Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity methods
Aim. Evidence-backed execution summary for Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity methods from Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity.
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Fecal pellet collection and DNA extraction
reagent used in the protocol.
- Use
- Extraction of DNA from fecal bacteria followed the protocol of Wang et al.. Fecal pellets were obtained from all mice at the start (prior to beginning the protocol, week 0), mid-point (week 6), and at week 12 of the diet and Ex protocol. Fecal pellets were collected directly from the mice and stored at -80...
Terminal Restriction Fragment Length Polymorphism
reagent used in the protocol.
- Use
- The T-RFLP analysis was performed as previously described. Briefly, the 16S rRNA encoding gene was amplified from fecal sample DNA using a broad-range universal primer set: 8F ( 5′-AGAGTT TGATCCTGGCTCAG-3′ ) with a 6′ carboxyfluorescein (6-FAM) label and 1492R ( 5′-GGTTACCTTGTTACGACTT-3̸...
Terminal Restriction Fragment Length Polymorphism
reagent used in the protocol.
- Use
- Purified 16S rDNA was digested using 20 U Msp I enzyme (New England Biolabs Inc.) in a total volume of 15 µl (including 1.5 µl NE Buffer 2, 1.5 µl enzyme, and 12 µl PCR product) at 37°C for 3 hours. To enhance the fluorescent signal, each digested T-RFLP sample was desalted by single drop an...
Quantitative PCR
reagent used in the protocol.
- Use
- The two gut bacterial phyla of interest, Bacteroidetes and Firmicutes, were targeted by modifying a quantitative PCR assay developed by Armougom et al. using primer and probe sets that recognized a conserved region of the 16S rRNA encoding gene unique to these taxonomic groups. This assay was tested by Armougom et...
Quantitative PCR
reagent used in the protocol.
- Use
- The assay was performed on the ABI-PRISM 7700 Sequence Detection System (Applied Biosystems, Life Technologies, Grand Island, NY). The methods on which this assay was based have been published previously,, but briefly the conditions for quantitative amplification for all qPCR runs were as follows: pre-amplificatio...
Sequencing
reagent used in the protocol.
- Use
- Microbial sequencing was performed on the MiSeq Illumina platform (Argonne National Laboratory, Institute for Genomics and Systems Biology, Next Generation Sequencing Core) as previously described by Caporaso et al.,. Briefly, the V4 region of 16S bacterial rDNA was amplified using the custom degenerate primer pai...
Quantitative polymerase chain reaction (q-PCR)
reagent used in the protocol.
- Use
- Quantitative PCR analysis of isolated fecal 16 s rRNA gene demonstrated no significant Ex effect on the content of the two major bacterial phyla, Bacteroidetes and Firmicutes, as demonstrated by the "fold change" in LF/Ex compared to LF/Sed and HF/Ex compared to HF/Sed using the 2 -ΔΔCt v...
Supporting Information
reagent used in the protocol.
- Use
- Identification of Amplicon Size for Bacteroidetes, Firmicutes and the Universal Primer Sets.
Methods
Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and use...
- Use
- Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and use...
Diet and activity protocol
Littermates were separated into groups as follows: LF/Sed, n = 12, LF/Ex, n = 10, HF/Sed, n = 14, and HF/Ex, n = 12. Two separate batches of mice were used: an initial batch of mice were studied for preliminary indications of a global microbiota shift (terminal-restric...
- Use
- Littermates were separated into groups as follows: LF/Sed, n = 12, LF/Ex, n = 10, HF/Sed, n = 14, and HF/Ex, n = 12. Two separate batches of mice were used: an initial batch of mice were studied for preliminary indications of a global microbiota shift (terminal-restric...
Terminal Restriction Fragment Length Polymorphism
Purified 16S rDNA was digested using 20 U Msp I enzyme (New England Biolabs Inc.) in a total volume of 15 µl (including 1.5 µl NE Buffer 2, 1.5 µl enzyme, and 12 µl PCR product) at 37°C for 3 hours. To enhance the fluorescent signal, each digested T-RFLP sample was desalted by single drop an...
- Use
- Purified 16S rDNA was digested using 20 U Msp I enzyme (New England Biolabs Inc.) in a total volume of 15 µl (including 1.5 µl NE Buffer 2, 1.5 µl enzyme, and 12 µl PCR product) at 37°C for 3 hours. To enhance the fluorescent signal, each digested T-RFLP sample was desalted by single drop an...
