Metabolomics reveals an essential role for peroxisome proliferator-activated receptor α in bile acid homeostasis.

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor that regulates fatty acid transport and metabolism. Previous studies revealed that PPARα can affect bile acid metabolism; however, the mechanism by which PPARα regulates bile acid homeostasis is not understood. In this study, an ultraperformance liquid chromatography coupled with electrospray ionization qua dru pole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-based metabolomics approach was used to profile metabolites in urine, serum, and bile of wild-type and Ppara-null mice following cholic acid (CA) dietary challenge. Metabolomic analysis showed that the levels of several serum bile acids, such as CA (25-fold) and taurocholic acid (16-fold), were significantly increased in CA-treated Ppara-null mice compared with CA-treated wild-type mice. Phospholipid homeostasis, as revealed by decreased serum lysophos phati dylcholine (LPC) 16:0 (1.6-fold) and LPC 18:0 (1.6-fold), and corticosterone metabolism noted by increased urinary excretion of 11β-hydroxy-3,20-dioxopregn-4-en-21-oic acid (20-fold) and 11β,20α-dihydroxy-3-oxo-pregn-4-en-21-oic acid (3.6-fold), were disrupted in CA-treated Ppara-null mice. The hepatic levels of mRNA encoding transporters Abcb11, Abcb4, Abca1, Abcg5, and Abcg8 were diminished in Ppara-null mice, leading to the accumulation of bile acids in the liver during the CA challenge. These observations revealed that PPARα is an essential regulator of bile acid biosynthesis, transport, and secretion.

dark cycle with water and a normal diet (NIH-31) provided ad libitum. Handling was in accordance with an animal study protocol approved by the National Cancer Institute Animal Care and Use Committee. Groups of 8-to 12-week-old male mice were put on a synthetic purifi ed diet (AIN-93G, Bio-Serv, NJ) for 5 days before experiment. Ppara -null mice and wild-type mice were fed with CA diet (AIN-93G supplemented with 1% CA) and control diet (AIN-93G) for 12 days based on previous studies (15)(16)(17). Urine samples were collected and food intake was measured from mice placed individually in metabowls (Jencons, Leighton Buzzard, UK) for 24 h, before treatment, and on days 1, 3, 6, 9, and 12 following the treatment. The mice had ad libitum access to diet and water in these metabolic chambers. All urine samples were stored at Ϫ 80°C until analyzed. The serum, bile, liver, and kidney tissue were harvested and frozen at Ϫ 80°C for further analysis at the end of the study.

UPLC-ESI-QTOFMS analysis
Samples for injection were prepared by diluting 20 µl urine with 180 µl 50% aqueous acetonitrile. Serum samples were prepared by 10 µl serum mixed with 190 µl 67% aqueous acetonitrile, and bile samples were prepared by 1 µl bile mixed with 1,000 µl 67% aqueous acetonitrile. The samples were vortexed for 5 min in shaker and centrifuged at 14,000 rpm for 20 min at 4°C to remove particulates and precipitate protein. The supernatant was transferred to an autosampler vial for analysis. A 5 µl aliquot of supernatant samples was injected into the system of ultraperformance liquid chromatography coupled quadrupole time-of-fl ight mass spectroscopy (UPLC-ESI-QTOFMS). The liquid chromatography system was ACQUITY UPLC® equipment (Waters) consisting of a reverse-phase 2.1 × 50 mm ACQUITY UPLC® BEH C18 1.7 µm column (Waters Corp., Milford, MA) with a gradient mobile phase comprising 0.1% formic acid solution (A) and acetonitrile containing 0.1% formic acid solution (B). The gradient was maintained at 100% A for 0.5 min, increased to 100% B over the next 7.5 min, and returned to 100% A in last 2 min. Data were collected in positive mode and negative mode on a Waters Q-TOF, which was operated in full-scan mode at m/z 100 to 1,000. Nitrogen was used as both cone gas (50 l/h) and desolvation gas (600 l/h). Source temperature and desolvation temperature were set at 120°C and 350°C, respectively. The capillary voltage and cone voltage were 3,000 and 20 V, respectively. Chlorpropamide (5 µM) was added in the sample as the internal standard.

