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JPET #160093
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Administration of ampicillin elevates hepatic primary bile acid synthesis through
suppression of ileal FGF15 expression
Masaaki Miyata, Yuki Takamatsu, Hideaki Kuribayashi and Yasushi Yamazoe
Division of Drug Metabolism and Molecular Toxicology, Graduate School of
Pharmaceutical Sciences, Tohoku University, Sendai, Japan (M.M.,Y.T., H.K., Y.Y.)
CRESCENDO, Tohoku University 21st Century “Center of Excellence” Program,
Sendai, Japan (Y.Y.)
JPET Fast Forward. Published on September 18, 2009 as DOI:10.1124/jpet.109.160093
Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: hepatic bile acid synthesis regulated by enterobacteria
Corresponding author: Masaaki Miyata, Ph.D.
Division of Drug Metabolism and Molecular Toxicology, Graduate School of
Pharmaceutical Sciences, Tohoku University
6-3, Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan
TEL: +81-22-795-6829
FAX: +81-22-795-6826
E-mail: miyata@mail.pharm.tohoku.ac.jp
Number of text pages: 30
Number of tables: 1
Number of figure: 5
Number of references: 40
Number of words in the Abstract: 214
Number of words in the Introduction: 503
Number of words in the Discussion: 1136
Abbreviations:ABPC, ampicillin; SHP, small heterodimer partner; FGF15, fibroblast
growth factor 15; FXR, farnesoid X receptor; LCA, lithocholic acid; TDCA,
taurodeoxycholic acid; BDL, bile duct-ligated; BC, bacitracin; NM, neomycin; SM,
streptomycin; PON1, paraoxonase 1; TβMCA, tauro-β-muricholic acid; TUDCA,
tauroursodeoxycholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic
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acid; PCR, polymerase chain reaction.
Recommended section: Gastrointestinal, Hepatic, Pulmonary, and Renal
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Abstract
Administration of the antibacterial drug ampicillin (ABPC) significantly increased
hepatic bile acid concentrations. In the present study, we investigated the mechanisms
for the elevation of bile acid levels in ABPC-treated mice. Hepatic microsomal
cholesterol 7α-hydroxylation as well as CYP7A1 mRNA level were increased 2.0-fold
in ABPC-treated mice despite higher bile acid levels in the liver and small intestinal
lumen. A significant change in hepatic small heterodimer partner (SHP) mRNA level
was not observed in ABPC-treated mice, whereas a marked decrease in ileal fibroblast
growth factor 15 (FGF15) mRNA level was observed (3% of vehicle-treated mice).
These phenomena were also observed in mice co-treated with
bacitracin/streptomycin/neomycin, which are barely absorbed from the intestine.
Primary bile acid contents in the small intestinal lumen were increased in ABPC-treated
mice whereas secondary bile acid, deoxycholic acid (DCA) contents were reduced to
below detection limits (<0.01 μmol). In ABPC-treated mice, co-treatment with
tauroDCA (TDCA) reversed reductions in ileal FGF15 mRNA level. Ileal SHP mRNA
level was, however, not decreased in ABPC-treated mice. ABPC administration to
farnesoid X receptor (Fxr)-null mice also decreased ileal FGF15 mRNA levels and
secondary bile acid content in the small intestinal lumen. These results suggest that
ABPC administration elevates hepatic primary bile acid synthesis, at least in part,
through suppression of ileal FGF15 expression.
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Introduction
Administration of antibacterial drugs for infectious diseases can cause side effects
such as diarrhea and hepatic injury (Beaugerie and Petit, 2004; Polson, 2007).
Administration of antibacterial drugs decreases enterobacterial populations, which have
a crucial role in symbiosis with the host through metabolism of endogenous compounds
such as bile acids as well as exogenous compounds. Antibacterial drug-mediated
alteration of enterobacterial populations is therefore an important issue concerning
human health and disease.
Some enterobacteria catalyze 7α-dehydroxylation of primary bile acids. This results
in the production of more hydrophobic secondary bile acids such as deoxycholic acid
(DCA) and lithocholic acid (LCA). In general, killing of enterobacteria by
administration of antibacterial drugs reduces secondary bile acids in the bile acid pool
(Hofmann, 1977). Administration of ampicillin (ABPC) significantly reduces DCA
levels in the bile acid pool of humans (Berr et al., 1996), but the physiological influence
of the reduction of DCA production remains unclear.