Quantitative PCR
The assay was performed on the ABI-PRISM 7700 Sequence Detection System (Applied Biosystems, Life Technologies, Grand Island, NY). The methods on which this assay was based have been published previously,, but briefly the conditions for quantitative amplification for all qPCR runs were as follows: pre-amplificatio...
- Use
- The assay was performed on the ABI-PRISM 7700 Sequence Detection System (Applied Biosystems, Life Technologies, Grand Island, NY). The methods on which this assay was based have been published previously,, but briefly the conditions for quantitative amplification for all qPCR runs were as follows: pre-amplificatio...
Sequencing
Microbial sequencing was performed on the MiSeq Illumina platform (Argonne National Laboratory, Institute for Genomics and Systems Biology, Next Generation Sequencing Core) as previously described by Caporaso et al.,. Briefly, the V4 region of 16S bacterial rDNA was amplified using the custom degenerate primer pai...
- Use
- Microbial sequencing was performed on the MiSeq Illumina platform (Argonne National Laboratory, Institute for Genomics and Systems Biology, Next Generation Sequencing Core) as previously described by Caporaso et al.,. Briefly, the V4 region of 16S bacterial rDNA was amplified using the custom degenerate primer pai...
Bioinformatics
Bioinformatic analysis of sequencing data was conducted using the QIIME 1.5.0 software suite. Reads from all samples were clustered at 97% sequence identity into operational taxonomic units (OTUs) then aligned to the October 12th, 2012 Greengenes bacterial reference tree. Following removal of OTUs composed of only...
- Use
- Bioinformatic analysis of sequencing data was conducted using the QIIME 1.5.0 software suite. Reads from all samples were clustered at 97% sequence identity into operational taxonomic units (OTUs) then aligned to the October 12th, 2012 Greengenes bacterial reference tree. Following removal of OTUs composed of only...
Statistical Analysis
Quantitative data is represented as the mean ± SEM, unless otherwise indicated. Differences between treatment groups were compared using an independent t-test (for running distance), a Pearson's r for correlation, a one-way ANOVA for qPCR data (on the ΔCt values) and a two-way ANOVA for all other data comp...
- Use
- Quantitative data is represented as the mean ± SEM, unless otherwise indicated. Differences between treatment groups were compared using an independent t-test (for running distance), a Pearson's r for correlation, a one-way ANOVA for qPCR data (on the ΔCt values) and a two-way ANOVA for all other data comp...
Bacterial 16S rRNA Amplicon Sequencing
Principal coordinate analysis (PCA) was performed based on the weighted UniFrac distance matrix generated from sequencing fecal 16S rRNA gene in samples from mice at week 0 and 12 of the diet and activity protocol. A. Clustering demonstrated by litter at week 0. B. No clustering demonstrated by litter at week 12. C....
- Use
- Principal coordinate analysis (PCA) was performed based on the weighted UniFrac distance matrix generated from sequencing fecal 16S rRNA gene in samples from mice at week 0 and 12 of the diet and activity protocol. A. Clustering demonstrated by litter at week 0. B. No clustering demonstrated by litter at week 12. C....
Bioinformatics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Bioinformatic analysis of sequencing data was conducted using the QIIME 1.5.0 software suite. Reads from all samples were clustered at 97% sequence identity into operational taxonomic units (OTUs) then aligned to the October 12th, 2012 Greengenes bacterial reference tree. Following removal of OTUs composed of only...
Statistical Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Quantitative data is represented as the mean ± SEM, unless otherwise indicated. Differences between treatment groups were compared using an independent t-test (for running distance), a Pearson's r for correlation, a one-way ANOVA for qPCR data (on the ΔCt values) and a two-way ANOVA for all other data comp...
Before you run
What should be confirmed before execution?
First confirmation
Equipment is listed but no product mappings are linked.
Confirm before execution
This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Methods
Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation.
Methods
All experimental protocols were approved by the Midwestern University Institutional Animal Care and Use Committee (IACUC) and all procedures were performed in accordance with Midwestern University's guidelines for the care and use of laboratory animals. Five week old, male C57BL/6 mice (n = 48) (Jackson Laboratories, Bar Harbor, ME, USA) were received as littermates (15-19 g) and allowed to acclimate in Midwestern University's animal facility for 1 week prior to their random assignment to one of 4 diet and activity treatment groups. During the acclimation period, mice were kept in their respective litters and provided standard chow (composition: 18 kcal% fat, 58 kcal% carbohydrate, 24 kcal% protein; 3.1 kcal/g total energy content, Teklad Diets, Madison WI) and reverse osmosis water ad libitum.
