PON3 knockout mice are susceptible to obesity, gallstone formation, and atherosclerosis

The FASEB Journal • Research Communication

PON3 knockout mice are susceptible to obesity, gallstoneformation, and atherosclerosis

Diana M. Shih,*,1,2 Janet M. Yu,†,1 Laurent Vergnes,‡ Nassim Dali-Youcef,§

Matthew D. Champion,{ Asokan Devarajan,k Peixiang Zhang,‡ Lawrence W. Castellani,*David N. Brindley,# Carole Jamey,** Johan Auwerx,†† Srinivasa T. Reddy,*,k David A. Ford,{

Karen Reue,‡,‡‡ and Aldons J. Lusis*,†,‡

*Division of Cardiology, Department of Medicine, †Department of Microbiology, Immunology, andMolecular Genetics, ‡Department of Human Genetics, kDepartment of Molecular and MedicalPharmacology, and ‡‡Department of Medicine and Molecular Biology Institute, University of Californiaat Los Angeles, Los Angeles, California, USA; §IGBMC, Illkirch and Hopitaux Universitaires deStrasbourg, and **Laboratoire de Toxicologie, Universitaires de Strasbourg, Strasbourg, France;{Department of Biochemistry and Molecular Biology, and Center for Cardiovascular Research, St. LouisUniversity School of Medicine, St. Louis, Missouri, USA; #University of Alberta, Edmonton, Alberta,Canada; and ††Laboratory for Integrative and Systems Physiology, School of Life Sciences, EcolePolytechnique Federale de Lausanne, Lausanne, Switzerland

ABSTRACT We report the engineering and character-ization of paraoxonase-3 knockout mice (Pon3KO). Themice were generally healthy but exhibited quantitativealterations inbile acidmetabolismanda37%increasedbodyweight compared to the wild-type mice on a high fat diet.PON3 was enriched in the mitochondria-associated mem-brane fraction of hepatocytes. PON3 deficiency resultedin impaired mitochondrial respiration, increased mito-chondrial superoxide levels, and increased hepatic ex-pression of inflammatory genes. PON3 deficiency didnot influence atherosclerosis development on an apoli-poprotein E null hyperlipidemic background, but it didlead to a significant 60% increase in atherosclerotic lesionsize in Pon3KOmice on the C57BL/6J background whenfed a cholate-cholesterol diet. On the diet, the Pon3KOhad significantly increased plasma intermediate-densitylipoprotein/LDL cholesterol and bile acid levels. Theyalso exhibited significantly elevated levels of hepatotox-icity markers in circulation, a 58% increase in gallstoneweight, a 40% increase in hepatic cholesterol level, andincreasedmortality. Furthermore, Pon3KOmice exhibiteddecreased hepatic bile acid synthesis and decreased bileacid levels in the small intestine compared with wild-typemice. Our study suggests a role for PON3 in the metab-olism of lipid and bile acid as well as protection againstatherosclerosis, gallstone disease, and obesity.—Shih,D.M., Yu, J. M., Vergnes, L., Dali-Youcef, N., Champion,M. D., Devarajan, A., Zhang, P., Castellani, L. W.,

Brindley, D. N., Jamey, C. Auwerx, J., Reddy, S. T., Ford,D. A., Reue, K., Lusis, A. J. PON3 knockout mice aresusceptible to obesity, gallstone formation, and athero-sclerosis. FASEB J. 29, 1185–1197 (2015). www.fasebj.org

