RESEARCH ARTICLE

The Effect of Diet and Exercise on Intestinal

Integrity and Microbial Diversity in Mice
Sara C. Campbell1☯*, Paul J. Wisniewski1‡, Michael Noji1‡, Lora R. McGuinness2‡, Max
M. Häggblom3☯, Stanley A. Lightfoot4‡, Laurie B. Joseph5☯, Lee J. Kerkhof2☯
1 Department of Exercise Science and Sports Studies, Rutgers University, New Brunswick, NJ 08901,
United States of America, 2 Department of Marine and Coastal Sciences, Rutgers University, New
Brunswick, NJ 08901, United States of America, 3 Department of Biochemistry and Microbiology, Rutgers
University, New Brunswick, NJ 08901, United States of America, 4 Chief Pathologist, Oklahoma City, OK,
United States of America, 5 Department of Pharmacology and Toxicology, Rutgers University, New
a11111 Brunswick, NJ 08901, United States of America

☯ These authors contributed equally to this work.

‡ These authors also contributed equally to this work.
* saracamp@[Link]

OPEN ACCESS
Abstract
Citation: Campbell SC, Wisniewski PJ, Noji M,
McGuinness LR, Häggblom MM, Lightfoot SA, et al.
(2016) The Effect of Diet and Exercise on Intestinal Background
Integrity and Microbial Diversity in Mice. PLoS ONE
11(3): e0150502. doi:10.1371/[Link].0150502 The gut microbiota is now known to play an important role contributing to inflammatory-
based chronic diseases. This study examined intestinal integrity/inflammation and the gut
Editor: François Blachier, National Institute of
Agronomic Research, FRANCE microbial communities in sedentary and exercising mice presented with a normal or high-fat
diet.
Received: September 23, 2015

Accepted: February 15, 2016

Published: March 8, 2016 Methods

Copyright: © 2016 Campbell et al. This is an open Thirty-six, 6-week old C57BL/6NTac male mice were fed a normal or high-fat diet for 12-
access article distributed under the terms of the weeks and randomly assigned to exercise or sedentary groups. After 12 weeks animals
Creative Commons Attribution License, which permits
were sacrificed and duodenum/ileum tissues were fixed for immunohistochemistry for
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are occludin, E-cadherin, and cyclooxygenase-2 (COX-2). The bacterial communities were
credited. assayed in fecal samples using terminal restriction fragment length polymorphism (TRFLP)
Data Availability Statement: All relevant data are analysis and pyrosequencing of 16S rRNA gene amplicons.
within the paper and its Supporting Information files.
GenBank KU644595-KU644645, 454 data we will be
depositing the sequences in BioProject # PRJNA Results
309613.
Lean sedentary (LS) mice presented normal histologic villi while obese sedentary (OS)
Funding: This work was also supported in part by
the National Institutes of Health CounterACT
mice had similar villi height with more than twice the width of the LS animals. Both lean (LX)
Program through the National Institute of Arthritis and and obese exercise (OX) mice duodenum and ileum were histologically normal. COX-2
Musculoskeletal and Skin Diseases (award expression was the greatest in the OS group, followed by LS, LX and OX. The TRFLP and
U54AR055073). This funding only supported
pyrosequencing indicated that members of the Clostridiales order were predominant in all
histochemistry. The funders had no role in study
design, data collection and analysis, decision to diet groups. Specific phylotypes were observed with exercise, including Faecalibacterium
publish, or preparation of the manuscript. Additional prausnitzi, Clostridium spp., and Allobaculum spp.

PLOS ONE | DOI:10.1371/[Link].0150502 March 8, 2016 1 / 17

 Exercise Enhances Intestinal Health

funds for these experiments came from start-up funds Conclusion

given to SCC. Undergraduate research funds
awarded to PJW, MN. These data suggest that exercise has a strong influence on gut integrity and host micro-
biome which points to the necessity for more mechanistic studies of the interactions
Competing Interests: The authors have declared
that no competing interests exist. between specific bacteria in the gut and its host.