Diet and activity protocol
Following the acclimation phase, mice were randomly assigned to diet and activity groups based on equal distribution of littermates (i.e. mice in each litter were distributed as equally as possible among the 4 groups). Dietary intake included either high fat diet (HF; composition 60 kcal% fat, 20 kcal% carbohydrates, 20 kcal% protein; 5.24 kcal/g total energy content) or low fat diet (LF; composition 10 kcal% fat, 70 kcal% carbohydrates, 20 kcal% protein; 3.85 kcal/g of food, Research Diets, Inc.; New Brunswick, NJ). Mice were individually housed in rat cages in the absence (sedentary; Sed) or presence (exercise; Ex) of a non-load bearing 14.6 cm hamster wheel (PetSmart, Phoenix, AZ) suspended from a metal rod based on a design reported by Konhilas et al.. The cages were modified to record wheel movement 24 h/day, 7 days/week, using Schwinn 17-function odometers (Schwinn, Madison, WI...
Diet and activity protocol
Littermates were separated into groups as follows: LF/Sed, n = 12, LF/Ex, n = 10, HF/Sed, n = 14, and HF/Ex, n = 12. Two separate batches of mice were used: an initial batch of mice were studied for preliminary indications of a global microbiota shift (terminal-restriction fragment length polymorphism (T-RFLP)) and measurement of gut transit (N = 24: LF/Sed, n = 6, LF/Ex, n = 4, HF/Sed, n = 8, and HF/Ex, n = 6) and a second batch for quantitative PCR (qPCR) and sequencing studies (N = 24; n = 6 all groups). All mice were followed on the protocol for 12 weeks for collection of fecal samples and then an additional two weeks for collection of blood samples and harvesting of tissues.
Weight and food intake measurements
Initial, weekly and final body weights were determined for all mice. Twenty-four hour food intake was measured at week 12 of the experimental period. For these measurements, mice were placed in cages without bedding and each mouse was given one pre-weighed pellet of their respective diet, either LF or HF food, in a ceramic dish designed to minimize food loss or contamination by feces. After 24 hours, the pellet and its remnants were weighed to determine food consumption. Food intake was expressed as kcal/24 hours.
Blood glucose measurements
Fasting blood glucose (FBG) and oral glucose tolerance (OGT) were measured at 13 weeks on the diet and activity program. Mice were fasted overnight in cages without bedding prior to measuring tail vein blood glucose levels using a OneTouch UltraMini® monitor (LifeScan, Inc. Milpitas, CA). Oral glucose tolerance was measured immediately following the FBG test. Mice were gavaged with 25% glucose in saline (1 g/kg body weight) followed by blood glucose measurements at 15, 30, 60 and 120 minutes post glucose challenge using the tail vein with a lancet stick according to the method of Golde et al..
Intestinal transit
Measurement of intestinal transit was performed as a terminal experiment. Mice were fasted for 16 h with water available ad libitum. At 30 minutes prior to euthanasia, 0.1 ml of 0.5 mmol/L 70 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich, St. Louis, MO) was administered by oral gavage,. After 30 minutes, mice were euthanized and the entire gastrointestinal tract, from stomach to anus, was removed. The small intestines were cut into 8 segments of equal length from the stomach to the cecum. The stomach and each segment were flushed with 3 ml of 50 mmol/l Tris buffered saline, the effluent was collected and centrifuged at 1200 rpm for 5 minutes to pellet any suspended debris. Fluorescent intensity in each sample was measured using a fluorescence spectrophotometer (emission wavelength = 518 nm; excitation wavelength = 492 nm; F-2000, Hitac...
