fragment Removal Procedure. After purification, the products were quantified by both Qubit (Lifetech, Carlsbad, CA) and qPCR, using the KAPA Biosystems Library Quantification Kit (Kapa-Biosystems, Woburn, MA). Products were pooled based on molar amounts, run ona 1% agarose gel and extracted. After clean-up with a QIAquick PCR Purificationkit (Qiagen, Valencia, CA), quality and quantity were assessed using a DNA 7500LabChip on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Qubit quantification. The sequencing was performed using a quarter Picotiter Plate on a 454 Life Sciences Genome Sequencer FLX + (Roche Diagnostics). qPCR was carried out using SYBR green PCR master mix in an ABI Prism 7900HT sequence detection system (Applied Biosystems). PCR conditions were50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 15S; and 60 °C f...
Full model: (1) Y i j k = u + Dose i + Study j + Error i j k with i = 1, 2, 3; j = 1,2; k = 1, 2, 3, 4
Reduced model: (2) Y i k = u + Dose i + Error i k with i = 1, 2, 3; k = 1, 2, 3, 4
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity methods",
"description": "Evidence-backed execution summary for Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity methods from Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity.",
"totalTime": "PT24106M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Tempol affects gut microbiota and BSH activity.",
"text": "Earlier studies indicated that tempol increased the urinary excretion of 2,8-dihydroxyquinoline and its glucuronide metabolites that are generated by bacteria residing in the gut,. These observations suggested that tempol might affect gut microbial metabolism or the composition of the gut microbiome. Indeed, significant phylum-level shifts from Firmicutes to Bacteroidetes in the gut microbiome composition are observed in mouse caecum following 5 days of tempol administered by gavage (250 mg kg -1 per day) to mice on a normal chow diet ( ). Heat map diagrams of 16S rRNA sequencing results indicate that tempol treatment dramatically decreases the family Lactobacillaceae ( ) and robustly reduces the genus Lactobacillus ( ). Similar to the results of acute treatment via gavage, quantitative real-time PCR (qPCR) analysis of suspected caecum microbes obtained from mice on a HFD reve..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Tempol enhances the intestinal taurine-conjugated bile acids.",
"text": "Previous studies demonstrated that alteration of the gut micro-biome affects the levels of bile acids in the liver, heart and kidney. To determine whether the composition of bile acids were changed in the tempol group, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-based metabolomics analysis was used to examine the bile-acid composition and their levels in the feces and intestinal tissue. Partial least-squares discriminant analysis (PLS-DA) modelling of the UPLC-ESI-QTOFMS-negative mode data from mouse feces and intestine reveal distinct clustering of the control and tempol groups ( ). Levels of the four ions (m/z, 453.2842 at 3.14min; m/z, 391.2841 at 4.11min; m/z, 407.2782 at 3.33 min; and m/z, 391.2838 at 4.06 min) are significantly reduced in the feces by tempol ( ), whereas four ions..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Intestine-specific FXR disruption reduces obesity.",
"text": "To further explore the role of intestinal FXR in lipid and glucose metabolism, male wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice were fed a HFD, revealing that the body weights of Fxr ΔIE mice are lower than Fxr fl/fl mice after 3 weeks of treatment ( ). NMR measurements confirm that the fat mass, the ratio of fat and body mass and the ratio of fat and lean mass of Fxr fl/fl mice on a HFD are higher than Fxr ΔIE mice (, ). Food intake measurements are comparable between Fxr fl/fl and Fxr ΔIE mice ( ). GTT reveals that Fxr ΔIE mice have improved glucose intolerance compared with Fxr fl/fl mice ( ) and ITT demonstrates that the insulin sensitivity in Fxr ΔIE mice is significantly increased ( ). Moreover, fasted serum insulin levels and the homoeostasis model assessment index are significantly reduced in Fxr ΔIE mice ( )..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Animal studies.",