Data processing and multivariate data analysis
Raw data from UPLC-ESI-QTOFMS system were processed using MarkerLynx software (Waters) to generate a data matrix consisting of peak areas corresponding to a unique m/z and retention time (RT) without normalization. After the generation of a multivariated data matrix, this data set was exported into SIMCA-P+12.0 (Umetrics, Kinnelon, NJ) for principal component analysis (PCA). The loadings scatter plots and the contribution lists were used to determine the candidate biomarkers in CA-fed Ppara -null mice compared with other group mice.

Identifi cation and quantitation of urinary and serum metabolites
To identify the structure of high-contribution score metabolites, metabolomics databases (Madison Metabolomics Consortium Database and METLIN) were searched to fi nd potential candidates. Seven Golden Rules ( 18 ) were used to calculate the mass error based on the elemental compositions of each metabolite. To confi rm the identities of markers, authentic standards at 5 to 20 M in 50% acetonitrile were compared with the urine and serum sample on the condition of MS/MS fragmentation with collision energy ramping from 15 to 35 V. Identities of the was reported that 0.5% bezafi brate treatment leads to a decrease in liver bile acid and Cyp7a1 expression in wild-type mice and Ppara -null mice ( 5 ). Others found that Cyp7a1 and cholesterol 27 ␣ -hydroxylase (Cyp27a1) were decreased in wild-type mice by ciprofi brate treatment but not in Ppara -null mice ( 6 ). These results indicate that the activation of PPAR␣ by fi brates can inhibit bile acid biosynthesis; however, bezafi brate and ciprofi brate show different effects on bile acid biosynthesis enzymes in Ppara -null mice. Thus, the mechanism by which PPAR␣ controls bile acid homeostasis remains uncertain. Additionally, limited studies revealed that PPAR␣ exert its effect on the bile acid conjugation and transport in the liver and intestine ( 7,8 ).
Recent studies have demonstrated the power of mass spectrometry-based metabolomics to profi le metabolic pathways to reveal the mechanism of action of nuclear receptors and the metabolic fate of drugs (9)(10)(11)(12). Metabolomics has revealed the downregulated tryptophan-nicotinamide pathways following Wy-14,643 treatment ( 9 ) and the decreased excretion of carnitine-conjugated metabolites by fenofi brate treatment in humans ( 13 ). Furthermore, an increase in the excretion of glycine conjugated metabolites was observed in Ppara -null mice ( 9 ), and the long-chain fatty acid carnitines, including palmitoylcaritine, myristolcarnitine, oleoylcarnitine, and palmitoleoylcarnitine, were elevated in the serum after suppression of PPAR␣ signal transduction by acetaminophen ( 14 ).
In the present study, cholic acid (CA)-treated wild-type and Ppara -null mice were used to evaluate the functional role of PPAR␣ in bile acid homeostasis. Metabolomics analysis showed that severe liver dysfunction in Ppara -null mice was induced during CA challenge, including the disruption of bile acids, phospholipids, and cholesterol homeostasis. Additionally, the excretion of several endogenous cationic and anionic metabolites was signifi cantly affected. Further results demonstrated that PPAR␣ exerted a role in bile acid homeostasis via regulation of bile acid biosynthesis, transport, and secretion.

Animal study
Ppara -null mice and wild-type mice on a C57BL/6N genetic background were maintained under a standard 12 h light/12 h The signifi cance of metabolite concentration and mRNA levels was determined using two-tailed Student t -test or the one-way ANOVA with Bonferroni correction for multiple comparisons. P values of less than 0.05 were considered signifi cant.