Introduction
Microbiome dysbiosis has proven to be a major contributor to chronic gut inflammatory dis-
eases, like Irritable Bowel Syndrome (IBS) and ulcerative colitis [1]. Interestingly, exercise has
been observed to improve the quality of life for IBS patients, but generally the influence of exer-
cise on intestinal health is poorly understood [2,3]. Other groups have explored the link
between diet and dysbiosis but there is limited evidence on the combined interaction of gut
microbiome, diet and exercise [4,5]. This work has sought to explore the symbiotic relationship
between host behavior, through diet and exercise, by investigating the histopathological alter-
ations in the gut through specific biomarkers of inflammation, gut integrity and gut microbial
ecology.
The human and animal gut microbiome has received widespread attention due its role in
energy harvesting and contributing to chronic systemic low grade inflammation [6–8]. The del-
icate balance between members of the phyla Firmicutes and Bacteroidetes are important in
determining the metabolic phenotype of the host [6, 9–11], This has been shown in animal
studies where high-fat diets alter the relative abundance of Firmicutes and Bacteroidetes [6,8,
11–13]. Recent studies suggest that the delicate balance between key opportunistic pathogens,
e.g. Enterobacter spp. [14] and favorable bacteria, e.g. Akkermansia muciniphilia [5,15] is espe-
cially critical for homeostasis.
Obesity has risen dramatically throughout the world and is often associated with inflamma-
tion, comorbidities and disability [16,17]. The term “leaky gut” was coined by Gummesson,
who related lower gastrointestinal (GI) leakiness and adiposity, which in turn produces low
grade systemic inflammation resulting from metabolic endotoxemia [18], while Cani observed
that leaky gut can also be associated with diabetes and obesity [12]. Leaky gut results from dys-
biosis in the gut microbiota. Dysbiois promotes the release of endotoxin and flagellin from
gram negative bacteria, which bind to TLR5, inducing inflammation and damage of the intesti-
nal epithelium [12,19]. Interestingly, altered intestinal integrity has also been associated with
high-fat diets and breakdown of tight junction proteins, occludin and zona-occludin-1 (ZO-1)
[12,20–22]. Regulation of this barrier by tight junction proteins is dynamic representing a bal-
ance between the pore pathway and leak pathway. The pore pathway regulates the permeability
of the tight junction to ions and small molecules, whereas the leak pathway regulates the per-
meability of macromolecules [23]. The pore pathway appears to be dependent upon the cad-
herins and claudins while the leak pathway is dependent upon the ZO-1 and the occludins
[24,25].
Obesity is associated with many pathological conditions and higher levels of inflammatory
cytokines. Interlukin-6 (IL-6) is one of the most prominent inflammatory cytokines in obesity
and diabetes research because its serum concentration positively correlates with increased fat
mass [26]. The secretion IL-6 is complex as evidence suggests that this cytokine can have both
detrimental effects in obesity and yet positive effects on tissue homeostasis and potential to
resolve inflammation [27]. The totality of evidence suggests that inflammation and dysmetabo-
lism seem to be more a consequence rather than a cause of obesity [28].

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 Exercise Enhances Intestinal Health

Exercise is often prescribed for weight loss and maintenance. Evidence suggests that chronic
exercise typically causes a partial but incomplete compensation in energy intake and this is
likely due to beneficial changes in appetite-regulating hormones [29]. Notably increases in pep-
tide YY (PYY) and decreases in ghrelin have been reported [30–33]. PYY mediates its effects
via G-protein coupled receptors and primarily acts in the brain to inhibit food intake [34].
Additionally, it has been shown to increase energy expenditure and fat oxidation rates [35].
Ghrelin levels are higher prior to meals and lower after meals, suggesting it may have a role in
weight gain and meal initiation. Since ghrelin is still considered one of the only circulating
appetite stimulants, the ability of exercise to reduce this hormone may promote weight control
by reducing ones desire to consume meals [29,36].
It has also been observed that exercise induces a diverse microbiome [5]. This is an impor-
tant observation, though exercise has not been thoroughly linked to gut integrity. Existing evi-
dence in this area suggests that intense, prolonged exercise, in the heat, will cause leaky gut in
endurance athletes [37]. This is an interesting observation since exercise normally reduces the
risk of GI cancer, reflux, and incidence of ulcers, fatty liver, IBS and diverticulitis [38]. In addi-
tion, exercise in older animals was shown to reduce expression of inflammatory mediators and
apoptotic markers in intestinal lymphocytes, suggesting a protective role for exercise in intesti-
nal health [39,40]. Furthermore, exercise may enhance butyrate producing cecal bacteria,
which are known to reduce inflammation and promote cell proliferation [41]. Exercise may
protect the morphology and integrity of the intestine, and reduce systemic inflammation, even
in the presence of a high fat diet.
Therefore, the purpose of this study was to examine intestinal integrity and gut microbial
ecology in sedentary and exercised animals on normal and high-fat diets. Our hypothesis was
two-fold: (1) exercise reduces intestinal inflammation in the high-fat fed animals; and (2) exer-
cise promotes an anti-obesogenic microbiome.

Methods
Animals, Diets and Exercise
All animals received humane care in compliance with the institution's guidelines, as outlined in
the Guide for the Care and Use of Laboratory Animals published by the National Institutes of
Health. Experiments were completed at Rutgers University and approved by the Rutgers Uni-
versity Institutional Animal Care and Use Committee (IACUC). C57BL/6NTac male 6-week
old mice (Taconic Farms, Germantown, NY) were housed 1/cage in an environmentally con-
trolled room with a 12 hour light/dark cycle and maintained on the specified diet and water ad
libitum. The animals were acclimated for two-weeks and upon acclimation randomly assigned
to one of four groups: (1) lean sedentary (LS); (2) diet-induced obesity (DIO) sedentary (OS);
(3) lean exercise (LX); and (4) DIO exercise (OX) groups. Animals in the lean groups con-
sumed a normal diet (D12450H, 10% kcal from fat, Research Diets, New Brunswick, NJ); and
in the DIO groups a high-fat diet (D12451, 45% kcal from fat, Research Diets, New Brunswick,
NJ). Food intake was monitored every other day, animals weighed once per week and body
composition was determined prior to sacrifice using Echo MRI (Houston, TX). Animals in the
exercise groups had access to a free running wheel (Coulbourn Instruments, Allentown, PA)
linked to ClockLab (Actimetrics, Willmette, IL) to determine exercise volume.
At the end of 12 weeks animals were sacrificed by live decapitation. All animals in the study
were healthy for the duration of the study; however humane endpoints were in place, per the
Rutgers University IACUC, if an animal would have become severely ill/moribund prior to the
experimental endpoint. Fecal pellets were collected from the distal colon of the mice, snap fro-
zen and stored at -80°C for later analysis. Blood was collected in EDTA coated tubes and