Key Words: bile acid • cholesterol • hyperlipidemia • mitochondria •

mitochondria-associated membrane

THE PARAOXONASE GENE FAMILY contains 3 members, PON1,PON2, and PON3, located as a cluster on mouse chromo-some 6 or human chromosome 7. PON1 is expressed pri-marily in the liver and is associatedwithHDLparticles in theblood(1, 2). PON2 isubiquitously expressedandassociatedwith endoplasmic reticulum (ER), nuclearmembrane, andmitochondria of the cell (3–7). PON3 is expressed pri-marily in the liver, although lower expression levels are alsoobserved in a variety of other tissues (8, 9). In humans andrabbits, low levels of PON3 are present on HDL (10, 11).However, murine PON3 is absent from the HDL and cir-culation, and it is entirely cell associated (12). Within thecells, PON3 was reported to be localized to ER and mito-chondria (13), although we show here that in liver it isprimarily in the mitochondria-associated membrane(MAM). All 3 PON members exhibit quorum-quenchinglactonase activities (14, 15). PON1also exhibits the capacityto hydrolyze organophosphates such as organophosphorusinsecticides (16). Studies with genetically engineeredmice and human epidemiologic studies suggest thatthe PON family members can inhibit oxidative stress,

Abbreviations: apoE, apolipoprotein E; CA, cholic acid; CC,cholate-cholesterol; CDCA, chenodeoxycholic acid; Cyp7a1,cytochrome P450, family 7, subfamily A, polypeptide 1; ER,endoplasmic reticulum; FCCP, carbonyl cyanide-p-trifluor-omethoxyphenylhydrazone; FXR, farnesoid X receptor; IDL,intermediate-density lipoprotein; KO, knockout; MAM,mitochondria-associated membrane; MCA, muricholic acid;

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1 These authors contributed equally to this study.2 Correspondence: Division of Cardiology, David Geffen

School of Medicine at UCLA, 10833 Le Conte Ave., A2-237CHS, Los Angeles, CA 90095-1679, USA. E-mail: [email protected]: 10.1096/fj.14-260570This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

0892-6638/15/0029-1185 © FASEB 1185

suppress inflammation, and protect against atheroscle-rosis (7, 17–24). While PON1 and PON2 have beenquite extensively characterized, the function of PON3 isless clear. Transgenic mice overexpressing PON3 wereshown to be protected against atherosclerosis and obesity(12), but the mechanism involved is unknown. Recently,PON3was shown to be up-regulated in cancer tissues andprotect against mitochondrial superoxide-mediated celldeath (13).

In the present report, we engineered mice that lackPON3 and used these mice to examine the functions ofPON3 under a variety of conditions. Although the micewere generally healthy, they showed quantitative alter-ations in mitochondrial functions, bile acid metabolism,and body fat. When stressed with a cholate-cholesterol(CC) diet, they exhibited substantial variation in choles-terol metabolism and increased atherosclerosis.

MATERIALS AND METHODS

Generation of Pon3KO mice, animal breeding, feeding, andatherosclerosis lesion development

A Pon3 targeting vector was constructed by subcloning a 3.6 kbClaI fragment containing exon 3 upstream and a 2.8 kb BamHIfragment containing exon 5 downstream of the neomycin-resistance gene expression cassette in the pMC1TKpA vector(Supplemental Fig. S1A). The gene targeting vector was linear-ized and electroporated into RW-4 embryonic stem cells derivedfrommouse strain 129X1/SvJ. G418/gancyclovir-resistant cloneswere screened by Southern blot analysis, and embryonic stemcells carrying the disrupted allele were microinjected into blas-tocysts of mouse strain C57BL/6J to produce chimeric mice.Chimeric mice were crossed with C57BL/6J mice to produce F1mice heterozygous for the PON3 null mutation. F1 PON3 het-erozygous mice were then backcrossed with C57BL/6J mice for9 more generations before intercrossing to produce mice ho-mozygous for the Pon3 mutation. To introduce the PON3 nullmutation onto the apolipoprotein E (apoE) knockout (KO)background, N5 mice carrying the PON3 null mutation werecrossed with apoEKOmice. The offspringmice heterozygous forboth the PON3 and apoE null mutations were then intercrossedto generate apoE KO mice and Pon3KO/apoE KO mice. Onlyfemale mice were included in the studies. ApoE KO andPon3KO/apoE KO mice were maintained on chow diet for ath-erosclerosis assessment.Diet induced atherosclerosis was assessedusing Pon3KO and wild-type (WT) mice on the C57BL/6J back-ground using a CC diet containing 15.8% fat, 1.25% cholesterol,and 0.5% sodium cholate (TD.90221; Harlan Laboratories,Indianapolis, IN, USA). Atherosclerotic lesion size at the aorticroot region was determined as previously described (25). Forobesity study, Pon3KO and WTmice were fed a high-fat Westerndiet (TD.88137;HarlanLaboratories) for 10wk.All animal studieswere approved by the UCLA Animal Research Committee.