Phenotypes of Ppara -null mice fed with CA diet
Male wild-type and Ppara -null mice fed a control diet showed no signifi cant differences in body weight. However, Ppara -null mice exhibited signifi cant body weight loss after feeding a CA diet for 12 days, whereas wild-type mice were slightly decreased ( Table 1 ). There were no signifi cant differences in food intake and urine volume between the four groups of mice (supplementary Fig. I-A, B). Histological analysis showed severe fatty liver and cholestasis in CA-treated Ppara -null mice ( Fig. 1 ). In contrast, no fatty liver and cholestasis was induced in wild-type mice fed the CA diet. Further examination indicated that the major lipid composition of hepatic steatosis in CA-treated Pparanull mice was cholesterol and triglyceride (supplementary Fig. II-A, B). In addition, serum chemistry analysis indicated that CA-treated Ppara -null mice showed higher ALP, ALT, and serum total bilirubin levels than CA-treated wildtype mice ( Table 1 ). These results indicate that Ppara -null mice are susceptible to liver injury induced by dietary CA overload.

Metabolomic analysis of mouse bile, urine, and serum
To gain an understanding for why Ppara -null mice were more sensitive to CA challenge than wild-type mice, metabolomic analysis was used to profi le the metabolites in the serum, urine, and bile from the four groups. Unsupervised PCA was used to analyze the data sets from the control and CA-fed groups in both wild-type and Ppara -null mice. The PCA model shows that bile samples between wild-type and Ppara -null mice fed with control or CA diet were distributed in four different quadrants. CA Treatment can signifi cantly increase bile volume in both wildtype and Ppara -null mice gallbladder; however, there were no signifi cant differences in bile volume between wild-type and Ppara -null mice or between CA-treated wild-type and ions were further confi rmed by comparison of fragmentation pattern and retention time with authentic compounds.
Quantitation of metabolites in urine and serum was performed using an ACQUITY UPLC system coupled with a XEVO triplequadrupole tandem mass spectrometer (Waters). The detection and quantitation of biomarkers were accomplished by multiple reaction monitoring (MRM) mass spectrometry.

Gene expression analysis
Total RNA was extracted from approximately 100 mg portions of frozen liver and kidney using TRIzol reagent (Invitrogen, Carlsbad, CA). Quantitative real-time PCR (qPCR) was carried out using SYBR green PCR master mix (Superarray) in an ABI Prism 7900HT sequence detection system (Applied Biosystems). QPCR primer sequences are shown in the supplementary Table I. Measured mRNA levels were normalized to those of ␤ -actin (liver) or 18S (kidney) rRNA and expressed as fold change relative to those of wild-type mice fed a control diet.

Histological analysis
Fresh livers were fi xed in 10% neutral-buffered formalin, and then subjected to dehydration in different concentrations of alcohol and xylene for paraffi n embedding. Four-micrometer serial sections were made through the entire tissue. Histological fi ndings were examined using a light microscope after hematoxylin and eosin staining.

Statistical analysis
Experimental values are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism (San Diego, CA). Comparison of diagnostic biomarkers between wild-type and Ppara -null mice fed with CA diet (n = 4). The percentage of body weight was calculated by comparing the body weight at day 12 to the starting weight at day 0. The liver/body mass was calculated by comparing the liver weight at day 12 to the body weight at day 12. a P < 0.01. b P < 0.05.
After PCA modeling of wild-type and Ppara -null mice in the CA-and control-fed groups, a loadings scatter plot revealed ions with the greatest contribution to separation between CA-fed Ppara -null mice and other groups. The signifi cant ions increased in Ppara -null mice after CA loading were in the fi rst and fourth quadrants, and they decreased in the second and third quadrants ( Fig. 3A, B ) ( Table 2 ).