Bile acids play an essential part not only in the intestinal absorption of lipids and
vitamins, but also in the activation of nuclear receptors and cell signaling pathways. It is
well recognized that bile acids serve as key signaling molecules that regulate a network
of metabolic pathways, including lipid and glucose as well as bile acids (Hylemon et al.,
2009). The size of the bile acid pool is therefore tightly controlled by negative feedback
regulations through several nuclear receptor and cell signaling pathways. The bile acid
pool size is mainly regulated by the synthesis of hepatic bile acid and reabsorption of
intestinal bile acid (Hofmann, 1994). The specific mRNA level of CYP7A1, the rate-
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limiting enzyme of the synthesis of hepatic bile acid, is negatively regulated by several
bile acid-mediated signaling pathways (Chiang, 2009). Initially, it was reported that
CYP7A1 transcription is suppressed by the hepatic FXR/SHP signaling pathway
activated by bile acids (Lu et al., 2000; Goodwin et al., 2000). However, the expression
of hepatic CYP7A1 and activity of cholesterol 7α-hydroxylase are elevated in bile
duct-ligated (BDL) mice in which hepatic bile acid concentration is increased (Dueland
et al., 1991). Furthermore, intraduodenal, but not intravenous or portal, administration
of bile acid significantly suppresses CYP7A1 expression (Pandak et al., 1991; Nagano
et al., 2004). These results suggested a role of the intestine in feedback regulation of the
synthesis of hepatic bile acids. Hepatic CYP7A1 levels are also negatively regulated by
the endocrine action of intestine-produced FGF15 (human ortholog, FGF19) signaling
pathway that is induced by intestinal bile acid-mediated FXR activation (Inagaki et al.,
2005). Thus, intestine-produced FGF15 signaling pathway, rather than hepatic FXR
pathway, is required for short-term repression of hepatic CYP7A1 (Kim et al., 2007).
In our preliminary experiment, we administered the antibacterial drug ABPC to
mice and observed elevation in hepatic bile acid concentration. In the present study, to
understand the mechanisms of this elevation, we focused on the intestinal role for
elevated primary bile acid synthesis. The present experiments suggest that ABPC
administration elevates hepatic primary bile acid synthesis, at least in part, through
suppression of expression of ileal FGF15.
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Materials and Methods
Materials
Ampicillin (ABPC) was purchased from Nacalai Tesque Inc. (Kyoto, Japan).
Bacitracin (BC) was purchased from Wako Pure Chemical Industries Ltd. (Osaka,
Japan). Neomycin trisulfate (NM) was purchased from MP Biomedicals Inc (Irvine,
CA). Streptomycin sulfate (SM), cholic acid (CA), taurocholic acid (TCA),
chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (TCDCA), deoxycholic
acid (DCA), taurodeoxycholic acid (TDCA), lithocholic acid (LCA) and
taurolithocholic acid (TLCA) were purchased from Sigma-Aldrich (St. Louis, MO).
β-Muricholic acid (βMCA), tauro-β-muricholic acid (TβMCA), ursodeoxycholic acid
(UDCA), tauroursodeoxycholic acid (TUDCA), etiocholan-3α, 17β-diol
(5β-androstan-3α, 17β-diol) and 7α-hydroxycholesterol were purchased from Steraloids
(Newport, RI). L-column ODS (2.1 × 150 mm) was obtained from Chemicals
Evaluation and Research Institute (Tokyo, Japan). Enzymepak 3α-HSD column was
purchased from Jasco (Tokyo, Japan).
Animal treatment and sample collection
C57BL/6N male mice (Charles River Japan Inc., Yokohama, Japan), farnesoid X
receptor (Fxr)-null mice (Sinal et al., 2000), and wild-type mice were housed under a
standard 12-h light /dark (9:00 AM to 9:00 PM) cycle. Before experimentation, mice
were fed standard rodent chow (CE-2; Clea Japan Inc., Tokyo, Japan) and water ad
libitum for acclimatization. Age-matched groups of 8- to 9-week-old mice were used for
all experiments. Mice were administered (p.o.) ABPC (100 mg/kg body weight,
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dissolved in saline) at 9:00 AM, bacitracin/neomycin/streptomycin (BC/NM/SM) (200
mg/kg body weight each agent, dissolved in saline) or ABPC + TDCA (100 or 500
mg/kg body weight, dissolved in saline) for 3 days. They were fasted for the last 12 h to
control bile acid secretion into the duodenum and euthanized 24 h after the last
administration (9:00 AM). For CA-treatment experiment, mice were fed a control diet
(CE-2) or control diet supplemented with 1% (w/w) CA for 6 days and euthanized at
9:00 AM. Liver samples were taken for biochemical assays. The small intestinal lumen
was washed with phosphate-buffered saline (PBS) and the washed solution collected.
