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Lactobacillus GG and Tributyrin Supplementation Reduce
Antibiotic-Induced Intestinal Injury
Gail Cresci, PhD, RD, LD1, Laura E. Nagy, PhD1, and Vadivel Ganapathy, PhD2
1Cleveland Clinic, Cleveland, Ohio
2Georgia Health Sciences University, Augusta, Georgia
Abstract
Background—Antibiotic therapy negatively alters the gut microbiota.
Lactobacillus
GG (LGG)
decreases antibiotic-associated diarrhea (AAD) symptoms, but the mechanisms are unknown.
Butyrate has beneficial effects on gut health. Altered intestinal gene expression occurs in the
absence of gut microbiota. We hypothesized that antibiotic-induced changes in gut microbiota
reduce butyrate production, varying genes involved with gut barrier integrity and water and
electrolyte absorption, lending to AAD, and that simultaneous supplementation with LGG and/or
tributyrin would prevent these changes.
Methods—C57BL/6 mice aged 6–8 weeks received a chow diet while divided into 8 treatment
groups (± saline, ± LGG, ± tributyrin, or both). Mice received treatments orally for 7 days with ±
broad-spectrum antibiotics. Water intake was recorded daily and body weight was measured.
Intestine tissue samples were obtained and analyzed for expression of genes and proteins involved
with water and electrolyte absorption, butyrate transport, and gut integrity via polymerase chain
reaction and immunohistochemistry.
Results—Antibiotics decreased messenger RNA (mRNA) expression (butyrate transporter and
receptor, Na+/H+ exchanger, Cl−/HCO3−, and a water channel) and protein expression (butyrate
transporter, Na+/H+ exchanger, and tight junction proteins) in the intestinal tract. LGG and/or
tributyrin supplementation maintained intestinal mRNA expression to that of the control animals,
and tributyrin maintained intestinal protein intensity expression to that of control animals.
Conclusion—Broad-spectrum antibiotics decrease expression of anion exchangers, butyrate
transporter and receptor, and tight junction proteins in mouse intestine. Simultaneous oral
supplementation with LGG and/or tributyrin minimizes these losses. Optimizing intestinal health
with LGG and/or tributyrin may offer a preventative therapy for AAD.
Keywords
probiotics; tributyrin; antibiotics; diarrhea; intestine
Trillions of bacteria consisting of more than 800 different bacterial species and 7000 strains
comprise the gut microbiota.1 The gut microbiota markedly influences the biology of the
host through several mechanisms, including energy balance, gene expression, immune
function, and disease processes; thus, any disturbances in the microbiota can lead to a
© 2013 American Society for Parenteral and Enteral Nutrition
Corresponding Author: Gail Cresci, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195. crescig@ccf.org.
Financial disclosure: This work was supported in part by the A.S.P.E.N. Rhoads Research Foundation Grant, the Case Western
Reserve University/Cleveland Clinic CTSA (UL1RR024989), and National Institutes of Health grants (UO1AA021890 and
1F32AA021044).
NIH Public Access
Author Manuscript
JPEN J Parenter Enteral Nutr
. Author manuscript; available in PMC 2014 November 01.
Published in final edited form as:
JPEN J Parenter Enteral Nutr
. 2013 ; 37(6): . doi:10.1177/0148607113486809.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
variety of pathogenic states.1–3 Antibiotic therapy is believed to represent such a condition.4
Antibiotic-associated diarrhea (AAD), defined as diarrhea associated with the administration
of antibiotics without another obvious cause, occurs in approximately 5%–25% of patients
receiving antibiotics, varying with the class of antibiotics used and patient risk factors.5
Overgrowth by the toxigenic bacterium
Clostridium difficile
is responsible for virtually all
cases of antibiotic-associated pseudomembranous colitis, which can lead to complications
such as paralytic ileus and colonic dilatation and perforation.6 However, it is estimated that
only 15%–25% of all cases of AAD are due to the overgrowth of
C difficile
.4 Alterations in
the composition and quantity of gut microbiota leading to losses of beneficial metabolic
activities of the normal colonic microbiota are associated with non–
C difficile
AAD.7–9
Short-chain fatty acids (SCFAs) are fermentation by-products of undigested polysaccharides
and some proteins by anaerobic bacteria formed in the intestinal tract of mammals.10,11 The
metabolism of undigested fiber and starch by colonic anaerobes to SCFAs, particularly
butyrate, is hypothesized to prevent osmotic diarrhea as well as provide a supply of the
preferred carbon and energy source to the colonic enterocytes.12–14 Recent metabolomic
studies have shown that depletion of Gram-positive bacteria by vancomycin disrupts
carbohydrate fermentation in mice; these changes increase quantities of unfermented
oligosaccharides in the feces and reduce concentrations of the SCFAs acetate, butyrate,
propionate, and lactate.7 Distinct gut microbiota diversity, including a marked decrease in
the prevalence of butyrate-producing bacteria, was found following administration of the
antibiotic amoxicillin-clavulanate.8 This alteration is of significant concern because among
the SCFAs, butyrate is highly important as it contributes to the differentiation of epithelial
cells, enhancement of electrolyte and water absorption, promotion of angiogenesis, and
modulation of the immune function.12–14
It is estimated that the average intraluminal concentration of SCFA is between 100 and 170