Disrupted bile acid homeostasis in CA-treated
Ppara -null mice Metabolomic analysis indicated that TCA was the most important ion increased in the serum of CA-fed Ppara -null mice. The level of TCA was increased from day 6 of treatment, and the concentration was nearly 16-fold higher at day 12 of treatment in CA-fed Ppara -null mice compared with CA-fed wild-type mice ( Fig. 4A ). Along with TCA, CA was markedly elevated in CA-fed Ppara -null mice from day 6 Ppara -null mice (supplementary Fig. I-C). These data suggested that different bile compositions were present in the four groups of mice ( Fig. 2A ). Signifi cant separation of urine and serum samples were observed in the CA-treated group and control group on day 12 ( Fig. 3A , B ). Compared with the urine sample, a more signifi cant difference was observed between CA-treated Ppara -null and wild-type mouse serum in component 1 (X-axis) of the PCA model, suggesting that serum metabolites were more directly affected by CA treatment in Ppara -null mice.  untreated Ppara -null mice ( Fig. 4C ). Two enzymes involved in the bile acid conjugation, bile acid-CoA synthetase (Bacs, also called Slc27a5) and bile acid-CoA amino acid N -acetyltransferase (Baat), were signifi cantly diminished in the liver of CA-fed Ppara -null mice. The bile acid transporters sodium-taurocholate cotransporting polypeptide (Ntcp), anion transporting polypeptide 1 (Oatp1), and anion transporting polypeptide 4 (Oatp4), which absorb bile acids from blood into liver, were decreased in Ppara -null mice, whereas the transporter Abcc4, which carries bile acids from liver to blood, was dramatically increased ( P < 0.05) to 12 ( P < 0.01) ( Fig. 4B ). On day 12, there was a 25-fold increase in CA-treated Ppara -null mice compared with CA-treated wild-type mice. In addition, the levels of serum CDCA, T-␣ / ␤ -MCA, and TCDCA were increased at day 12 of treatment in Ppara -null mice fed with CA diet (data no shown). Further gene expression analysis indicated that the major enzymes involved in the bile acid biosynthesis, including Cyp7a1, Cyp7b1, Cyp8b1, Cyp27a1, and hydroxy-delta-5-steroid dehydrogenase 3b7 (Hsd3b7) were decreased in the CA-fed Ppara -null mice at day 12 of treatment compared with CA-treated wild-type mice and  LPC 16:0 and LPC 18:0 in the serum of CA-treated Ppara -null mice. Triplequadrupole quantitation further confi rmed that the level of LPC 16:0 was decreased in CA-treated Ppara -null mice from day 3 ( P < 0.05) to 12 ( P < 0.01) ( Fig. 5A ). Compared with LPC 16:0, the level of LPC 18:0 was signifi cantly reduced in CA-treated Ppara -null mice on day 12 ( P < 0.05) ( Fig. 5B ). On day 12, there was a 1.6-fold depletion of both phospholipids in CA-treated Ppara -null mice compared with CA-treated wild-type mice. Phospholipids have been reported to be biomarkers for LCA induced cholestasis ( 19 ). Liver gene expression indicated that the mRNAs encoding enzymes involved in the LPC biosynthesis [lecithin-cholesterol acyltransferase (Lcat), phosphate cytidylyltransferase 1 (Pcyt1 ␣ ), choline phosphotransferase 1 (Chpt1), and choline kinase ␤ (Chk ␤ )] were signifi cantly decreased in CA-treated Pparanull mice compared with CA-treated wild-type mice and untreated Ppara -null mice ( Fig. 5C ). Similarly, the expression of mRNAs encoding proteins associated with LPC metabolism [lysophosphatidylcholine acyltransferase 3 (Lpcat3), lysophospholipase A1 (Lyp1a1), and ectonucleotide pyrophosphatase/ phosphodiesterase 2 (Enpp2)] were reduced in CA-treated Ppara -null mice ( Fig. 5C ). In addition, the mRNA encoding the phospholipid transporter ATPbinding cassette subfamily B member 4 (Abcb4) located in the liver canaliculus, was diminished in CA-treated Pparanull mice compared with CA-treated wild-type mice. These data indicate that phospholipids homeostasis was also disrupted in CA-treated Ppara -null mice.
( Fig. 4D ). In addition, the mRNA encoding bile acid transporter ATP-binding cassette subfamily B member 11 (Abcb11, also called Bsep) located in liver canaliculus that transports bile acids from liver to bile duct, was increased by CA treatment in wild-type and Ppara -null mice. However, lower levels of Abcb11 were observed in CA-treated Ppara -null mice compared with CA-treated wild-type mice. Metabolomic analysis of bile indicated that CA treatment signifi cantly changes the bile acid composition of bile in both wild-type and Ppara -null mice, notably T-␣ / ␤ -MCA, TUDCA, and TMDCA ( Fig. 2B ).
Since CA treatment signifi cantly altered the bile volume in both wild-type and Ppara -null mice, the relative concentrations of bile acids in bile were determined (Fig. 2B). However, compared with CA-treated wild-type mice, TCA and TCDCA were lower in CA-treated Ppara -null mice. These results suggested that the bile acid homeostasis was markedly disrupted in CA-treated Ppara -null mice compared with CAtreated wild-type mice and control mice.

