Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity methods
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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...
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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...
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