mM, and acetate, propionate, and butyrate are present in a nearly constant molar ratio of
60:25:15.11 Butyrate can reach concentrations up to 20 mM in the colon and feces of
mammals with normal gut health.10 In the adult human with a fecal output of 80–230 g/d,
SCFAs are excreted only at a rate of 5–20 mM/d, so most SCFAs (95%) are absorbed.15
Butyrate is also available in the diet with low levels in many fruits and vegetables, and milk
fat, which contains 3%–4% butyrate in a complex of glycerides or esters of glycerol, is also
a good source of butyrate.16 Another source of butyrate, glyceryl tributyrate (tributyrin), is a
triglyceride with glycerol esterfied with butyrate at the 1, 2, and 3 positions.10 Tributyrin is
neutral, chemically stable, and rapidly hydrolyzed by pancreatic and gastric lipases to
glycerol and 3 butyrate molecules.10 There are several mechanisms by which butyrate exits
the gut lumen. Lipid-soluble protonated SCFAs diffuse readily across cell membranes, but
ionized SCFAs do not and require various anion exchangers for diffusion.10 Recent studies
have identified an Na+-coupled transporter for butyrate and other SCFAs.17,18 SLC5A8 is
expressed in the apical membrane throughout the intestinal tract and most abundantly in the
ileum and colon.18 SLC5A8 transports butyrate via an Na+-dependent electrogenic process,
and the expression of the transporter is reduced markedly in colon cancer and germ-free
(GF) mice.18,19 Other monocarboxylate transporters are also present throughout the
intestinal tract.20,21
Providing butyrate can be challenging for several reasons, including short metabolic half-
life, toxicity, and patient intolerance. Butyrate has been provided via several routes:
intravenously, rectally as enemas, and orally. There are limitations to providing butyrate
intravenously (500 mg/kg body weight) in that large volumes are required, and the
metabolic half-life is very short, with blood levels peaking about 6 minutes after delivery.10
Providing higher rates of intravenous (IV) butyrate infusion is undesirable due to risk of
toxicity from sodium overload. Rectal enemas (100 mmol/L) have been successful in
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reversing negative gastrointestinal (GI) effects in patients with inflammatory bowel disease;
however, this mode of delivery lends to very poor patient compliance.10 Tributyrin
overcomes many of the problems of the parent compound. Tributyrin delivered orally in
animals has a plasma half-life of 40 minutes.16 In humans, oral delivery provided once daily
for 3 weeks was without severe toxicity, and peak plasma butyrate concentrations occurred
between 0.25 and 3 hours after dose and ranged from 0–0.45 mM, which is near those found
to be effective in vitro (0.5–1 mM).22
Probiotics, defined as “live microorganisms which, when consumed in adequate amounts,
confer a health benefit on the host,” have been used in the treatment and prevention of AAD
as well as in the prevention of relapses of
C difficile
–associated diarrhea.23 The exact
mechanism of how probiotics prevent AAD is unknown, but they are believed to compete
with pathogenic microbes for available nutrients and epithelial binding sites; decrease GI
luminal pH, making it less favorable for pathogenic bacteria; modulate the immune
response; and reestablish the intestinal barrier function.24 A meta-analysis of randomized
controlled trials found a moderate beneficial effect of
Lactobacillus
GG (LGG),
Saccharomyces boulardii
, and a combination of
Bifidobacterium lactis
and
Streptococcus
thermophilus
in preventing AAD.25 A Cochrane review of 10 randomized controlled trials
with probiotics found a significant reduction in the incidence of AAD, confirming the
efficacy of LGG and
S boulardii
.26
The expression of genes in the ileum and colon is altered markedly in GF mice compared
with mice raised under conventional conditions.19,27,28 DNA microarray analysis showed
that ~700 genes were affected (increased or decreased) by more than 2-fold in the colon
from GF mice compared with the colon from conventional mice, and these changes were
completely reversed when the colon was recolonized.19 Most notable among the genes that
were downregulated in GF mouse colon compared with conventional mouse colon were
those involved in immune development and antimicrobial defense, with some
downregulated more than 20-fold. Transporters involved with water and electrolyte
exchange were also down-regulated, including SLC5A8 (sodium-coupled butyrate
transporter), SLC26A3 (chloride-bicarbonate exchanger), aquaporin 4 (AQP4, water
channel), NHE3 (sodium-hydrogen exchanger), and a butyrate receptor involved with
inflammation (GPR109a).19 It is very interesting and potentially clinically relevant that
genes involved with water and electrolyte absorption were downregulated in GF mouse
ileum and colon, suggesting that conventional gut microbiota play an active role in the
control of water and electrolyte absorption.
Since antibiotic usage can cause profound changes in gut microbiota, it is likely that there is
a consequential reduction of butyrate produced in the GI tract. We hypothesize that altered
gut microbiota from antibiotic therapy affects expression of genes involved with water and
electrolyte absorption as seen in GF mice, as well as those dependent on butyrate for
expression. The objective of this work was to explore the efficacy and mechanism of
probiotics and/or tributyrin provision as a clinically feasible method for mitigating AAD
through the preservation of physiologic responses in the intestine.