Alteration of phospholipid homeostasis in CA-treated Ppara -null mice
Along with the accumulation of bile acids, metabolomic analysis showed the decreased levels of two phospholipids,

Change in cholesterol and corticosterone metabolism in CA-treated Ppara -null mice
The generation of bile acids from cholesterol is a metabolic pathway for cholesterol degradation in vivo. To determine whether other metabolic pathways involving cholesterol were also affected, the metabolites associated with cholesterol and steroid hormone metabolism were examined in serum and urine. Although there was no signifi cant change in serum corticosterone and progesterone levels (supplementary Fig. III), two corticosterone derivatives, HDOPA and DHOPA, showed a signifi cant increase in urine of CA-treated Ppara -null mice ( Fig. 6A , B ). Both metabolites were signifi cantly increased from day 6 to 12. On day 12, HDOPA and DHOPA were increased by 20and 3.6-fold, respectively, in CA-treated Ppara -null mice compared with CA-treated wild-type mice. In addition, hepatic expression levels of the PPAR␣ target gene aldo-keto reductase family 1c18 (Akr1c18) was signifi cantly diminished in the control diet-treated Ppara -null mice compared with wild-type mice ( 20 ). The mRNA encoding Akr1c18 involved in the generation of HDOPA and DHOPA was greatly increased (6-fold) in CA-treated Ppara -null mice ( Fig. 6C ), whereas genes associated with corticosterone metabolism, such as aldo-keto reductase family 1c14 (Akr1c14), steroid 5 ␣ -reductase 1 (Srd5a1), hydroxysteroid (17-␤ ) dehydrogenase 3 (Hsd17b3), and aldehyde dehydrogenase family 3, subfamily A2 (Aldh3a2), were decreased (supplementary Fig. IV). These data suggested that enhanced Akr1c18 expression was responsible for the increase of HDOPA and DHOPA in the CA-treated Ppara - null mice urine. In addition, three cholesterol transporters, ATP-binding cassette subfamily A member 1 (Abca1), ATP-binding cassette subfamily G member 5 (Abcg5), and ATP-binding cassette subfamily G member 8 (Abcg8), were signifi cantly decreased in CA-treated Ppara -null mice compared with CA-treated wild-type mice. Although the mRNA levels of Abcg5 and Abcg8 are elevated by CA treatment in wild-type mice, there was no signifi cant difference between CA-treated and untreated Ppara -null mice.