Lipid and bile acid levels, serum chemistry, liver lipidextraction and analysis by mass spectrometry, andlovastatinase activity assay

For plasma lipid and lipoprotein level determinations, mice werefasted for 16hbefore bleeding.Total cholesterol,HDLcholesterol,unesterified/free cholesterol, triglycerides, and free fatty acidlevels were determined by enzymatic colorimetric assays (25).Phosphatidylcholine levels were assayed using an enzymaticcolorimetric assay from Wako (Richmond, VA, USA). Serumchemistry tests were performed by Pathology and LaboratoryMedicine Services of the Department of Laboratory AnimalManagement atUCLA.Total bile acids levels were assayedusinga kit fromDiazymeLaboratories (Poway,CA,USA) according tothemanufacturer’s protocol. This kit measures the 3a-hydroxylgroup of bile acids. For lipid extraction, 50 mg of liver werehomogenized in PBS. The lipids in the homogenate were thenextracted using the Folch method (26). The extracted lipidswere dried down and resuspended in 1% Triton X-100 beforelipid assays were performed as described above. Tissue homo-genates made from the livers of Pon3KO and WT mice wereused in lovastatinase activity assay as previously described (12).In other analyses, mass spectrometry was performed using lipidextracts prepared by Bligh-Dyer extraction (27) with eitherwater or water enriched with 1% acetic acid (for phosphatidicacid analyses) and using lipid class specific internal standards(1,2,3-triheptadecenoyl-sn-glycerol, heptadecanoyl cholesterylester,1,2-diarachidoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycerol, and 1,2-dimyristoyl-sn-glycero-3-phosphate). Elec-trospray ionization mass spectrometry (Quantum Ultra;Thermo-Fisher, Waltham, MA, USA) with lipid class (or mo-lecular species)–specific tandem mass spectrometric analysiswas utilized to quantify liver triglyceride [neutral loss scanning(NLS) of fatty acid losses with fingerprinting] (28), cholesterylester (NLS 368.5) (29), phosphatidylcholine (NLS 59.1) (30),diacylglycerol (selected reaction monitoring) (29), and phos-phatidic acid (PA) (product ion scanningm/z = 183) (30) levels.

Phosphatidic acid phosphatase activity assay

Phosphatidic acid phosphatase (PAP) activity was measured onliver tissue extracts as described previously (31, 32). Briefly, freshmouse tissues were directly homogenized in lysis buffer [250 mMsucrose, 2mMDTT,proteinphosphatase inhibitormixtures2and3 (Sigma-Aldrich, St. Louis,MO,USA), protease inhibitormixture(Roche Diagnostics, Basel, Switzerland), and 0.15% Tween-20].PAP-1 activity wasmeasured in a final volume of 0.1ml containing100 mMTris maleate buffer, pH 7.5, 5 mMMg2+, and 0.6 mM PAlabeled with [3H]palmitate (about 75 Ci/mol) dispersed in0.4 mM phophatidylcholine and 1 mM EDTA, and the reactionswere incubatedat37°C.Chloroformcontaining0.08%oliveoilwasadded to stop the reaction. Then basic alumina was added toabsorb the PA and any [3H] palmitate formed by phospholipaseA–typeactivities.The[3H]diacylglycerolproductwas then isolatedand quantified by scintillation counting. Three different proteinconcentrations were analyzed for each sample to ensure the pro-portionality of the assay. Parallel analyses were done in the pres-enceof excessN-ethylmaleimide (5mM) toassess the contributionof lipid phosphate phosphatase activity, and this latter activity wassubtracted from the total activity to yield true PAP activity values.