Fecal pellet collection and DNA extraction
Extraction of DNA from fecal bacteria followed the protocol of Wang et al.. Fecal pellets were obtained from all mice at the start (prior to beginning the protocol, week 0), mid-point (week 6), and at week 12 of the diet and Ex protocol. Fecal pellets were collected directly from the mice and stored at -80°C prior to lysis in 1 ml extraction buffer [50 mM Tris (pH 7.4), 100 mM EDTA (pH 8.0), 400 mM NaCl, 0.5% SDS] containing 20 µl proteinase K (20 mg/ml). Bacterial disruption was achieved using 0.1-mm-diameter zirconia/silica beads (BioSpec Products, Bartlesville, OK) and a Mini-Beadbeater-8 k Cell Disrupter (BioSpec Products). The homogenized samples were incubated overnight at 55°C with shaking. The beads were then spun down by centrifugation and 200 µl of room temperature saturated NaCl (175.3 g/500 ml) was added to 500 µl of the supernatant. After 2...
Measurement outputs
What raw and processed outputs should exist?
Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex...
- 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
Following the acclimation phase, mice were randomly assigned to diet and activity groups based on equal distribution of littermates (i.e. mice in each litter were distributed as...
- 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
Littermates were separated into groups as follows: LF/Sed, n = 12, LF/Ex, n = 10, HF/Sed, n = 14, and HF/Ex, n = 12. Two separate...
- 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
The T-RFLP analysis was performed as previously described. Briefly, the 16S rRNA encoding gene was amplified from fecal sample DNA using a broad-range universal primer set: 8F...
- 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
Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex...; Following the acclimation phase, mice were randomly assigned to diet and activity groups based on equal distribution of littermates (i.e. mice in each litter were distributed as...; Littermates were separated into groups as follows: LF/Sed, n = 12, LF/Ex, n = 10, HF/Sed, n = 14, and HF/Ex, n = 12. Two separate...; The T-RFLP analysis was performed as previously described. Briefly, the 16S rRNA encoding gene was amplified from fecal sample DNA using a broad-range universal primer set: 8F....
from paperStatistical comparison
Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex...; Bioinformatic analysis of sequencing data was conducted using the QIIME 1.5.0 software suite. Reads from all samples were clustered at 97% sequence identity into operational ta...; Quantitative data is represented as the mean ± SEM, unless otherwise indicated. Differences between treatment groups were compared using an independent t-test (for running...; demonstrates the weekly body weight for each group. Mice were 5 weeks old at the start of the diet and Ex protocol (Time 0; ), with comparable starting body weights ( ). By week...
from paperReporting output
Report representative outputs alongside summary comparisons for Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex..., Following the acclimation phase, mice were randomly assigned to diet and activity groups based on equal distribution of littermates (i.e. mice in each litter were distributed as..., Littermates were separated into groups as follows: LF/Sed, n = 12, LF/Ex, n = 10, HF/Sed, n = 14, and HF/Ex, n = 12. Two separate..., The T-RFLP analysis was performed as previously described. Briefly, the 16S rRNA encoding gene was amplified from fecal sample DNA using a broad-range universal primer set: 8F....
inferred from protocolStructured statistical methods
Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex...; Bioinformatic analysis of sequencing data was conducted using the QIIME 1.5.0 software suite. Reads from all samples were clustered at 97% sequence identity into operational ta...; Quantitative data is represented as the mean ± SEM, unless otherwise indicated. Differences between treatment groups were compared using an independent t-test (for running...; demonstrates the weekly body weight for each group. Mice were 5 weeks old at the start of the diet and Ex protocol (Time 0; ), with comparable starting body weights ( ). By week...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation.
All experimental protocols were approved by the Midwestern University Institutional Animal Care and Use Committee (IACUC) and all procedures were performed in accordance with Midwestern University's guidelines for the care and use of laboratory animals. Five week old, male C57BL/6 mice (n = 48) (Jackson Laboratories, Bar Harbor, ME, USA) were received as littermates (15-19 g) and allowed to acclimate in Midwestern University's animal facility for 1 week prior to their random assignment to one of 4 diet and activity treatment groups. During the acclimation period, mice were kept in their respective litters and provided standard chow (composition: 18 kcal% fat, 58 kcal% carbohydrate, 24 kcal% protein; 3.1 kcal/g total energy content, Teklad Diets, Madison WI) and reverse osmosis water ad libitum.