Disorder of cation and anion transport in CA-treated Ppara -null mice
Along with the increase in the HDOPA and DHOPA excretion, metabolomic analysis revealed altered levels of endogenous cationic and anionic metabolites, such as nicotinamide 1-oxide and xanthurenic acid, in urine of CA-treated Ppara -null mice. As shown in Fig. 7A , the excretion of creatinine was signifi cantly decreased in CA-treated Ppara -null mice. Therefore, the level of urinary metabolites was presented as total mass in 24 h. The excretion of creatine, one endogenous cation, was signifi cantly increased in CA-treated Ppara -null mice from day 3 ( P < 0.05) to 12 ( P < 0.05) ( Fig. 7B ), whereas creatinine excretion was decreased on day 12 ( P < 0.05) ( Fig. 7A ). The excretion of another cation nicotinamide 1-oxide also was signifi cantly reduced from day 3 ( P < 0.05) to 12 ( P < 0.05) ( Fig. 7C ). Similarly, the urinary excretion of two endogenous anion metabolites, phenylacetylglycine (3.9-fold) and xanthurenic acid (3.2-fold), was decreased in CAtreated Ppara -null mice on day 12 ( Fig. 7D, E ). qPCR analysis indicated that the expression of several organic cation transporters (Octs) and organic anion transporters (Oats) was dramatically diminished in the liver and kidney of CAtreated Ppara -null mice compared with untreated Pparanull mice and CA-treated wild-type mice, including organic cation transporter 1 (Oct1/Slc22A1), organic cation transporter 2 (Oct2/Slc22A2), organic cation transporter 3 (Oct3/Slc22A3), organic cation/carnitine transporter 1 (Octn1/Slc22A4), organic cation/carnitine transporter 2 (Octn2/Slc22A5), Oatp1, and Oatp4 ( Figs. 3D and 7F and supplementary Fig. V). These results demonstrated that CA challenge also leads to a disruption of endogenous cation and anion transport in Ppara -null mice.