RNA isolation and quantitative RT-PCR analyses

TotalRNAsamples fromtissueswere isolatedusingTrizol reagent(Life Technologies) according to the manufacturer’s protocol.The cDNA was synthesized using the High Capacity cDNA Re-verse Transcription Kit (Applied Biosystems, Foster City, CA,USA). Quantitative PCR was performed using gene-specific

NLS, neutral loss scanning; Ntcp, Na+-taurocholate cotrans-porting polypeptide; OCR, oxygen consumption rate; Ost,organic solute transporter; PA, phosphatidic acid; PAP,phosphatidic acid phosphatase; Pon3, paraoxonase-3; Shp,small heterodimer partner; TIGM, Texas A&M Institute forGenomic Medicine; UDCA, ursodeoxycholic acid; VDAC,voltage-dependent anion channel; VLDL, very low-density li-poprotein; WT, wild type

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primers (Supplemental Table S1) and the Roche SYBR greenmaster mix in a Roche Lightcycler 480 system. The mRNA levelsof specific genes were normalized to the mRNA levels of thehousekeeping gene, Rpl13a, of the same sample.

Quantification of gallstones, collection of gallbladder andhepatic bile, bile lipid analysis, and bile acid compositionanalysis by mass spectrometry

After cholecystectomy, the gallbladder was cut at the fundus tocollect bile and gallstones. After drying overnight at room tem-perature, stones were weighed. Hepatic bile was collected for30 min as described (33). Bile samples were diluted with watercontaining 1% Triton X-100, followed by determination of cho-lesterol, phospholipid, and total bile acid concentrations usingcolorimetric assays as described above. The bile acid compositionof plasma, liver, gallbladder bile, and small intestine samples wasexamined and quantified using mass spectrometry as previouslydescribed (34).

Determination of average adipocyte size

Hematoxylin and eosin–stained histologic sections of fat padswere used for determination of adipocyte size as previously de-scribed (35).

Isolation of organelles and immunoblotting

Four mice from each group were fasted overnight before killingand collection of livers. The pooled liver samples were then usedfor isolation of organelles by ultracentrifugation according to theprotocol of Wieckowski et al. (36). For immunoblotting, equalamounts of purified organelles or liver lysates were fractionatedby SDS-PAGE, transferred onto a nylon membrane, incubatedwith various primary antibodies, washed, incubated with a sec-ondary antibody, and detected using electrochemiluminescence(GEHealthcare Bio-Sciences, Piscataway, NJ, USA). The primaryantibodies againstPON3andcalnexinwerepurchased fromR&DSystems (Minneapolis, MN, USA) and Santa Cruz Technology(Santa Cruz, CA, USA), respectively. Primary antibodies againstcytochrome c and voltage-dependent anion channel (VDAC)were purchased from Cell Signaling Technology (Danvers, MA).Primary antibody against Cyp7a1 (cytochrome P450, family 7,subfamily A, polypeptide 1) was a gift from Simon Hui of UCLA.Lipin-1 antibodywas a gift fromMarounBouKahlil (University ofOttawa, Ottawa, ON, Canada). Lipin-2 antibody was a gift fromBrian Finck (Washington University, St. Louis, MO, USA).

Mitochondrial functional assays and TUNEL assay

Mitochondria were isolated from mouse liver as described byRogers et al. (37). The mitochondrial function of isolated mito-chondria (5 mg per well) was determined using an XF24-3 Extra-cellular Flux Analyzer (Seahorse Bioscience) as described (37).Succinate (10 mM), rotenone (2 mM), ADP (2 mM), oligomycin(25 mM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone(FCCP; 40 mM), and antimycin A (15 mg/ml) were used to assaycomplex II dependent respiration. Oxygen consumption rate(OCR) in fresh tissues were measured with the XF24 Analyzer asdescribed (38). Briefly, freshly isolated tissues were minced andrinsed in PBS, placed in a Seahorse Islet Plate (3–5 mg per well),and incubated with 625 ml of unbuffered DMEM (containing25mMglucose) 1hprior tomeasurements.Oxygen consumptionwas measured after 200 mMpalmitate or 0.75 mMFCCP injectionand expressed as percentage OCR of baseline. For OCR in pri-mary white adipocyte, stromal vascular cells were collected, cul-tured, and differentiated as previously described (39). OCR was

measured at baseline and after the sequential injection of 1 mMoligomycin, 0.75mMFCCPand1mMrotenone/myxothiazol.TheTUNEL assay was performed on frozen sections of liver samples,using the In situ Cell Death Detection kit purchased from Roche,according to the manufacturer’s manual.