All experiments were performed in accordance with the Guidelines for Animal
Experiments of Tohoku University.
Quantification of bacterial DNA in feces
The fecal content of bacteria in stools collected over the last 24 h in vehicle and
ABPC-treated mice was determined. DNA was extracted from 200 mg of feces using a
QIAamp DNA Stool Mini Kit (QIAGEN K.K., Tokyo, Japan). The extracted DNA was
diluted in Tris-EDTA buffer, and the purity confirmed by measuring absorbance at 260
and 280 nm using a DU800 spectrophotometer (Beckman Coulter, Fullerton, CA).
These DNA samples were subjected to real-time polymerase chain reaction (PCR) using
SYBR Green 1 with an ABI PRISM 7000 Sequence Detection System (Applied
Biosystems, Foster City, CA). Relative DNA levels were calculated by the comparative
threshold cycle method. The following specific forward and reverse primers were used
for real-time quantitative PCR: Bacteroides flagilis sense,
5’-CTGAACCAGCCAAGTAGCG-3’ and antisense,
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5’-CCGCAAACTTTCACAACTGACTTA-3’; Clostridium scidens sense,
5’-GCAACCTGCCTGCACT-3’ and antisense, 5’-ACCGAATGGCCTTGCCA-3’;
Clostridium sordelli sense, 5’-TCGAGCGACCTTCGG-3’ and antisense,
5’-CACCACCTGTCACCAT-3’.
Bile acid composition in the liver and small intestinal lumen
Bile acid composition in the liver and small intestinal lumen was measured by
high-performance liquid chromatography (HPLC) as described previously (Kitada et al.,
2003; Miyata et al., 2006). The contents of βMCA, TβMCA, UDCA, TUDCA, CA,
TCA, CDCA, TCDCA, DCA, TDCA, LCA and TLCA were measured.
Cholesterol 7α-hydroxylase activity
Cholesterol 7α-hydroxylase activity was measured by HPLC. We modified the
previously reported method (Chiang, 1991). The reaction mixture consisted of 0.1 M
potassium phosphate, pH 7.4, 50 mM NaF, 5 mM DTT, 0.015% CHAPS
(3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate), 0.1 mM cholesterol and
0.5 mg of hepatic microsomes in a final volume of 0.5 mL. The mixture was
pre-incubated at 37°C for 5 min, and 10 μL of 100 mM NADPH was added to initiate
the reaction. After incubation at 37°C for 15 min, the reaction was terminated by
addition of 10 μL of 25% CA, and 2.5 nmol of 20α-hydroxycholesterol was added as an
internal standard. Then, one unit of cholesterol oxidase in 10 mM potassium phosphate
buffer, pH 7.4, containing one mM DTT and 20% glycerol was added to the reaction
mixture. After incubation at 37°C for 10 min, the reaction was stopped by the addition
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of 3 mL of ice-cold hexane. The mixture was then centrifuged at 2,000×g, and 2 ml of
supernatant was collected. The collected supernatant was evaporated to dryness,
dissolved in 100 μL of acetonitrile/methanol (92:8), and then analyzed by HPLC. The
metabolites were separated at 37°C with a ChemcoPak® HYPERSIL ODS-5 (4.6×250
mm) (Chemco Scientific Co. Ltd., Osaka, Japan). The metabolites were eluted using
acetonitrile/methanol (92:8) at a flow rate of 0.6 mL/min and monitored at 240 nm.
Analysis of mRNA levels
Total RNA was prepared from the liver and ileum using an RNAgents total isolation
system (Promega, Madison,WI). RNA concentration was determined by measuring
absorbance at 260 nm using a DU800 spectrophotometer (Beckman Coulter, Fullerton,
CA). Single-stranded cDNA were synthesized using an oligo (dT) primer and a
Ready-To-Go You-Prime First-Strand Beads kit (GE Healthcare Bio-Sciences Corp.,
Piscataway, NJ). These cDNA templates were subjected to real-time PCR using SYBR
Green 1 with ABI PRISM 7000 Sequence Detection System (Applied Biosystems).