Following the acclimation phase, mice were randomly assigned to diet and activity groups based on equal distribution of littermates (i.e. mice in each litter were distributed as equally as possible among the 4 groups). Dietary intake included either high fat diet (HF; composition 60 kcal% fat, 20 kcal% carbohydrates, 20 kcal% protein; 5.24 kcal/g total energy content) or low fat diet (LF; composition 10 kcal% fat, 70 kcal% carbohydrates, 20 kcal% protein; 3.85 kcal/g of food, Research Diets, Inc.; New Brunswick, NJ). Mice were individually housed in rat cages in the absence (sedentary; Sed) or presence (exercise; Ex) of a non-load bearing 14.6 cm hamster wheel (PetSmart, Phoenix, AZ) suspended from a metal rod based on a design reported by Konhilas et al.. The cages were modified to record wheel movement 24 h/day, 7 days/week, using Schwinn 17-function odometers (Schwinn, Madison, WI.). The odometers were hand-tested for reliability of time of movement and distance covered. Exercise was recorded continuously (kilometers) and the cages were visually checked at least 3 times/week to ensure the odometers were working correctly and the wheels were functioning properly. Mice were ob...
Littermates were separated into groups as follows: LF/Sed, n = 12, LF/Ex, n = 10, HF/Sed, n = 14, and HF/Ex, n = 12. Two separate batches of mice were used: an initial batch of mice were studied for preliminary indications of a global microbiota shift (terminal-restriction fragment length polymorphism (T-RFLP)) and measurement of gut transit (N = 24: LF/Sed, n = 6, LF/Ex, n = 4, HF/Sed, n = 8, and HF/Ex, n = 6) and a second batch for quantitative PCR (qPCR) and sequencing studies (N = 24; n = 6 all groups). All mice were followed on the protocol for 12 weeks for collection of fecal samples and then an additional two weeks for collection of blood samples and harvesting of tissues.
Initial, weekly and final body weights were determined for all mice. Twenty-four hour food intake was measured at week 12 of the experimental period. For these measurements, mice were placed in cages without bedding and each mouse was given one pre-weighed pellet of their respective diet, either LF or HF food, in a ceramic dish designed to minimize food loss or contamination by feces. After 24 hours, the pellet and its remnants were weighed to determine food consumption. Food intake was expressed as kcal/24 hours.
Fasting blood glucose (FBG) and oral glucose tolerance (OGT) were measured at 13 weeks on the diet and activity program. Mice were fasted overnight in cages without bedding prior to measuring tail vein blood glucose levels using a OneTouch UltraMini® monitor (LifeScan, Inc. Milpitas, CA). Oral glucose tolerance was measured immediately following the FBG test. Mice were gavaged with 25% glucose in saline (1 g/kg body weight) followed by blood glucose measurements at 15, 30, 60 and 120 minutes post glucose challenge using the tail vein with a lancet stick according to the method of Golde et al..
Measurement of intestinal transit was performed as a terminal experiment. Mice were fasted for 16 h with water available ad libitum. At 30 minutes prior to euthanasia, 0.1 ml of 0.5 mmol/L 70 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich, St. Louis, MO) was administered by oral gavage,. After 30 minutes, mice were euthanized and the entire gastrointestinal tract, from stomach to anus, was removed. The small intestines were cut into 8 segments of equal length from the stomach to the cecum. The stomach and each segment were flushed with 3 ml of 50 mmol/l Tris buffered saline, the effluent was collected and centrifuged at 1200 rpm for 5 minutes to pellet any suspended debris. Fluorescent intensity in each sample was measured using a fluorescence spectrophotometer (emission wavelength = 518 nm; excitation wavelength = 492 nm; F-2000, Hitachi, Inc., Japan). Intestinal geometric center (IGC) was calculated as: (fraction of amount of FITC in each segment) × (segment number). The dextran concentration in each segment was expressed as a fraction of total dextran recovery.