DISCUSSION
To clarify the functional role of PPAR␣ in bile acid homeostasis, the present study investigated the change in endogenous metabolites and associated genes in Ppara -null mice during exposure to a CA diet. Metabolomics indicated disruption of bile acid homeostasis, phospholipid homeostasis, cholesterol metabolism, and endogenous cations and anions transport in CA-treated Ppara -null mice. Further gene expression analysis revealed that the bile acid disorder in CA-treated Ppara -null mice might be due to a defi ciency in expression of the Abc transporters (Abcb11, Abcb4, Abca1, Abcg5, and Abcg8) in the liver canliculus. Additionally, the levels of bile acid synthesis enzymes (Cyp7a1, Cyp27a1, and Hsd3b7) were lower in untreated are important components in bile that are sensitive to liver bile acid levels. In the clinic, abnormal levels of serum LPCs are typically associated with liver injury ( 24,25 ). Similarly, it was reported that a series of LPCs were significantly decreased in cholestasis induced by LCA treatment and nonalcoholic steatohepatitis induced by methionineand choline-defi cient diet treatment ( 19,26 ). Third, the excretion of HDOPA and DHOPA was increased in the CA-treated Ppara -null mice urine. HDOPA and DHOPA were generated from corticosterone under the effect of cytochrome P450s (Cyps), which was confi rmed by in vitro and in vivo experiments ( 9,20 ). Recently, HDOPA and DHOPA were reported as biomarkers for PPAR␣ activation ( 9 ). However, the expression of PPAR␣ target genes suppressed in Ppara -null mice suggested that the increase in both metabolites in this study was not likely due to the activation of PPAR␣. It was reported that in cholestastic animals and humans, the hypothalamic-pituitary-adrenal axis can be activated by endotoxin and cytokines, such as tumor necrosis factor ␣ (TNF ␣ ), resulting in the elevation of glucocorticoids ( 27 ). Two studies indicated that the excretion of HDOPA and DHOPA was increased in the hepatotoxicity model of hepatocyte nuclear factor 1 ␣ ( Hnf1 ␣ )-null mice ( 28 ) and CA-treated Fxr -null mice ( 21 ). Here, the expression of TNF ␣ was activated in CA-treated Ppara -null mice, suggesting that accelerated corticosterone metabolism in CA-treated Ppara -null mice also results from liver Ppara -null mice than in untreated wild-type mice. These results demonstrated that PPAR␣ regulates bile acid homeostasis not only via its effect on bile acid biosynthesis and transport but also via its effect on biliary secretion.
When Ppara -null mice were treated with a CA diet, one signifi cant observation was liver injury. The change in phenotype of the CA-treated Ppara -null mice included body weight loss, enhanced serum ALP and ALT levels, and cholestasis. These phenotypes are similar with those previously reported for the farnesoid X receptor ( Fxr )-null mice after feeding CA ( 21 ). In addition, higher total bilirubin and lower glucose levels were also observed in CA-fed Pparanull mice. Further metabolomic analysis revealed an alteration of three important metabolic pathways, bile acid metabolism, phospholipid metabolism, and cholesterol metabolism, in CA-treated Ppara -null mice. First, a high level of bile acids, including CA, CDCA, TCA, T-␣ / ␤ -CA, and TCDCA, were noted in CA-treated Ppara -null mouse serum, and the genes involved in bile acid synthesis and transport were diminished in the liver, thus suggesting signifi cant liver injury after feeding Ppara -null mice CA. Numerous studies have demonstrated that increased levels of serum bile acids were induced in cholestasis models ( 16,22,23 ). Second, two phospholipids, LPC 16:0 and LPC 18:0, were decreased in CA-treated Ppara -null mice serum. LPCs, which are derived from phosphatidylcholine by hydrolysis, are the major phospholipid species in serum. Phospholipids Although earlier studies showed that ciprofi brate ( 43 ) and perfl uorinated fatty acids ( 7 ) cause a decrease in hepatic transporters Ntcp and Oatp1, the level of Ntcp and Oatps were unchanged in control diet-treated Ppara -null mice compared with wild-type mice. In the liver, the expression of Abcb11 was decreased in control diet-treated Ppara -null mice, consistent with the observations that Ppara -null mice show lower hepatic Abcb11 level than wild-type mice ( 5 ). Thus, the defi ciency of PPAR␣ can directly affect the bile acid transporter Abcb11 in the liver. More importantly, the mRNA levels of the phospholipid transporter Abcb4 and cholesterol transporters Abca1, Abcg5, and Abcg8 also were diminished in the liver following knockout of PPAR␣. Various studies have indicated that treatment with Wy-14,643 and other PPAR␣ activators can stimulate the expression of Abcb4, Abca1, Abcg5, and Abcg8 (43)(44)(45). Actually, it was reported that PPAR␣ can regulate hepatic lipid homeostasis via its target effect on these Abc transporters ( 46,47 ). During CA challenge, the reduced mRNA expression of these Abc transporters greatly affected the transport of bile acids in Ppara -null mice, fi nally causing severe liver cholestasis. Taken together, the defi ciency of PPAR␣ can cause decreased bile acid biosynthesis, transport, and secretion.
