Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity methods
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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
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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
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