Relative mRNA levels were calculated by the comparative threshold cycle method. The
following specific forward and reverse primers were used for real-time quantitative
PCR: CYP7A1 sense, 5’-AGCAACTAAACAACCTGCCAGTACTA-3’ and antisense,
5’-GTCCGGATATTCAAGGATGCA-3’; SHP sense,
5’-AAGATACTAACCATGAGCTCCG-3’ and antisense,
5’-GTCTTGGCTAGGACATCCAAG-3’; FGF15 sense,
5’-GAGGACCAAAACGAACGAAATT-3’ and antisense,
5’-ACGTCCTTGATGGCAATCG-3’; glyceraldehyde-3-phosphate dehydrogenase
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(GAPDH) sense, 5’-TGTGTCCGTCGTGGATCTGA-3’ and antisense,
5’-CCTGCTTCACCACCTTCTTGAT-3’; β-actin sense,
5’-ACCCTGTGCTGCTCACCGA-3’ and antisense,
5’-CTGGATGGCTACGTACATGGCT-3’.
Statistical analysis
Values are mean ± S.D. Data were analyzed by unpaired Student’s t-test or by
analysis of variance followed by Dunnett’s method using Prism 4.0 software (GraphPad
Software Inc., San Diego, CA) for significant differences between the mean values of
each group. p < 0.05 was considered to be statistically significant.
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Results
Level of hepatic bile acids and synthesis of primary bile acids in ABPC-treated
mice
When ABPC (100 mg/kg) corresponding to twice the clinical dose was
administered to mice for 3 days, the hepatic bile acid concentration was significantly
increased in ABPC-treated mice (2.5-fold relative to vehicle-treated mice) (Fig. 1A).
Analysis of hepatic bile acid composition revealed significant increases in primary bile
acid (βMCA, TβMCA and TCA) contents in ABPC-treated mice compared with
vehicle-treated mice (4.8-, 2.4- and 2.3-fold, respectively).
Hepatic bile acid synthesis was estimated to understand the mechanisms of
accumulation of hepatic bile acids. Hepatic microsomal activity of cholesterol
7α-hydroxylase, which catalyzes the rate-limiting step in bile acid synthesis, was
increased in ABPC-treated mice (2.0-fold relative to vehicle-treated mice)(Fig. 1B).
Influence of ABPC or CA treatment on CYP7A1, SHP and FGF15 mRNA levels
To identify the mechanistic basis for increased microsomal cholesterol
7α-hydroxylation in ABPC-treated mice, hepatic CYP7A1 mRNA levels were measured
by real-time PCR. CYP7A1 mRNA levels were significantly higher in ABPC-treated
mice compared with vehicle-treated mice (Fig. 2A). To further explore the mechanistic
basis for the increase in CYP7A1 mRNA levels despite the elevation of hepatic bile acid
levels, mRNA levels of the gene involved in the negative regulation of CYP7A1
expression were analyzed by real-time PCR. Significant changes in hepatic SHP
mRNA levels were not observed in ABPC-treated mice. For comparison, mRNA levels
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of the gene were measured in CA-fed mice in which elevation of hepatic bile acid levels
and the negative regulation of CYP7A1 expression are found. Consistent with previous
report (Sinal et al., 2000; Miyata et al., 2005), CYP7A1 mRNA levels were markedly
reduced in CA-fed mice (7% of control group), whereas mRNA levels of hepatic SHP
and ileal FGF15 were significantly increased in CA-fed mice (3- and 100-fold relative
to the control diet, respectively) (Fig. 2B). In ABPC-treated mice, marked decreases in
ileal FGF15 mRNA levels were observed (3% of vehicle-treated mice)(Fig. 2A).
Hepatic paraoxonase 1 (PON1) as well as CYP7A1 are also decreased by bile acids
acting via FXR to increase FGF15 expression (Gutierrez et al., 2006; Shih et al., 2006).
Significant increases in PON1 mRNA levels were also observed in the livers of
ABPC-treated mice (data not shown).