Materials and Methods
Materials
Antibiotics, sucrose, tributyrin, MRS broth and agar, and Mueller Hinton media were
purchased from Sigma-Aldrich (St Louis, MO);
Lactobacillus rhamnosus
strain GG (LGG)
was purchased from ATCC (ATCC, Rockville, MD); RNA extraction reagent (TRIzol) was
from Invitrogen-GIBCO (Carlsbad, CA); GeneAmp reverse transcription polymerase chain
reaction (RT-PCR) kit was from Applied Biosystems (Foster City, CA); and Taq polymerase
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kit was from TaKaRa (Tokyo, Japan). All primers for real-time RT-PCR were synthesized
by Integrated DNA Technologies (Coralville, IA). Primary antibodies were purchased from
the following companies: Abcam (Cambridge, MA) for SLC5A8, NHE3, and zonula
occludens 1 (ZO-1) and Hycult Biotech (Plymouth Meeting, PA) for occludin.
Animals
Studies were performed at 2 institutions: the Medical College of Georgia (Augusta, GA) and
the Cleveland Clinic (Cleveland, OH). Female C57BL6 mice (6–8 weeks old) were
purchased from Jackson Laboratory (Bar Harbor, ME) or the National Cancer Institute
(NCI, Frederick, MD). All mice were housed, maintained, and studied in accordance with
approval from the National Institutes of Health (NIH), Medical College of Georgia, and/or
the Cleveland Clinic Institutional Animal Care and Use Committee. Upon arrival, the
animals were acclimated, and during this time, the animals had access ad libitum to tap
water and regular unsterilized food. The animals were divided into 8 treatment groups and
housed together with 4 mice per cage (see below). Feeding trials were repeated for adequate
statistical power.
Antibiotic-Free Groups
Group 1 control (provided with sodium bicarbonate, plain broth, or saline for 7
days)
Group 2 LGG group (provided 106 colony-forming units [CFU] LGG for 7 days)
Group 3 Tributyrin group (provided 5 mM tributyrin for 7 days)
Group 4 LGG and tributyrin group (provided 106 CFU LGG and 5 mM tributyrin
for 7 days)
Antibiotic Therapy Groups
Group 5 metronidazole, neomycin sulfate, vancomycin (MNV) group only control
(provided with sodium bicarbonate, plain broth, or saline for 7 days)
Group 6 LGG group (provided 106 CFU LGG for 7 days)
Group 7 Tributyrin group (provided 5 mM tributyrin for 7 days)
Group 8 LGG and tributyrin group (provided 106 CFU LGG and 5 mM tributyrin
for 7 days)
Fresh stool samples were obtained on days 0 and 7. Following antibiotic therapy and
probiotic and tributyrin treatments (see below), mice were euthanized and the proximal
jejunum, terminal ileum, and proximal and distal colon were removed for preparation of
RNA and tissue sections.
Antibiotic Delivery
Antibiotics were provided as described previously.29 For antibiotic treatment, mice were
provided metronidazole (1 g/L), neomycin sulfate (500 mg/L), and vancomycin (1 gm/L) in
their water supply daily for 7 days. The water supply for both the antibiotic-treated and
control (antibiotic-free) groups contained 15% sucrose concentration to encourage
consumption. The amount of water consumed was recorded daily, and animal weight was
measured and recorded on days 0, 3, and 7 of the treatments.
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Oral Inoculation of LGG and Assay for Fecal Excretion
The colonization of mice with LGG was performed as described previously for other
bacteria.30,31 Briefly, 6- to 8-week-old C57BL/6 mice were inoculated orally daily for 7
days throughout the antibiotic therapy with LGG as follows. Single-colony LGG was
cultured in MRS broth at 37°C in an atmosphere of 5% (v/v) CO2 in air for 18–20 hours
prior to the inoculation. Mice were given 0.15 mL of 5% sodium bicarbonate by oral gavage
to buffer stomach acidity. The mice were then provided a dose of 1 × 106 CFU in 0.15 mL
LGG broth by oral gavage. Control animals received sodium bicarbonate and LGG broth
only. Tributyrin (5 mM/L) was provided in a similar manner as the LGG. One 0.15-mL
bolus was provided for the mice that received both tributyrin and LGG. Colonization with
LGG was determined by viable counts of LGG bacteria in fecal pellets, which was
enumerated on selective media (MRS agar; Sigma-Aldrich). The presence of the most
commonly encountered aerobic and facultative anaerobic bacteria was determined by viable
counts of bacteria in fecal pellets enumerated on Mueller Hinton agar (Sigma-Aldrich).
RT-PCR
RNA was prepared as previously described from antibiotic-treated and control mouse ileum
and colon, which were used for RT-PCR.32 The PCR primers for gene-specific products
were designed based on the nucleotide sequences available in GenBank (Table 1). The level
of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) was
used as the internal control in RT-PCR. PCR products were size fractionated on agarose
gels. Bands were visualized by ethidium bromide signals quantified using the STORM
phosphorimaging system (Global Medical Instrumentation, Inc, Ramsey, MN). RT-PCR was
carried out with 3 or 4 biological replicates, and PCR was repeated at least twice with each
RNA sample. The band intensity of each PCR product was normalized using GAPDH
mRNA as an internal control.
