administration of ampicillin elevates hepatic primary bile...

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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.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on September 18, 2009 as DOI: 10.1124/jpet.109.160093

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Running title: hepatic bile acid synthesis regulated by enterobacteria

Corresponding author: Masaaki Miyata, Ph.D.


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: [email protected]

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.


Pharmaceutical Sciences, Tohoku University

6-3, Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan

e-mail: [email protected]


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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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