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 Exercise Enhances Intestinal Health

centrifuged at 4°C to obtain plasma which was stored at -80°C for later analysis. Tissue analysis
was as described below.

Histochemistry and Immunohistochemistry

Histochemistry. Duodenal and ileal intestinal segments were harvested and denoted as
follows: the first 4–6 cm from the pyloric sphincter was collected as duodenum and the 4–6 cm
above the ileocecal junction was collected as ileum. Tissue was fixed overnight at 4°C in 3%
paraformaldehyde and 2% sucrose then embedded in paraffin. For histological analysis, 5 μm
intestinal tissue sections were stained with hematoxylin and eosin (H & E) (Goode Histolabs,
New Brunswick, NJ). For histomorphometry all tissues sections were blindly scored by a board
certified pathologist (Dr. Stanley Lightfoot).
Immunohistochemistry. For immunohistochemical studies, 5 μm intestinal sections were
deparaffinized, rehydrated and subsequently blocked with 10%, 25% or 100% normal goat
serum at room temperature for 2 hr. The tissue sections were then incubated overnight at 4°C
with primary rabbit affinity purified polyclonal antibodies against structural components of the
intestine as described below, or with rabbit IgG (ProSci Inc., Poway, CA) as a control. Duodenal
segments were stained for cyclooxygenase-2 (COX-2, 1:200, Abcam, Cambridge, MA) expres-
sion. Ileal segments were stained for COX-2 (1:200, Abcam), occludin (1:50, Thermo Fisher Sci-
entific, Waltham, MA), and E-cadherin (1:300, Abcam). Tissue sections were then incubated for
30 min with a biotinylated goat anti-rabbit secondary antibody (Vector Labs, Burlingame, CA).
Antibody binding was visualized using a DAB Peroxidase Substrate Kit (Vector Labs). Tissue
sections were photographed using the VS120-S5 System (Olympus, Center Valley, PA). For his-
tomorphometry all tissues sections were blindly scored by a board certified pathologist (Dr. Stan-
ley Lightfoot). Slide staining was reported using a dual number system (#X#). The first number
was the intensity of the stain and the second number was the amount of stain present in the spec-
imen. The intensity was graded on 1–3 and the amount indicated as follows- 1-<10%; 2-11-
40%; 3-41-60%; and 4->60%. The total score was obtained by multiplying the two numbers.

Gut Microbial Community Analysis

DNA purification, 16S rRNA gene profiling, and pyrosequence analysis of fecal bacte-
ria. The gut bacteria were assayed by terminal restriction fragment length polymorphism
(TRFLP) in a single stool sample from triplicate animals in the experimental treatments [42],
followed by pyrosequence analysis of 16S rRNA genes of pooled DNA samples per experimen-
tal treatment. Genomic DNA was extracted by phenol/chloroform methodology [43]. Small
subunit rRNA genes were amplified using 10 ng of genomic DNA, the forward primer 27F
((5’-AGAGTTTGATCCTGGCTCAG-3’; labeled with the fluorochrome 6’-carboxyfluorescein)
and the bacterial-specific primer 1100R (5’-AGGGTTGCGCTCGTTG-3’) for TRFLP analysis
[43]. Fifteen ng of amplicon was digested with Mnl-I endonuclease (New England Biolabs, Bev-
erly, MA) for 6 h and sized using an ABI 310 genetic analyzer (Applied Biosystems, Foster
City, CA) with Genescan™ software and an internal size standard. Peak detection was set at 25
arbitrary fluorescent units and only peaks representing >0.5% of the overall community profile
area were considered for further analysis. Cluster analysis of the profiles was performed by con-
versions of presence/absence data to A/T and FastTree methods with bootstrap support using
Geneious™ phylogenetic software, similar to Clark and Warwick [44]. Sorting of the terminal
restriction fragment (TRF) areas for heat mapping was initially done in ascending order by
counting peaks present in all experimental treatments (LS, OS, LX, OX) and then sub-sorting
per treatment in descending order of peak area for ease of comparison. Color coding of the
data was in increments of 1000–5000 arbitrary fluorescent units.