Although the expression of several genes involved in the bile acid metabolism was reduced in Ppara -null mice, metabolomics did not reveal abnormal bile acid levels in untreated urine and serum in these mice. To better evaluate the role of PPAR␣ in the regulation of bile acid homeostasis, CA challenge was conducted in Ppara -null mice. The results showed severe hepatotoxicity in Ppara -null mice during CA challenge that was not observed in wildtype mice. Numerous studies have reported that CA challenge can lead to severe liver injury in transgenic mice in which specifi c genes involved in bile acid metabolism had been knocked out, such as Fxr -null mice ( 48 ), Abcb11 -null mice ( 49 ), and Shp -null mice ( 50 ). The current studies demonstrated a key role of PPAR␣ in the regulation of bile acid homeostasis. The hepatotoxicity in CA-treated Ppara -null mice might be due to the decrease in expression of the Abc transporters in the liver canaliculus. The reduced biliary secretion causes the accumulation of bile acids in Ppara -null mice liver during CA exposure. Indeed, a metabolic disorder was observed in CA-treated Pparanull by metabolomics, including altered bile acid metabolism, phospholipid metabolism, cholesterol metabolism, and transport of endogenous cations and anions. The data presented herein demonstrate the power of mass spectrometry-based metabolomic analysis in combination with traditional serum chemistry analysis and histological examination using a transgenic mouse model to profi le metabolic pathways and uncover the physiological function of nuclear receptor.
cholestasis. The change in the bile acids, phospholipids, and cholesterol homeostasis provides compelling evidence that hepatotoxicity was induced in Ppara -null mice in response to CA challenge.
Another observation in this study is the disrupted transport of endogenous cations and anions in the liver and kidney. The possibility exists that altered levels of creatine, creatinine, and nicotinamide 1-oxide observed in the CAtreated Ppara -null mice is due to inhibition of Octs function in vivo. In the liver and kidney, the Octs family mediates the reabsorption and excretion of various cationic compounds, including endogenous cations (choline and carnitine), cationic toxins (1-methyl-4-phenylpyridium), and cationic drugs (quinidine and procainamide) ( 29 ). Several studies have shown that Oct2 plays an important role in renal secretion of creatinine in humans and mice ( 30,31 ). Additionally, methylnicotinamide, used as a model substrate for studying organic cation transport, can be regulated by Oct1 and Oct2 ( 32,33 ). In vivo, nicotinamide 1-oxide and methylnicotinamide are derived from nicotinamide. These studies suggest that inhibition of Octs might directly affect the transport of creatinine, creatine, and nicotinamide 1-oxide in vivo. Additionally, two organic anionic metabolites, phenylacetylglycine and xanthurenic acid, were signifi cantly decreased in CA-treated Ppara -null mice. A recent study using untargeted metabolomics revealed that both metabolites were identifi ed as biomarkers associated with the defi ciency of Oat1 ( 34 ). Another earlier study conducted by gas chromatography-mass spectrometry (GC-MS), also indicated that the excretion of endogenous organic anions was decreased in Oat1 knockout mouse urine ( 35 ). Here, CA treatment decreased Oatp1 and Oatp4 in CA-fed Ppara -null mouse liver and kidney. Oatp1 mRNA is found in various tissues ( 36 ) including the liver, kidney, and brain, whereas Oatp4 is exclusively in the liver ( 37 ). Along with the transport of bile acids, both transporters play a major role in the uptake and secretion of other organic anions in the kidney and liver, such as bromosulfophthalein and estrone sulfate ( 38,39 ). Thus, it is likely that the diminished expression of Oatp1 and Oatp4 in the liver and kidney is responsible for the decrease of phenylacetylglycine and xanthurenic acid in urine.
Previous studies have demonstrated that the activation/ repression of PPAR␣ can affect bile acid biosynthesis. However, different activators, such as Wy-14,643 and fibrates, show completely different effects on the rate-limiting enzyme in bile acid synthesis enzyme Cyp7a1 (3)(4)(5) . Compared with fenofi brate and other fi brates, Wy-14,643 is a more potent ligand of PPAR␣. One possible reason for different effects is that the level of Cyp7a1 might be regulated by other nuclear receptors, such as liver X receptor (LXR) ( 40 ) and hepatocyte nuclear factor 4 ␣ (HNF4 ␣ ), during the activation of PPAR␣ ( 41,42 ). In this study, in comparison with control diet-treated wild-type mice, the enzymes involved in biosynthesis of bile acids from cholesterol were reduced in control diet-treated Ppara -null mice, including Cyp7a1, Cyp27a1, and Hsd3b7. One early study reported that the mRNA level of Cyp7a1 also could be reduced in Ppara -null mice compared with wild-type mice ( 3 ).