Statistical analyses

Log-rank (Mantel-Cox) test was used for comparison of survivalcurves shown in Fig. 1B. For the rest of the study, Student’s t testwas used for comparison of means.

RESULTS

General characteristics of mice deficient in PON3

We constructed mice targeted for Pon3 using classic genetargeting approaches (Supplemental Fig. S1). The strategyis shown in Supplemental Fig. S1A. Supplemental FigureS1B shows the expected Southern blot pattern followingcleavage with PstI in heterozygous targeted mice. The ho-mozygous Pon3KO mice lacked PON3 mRNA (Supple-mental Fig. S1C), and protein (Supplemental Fig. S1D) inliver. We previously showed that PON3 is able to hydrolyzelovastatin (12). The Pon3KOmice had no lovastatinase ac-tivity (Supplemental Fig. S1E) in liver, indicating that PON3is entirely responsible for lovastatin hydrolysis. The dataindicate that the targeting creates a PON3 null mutation.

We generated the Pon3KO on the background of strain129X1/SvJ. Since strain 129 mice are difficult to breed, wetransferred thenullmutationonto thegeneticbackgroundof C57BL/6J through a series of backcrosses for 10 gen-erations.ThePon3KOmicedidnot exhibit anydiscernibleeffects on appearance, fertility, or life span. When main-tained on a chow diet, there was no evidence of liver pa-thology. However, small but significant differences inplasma triglycerides and free fatty acid levels wereobservedbetween Pon3KO andWTmice (Supplemental Table S2).

PON3 deficiency does not influence atherosclerosis ina hyperlipidemic apoE null background

Previously, we had shown that transgenic mice over-expressing PON3 in liver exhibited reduced atherosclero-sis on a LDL receptor null background (12). To testwhether the Pon3KO mice would exhibit increased ath-erosclerosis, we bred PON3KOmice onto the backgroundof apoE null mice and examined the double KO mice foreffects on atherosclerotic lesion development. Surpris-ingly, there were no discernible differences effects onatherosclerosis (Supplemental Fig. S1F). Plasma lipopro-tein and lipid levels were similar between the double KOand apoE KOmice, except small but significant decreasesof HDL and free fatty acid levels in the double KO micecompared to the apoE KO (Supplemental Table S3).

Pon3KO mice exhibit significant alterations in lipidmetabolism and atherosclerosis when fed a dietcontaining cholic acid and cholesterol

We had previously shown that transgenic PON3 mice feda CC diet, exhibited significantly reduced atherosclerosis

PON3 KNOCKOUT MICE 1187

compared to control mice (12). We, therefore, testedwhether the Pon3KO mice would also exhibit altered ath-erosclerosis when fed the CC diet. When fed the diet for16wk, Pon3KOmiceexhibiteda significant 60% increase inatherosclerotic lesion size compared to WTmice (Fig. 1A).During thecourseof these studieswenoticed thatwhile96%(23 out of 24) of the WT mice survived, the Pon3KO miceexhibited significant mortality with only 65% (17 out of 26)survival (Fig. 1B). Furthermore, there were significantincreases in liver total cholesterol (40%), cholesterol ester(44%), and unesterified cholesterol (22%) levels, and a de-crease in liver triglyceride (56%) level in the Pon3KOmice(Fig. 1C).