Immunofluorescence
Cryosections of mouse intestinal sections were fixed at room temperature in 4%
paraformaldehyde for 20 minutes at room temperature and then washed with phosphate-
buffered saline (PBS). Sections were then blocked with 2% bovine serum albumin (diluted
in PBS) containing 0.1% Triton X-100 for 1 hour, followed by overnight incubation at 4°C
with the primary antibody (anti-SLC5A8, 1:2000; anti-NHE3, 1:1000; anti-occludin, 1:50;
or anti–ZO-1, 1:100). Negative control sections were treated identically except that primary
antibody was substituted with PBS for overnight incubation. All sections were rinsed with
PBS (3 times for 5 minutes each), incubated with the fluorochrome-conjugated secondary
antibody for 2 hours in the dark at room temperature (for detection of SLC5A8 labeling,
sections were incubated with 1:250 goat anti–rabbit IgG Alexa Fluor 568; ZO-1 and NHE3
with 1:250 goat anti–rabbit IgG Alexa Fluor 488; occludin with 1:250 goat anti–guinea pig
IgG Alexa Fluor 568; Invitrogen, Grand Island, NY), washed again in PBS, and mounted
with VECTASHIELD containing DAPI and antifade reagent (Vector Laboratories,
Burlingame, CA). Fluorescent images were acquired using an inverted fluorescent
microscope (Leica, Cologne, Germany). No specific immunostaining was seen in sections
incubated with PBS.
Statistical Analysis
All values presented represent means ± standard error of the mean (SEM), with n = 4–12
experimental points (per site). Data were analyzed by analysis of variance using the general
linear models procedure (SAS Institute, Cary, NC) and per experimental site. Data were log-
transformed, if needed, to obtain a normal distribution. Follow-up comparisons were made
by least squares means testing. A
P
value of <.05 was considered statistically significant.
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Results
Effects of Treatments on Mouse Health
The mice tolerated the supplemental oral gavage treatments well. The dose of LGG was
sufficient to survive transit through the gut of mice; mice receiving LGG had 103–104 CFU
LGG in their fecal pellets compared with no detectable LGG in animals receiving saline,
broth, or only tributyrin (Table 2). All groups were colonized with bacteria, but the total
number of bacteria in the antibiotic-saline group was minimal. At the study start (day 0), for
animals enrolled at both study sites, mouse weights did not differ between treatment groups
(Table 3). However, by day 3, the mice in antibiotic-treated groups weighed less than those
not receiving antibiotics. Interestingly, despite consuming less water each day (Figure 1A),
for experiments conducted at the Cleveland Clinic, tributyrin restored body weight to that of
antibiotic-free mice by day 7. Mice mortality was highest in the antibiotic-treated broth/
saline groups (n = 2–3) compared with any other treatment group (n ≤ 1). Following 7 days
of treatments, the gross appearance of the cecum in animals receiving antibiotics was
visually markedly enlarged compared with the antibiotic-free groups (Figure 1B). Tributyrin
and, to a lesser extent, LGG supplementation reduced the antibiotic enlargement of the
cecum.
Expression of Butyrate Receptor GPR109A and Butyrate Transporter SLC5A8
We investigated whether the provision of antibiotics influences the expression of the
butyrate transporter SLC5A8 and the butyrate receptor GPR109A in the intestinal tract and
if LGG and/or tributyrin supplementation affects these changes. The steady-state levels of
SLC5A8 and GPR109A mRNA in the colon and ileum were reduced markedly in mice
receiving antibiotics compared with antibiotic-free mice (Figure 2A, B). In the antibiotic-
saline–treated mice, there was an 80% and 53% reduction in mRNA expression for the
butyrate receptor and transporter, respectively. Supplementation with LGG and/or tributyrin
prevented the reduced butyrate receptor and transporter mRNA expression. Protein
expression for the butyrate transporter SLC5A8 was predominantly seen on the lumen-
facing apical membrane of the ileal and colonic epithelial cells in antibiotic-free mice
(Figure 2C). The immunoreactive SLC5A8 was visibly reduced in antibiotic-saline–treated
mice. These changes were absent in mice supplemented with tributyrin.
Expression of Ion Exchangers SLC26A3 and NHE3 and Water Channel AQP4
SLC26A3 is an anion exchanger, mediating chloride-bicarbonate exchange and thus serving
an important role in electrolyte absorption in the intestinal tract. NHE3 is a sodium-
hydrogen ion antiporter. AQP4 is a water channel responsible for water reabsorption in the
gut. Both ion exchangers and water channel are expressed predominantly in the apical
membrane of the intestinal enterocytes.33 Steady-state levels of SLC26A3, AQP4, and
NHE3 mRNA were reduced by 47%, 40%, and 20%, respectively, in antibiotic-saline–
treated mice (Figure 3A–C). There was no difference in the mRNA levels of SLC26A3 and
NHE3 in the antibiotic-treated mice that received any of the 3 supplement treatments—
LGG, tributyrin, or LGG/tributyrin—compared with all the antibiotic-free mouse groups.
AQP4 mRNA expression was protected in the antibiotic-treated animals receiving LGG but
not the animals receiving tributyrin (Figure 3C). NHE3 protein was expressed in the apical
membrane of ileal and colonic epithelial cells in antibiotic-free mice (Figure 3D). The
staining intensity for NHE3 decreased in antibiotic-saline treated mice, but the intensity was
not affected in antibiotic-treated mice receiving tributyrin supplementation.