Extraction of DNA from fecal bacteria followed the protocol of Wang et al.. Fecal pellets were obtained from all mice at the start (prior to beginning the protocol, week 0), mid-point (week 6), and at week 12 of the diet and Ex protocol. Fecal pellets were collected directly from the mice and stored at -80°C prior to lysis in 1 ml extraction buffer [50 mM Tris (pH 7.4), 100 mM EDTA (pH 8.0), 400 mM NaCl, 0.5% SDS] containing 20 µl proteinase K (20 mg/ml). Bacterial disruption was achieved using 0.1-mm-diameter zirconia/silica beads (BioSpec Products, Bartlesville, OK) and a Mini-Beadbeater-8 k Cell Disrupter (BioSpec Products). The homogenized samples were incubated overnight at 55°C with shaking. The beads were then spun down by centrifugation and 200 µl of room temperature saturated NaCl (175.3 g/500 ml) was added to 500 µl of the supernatant. After 2 minutes of vigorous shaking, the samples were spun for 15 minutes to pellet the protein and debris. Five hundred µl of phenol:chloroform:isoamylalcohol (25∶24∶1;4°C; Ambion) was added to 500 µl of the supernatant. Following shaking and centrifugation, 500 µl of the t...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity methods",
"description": "Evidence-backed execution summary for Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity methods from Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity.",
"totalTime": "PT79232M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Methods",
"text": "Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Methods",
"text": "All experimental protocols were approved by the Midwestern University Institutional Animal Care and Use Committee (IACUC) and all procedures were performed in accordance with Midwestern University's guidelines for the care and use of laboratory animals. Five week old, male C57BL/6 mice (n = 48) (Jackson Laboratories, Bar Harbor, ME, USA) were received as littermates (15-19 g) and allowed to acclimate in Midwestern University's animal facility for 1 week prior to their random assignment to one of 4 diet and activity treatment groups. During the acclimation period, mice were kept in their respective litters and provided standard chow (composition: 18 kcal% fat, 58 kcal% carbohydrate, 24 kcal% protein; 3.1 kcal/g total energy content, Teklad Diets, Madison WI) and reverse osmosis water ad libitum."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Diet and activity protocol",
"text": "Following the acclimation phase, mice were randomly assigned to diet and activity groups based on equal distribution of littermates (i.e. mice in each litter were distributed as equally as possible among the 4 groups). Dietary intake included either high fat diet (HF; composition 60 kcal% fat, 20 kcal% carbohydrates, 20 kcal% protein; 5.24 kcal/g total energy content) or low fat diet (LF; composition 10 kcal% fat, 70 kcal% carbohydrates, 20 kcal% protein; 3.85 kcal/g of food, Research Diets, Inc.; New Brunswick, NJ). Mice were individually housed in rat cages in the absence (sedentary; Sed) or presence (exercise; Ex) of a non-load bearing 14.6 cm hamster wheel (PetSmart, Phoenix, AZ) suspended from a metal rod based on a design reported by Konhilas et al.. The cages were modified to record wheel movement 24 h/day, 7 days/week, using Schwinn 17-function odometers (Schwinn, Madison, WI..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Diet and activity protocol",
"text": "Littermates were separated into groups as follows: LF/Sed, n = 12, LF/Ex, n = 10, HF/Sed, n = 14, and HF/Ex, n = 12. Two separate batches of mice were used: an initial batch of mice were studied for preliminary indications of a global microbiota shift (terminal-restriction fragment length polymorphism (T-RFLP)) and measurement of gut transit (N = 24: LF/Sed, n = 6, LF/Ex, n = 4, HF/Sed, n = 8, and HF/Ex, n = 6) and a second batch for quantitative PCR (qPCR) and sequencing studies (N = 24; n = 6 all groups). All mice were followed on the protocol for 12 weeks for collection of fecal samples and then an additional two weeks for collection of blood samples and harvesting of tissues."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Weight and food intake measurements",
"text": "Initial, weekly and final body weights were determined for all mice. Twenty-four hour food intake was measured at week 12 of the experimental period. For these measurements, mice were placed in cages without bedding and each mouse was given one pre-weighed pellet of their respective diet, either LF or HF food, in a ceramic dish designed to minimize food loss or contamination by feces. After 24 hours, the pellet and its remnants were weighed to determine food consumption. Food intake was expressed as kcal/24 hours."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Blood glucose measurements",