Influence of bacitracin/neomycin/streptomycin-treatment on hepatic bile acid
levels and gene expression
To test whether these changes in gene expression were found in mice treated with
other antibacterial drugs that are not absorbed from the intestine, mice were orally
treated with bacitracin/neomycin/ streptomycin (BC/NM/SM, 200 mg/kg each agent for
3 days) (Fig. 3). Treatment of mice with these antibiotics reduces levels of
enterobacteria to less than 0.001% (Kinouchi et al., 1993). Consistent with
ABPC-treated mice, hepatic bile acid levels were elevated in mice treated with
BC/NM/SM (Fig. 3A). Hepatic CYP7A1 mRNA level was increased in
BC/NM/SM-treated mice (2.5-fold relative to vehicle-treated mice) whereas ileal
FGF15 mRNA level was markedly decreased in BC/NM/SM-treated mice (10% of
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vehicle-treated mice) as well as ABPC-treated mice (Fig. 3B).
Influence of treatment by ABPC or CA on bile acid composition in the small
intestinal lumen
Bile acid composition in the small intestinal lumen was measured to identify the
reason for the differential response of FGF15 mRNA levels between ABPC- and
CA-treated mice (Table 1). Total bile acid contents were increased in both ABPC- and
CA-treated mice (1.6- and 2.3-fold, respectively). Primary bile acid contents were
increased in both ABPC- and CA-treated mice (1.7- and 1.8-fold, respectively). A
decrease in TβMCA content was observed in CA-fed mice suggesting reduction of de
novo synthesis of primary bile acids. DCA and TDCA contents were reduced to below
detection limits (<0.01 μmol) in ABPC-treated mice, whereas they were increased in
CA-treated mice (12- and 66-fold, respectively, relative to the control diet).
Fecal bacterial DNA was quantified by real-time PCR to identify the mechanism for
the reduction of DCA levels in the small intestinal lumen of ABPC-treated mice.
Consistent with the reduction of DCA levels in the small intestinal lumen, the levels of
Clostridium scidens, Clostridium sordelli and Bacteroides flagilis which exhibit
7α-dehydroxylase activity of bile acids (Kitahara et al., 2001; Ridlon et al., 2006) were
markedly reduced to 8.8%, 1.4% and <0.1%, respectively, of vehicle-treated controls
compared to ABPC-vehicle-treated mice.
Influence of TDCA co-treatment on FGF15 mRNA levels in ABPC-treated mice
To assess the influence of TDCA co-treatment on ileal FGF15 mRNA level in
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ABPC-treated mice, ABPC and TDCA were administered to mice. TDCA content in the
small intestinal lumen and ileal FGF15 mRNA level were markedly decreased in
ABPC-treated mice (<0.01 μmol and 2% of vehicle-treated mice, respectively).
Co-treatment with increasing amounts of TDCA reversed reductions in the TDCA
content in the small intestinal lumen and ileal FGF15 mRNA level of ABPC-treated
mice in a dose-dependent manner (Fig. 4A, B).
Influence of ABPC treatment on FGF15 mRNA levels in Fxr-null mice
To clarify whether antibiotic-mediated suppression of ileal FGF15 expression is
dependent on FXR signaling, ileal FXR target gene SHP mRNA levels were measured
in ABPC-treated mice. Significant decreases in ileal SHP mRNA levels were not
observed in ABPC-treated mice although marked decreases in ileal FGF15 mRNA
levels were noted (data not shown). Furthermore, Fxr-null mice were treated with
ABPC to clarify the involvement of FXR-independent signaling. Ileal FGF15 mRNA
levels in Fxr-null mice were 10 times lower than those in wild-type mice (Fig. 5A).
ABPC administration to Fxr-null mice decreased ileal FGF15 mRNA levels. Secondary
bile acid (TDCA, DCA, TLCA and LCA) contents in the small intestinal lumen were
reduced to below detection limits in ABPC-treated Fxr-null mice despite increases in
primary bile acid contents (Fig. 5B).
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DISCUSSION
In the present study, administrations of antibacterial drug suppressed ileal
FGF15 expression resulting in elevation of synthesis of hepatic primary bile acids.