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Expression of Tight Junction Proteins Zonula Occludens and Occludin
Butyrate is known to have an important role in maintaining gut integrity.34,35 Since
antibiotic therapy negatively alters butyrate-producing bacteria and likely butyrate levels in
the gut lumen, as well as expression levels of genes and proteins involved with gut
physiology, we tested the hypothesis that antibiotics also would alter proteins integral to
maintaining gut integrity. Immunofluorescence localization of ZO-1 and occludin
demonstrated an intact tight junctional protein network in the ileum, proximal colon, and
jejunum (not shown) in antibiotic-free mice (Figure 4). The staining intensity for ZO-1 and
occludin was reduced in the antibiotic-saline–treated mice. Interestingly, tight junction
protein staining intensity was preserved in mice receiving antibiotics and supplemental
tributyrin.
Discussion
The present study evaluated the effect of LGG and tributyrin oral supplementation on
antibiotic therapy–induced changes in expression levels of water and electrolyte exchangers
and intestinal epithelial cell permeability markers. Although probiotics, such as LGG, are
known to decrease the duration and severity of symptoms of AAD, the mechanisms are not
fully understood. We demonstrated that simultaneous treatment with LGG and tributyrin
prevents antibiotic-induced downregulation of genes and proteins involved with intestinal
fluid and electrolyte homeostasis and intestinal barrier function.
The suppression and elimination of microbial pathogens by antibiotics is a time-tested
approach in medical management. Recent studies have highlighted the profound changes in
microbial populations that result from applications of antimicrobial agents. AAD is a
significant adverse effect of antimicrobial administration. A critical factor in the
pathogenesis of AAD is believed to be an alteration in the normal GI microbiota.9 Changes
in the human-associated microbiota are usually temporary, but long-term microbial
population fluctuations have been reported in healthy adults.36 Vancomycin, neomycin, and
metronidazole eliminate Gram-positive, Gram-negative, and anaerobic commensal
bacteria.29 Administration of this antibiotic combination not only depletes and alters the gut
microbiota community richness and structure,37 thus providing space and nutrients for
opportunistic pathogenic bacteria, but also impairs mucosal innate immune defenses.29 This
antibiotic combination is not reported to cause diarrhea in mice, but we chose this antibiotic
combination due its ability to deplete the gut microbiota and therefore create a clinically
relevant “germ-free” gut microenvironment. The antibiotic therapy in this study did deplete
total bacteria in fecal pellets. Our results corroborate previous data in GF mice showing that
altered gut microbiota by antibiotic therapy affects the expression levels of genes and
proteins involved with water and electrolyte absorption in the gut.19,27–29
Alterations in commensal gut microbiota impair the concentration and distribution of
organic compounds such as carbohydrates, SCFAs, and bile acids.9 The most numerous
butyrate-producing bacteria in the gut have been found to belong to the clostridial clusters
IV and XIVa; absences of these commensals were identified following antibiotic treatment
for sinusitis.9 Since butyrate is an important molecule for gut homeostasis, it is likely that
antibiotic therapy compromises butyrate actions in the intestine by altering the levels of
butyrate-producing bacteria, thus limiting the availability of luminal effects of butyrate.
Although the literature supports provision of probiotics, specifically LGG, for mitigating
AAD, the end products of most probiotics do not include butyrate, which raises questions
about their effectiveness in promoting bowel health in adults.38 There are no reports in the
literature for providing tributyrin to improve symptoms of AAD caused by broad-spectrum
antibiotics.
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The experimental treatments were carefully selected for their significance to current
literature and future clinical application. LGG was selected as a clinically relevant,
commercially available probiotic that is well studied in AAD.23–26 LGG administration is
safe and well tolerated; the dosage provided augmented colonization of gut microbiota in
treated animals. Tributyrin has been provided at various dosages in vitro and in vivo, in
animals and humans, with the goal end point of plasma butyrate levels being >0.5 mM.12
The molar ratio of propionate and acetate in the blood is much higher than butyrate at
physiologic conditions.11 This is because butyrate is the primary energy source for the
colonic mucosal enterocytes, accounting for 70% of their oxygen consumption; butyrate is
preferentially oxidized over propionate and acetate in a ratio of 90:30:50.10 In vitro studies
have shown beneficial effects of butyrate when provided at concentrations of 1–10 mM/
L.11,32 Although the in vivo physiologic concentration is proposed to be 10–15 mM/L,
knowing that tributyrin can have cytotoxic effects and that 1 mole of tributyrin yields 3
moles of butyrate, we chose to dose tributyrin at 5 mM/L initially to determine potential
beneficial effects. Indeed, we found this dose of tributyrin to be well tolerated by mice and
to have positive benefits.