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 Exercise Enhances Intestinal Health

Clone libraries from pooled fecal DNA samples from each treatment group were established
using gel purified 16S rRNA gene amplicons (GeneClean1 II kit; MP Biomedicals Ca. No.
111001400), and the pGEM1-T Easy Vector System I kit (Promega Ca. No. A1360). Clones
matching specific peaks were identified by TRFLP and sequenced using Sanger methods (Gene-
Wiz, Plainfield, NJ) and are available in Genbank (Ascension numbers: KU644595-KU644645).
Phylogenetic analysis was performed using likelihood methods for 379 unambiguously aligned
positions with the Geneious™ software package.
Pooled fecal DNA samples from each treatment group were also processed for pyrosequen-
cing and community analysis. 16S rRNA genes of the bacterial community were amplified with
27F and 519R primers, then sequenced and analyzed by Molecular Research LP (Shallowater,
TX) using a Roche 454 Genome Sequencer following the manufacturer’s guidelines. The
sequence data was processed using a proprietary analysis pipeline (MR DNA, Molecular
Research LP). Briefly, the barcodes, primers, short sequences (< 200bp) and the sequences
containing ambiguous base calls and homopolymer runs (>6bp) were removed to ensure the
quality of reads. The remaining sequences were denoised and then chimeras and singleton
sequences were removed. Operational taxonomic units (OTUs) were clustered using a
sequence similarity threshold of 97% and taxonomically classified by BLASTN against a
curated GreenGenes database as described previously [45,46]. These sequences are presented
in S1 Table and all 454 sequences are available in Genbank (BioProject# PRJNA 309613).

Plasma Blood Samples

Plasma samples were screened for metabolic markers using a mouse metabolic hormone kit
(Millipore, MMHMAG-44K) on a Luminex MagPix; specific markers analyzed included leptin,
ghrelin, insulin, IL-6, C-peptide 2, PYY and pancreatic polypeptide (PP).

Statistical analysis
Anthropometric data and blood markers were analyzed using one-way ANOVA and Tukey
post-hoc test (SPSS 23, IBM SPSS Statistics, Armonk, NY). A difference of mean with a p value
of  0.05 was considered statistically significant.

Results
Animals, Body Weights and Exercise
For the exercise groups, LX and OX, wheel counts were not significantly different from each
other (Table 1). Animals in the LX group consumed significantly more food per day than the
other groups. Furthermore, animals in the OX group consumed significantly more that the sed-
entary groups which equated to the highest caloric intake of any of the groups. Despite this
higher kcal consumption OX animals had lower percent body fat, less total fat mass and
reduced body weights compared to their sedentary counterparts (OS) (Table 1). There were no
significant differences between the groups with regards to lean mass.
Animals in the LX group had the lowest body weights followed by LS, although these were
not significantly different from each other. The OS group had significantly higher body weights
compared to the lean groups. It is important to note that the body weights were variable in the
OX group, as were the wheel counts per day. Our results indicated that some of the mice con-
sumed on average the same as their exercising counterparts. This was despite a lower exercise
volume only ~6,000 counts per day, significantly less than the group average. We also noted
that 4 of our 9 OX mice were very lean, averaging 29 grams, (similar to the LX group) and exer-
cised substantially. This observation, anecdotally, appears to be similar to a human population

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 Exercise Enhances Intestinal Health

Table 1. Animal Characteristics.

Lean-Sedentary Lean-Exercise DIO-Sedentary DIO-Exercise

a a b
Body weight (g) 30.4±3.2 29.7±5.5 42.7±3.2 35.3±7.4c
a a b
Food Intake (g/day) 3.3±0.3 4.2±0.4 3.3±0.4 3.7±0.5c
Body Fat (%) 22.9±6.0a 15.3±8.9b 42.4±2.5c 32.8±11.7d
Lean Mass (g) 21.5±1.1 22.8±1.6 22.8±1.2 22.4±2.0
Fat Mass (g) 7.1±2.5a 4.9±4.2a 18.1±2.1b 12.2±5.9c
a b b
Kcal/day 12.3±1.0 16.0±1.4 15.0±1.7 17.0±2.1b
a b a
Exercise volume (counts/day) 0 17,390±6,890 0 16,480±6,930b

Values are mean ± SD, n = 9 in each group.

Values that do not share the same superscript letters are significantly (p<0.05) different from each other.

doi:10.1371/[Link].0150502.t001

where some may consume a higher fat diet and with exercise can maintain a healthier weight,
while others do not.