ABPC is absorbed into the small intestine and secreted into bile, whereas BC, NM, and
SM are barely absorbed from the small intestine. The influence of BC, NM, and SM are
therefore largely restricted within the intestinal lumen. Elevation of the synthesis of
hepatic primary bile acids was observed in both ABPC-treated mice and
BC/NM/SM-treated mice, so indirect effects through enterobacteria are probably
involved in the elevation of hepatic primary bile acid synthesis in antibacterial
drug-treated mice. ABPC-treated mice showed increased activity of hepatic cholesterol
7α-hydroxylase and CYP7A1 mRNA levels, and also showed reduced ileal mRNA
levels of FGF15. FGF15 was shown to be a potent negative regulator of CYP7A1. In
ABPC-treated mice, changes in ileal FGF15 mRNA levels, not hepatic SHP mRNA
levels, were inversely related with hepatic CYP7A1 mRNA levels. Hepatic CYP7A1
mRNA levels were increased in Fgf15-null mice (Kim et al., 2007) and in BDL-mice in
which ileal FGF15 mRNA levels were reduced (Inagaki et al., 2005). Antibacterial
drug-mediated suppression of ileal FGF15 expression is probably, at least in part,
involved in the elevation of hepatic CYP7A1 expression resulting in increases in
hepatic primary bile acid synthesis and bile acid concentration. Elevation of PON1
mRNA levels in ABPC-treated mice also supports this hypothesis. In the present study,
role of SHP in the antibacterial drug-mediated elevation of hepatic CYP7A1 expression
has remained unclear. Decreases in hepatic SHP protein level through attenuation of
FGF15 signaling might be involved in the elevation of hepatic CYP7A1 expression
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(Miao et al., 2009).
In gallstone patients, ABPC treatment elevated total bile acid pool size and CA
synthesis rate compared with before the treatment (Carulli et al., 1981; Berr et al., 1996)
although the mechanisms have remained unclear. The results of the present study raise
the possibility that administration of antibacterial drugs to humans reduces serum levels
of FGF19 and elevates the activity of hepatic cholesterol 7α-hydroxylase.
An antibiotic-treated model is similar to a BDL model that elevates hepatic bile
acid levels and CYP7A1 expression and reduces ileal FGF15 expression (Dueland et al.,
1991; Inagaki et al., 2005). However, it is of interest to note that bile acid contents in the
intestinal lumen were increased in ABPC-treated mice, whereas they were decreased in
BDL mice due to blocking of bile flow into the intestine. Despite the increase in bile
acid contents in the intestinal lumen, ileal FGF15 mRNA levels were reduced in
ABPC-treated mice concomitant with decreased levels of DCA contents. FGF15 mRNA
levels were elevated in CA-treated mice in which elevation of DCA content in the small
intestinal lumen were observed (Table 1). Furthermore, co-treatment with TDCA
reversed the decreases in ileal FGF15 mRNA levels in ABPC-treated mice. These
observations raise the possibility that ABPC-mediated reduction of ileal FGF15
expression is related to DCA contents rather than primary bile acid contents in mice. In
the present study, we focused on DCA in a mouse model. In humans, LCA as well as
DCA is the main secondary bile acid. Further studies are necessary to understand the
role of LCA in ileal FGF15 expression. The possible roles of non-bile acid-mediated
mechanisms for the reduction of ileal FGF15 expression in ABPC-treated mice should
not be excluded. Ileal FGF15 expression might be affected by endotoxin-mediated
signaling produced by enterobacteria killing.
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In general, it is considered that suppression of FXR signaling is involved in the
reduction of ileal FGF15 mRNA levels (Holt et al., 2003). Based on this hypothesis, bile
acid composition in the intestinal lumen of ABPC-treated mice raises the possibility that
DCA, but not CA and βMCA, is crucially involved in activation of FXR signaling in the
mouse intestine. Reports indicated that hydrophobic bile acids such as CDCA, DCA and
LCA exhibit higher capacity of human and rodent FXR activation in vitro reporter assay
than hydrophilic bile acids such as CA, βMCA and UDCA (Makishima et al., 1999;
Parks et al., 1999; Wang et al., 1999). Thus, the reduction in ileal FGF15 expression
may be, at least in part, explained by suppression of FXR signaling due to reduction of
DCA levels in the intestine. However, SHP mRNA levels were not decreased in
ABPC-treated mice. Significant decreases in the ileal FGF15 mRNA level in
ABPC-treated Fxr-null mice suggest FXR-independent mechanisms for ileal FGF15
expression. Various bile acid-mediated signals independent of FXR have been reported
(Nguyen and Bouscarel, 2008). Wistuba et al reported that LCA induction of FGF19
promoter in intestinal cells is mediated by PXR (Wistuba et al., 2007). Involvement of
secondary bile acid-mediated signaling of other nuclear receptors and cellular kinases
on ileal FGF15 expression in vivo may need to be evaluated.