SCFAs, including butyrate, are the end products of anaerobic bacterial fermentation of
undigested carbohydrates in the distal intestine.14 The total concentration and relative molar
concentrations of individual SCFAs are greatly influenced by the diet. The average
intraluminal concentration of SCFAs is estimated to be between 100 and 170 mM, with
butyrate representing approximately 15% of the colonic SCFAs.10 Although it is feasible to
manipulate different dietary substrates to achieve desired ratios of SCFAs, the composition
of the commensal microbiota is an important factor if a particular SCFA is desired to be
present in the colon.14 Since a decreased number of total gut microbiota, particularly
butyrate-producing bacteria, are noted with antibiotic therapy, we opted to provide butyrate
directly to ensure its availability due to the uncertainty that modulation of dietary fiber/
carbohydrate may or may not yield adequate butyrate during antibiotic therapy. Butyrate has
been provided via several routes (eg, intravenously, rectally, and orally), all having
limitations.10 Tributyrin overcomes many of the problems of the parent compound. Provided
orally, tributyrin is hydrolyzed by pancreatic and gastric lipases, yielding glycerol and 3
butyrate molecules10; has a longer half-life compared with IV delivery16; and is safe when
provided at lower doses but can be cytotoxic at higher doses (eg, in vivo, ≥ 10.3 g/kg; in
vitro, >10 mM).10,22,32,39,40 Although the liberated butyrate molecules can exit the proximal
intestinal lumen by passive diffusion, butyrate transporters are also present in the proximal
intestinal tract and therefore available for active transport of butyrate across the apical
membrane.17,18,20 Interestingly, although tributyrin is rapidly hydrolyzed in the proximal
intestinal tract, we found beneficial effects on mRNA and protein expression in the distal
intestine. Others have shown similar benefits of not only a direct trophic effect of butyrate
provision but also trophic effects on unexposed adjacent intestinal tissue.11,41,42
Jejunotrophic effects of cecally infused SCFAs were mediated afferently by the autonomic
nervous system and associated with increased jejunal gastrin.43 It is also possible that, with
tributyrin administration orally, butyrate reaches the colon at sufficient concentrations to
elicit the changes on gene expression. Since the mechanisms of butyrate action on
colonocytes at least partly involve inhibition of histone deacetylation, a process that is seen
at micromolar concentrations of butyrate, such levels of butyrate can easily be reached in the
colon with oral administration of tributyrin.
In these experiments, the animals tolerated oral tributyrin supplementation well without any
adverse events. Antibiotic-treated animals consumed significantly less water than the
antibiotic-free mice for experiments performed at both research sites, likely because the
antibiotics are unpalatable and have a bitter taste. Adverse effects for oral delivery of these
antibiotics include nausea, diarrhea, appetite loss, and stomach cramps. Although there was
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no significant difference in body weight for antibiotic-treated mice supplemented with LGG
and/or tributyrin for the experiments performed at the Medical College of Georgia, the mice
treated at the Cleveland Clinic receiving tributyrin were comparable in body weight and
activity level to the antibiotic-free animals at day 7. Similar effects have been noted in
clinical studies in which cancer patients treated with tributyrin reported an improved sense
of well-being, appetite, and pain control.39
It is known that GF mice have striking abnormalities that interfere with normal histologic
development of the intestinal epithelium, which brings about a gross enlargement of the
cecum.44,45 These abnormalities are rapidly corrected when GF animals are associated with
some components of the normal gut microbiota.44 These components may be not just the
bacteria but also fermentation end products (eg, SCFAs). The gut microbiota uses specific
glycoconjugates on the enterocyte surface as receptors to colonize a region of the gut,
lending these glycoconjugates to likely determine the colonization of gut microbiota.46
Modification of glycosylation could feasibly result in an opportunity for pathogens to attach
on the luminal surface, enabling colonization and invasion of the gut barrier, leading to
inflammatory responses. Our data corroborate GF data in that an antibiotic treatment known
to alter and deplete the gut microbiota enlarges the cecum of mice. Particularly noteworthy
is that LGG and/or tributyrin supplementation diminishes this response, but the response is
more dramatic with tributyrin. Original experiments with GF mice recolonized with
lactobacilli and anaerobic streptococci corrected cecal enlargement, but slowly and
imperfectly; the response was more rapid and remarkable when GF mice were associated
with
Bacteroides
bacteria.44 Specific strains of bacteria (eg,
Bacteroides thetaiotaomicron
)
have been shown to modulate the expression of host genes related to important intestinal
functions, including nutrient absorption, mucosal barrier function, and intestinal
maturation.27 Likewise, similar effects are known to occur with butyrate provision.12 Our
data show that tributyrin alone can exhibit positive effects on the cecum of antibiotic-treated
mice. Butyrate is a major metabolic fuel for colonocytes and promotes a normal phenotype
in these cells. Butyrate interacts not only with its transporters but also with butyrate
receptors localized in the apical membrane of the intestine. GPR109a is a butyrate receptor
known to have anti-inflammatory properties upon interaction with its ligand.32 It is
unknown from these data whether butyrate provided during antibiotic therapy produces a
less inflammatory environment through interaction with GPR109a, causing a pattern shift in
glycoconjugate expression, thus decreasing the cecum size of antibiotic-treated mice, but
this may warrant further investigation.
As an inhibitor of histone deacetylases, butyrate has the ability to influence gene expression
in the colon. This can lead to hyperacetylation of histones that is followed by increased gene
expression. Two potential mediators of the biologic effects of butyrate are SLC5A8, an Na+-
coupled transporter for butyrate, and GPR109A, a G-protein–coupled receptor; both are
expressed in the lumen-facing apical membrane of colonic epithelial cells.18,19,32 Prior work
shows that the gut microbiota is obligatory for optimal expression of these 2 genes and their
protein products as well as hundreds of others.19 Fascinating and clinically relevant is that
our results corroborate this prior work; gene expression of the butyrate transporter and
receptor, several ion exchangers, and a water channel were significantly downregulated in
antibiotic-treated animals. Supplemental treatments, LGG, tributyrin, and their combination
were able to preserve the expression of these genes and/or their protein products. The dose
of LGG provided was able to survive and reach the colon as indicated by growth patterns in
the fecal pellets of animals supplemented with LGG. It is thus likely that supplemental LGG
contributed to a positive influence on gene and protein expression. Appealing is that
provision of tributyrin alone was also able to maintain gene and protein expression. Others
have reported transporter regulation via luminal nutrient sensing through interaction with
cell surface receptors.21,47 GPR109A, which has a higher affinity for butyrate than SLC5A8,
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was recently associated with the trafficking of monocarboxylate transporters to the apical
membrane in response to the presence of butyrate.21 It is unknown from these data how
much of the liberated butyrate from tributyrin supplementation was able to reach the colon.