High-Fat Diets Alter Intestinal Morphology

Sedentary Animals—The LS villous histology was normal, exhibiting a single layer of epithe-
lium covering the villi and lamina propria. The OS villi were the same height as the LS
(~300 μm tall) while the width was twice that of the LS animals (95 μm vs 44 μm, respectively)
(Fig 1); as a result OS villi were crowded. Major causes of villi widening include an increase in
inflammatory cells present in lymph and plasma cells and an increase in fat cells.
Exercised Animals—LX and OX villi were histologically normal with open lumens. The villi
were well-formed and thicker than their sedentary counterparts, which was primarily due to
vessel dilation. In the OX group, the plasmacystoid and lymphocytic infiltrate was absent in
contrast to their sedentary counterparts. These results suggest that exercise prevented the mor-
phological changes that were associated with high fat feedings and reduced inflammatory
infiltrate.
Structural Distal Small Intestine Integrity (occludin and E-cadherin). Sedentary ani-
mals—Occludin expression was up-regulated in the OS group compared to the LS (Fig 2). Con-
trary, E-cadherin expression was up-regulated in the LS group compared to the OS counterpart
(Fig 3).
Exercised Animals—Occludin expression in the LX and LS mice was similar. Interestingly,
occludin expression had decreased in the OX animals as compared to the LX, LS and OS
groups (Fig 2). E-cadherin expression in the LX group was lower than its sedentary counterpart
with lowest expression observed in the OX group (Fig 3). These results initially seem confusing
as we expected occludin expression to be highest in the exercised groups, not the obese seden-
tary group. However, the higher E-cadherin expression in the groups with lower occludin
expression supports the notion that barrier function is dynamic and that regulation of its func-
tion may not be dependent upon the protein expression but other factors like plasma mem-
brane channels and transporters [23].
Exercise Reduces Intestinal Inflammation in Proximal and Distal Gut. Sedentary ani-
mals—COX-2 expression was increased in the duodenum (Fig 4a) and ileum (Fig 4b) in the
OS animals compared to the LS ones suggesting that high fat diets increased intestinal inflam-
mation, which is in agreement with the H&E staining.
Exercised animals—Both LX and OX groups had COX-2 expression similar to the LS group.
Furthermore, the OX group had less COX-2 expression compared to the sedentary

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 Exercise Enhances Intestinal Health

Fig 1. The Effects of Exercise on Duodenal Morphology. Widening in obese sedentary compared to lean
sedentary animal (black arrows) due to: increase in inflammatory cells, plasmacystoid and lymphoid cells (red
arrow); and infiltration of fat cells (yellow arrow). Exercise appears to protect the villi from widening. Obese
exercise villi contain wider lumen (asterisk) and are devoid of fat cell infiltrate. Lean Sedentary (A), Obese
Sedentary (B), Lean Exercise (C), Obese Exercise (D).
doi:10.1371/[Link].0150502.g001

counterpart, OS (Fig 4a and 4b). These data support the observed H&E findings that exercise
reduced inflammation due to a high-fat diet.
Exercise Influences Gut Microbiota. A 16S rRNA gene analysis was performed and an
example of the community profiles is shown in S1 Fig. Terminal restriction fragment length
polymorphism (TRFLP) detected 100 peaks (OTUs) in all experimental treatments. A cluster
analysis of the profiles of fecal pellets from individual animals demonstrated distinct groupings
within the experimental treatments supported by bootstrap values >0.72 (S2 Fig). A heat map
(Fig 5) of the average TRFLP peak areas indicated that 35 terminal restriction fragments
(TRFs) were only found within a single experimental treatment, with approximately half of the
unique TRFs observed in the normal diet/exercised animals (e.g. TRFs 298 and 161; Fig 5; S3
Fig) vs. the sedentary and exercised lean diet animals (S4 Fig). The other remaining TRFs were
detected in nearly all the animals regardless of diet or exercise (S5 Fig). In order to identify spe-
cific bacterial 16S rRNA genes associated with the various TRFs, clone libraries were generated
for all experimental groups (S1 Fig).
DNA from replicate samples were combined for pyrosequencing analysis which resulted in
the detection of over 440 OTUs with 90% of the OTUs present at <0.1% within the whole

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 Exercise Enhances Intestinal Health

Fig 2. The Effects of Exercise on Occludin Expression in Ileum. Sections prepared after 12 weeks of
treatment lean sedentary (A), obese sedentary (B), lean exercise (C), obese exercise (D),were stained with
anti-occludin antibody. Binding was visualized using a Vectastain Elite ABC kit (original magnification x 400).
One representative section from 6 mice/treatment group is shown.
doi:10.1371/[Link].0150502.g002

Fig 3. The Effects of Exercise on E-cadherin Expression in Ileum. Sections prepared after 12 weeks of
treatment lean sedentary (A), obese sedentary (B), lean exercise (C), obese exercise (D),were stained with
anti-E-cadherin antibody. Binding was visualized using a Vectastain Elite ABC kit (original magnification x
400). One representative section from 6 mice/treatment group is shown.
doi:10.1371/[Link].0150502.g003

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 Exercise Enhances Intestinal Health