Several studies have provided evidence that bile acid metabolism is implicated in
the regulation of not only cholesterol metabolism, but also lipid and glucose
metabolisms (Watanabe et al., 2004; Ma et al., 2006; Zhang et al., 2006). FGF19
signaling is also involved in the regulation of glucose and lipid metabolism as well as
bile acid metabolism (Bhatnagar et al., 2009; Shin and Osborne, 2009). Administration
of human FGF19 or transgenic expression of human FGF19 in mice enhanced energy
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expenditure and fatty acid oxidation and reduced hepatic, serum triglyceride and
glucose levels (Tomlinson et al., 2002; Fu et al., 2004). Thus, long-term administration
of an antibacterial drug, which decreases ileal FGF15 expression may cause disruption
of lipid and glucose homeostasis resulting in metabolic diseases such as diabetes and
hyperlipidemia. The relationship between enterobacteria and human disease has been
recently investigated (Hecht, 2009). Human health and disease may be influenced by
antibacterial drug-mediated alteration of the enterobacteria population.
Although the physiological significances of enterobacteria-mediated secondary bile
acid production is unclear, the present study suggests that secondary bile acid contents
may alter hepatic synthesis of primary bile acids through changes in FGF15 level.
Enterobacteria convert primary bile acids in the colon that have escaped ileal
reabsorption to secondary bile acids that are signalling molecules to suppress the
synthesis of primary bile acids. Enterobacteria may serve as bile acid sensors
cooperating with FXR and FGF15 signaling.
The present study suggests that administration of antibacterial drug suppresses ileal
FGF15 levels, resulting in the elevation of hepatic primary bile acid synthesis. Elevation
in the activity of hepatic cholesterol 7α-hydroxylase is probably involved, at least in
part, in the elevation of hepatic bile acid levels in ABPC-treatment mice. The
mechanism for the reduction of ileal FGF15 mRNA levels in antibiotic-treated mice is
unclear. However, the disappearance of enterobacteria-produced secondary bile acids
may be involved in the reduction in FGF15 mRNA levels via the FXR and other
signaling pathways. Further studies are necessary to understand the relationship
between primary bile acids and secondary bile acids in the regulation of ileal FGF15
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expression under physiological conditions.
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FOOTNOTES
This study was supported by a Grant-in-Aid from the Ministry of Education, Culture,
Sports Science and Technology of Japan [Grants 21390039, 20590137].
Reprint requests to: Masaaki Miyata Ph.D.
Division of Drug Metabolism and Molecular Toxicology, Graduate School of
Pharmaceutical Sciences, Tohoku University
6-3, Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan
e-mail: miyata@mail.pharm.tohoku.ac.jp
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Figure legends
Fig. 1 Influence of ABPC treatment on hepatic bile acid levels and microsomal
cholesterol 7α-hydroxylation A, hepatic bile acid levels. B, microsomal cholesterol
7α-hydroxylation. Liver homogenates and microsomes were prepared from mice treated
with ABPC (100 mg/kg) for 3 days and fasted for the last 12 h. Hepatic bile acid
compositions and microsomal cholesterol 7α-hydroxylase activities were determined by
HPLC. Data are mean ± S.D. (n = 5–6). Significant differences from vehicle-treated
group are expressed with an asterisk (**, p < 0.01).
Fig. 2 Influence of ABPC or CA treatment on hepatic CYP7A1, SHP, and ileal
FGF15 mRNA levels A, mRNA levels in ABPC-treated mice. B, mRNA levels in
CA-treated mice. Real-time PCR analysis was performed on cDNA prepared from total
RNA of the liver and ileum in mice treated with ABPC (100 mg/kg body weight) for 3
days and fasted for the last 12 h or mice fed a diet supplemented with 1% CA for 6 days.
Hepatic CYP7A1 and SHP mRNA levels were normalized to GAPDH. Ileal FGF15
mRNA level was normalized to β-actin. Data are relative values to the mRNA levels of
the vehicle-treated group or a control diet-fed group, and shown as mean ± S.D. (n =
5–6). Significant differences from vehicle-treated group are expressed with an asterisk
(**, p < 0.01; *** p < 0.001).