However, only micromolar concentrations of butyrate are needed to inhibit histone
deacetylases, even though millimolar concentrations are needed to activate GPR109A. It is
likely that butyrate liberated from tributyrin reaches the colon at least at levels sufficient to
affect histone acetylation and hence gene expression.
During acute cholera, in addition to a decrease in colonic anaerobes, there is also reduced
production of SCFAs and decreased absorption of electrolytes.33 SLC26A3, NHE3, and
AQP4 are important for water and electrolyte homeostasis. Intestinal ion transport and the
pathophysiology of diarrhea are complex and reviewed elsewhere.35 SLC5A8 functions as
an Na+-coupled transporter for butyrate with an Na+/butyrate stoichiometry of 2:1;
therefore, the transporter may promote Na+ absorption in the colon in the presence of the
bacterial fermentation product butyrate. GPR109A is coupled to Gi, the inhibitory G-
protein. Activation of the receptor by butyrate or other agonists leads to a decrease in
intracellular levels of cAMP. This cyclic nucleotide is one of the major signaling molecules
in the intestinal tract that controls electrolyte and water absorption; elevation of intracellular
levels of cAMP in the intestinal tract causes secretory diarrhea.48 Studies have investigated
the effects of SCFAs on enterotoxin-induced electrolyte and fluid secretion. SCFAs,
particularly butyrate, reduce cholera toxin–induced water and electrolyte secretion.49 Our
data are suggestive that benefits, such as decreased duration and severity of diarrheal
symptoms, associated with LGG and/or butyrate supplementation during antibiotic therapy
are linked with preservation of genes and proteins involved with electrolyte and water
homeostasis.
In addition to its role in stimulating intestinal NaCl absorption and inhibiting the
prosecretory action of several cAMP-generating secretagogues, butyrate is also known to
improve the barrier function of the gut epithelia.34 The barrier function of the intestinal
mucosa is critical to maintain beneficial relationships between the host and the gut
microbiota. The tight junction between the mucosal epithelial cells is the primary physical
barrier in the intestines. The tight junction is composed of several transmembrane proteins
such as claudins, zonula occludens, and occludin.35 Butyrate promotes transepithelial
resistance and reduced permeability, which is attributed to reorganization of the tight
junction molecules ZO-1 and occludin.50 Our data reveal that antibiotic therapy disrupts the
organization and expression of tight junction proteins throughout the intestinal tract and that
tributyrin supplementation preserves the epithelial barrier. Beneficial effects of butyrate
have been suggested in acute gastroenteritis, cholera, congenital chloride diarrhea, and
inflammatory bowel disease.34 The link with butyrate and inflammatory bowel disease
predominantly surrounds the involvement of tight junction protein alterations. Thus,
preservation of tight junction proteins with tributyrin therapy during antibiotic therapy may
also be clinically relevant.
In summary, this work indicates that oral supplementation with LGG and/or tributyrin
counteracts the negative effects induced by antibiotic therapy on expression of genes and
their protein products involved with water and electrolyte absorption and gut barrier
function in the intestinal tract. The fact that tributyrin alone was able to exhibit these
beneficial effects is intriguing as many factors can impair efficacy of probiotic provision
(eg, viability, dosing, timing, colonization, storage temperature). These issues surrounding
the efficacy of probiotic therapy should be considered if a treatment effect of probiotics is
not found. If tributyrin supplementation alone achieves the desired outcomes of improved
gut integrity and preservation of genes and proteins involved with water and electrolyte
homeo-stasis, then this therapy may prove more attractive to clinicians and patients. Further
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work investigating whether the same effects are found with various antibiotics and/or their
dosing or whether benefits are found in humans would be interesting.
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Figure 1.
Treatment effects on mouse health. (A) Water consumed (mL) per day per mouse. Different
letters over bars indicate a statistically significant difference (
P
< .05). (B) Representative
photo of mouse cecum on day 7. Control and antibiotic ± tributyrin,
Lactobacillus
GG
(LGG), and tributyrin/ LGG treatment groups. Data are expressed as mean ± SEM.
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Figure 2.
Expression levels of butyrate receptor and transporter. (A, B) Levels of messenger RNA
(mRNA) for GPR109A and SLC5A8 in the proximal colon of mice treated with or without
antibiotics,
Lactobacillus
GG (LGG), tributyrin, or combined LGG and tributyrin. Different
letters over bars indicate a statistically significant difference (
P
< .0004). (C) Levels of
SLC5A8 protein (red) and its localization in the ileum and proximal colon of mice ±
antibiotics and ± tributyrin. DAPI (blue) was used as a nuclear stain. Magnification of ×40
for ileum and ×20 for proximal colon.
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Figure 3.