Fig 4. The Effects of Exercise on COX-2 Expression in Duodenum. Duodenal (a) and Ileal (b) sections prepared after 12 weeks of treatment lean
sedentary (A), obese sedentary (B), lean exercise (C), obese exercise (D),were stained with anti-COX-2 antibody. Binding was visualized using a Vectastain
Elite ABC kit (original magnification x 400). One representative section from 6 mice/treatment group is shown.
doi:10.1371/[Link].0150502.g004

sample set. A pie-chart of the top 20 OTUs (representing OTUs >1% of the sample set; S6 Fig)
indicated that Roseburia intestinalis, Allobaculum spp., Enterorhabdus spp., and Blautia spp.
were the predominant bacteria observed in all groups. Allobaculum spp. and Clostridiales were
enriched within the exercised groups.
A phylogenetic analysis of the 76 OTUs detected by pyrosequence analysis and the 57
TRFLP clones demonstrated broad agreement (Fig 6). Both the clonal and pyrosequencing
analyses revealed that few Bacterioides family members were observed in the fecal microbiota.
Rather, Clostridiales dominated the fecal microbiota and this was observed in all animal
groups. Interestingly, Faecalibacterium prausnitzi was only detected in exercised animals
(green bar), while animals on the normal diet, regardless of exercise, had large clusters of
sequences related to Lachnospiraceae spp. that were not present in the high fat fed animals
(blue and red bars). Allobaculum spp. and Clostridium spp. were enriched in the exercise ani-
mals on a normal diet (LX) whereas the high fat fed animals (OS and OX) had microbial clus-
ters related to Peptococcus spp. (pink bar).

Blood Profiles of Sedentary and Exercised Animals on Normal and High-

Fat Diets
Lean sedentary animals had lower levels of IL-6 and insulin compared to the obese sedentary
counterparts (Table 2). Furthermore, exercised animals showed lower ghrelin levels compared
to both sedentary groups. In addition, the exercise groups had lower IL-6 compared to the OS
group but higher values than the LS group and were not significantly different from each other.
Both PYY and PP was highest in the LX group followed by the OX group and these values were
higher compared to both sedentary groups. Insulin values were lowest in the LX group and
highest in the OS group with LS and OX being similar and not different from each other, but
significantly lower that OS. Finally, amylin values were higher in both exercise groups com-
pared to sedentary. OX had a significantly higher amylin concentration compared to LX.

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 Exercise Enhances Intestinal Health

Fig 5. Heat Map. Heat map of average peak area within TRFLP profiles of fecal pellet bacterial community
grouped by treatment (L, lean diet; O, high fat diet; S, sedentary; X, exercise). The major peaks in the lean
exercise treatment are indicated.
doi:10.1371/[Link].0150502.g005

Discussion
The major findings of these studies indicate that: (1) high-fat diets altered intestinal morphol-
ogy particularly of the duodenum; (2) exercise protected duodenal morphology in the presence
of a high-fat diet; (3) high-fat diets increased intestinal inflammation and exercise reduced it;
(4) exercise manifested a unique microbiome independent of diet; (5) exercise reduced blood
levels of IL-6, insulin and ghrelin and increased levels of satiety related hormones (Fig 7). We
observed that high fat diets accompanied with sedentary behavior increased the width of duo-
denal villi. We are the first using IHC to substantiate in situ inflammation and loss of intestinal
integrity due to high fat diet and sedentary lifestyle in mice. These data correlate with results
from others in that animals fed a high-fat diet had a three-fold increase in TG accumulation in
intestinal mucosa and an up-regulation of genes for TG synthesis, chylomicron secretion and
uptake, oxidation and de novo synthesis of fatty acids [47]. These observations, accompanied
with an impaired oral fat tolerance, suggest a reduced rate of intestinal lipid secretion led to
increases in mucosal TG accumulation and contributed to the shortening and widening of the
villi. Analysis of H&E stained tissue revealed significant amounts of lipid infiltration, inflam-
matory lymphocytes, plasma cells and macrophages. This was further supported by the
increased expression of COX-2 in the high fat fed sedentary animals. An important finding of

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 Exercise Enhances Intestinal Health

Fig 6. Phylogenetic Tree. Maximum likelihood phylogenetic tree from TRFLP/clones and pyrosequencing
based on 379 unambiguously aligned bases. Color coding for the experimental treatments is indicated.
doi:10.1371/[Link].0150502.g006

our work suggests that exercise prevented high-fat triggered morphological changes by reduc-
ing COX-2 expression in both proximal and distal gut.
The high fat diet induced changes in intestinal morphology and inflammation were negated
by exercise, however, mechanisms associated with these observations need to be elucidated.

Table 2. Plasma Systemic Biomarkers.

Biomarker (pg/mL)* Lean-Sedentary Lean-Exercise DIO-Sedentary DIO-Exercise

a b a
Ghrelin 84.0±3.4 33.8±4.5 87.4±4.1 32.1±6.3b
a b c
IL-6 22.0±0.7 39.0±7.2 85.5±3.8 31.0±5.6b
a,c a b
Insulin 13.1±5.2 7.1±2.5 31.0±4.1 15.7±6.0c
PYY 8.9±3.7 36.0±9.6 10.8±6.4 25.7±3.6
PP 5.4±1.7a 19.6±7.3b 7.6±6.9a 15.2±4.3b
Amylin 25.0±0.7 43.4±7.2a 26.8±2.7 71.1±5.1b

*Values are mean ± SEM, n = 9 in each group.