Fig. 3 Influence of BC/NM/SM treatment on hepatic bile acid level and hepatic
CYP7A1, SHP and ileal FGF15 mRNA levels A, hepatic bile acid levels. B, mRNA
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levels. Hepatic bile acid compositions were determined by HPLC. mRNA levels were
measured by quantitative real-time PCR. Liver homogenates, liver and ileal total RNAs
were prepared from mice treated with BC/NM/SM (200 mg/kg each) for 3 days and
fasted for the last 12 h. Data are mean ± S.D. (n = 5). Significant differences from
vehicle-treated group are expressed with an asterisk (*, p < 0.05; *** p < 0.001).
Fig. 4 Influence of co-treatment with TDCA on FGF15 mRNA levels and TDCA
amounts in the small intestinal lumen A, FGF15 mRNA levels. B, TDCA amounts in
the small intestinal lumen. Mice were orally treated with vehicle, ABPC (100 mg/kg) or
ABPC+TDCA (100 or 500 mg/kg) for 3 days and fasted for the last 12 h. Real-time
PCR analysis was performed on cDNA prepared from total RNA of the ileum. All
mRNA levels were normalized to β-actin and presented as a relative value to the mRNA
level of vehicle-treated mice. TDCA amounts in the small intestinal lumen were
determined by HPLC. N.D. represents < 0.01 μmol. Data are mean ± S.D. (n = 4–5).
Significant differences from the ABPC-treated group are expressed with an asterisk (**,
p < 0.01; ***, p < 0.001).
Fig. 5 Influence of ABPC treatment on ileal FGF15 mRNA levels and bile acid
amounts in the small intestinal lumen in Fxr-null mice A, FGF15 mRNA levels. B,
bile acid amounts in small intestinal lumen. Fxr-null and wild-type mice were treated
with ABPC (100 mg/kg) for 3 days and fasted for the last 12 h. Ileal FGF15 mRNA
levels were measured by quantitative real-time PCR. Bile acid composition in the small
intestinal lumen was determined by HPLC. Primary bile acid (BA), βMCA, TβMCA,
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UDCA, TUDCA, CA, TCA, CDCA and TCDCA; Secondary bile acid (BA), DCA,
TDCA, LCA and TLCA. Data are mean ± S.D. (n = 5). Significant differences from the
vehicle-treated group are expressed with an asterisk (*, p < 0.05; *** p < 0.001).
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Table 1 Influence of ABPC or CA treatment on bile acid composition in the small intestinal
lumen
Vehicle ABPC Untreatment CA
Primary bile acid (μmol)
βMCA 0.32 ± 0.32 N.D. 0.40 ± 0.08 0.02 ± 0.02***
TβMCA 3.43 ± 1.57 6.63 ± 0.60** 3.40 ± 1.04 0.40 ± 0.22**
UDCA N.D. N.D. N.D. N.D.
TUDCA 0.10 ± 0.07 0.32 ± 0.03*** 0.14 ± 0.06 0.06 ± 0.02*
CA 0.54 ± 0.46 N.D. 0.64 ± 0.30 1.14 ± 1.02
TCA 3.05 ± 0.86 5.39 ± 0.42** 3.20 ± 0.50 12.38 ± 3.98***
CDCA 0.01 ± 0.01 0.01 ± 0.01 N.D. N.D.
TCDCA 0.04 ± 0.03 0.17 ± 0.04*** 0.02 ± 0.02 N.D.
Total 7.49 ± 1.87 12.50 ± 0.95** 7.80 ± 1.82 14.00 ± 4.78*
Secondary bile acid (μmol)
DCA 0.04 ± 0.06 N.D. 0.02 ± 0.02 0.24 ± 0.38
TDCA 0.21 ± 0.09 N.D. 0.06 ± 0.02 3.96 ± 1.26***
LCA N.D. N.D. N.D. N.D.
TLCA N.D. N.D. N.D. N.D.
Total 0.26 ± 0.09 N.D. 0.08 ± 0.02 4.18 ± 1.14***
Total bile acid 7.74 ± 1.81 12.50 ± 0.95** 7.88 ± 1.80 18.20 ± 5.86*
Data are shown as mean ± S.D. (n=4-5). N.D. represents <0.01μmol. Significant differences from
the vehicle-treated group are expressed with an asterisk (*, p<0.05; **, p<0.01; ***, p<0.001).
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