"text": "The wild-type ( Fxr +/+ ) mice and whole-body Fxr -null Fxr( -/- ) mice, and, wild-type (Fxr fl/fl ) mice and intestine-specific Fxr -null (Fxr ΔIE ) mice are all on a C57BL/6N genetic background,. Fxr +/+ and Fxr -/- mice were backcrossed with C57BL/6N mice for over four generations. Fxr fl/fl and Fxr ΔIE mice were backcrossed with C57BL/6N mice for over 10 generations. The mice were housed in temperature- and light-controlled rooms and given water and pelleted NIH-31 chow ad libitum. All animal studies were performed in accordance with Institute of Laboratory Animal Resources guidelines and approved by the National Cancer Institute Animal Care and Use Committee. In the tempol study, male mice were fed a HFD (Bio-Serv, Frenchtown, NJ; 60kcal% fat) from 6 weeks of age and administered 0.064% (w/v) tempol in the drinking water. For the chow diet stu..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Metabolic assays.",
"text": "For GTT, mice were fasted for 16 h, blood was drawn and mice were injected intraperitoneally with 1 g kg -1 glucose. For ITT, mice were fasted for 4 h, blood was drawn and then injected intraperitoneally with insulin (Eli Lilly, Washington, DC) 1 U kg -1 bodyweight. Blood samples were taken from the tail at 15, 30, 60 and 90 min after injection, and glucose was measured using a glucometer (Bayer, Pittsburgh, PA). Fasted serum insulin was measured using an enzyme-linked immunosorbent assay kit (Crystal Chem Inc., Downers Grove, IL)."
},
{
"@type": "HowToStep",
"position": 6,
"name": "16S rRNA gene sequencing of the intestinal microbiome.",
"text": "The bacteria in feces and caecum content were extracted using PowerSoil DNA Isolation Kit (Mo Bio laboratory, Inc., Carlsbad, CA). The PCR products (~ 1,000 bp) were purified using the AgencourtAMPure technology (Beckman Coulter, Brea, CA) as described in454 Technical Bulletin number 2011-002, Short Fragment Removal Procedure. After purification, the products were quantified by both Qubit (Lifetech, Carlsbad, CA) and qPCR, using the KAPA Biosystems Library Quantification Kit (Kapa-Biosystems, Woburn, MA). Products were pooled based on molar amounts, run ona 1% agarose gel and extracted. After clean-up with a QIAquick PCR Purificationkit (Qiagen, Valencia, CA), quality and quantity were assessed using a DNA 7500LabChip on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Qubit quantification. The sequencing was performed using a quarter Picotiter Plate on a..."
},
{
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"name": "Metagenomic data analysis.",
"text": "Full model: (1) Y i j k = u + Dose i + Study j + Error i j k with i = 1, 2, 3; j = 1,2; k = 1, 2, 3, 4"
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"text": "Reduced model: (2) Y i k = u + Dose i + Error i k with i = 1, 2, 3; k = 1, 2, 3, 4"
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"name": "Tempol affects gut microbiota and BSH activity."
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"name": "RNA analysis."
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{
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"name": "16S rRNA gene sequencing of the intestinal microbiome."
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{
"@type": "HowToTool",
"name": "Metagenomic data analysis."
},
{
"@type": "HowToTool",
"name": "Metagenomic data analysis."
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{
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"name": "Intestine-specific FXR disruption reduces obesity."
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{
"@type": "HowToSupply",
"name": "Intestine-specific FXR disruption reduces obesity."
},
{
"@type": "HowToSupply",
"name": "Tempol improves obesity via inhibition of intestinal FXR."
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"name": "Methods"
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{
"@type": "HowToSupply",
"name": "Preparation and culture of primary hepatocytes."
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"@type": "HowToSupply",
"name": "RNA analysis."
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"name": "Luciferase assays."
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{
"@type": "HowToSupply",
"name": "Metabolic assays."
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"headline": "Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity",
"datePublished": "2013",
"author": [
{
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"name": "Fei Li"
},
{
"@type": "Person",
"name": "Changtao Jiang"
},
{
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{
"@type": "Person",
"name": "Yunfei Li"
},
{
"@type": "Person",
"name": "Istvan Albert"
},
{
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"name": "Haiping Hao"
},
{
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"name": "Kristin M. Fabre"
},
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{
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"identifier": "10.1038/ncomms3384"
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