Values that do not share the same superscript letters are significantly (p<0.05) different from each other.

doi:10.1371/[Link].0150502.t002

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 Exercise Enhances Intestinal Health

Fig 7. Experimental Results Summary. Impacts of high-fat diet (HFD) and exercise on intestinal tissue,
microbiome and systemic biomarkers. Results indicate exercise can protect intestinal morphology in the
presence of a HFD, promote a diverse microbiome that has microbes that promote intestinal health and
reduce systemic inflammation while promoting satiety.
doi:10.1371/[Link].0150502.g007

There are a couple of plausible accepted explanations related to lipid metabolism for these
observations; 1) regular exercise is associated with reduced postprandial lipemia; and 2) sys-
temic metabolic adaptations to promote greater reliance on fat utilization during exercise and
at rest [48,49]. Cross-sectional and longitudinal studies show that regular aerobic exercise
reduced postprandial lipemia in the presence or absence of weight loss which may be intensity
and dose-dependent (extensively reviewed in [50]). Exercise reduced postprandial lipemia by 3
possible mechanisms acting alone or in combination: (1) decreased appearance of chylomi-
cron-TG concentrations from the gut, (2) increased clearance of TG-rich lipoproteins (VLDL
and/or CM) via exercise- mediated increases in skeletal muscle and/or adipose tissue LPL activ-
ity, and (3) decreased hepatic VLDL-TG secretion (for review see [51]). Regardless of the sub-
strate used during the exercise bout, the hepatic and skeletal muscle program following
exercise is suggested to preferentially use fatty acids from TRLs to replenish ATP, thus sparing
glucose [48,49]. Furthermore, in our studies there was a reduction of body weight in the high
fat fed exercised animals. thus the energy deficit created by exercise appears to be a primary
mediator of the exercise-induced decline in postprandial lipemia. These explanations may pro-
vide a rationale for normal morphology of the villi in the high fat fed exercised animals, sug-
gesting that exercise promoted reduced postprandial lipemia and alterations in substrate use
that favored fatty acids utilization to replenish energy stores between the exercise bouts.
Another plausible explanation for why exercise reduced intestinal inflammation is that exer-
cise has been shown to increase antioxidant enzymes (glutathione peroxidase and catalase),
anti-inflammatory cytokines (IL-10), and anti-apoptotic proteins (Bcl-2) in intestinal lympho-
cytes [39,40]. It was also observed that exercise decreased TNF-α, pro-apoptotic proteins (cas-
pase 3 and 7) and the pro-inflammatory cytokine IL-17, suggesting that exercise can modulate
the intestinal immune response [39,40]. These data correlate with our studies suggesting that
exercise, through laminar shear stress activation, may decrease superoxide anion production,
which in turn decreases ROS (reactive oxygen species) generation, and preserves endothelial
NO bioavailability [52–55].
Occludins are integral membrane proteins crucial for tight junctions and the adherens are
involved in cell to cell adhesion as well as communication [23]. Our studies showed that high-

PLOS ONE | DOI:10.1371/[Link].0150502 March 8, 2016 12 / 17

 Exercise Enhances Intestinal Health

fat fed animals had reduced E-cadheren expression while interestingly the opposite was true
for occludin. The occludin response differs from previous published data, suggesting impaired
barrier function with high-fat diets [10,12]. One animal in the OX group had influenced our
thoughts on these results. This animal’s exercise volume was only 50% of the other animals in
the group and weighed 48 g at sacrifice. We observed that this mouse’s COX-2 and occludin
expression was up-regulated compared to the other mice and interestingly E-cadherin expres-
sion was also up-regulated compared to the animals which exercised “normally”. These data
suggest that the increased expression of occludin may not imply intact tight junctions but may
be compensatory expression due to inflammation (COX-2) induced damage (S7 Fig). Research
on exercise and barrier function suggests that the more strenuous the exercise the greater bar-
rier disruption, due to changes in blood flow leading to insufficient removal of metabolites and
delivery of nutrients [36]. Though the exercise protocol in these studies is not considered stren-
uous; investigation of blood flow changes, particularly in the obese gut, would be informative
as there is a scarcity of literature in this area. Further, claudins play a critical role in barrier
function by sealing neighbor epithelial cells and these were not examined. Information about
their expression may help to fully understand the intricate relationship of the epithelial barrier.
Finally, the microbiome findings in this study appear to be similar (with some distinct dif-
ferences) to a separate study conducted by Evans et al. on exercise and diet in mice [4]. Primar-
ily, it was observed in both studies that exercise in the presence of a high fat diet (60% in the
Evans study and 40% in the current study) maintains body weight with feedings [4]. Likewise,
both studies demonstrated clustering of the microbial communities by experimental treatment
using a TRFLP approach, despite the differences in DNA extraction procedures, PCR priming
sets, and the restriction enzymes being used. However, Evans et al. [4] demonstrated that both
the lean exercise and obese sedentary groups had statistically higher abundance of Bacteroi-
detes and lower abundance of Firmicutes. In contrast, Firmicutes were detected within the fecal
samples from the high fat fed mice, which may be explained by the 2 different sources of mice
(Jackson Laboratories and Taconic Laboratories), the differences in DNA extraction (bead
beating/12 hrs at 55°C vs. 5 freeze/thaws and immediate extraction), or the use of different
primers (group specific vs. universal primers) for amplifying the target 16S rRNA genes. Addi-
tionally, in our study the microbiota was analyzed at the genus level rather than the phylum/
class or family level. Our findings which are in agreement with Khan et al. [56], suggesting that
bacteria related to Faecalibacterium prausnitzi are present in exercised mice and may provide
protection to the gut through oxygen detoxification by a flavin/thiol electron shuttle in F.
prausnitzi. Furthermore, the integrity of gut immunological markers observed in our study is
consistent with the hypothesis that F. prausnitzi promotes a healthy digestive tract by produc-
ing butyrate and lowering the oxygen tension in the lumen as described in humans [57]. Lach-
nospiraceae, as a cluster of Clostridia, in the exercise groups alone (LX and OX; Fig 7; blue/red
bars) contrast the sedentary group which had different set of clostridia related to Lachnospira-
ceae (Fig 7; pink bars). The concept that various Lachnospiraceae may be beneficial to the gut
while other closely related strains are not beneficial is supported by a study of 30 Lachnospira-
ceae genomes, where less than half of the strains were found to possess the genes for butyrate
production [58]. Our data suggests that further examination of the physiology of distinct Clos-
tridiales species within the mouse gut will be needed to resolve the role of butyrate production
or other mechanisms in the promotion of a healthy gut microbiome.
The mouse blood work results suggest that high fat diets promote systemic inflammation as
evident by the elevated plasma IL-6. While IL-6 is often used as a marker for obesity-associated
‘meta-inflammation [26]’ measurement of plasma endotoxin levels (LPS), which were not
assessed would be ideal and should be done in future studies. Of note, our lean exercise animals
had higher levels of IL-6 compared to the sedentary counter parts and this may be related to