"text": "Fasting blood glucose (FBG) and oral glucose tolerance (OGT) were measured at 13 weeks on the diet and activity program. Mice were fasted overnight in cages without bedding prior to measuring tail vein blood glucose levels using a OneTouch UltraMini® monitor (LifeScan, Inc. Milpitas, CA). Oral glucose tolerance was measured immediately following the FBG test. Mice were gavaged with 25% glucose in saline (1 g/kg body weight) followed by blood glucose measurements at 15, 30, 60 and 120 minutes post glucose challenge using the tail vein with a lancet stick according to the method of Golde et al.."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Intestinal transit",
"text": "Measurement of intestinal transit was performed as a terminal experiment. Mice were fasted for 16 h with water available ad libitum. At 30 minutes prior to euthanasia, 0.1 ml of 0.5 mmol/L 70 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich, St. Louis, MO) was administered by oral gavage,. After 30 minutes, mice were euthanized and the entire gastrointestinal tract, from stomach to anus, was removed. The small intestines were cut into 8 segments of equal length from the stomach to the cecum. The stomach and each segment were flushed with 3 ml of 50 mmol/l Tris buffered saline, the effluent was collected and centrifuged at 1200 rpm for 5 minutes to pellet any suspended debris. Fluorescent intensity in each sample was measured using a fluorescence spectrophotometer (emission wavelength = 518 nm; excitation wavelength = 492 nm; F-2000, Hitac..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Fecal pellet collection and DNA extraction",
"text": "Extraction of DNA from fecal bacteria followed the protocol of Wang et al.. Fecal pellets were obtained from all mice at the start (prior to beginning the protocol, week 0), mid-point (week 6), and at week 12 of the diet and Ex protocol. Fecal pellets were collected directly from the mice and stored at -80°C prior to lysis in 1 ml extraction buffer [50 mM Tris (pH 7.4), 100 mM EDTA (pH 8.0), 400 mM NaCl, 0.5% SDS] containing 20 µl proteinase K (20 mg/ml). Bacterial disruption was achieved using 0.1-mm-diameter zirconia/silica beads (BioSpec Products, Bartlesville, OK) and a Mini-Beadbeater-8 k Cell Disrupter (BioSpec Products). The homogenized samples were incubated overnight at 55°C with shaking. The beads were then spun down by centrifugation and 200 µl of room temperature saturated NaCl (175.3 g/500 ml) was added to 500 µl of the supernatant. After 2..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Methods"
},
{
"@type": "HowToTool",
"name": "Diet and activity protocol"
},
{
"@type": "HowToTool",
"name": "Terminal Restriction Fragment Length Polymorphism"
},
{
"@type": "HowToTool",
"name": "Quantitative PCR"
},
{
"@type": "HowToTool",
"name": "Sequencing"
},
{
"@type": "HowToTool",
"name": "Bioinformatics"
},
{
"@type": "HowToTool",
"name": "Statistical Analysis"
},
{
"@type": "HowToTool",
"name": "Bacterial 16S rRNA Amplicon Sequencing"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Fecal pellet collection and DNA extraction"
},
{
"@type": "HowToSupply",
"name": "Terminal Restriction Fragment Length Polymorphism"
},
{
"@type": "HowToSupply",
"name": "Terminal Restriction Fragment Length Polymorphism"
},
{
"@type": "HowToSupply",
"name": "Quantitative PCR"
},
{
"@type": "HowToSupply",
"name": "Quantitative PCR"
},
{
"@type": "HowToSupply",
"name": "Sequencing"
},
{
"@type": "HowToSupply",
"name": "Quantitative polymerase chain reaction (q-PCR)"
},
{
"@type": "HowToSupply",
"name": "Supporting Information"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity",
"datePublished": "2014",
"author": [
{
"@type": "Person",
"name": "Christian C. Evans"
},
{
"@type": "Person",
"name": "Kathy J. LePard"
},
{
"@type": "Person",
"name": "Jeff W. Kwak"
},
{
"@type": "Person",
"name": "Mary C. Stancukas"
},
{
"@type": "Person",
"name": "Samantha Laskowski"
},
{
"@type": "Person",
"name": "Joseph Dougherty"
},
{
"@type": "Person",
"name": "Laura Moulton"
},
{
"@type": "Person",
"name": "Adam Glawe"
},
{
"@type": "Person",
"name": "Yunwei Wang"
},
{
"@type": "Person",
"name": "Vanessa Leone"
},
{
"@type": "Person",
"name": "Dionysios A. Antonopoulos"
},
{
"@type": "Person",
"name": "Dan Smith"
},
{
"@type": "Person",
"name": "Eugene B. Chang"
},
{
"@type": "Person",
"name": "Mae J. Ciancio"
}
],
"identifier": "10.1371/journal.pone.0092193"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity methods",
"item": "https://replicatescience.com/experiments/exercise-prevents-weight-gain-and-alters-the-gut-microbiota-in-a-mouse-model-of-high-fat-diet-induced-obesity-methods-christian-c-evans-pmc3966766/exercise-prevents-weight-gain-and-alters-the-gut-microbiota-in-a-mouse-model-of-high-fat-diet-induce-mlph7yji"
}
]
}
]