Expression levels of ion exchangers and water channel. (A–C) Levels of messenger RNA
(mRNA) for SLC26A3, NHE3, and AQP4 in the ileum of mice treated with or without
antibiotics,
Lactobacillus
GG (LGG), tributyrin, or combined LGG and tributyrin. Different
letters over bars indicate a statistically significant difference (
P
< .05). (D) Levels of NHE3
protein (green) and its localization in the jejunum and ileum of mice ± antibiotics and ±
tributyrin. DAPI (blue) was used as a nuclear stain. Magnification of ×40 for jejunum and
ileum. AQP4, aquaporin 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Figure 4.
Expression of tight junction proteins. Levels of zonula occludens 1 (ZO-1; green) and
occludin (red) proteins and their localization in the ileum (A) and proximal colon (B) of
mice ± antibiotics and ± tributyrin. DAPI (blue) was used as a nuclear stain. Magnification
of ×40 for ileum and ×20 for proximal colon.
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Table 1
List of Primers Used in This Study
Gene (Genbank
Accession No.) Primer Sequence Position Product Size, bp
Annealing
Temperature,
Cycle No.
GPR109A (NM_177551) Sense: 5′-CGAGGTGGCTGAGGCTGGAATTGGGT-3′325–347 646 60°C, 30
Antisense: 5′-ATTTGCAGGGCCATTCTGGAT-3′950–970
SLC5A8 (NM_145423) Sense: 5′-GGGTGGTCTGCACATTCTACT-3′371–392 351 60°C, 30
Antisense: 5′-GCCCACAAGGTTGACATAGAG-3′700–721
NHE3 (NM_009700) Sense: 5′-TGG CCG GGC TTT CGA CCA CA-3′1425–1445 248 60°C, 30
Antisense: 5′-GGG ACC CAC GGC GCT CTC CCT-3′1651–1672
AQP4 (NM_009700) Sense: 5′-ACTATTTTTGCCAGCTGTGATTCCAAACGA-3′517–547 423 61°C, 24
Antisense: 5′-TTCCCCTTCTTCTCTTCTCCACGGTCA-3′912–939
SLC26A3 (NM_021353) Sense: 5′-CACAAATTCAGAAGACGAACATCGCAGACC-3′734–764 607 61°C, 24
Antisense:5′-GCATCAGCATTCCCTTTAAGTTTCCGAGTG-3′1310–1340
GAPDH (NM_008084) Sense: 5′-CTCTGGAAAGCTGTGGCGTGAT-3′567–589 122 61°C, 24
Antisense: 5′-CATGCCAGTGAGCTTCCCGTTCAG-3′664–688
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Table 2
Final Fecal Bacterial Patterns
Treatment Groups LGG Total Bacteria
Antibiotic free, saline ND +++
Antibiotic free, LGG 1.3 × 104 CFU +++
Antibiotic free, tributyrin ND +++
Antibiotic free, LGG/tributyrin 1.3 × 105 CFU +++
Antibiotic, saline ND +
Antibiotic, LGG 2.5 × 104++
Antibiotic, tributyrin ND ++
Antibiotic, LGG/tributyrin 1 × 103 CFU ++
CFU, colony-forming units; LGG,
Lactobacillus
GG; ND, none detected; +, ≤ 102 CFU; ++, 103 CFU; +++, ≥ 104 CFU.
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Table 3
Body Weight, g
Treatment Groups Day 0 Day 3 Day 7
Medical College of Georgia
Antibiotic free, broth 23.5 ± 0.5a (n = 7) 24.0 ± 0.6a (n = 7) 23.9 ± 0.7a (n = 7)
Antibiotic free, LGG 25.8 ± 0.5a (n = 8) 25.4 ± 0.6a (n = 8) 25.1 ± 0.7a (n = 8)
Antibiotic free, TB 24.9 ± 0.9a (n = 8) 24.9 ± 0.7a (n = 8) 25.5 ± 0.8a (n = 8)
Antibiotic free, LGG/TB 24.8 ± 0.8a (n = 8) 24.5 ± 0.7a (n = 8) 22.5 ± 0.5a (n = 8)
Antibiotic, broth 24.6 ± 0.5a (n = 7) 21.0 ± 0.7b (n = 4) 20.5 ± 0.5b (n = 4)
Antibiotic, LGG 24.0 ± 1.0a (n = 8) 18.8 ± 1.0b (n = 8) 19.6 ± 1.4b (n = 8)
Antibiotic, TB 24.3 ± 0.7a (n = 8) 19.6 ± 0.8b (n = 8) 19.9 ± 0.9b (n = 7)
Antibiotic, LGG/TB 24.9 ± 0.8a (n = 8) 20.1 ± 0.7b (n = 8) 19.7 ± 0.6b (n = 8)
Cleveland Clinic
Antibiotic free, saline 16.2 ± 0.8a (n = 6) 15.9 ± 0.5a (n = 6) 16.7 ± 0.6a (n = 6)
Antibiotic free, TB 16.5 ± 0.5a (n = 6) 16.5 ± 0.4a (n = 6) 16.8 ± 0.5a (n = 6)
Antibiotic, saline 16.2 ± 0.3a (n = 12) 14.2 ± 0.4b (n = 11) 14.0 ± 0.5b (n = 10)
Antibiotic, TB 16.0 ± 0.6a (n = 10) 14.4 ± 0.6b (n = 10) 14.6 ± 0.7a (n = 9)
LGG,
Lactobacillus
GG; TB, tributyrin. Values with different superscripts at each time point are significantly different with
P
< .05.
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