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 Exercise Enhances Intestinal Health

the duality of IL-6 function. It is accepted that exercising skeletal muscle produces IL-6 and
that this net release from muscle can increase circulating concentrations leading to hepatic glu-
cose output and lipolysis [58]. This may indicate that there may be a link to IL-6 released by
exercising muscle and exercise-related metabolic changes. Furthermore, high fat fed animals
had higher levels of insulin and leptin, which are common for this model and exercise regulated
this along with hormones of satiety and glucose control.
These studies support the hypothesis that exercise manifests a unique microbiome indepen-
dent of diet. Exercise reduced the intestinal inflammatory response due to high-fat diet which
lead to morphology similar to the sedentary animals and promoted satiety biomarkers. Taken
together these data suggest that exercise is a safe and efficacious strategy to combat obesity
through positive changes in microbial ecology and intestinal health.

Supporting Information
S1 Fig. Example TRFLP profiles of fecal pellet bacterial community. The biological replicate
for each treatment is indicated. Those TRFLP peaks represented in clone libraries from each
treatment are highlighted in black.
(TIFF)
S2 Fig. Cluster analysis of replicate TRFLP profiles from the different experimental treat-
ments (L, lean diet; O, high fat diet; S, sedentary; X, exercise). The numbers indicate boot-
strap support for the groupings.
(TIFF)
S3 Fig. Histogram of TRF average peak areas found in 1 diet/treatment as indicated. Error
bars indicate standard deviation of the biological replicates in positive direction only. Note the
difference in vertical scale.
(TIFF)
S4 Fig. Histogram of TRF average peak areas found in 2 diet/treatments as indicated. Error
bars indicate standard deviation of the biological replicates in positive direction only.
(TIFF)
S5 Fig. Histogram of TRF average peak areas for all diet/treatments. Error bars indicate
standard deviation of the biological replicates in positive direction only.
(TIFF)
S6 Fig. Pyrosequencing analysis of the different microbial communities from the experi-
mental treatments. The OTUs in the exercise treatment that are nearly undetectable in the
sedentary treatment have been pulled away from the center. Only those OTUs greater than 4%
have values shown and can be read clockwise to coordinate with the key.
(TIFF)
S7 Fig. The Effects of Minimal Exercise on Ileum in Obese Gut. Section was prepared after
12 weeks of feeding a high-fat diet and limited usage of free running wheel (exercise volume
was 50% less than animals in this cohort). Binding was visualized using a Vectastain Elite ABC
kit (original magnification x 400). Representative section is shown COX-2 (A), Occludin (B),
E-Cadherin (C).
(TIFF)
S1 Table. Pyrosequences used in the phylogenetic analysis.
(DOCX)

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 Exercise Enhances Intestinal Health

Acknowledgments
The authors would like to thank Dr. Dipak Sarkar (Rutgers University, Department of Animal
Sciences for the use of the running wheels for this experiment. The authors would also like to
thank Dr. David Feigley for providing help with statistics.

Author Contributions
Conceived and designed the experiments: SCC. Performed the experiments: SCC PJW MN LBJ
LRM MMH LJK. Analyzed the data: SCC LJK MMH LRM LBJ SAL. Contributed reagents/
materials/analysis tools: SCC LJK MMH LBJ. Wrote the paper: SCC. Provided editorial input
on the manuscript: LJK MMH LBJ.

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