Because of the mortality and the altered liver lipids, wecarried outmore detailedmetabolic studies of themice onthe CC diet. When maintained on the CC diet for 16 wk,PON3 mice exhibited significant alterations in plasmalipids and bile acid levels. The Pon3KO mice exhibitedsignificant increases in total cholesterol (320%), very low-density lipoprotein (VLDL)/intermediate-density lipo-protein (IDL)/LDL cholesterol (466%), unesterifiedcholesterol (896%), triglycerides (267%), and bile acids(273%) in the plasma (Table 1). These lipid effects couldcontribute to the increased atherosclerosis in Pon3KOmice compared to WTmice.

Given that the Pon3KO mice exhibited significantly in-creased mortality, we also examined evidence of hepatic

toxicity using plasma levels of alanine aminotransferase,aspartate aminotransferase, and direct bilirubin (Table 2).Compared to WT mice, the Pon3KO mice exhibiteda dramatic increase in all 3 parameters, consistent with in-creased hepatoxicity in response to the CC diet comparedto WT mice.

PON3 deficiency alters lipid and bile acid metabolism

To determine the basis of the differences in liver lipidsobserved in the atherosclerosis studies, we examined thegeneexpressionprofiles in liverof several genes involved ineither bile acid metabolism or inflammation. As shown inFig. 1D, in the livers of Pon3KOmaintained on the CC dietfor 16 wk, the levels of Cyp7a1 mRNA were decreased byabout 90% compared toWTmice. The Pon3KOmice alsoexhibited a substantial decrease in the levels of CYP7A1protein (Fig. 1E), the rate limiting enzyme in bile acidsynthesis. The mRNA levels of the alternative bile acidsynthesis pathway mediated by Cyp27a1 (cytochromeP450, family 27, subfamily A, polypeptide 1) were alsosignificantly reduced, as was the bile acid transporter Ntcp(Na+-taurocholate cotransporting polypeptide) (Fig. 1D).On the other hand, the transcriptional repressor of bileacid synthesis Shp (small heterodimer partner), exhibitedsignificantly increased expression (Fig. 1D). Thus, the

Figure 1. Pon3KO mice exhibit increased atherosclerosis, premature death, increased hepatic cholesterol accumulation, andaltered hepatic gene expression when fed a CC diet. Three-month-old WT and Pon3KO mice were maintained on a CC diet for16 wk before (A) atherosclerotic lesion at the aortic root region, (B) survival, (C) liver lipid content, (D) hepatic gene expression,and (E) hepatic Cyp7A1 protein levels were determined. CE, cholesterol ester; TC, total cholesterol; TG, triglyceride; UC,unesterified cholesterol. *P , 0.05; **P , 0.01; ***P , 0.001 between the 2 genotype groups.

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increased cholesterol in livers of Pon3KO mice fed theCC diet likely results in part from reduced bile acidsynthesis.

Atherosclerosis and inflammation in Pon3KO mice

Atherosclerosis is known to be enhanced by local or sys-temic inflammation (40). To test whether hepatic in-flammation might contribute to the increased lesiondevelopment that we observed in Pon3KO mice, we ex-amined the expression ofMcp-1, a chemokine, and Il-6, aninflammatory cytokine. Both were substantially increasedin Pon3KO mice (Fig. 1D). We also showed that Atf3 (ac-tivating transcription factor 3), a member of the unfoldedprotein response, was increased (Fig. 1D).

Lipid and lipoprotein metabolism in Pon3KO mice

We conducted another 10 wk CC diet feeding study toavoid mortality in the Pon3KO mice and further examinelipoprotein and lipid metabolism. The Pon3KO miceexhibited significantly increased total, VLDL/IDL/LDLcholesterol, and total bile acid levels, and decreased HDLcholesterol levels compared to WT mice (Table 3). Theplasma lipoprotein profile was further examined by FPLC.As shown in Fig. 2A, the Pon3KO exhibited higher IDL/LDL and lower HDL cholesterol levels compared to WTmice. Mass spectrometry analysis of liver lipid extractsshowed decreased triglycerides and diacylglycerol, but in-creased phosphatidate levels in the Pon3KO liver com-pared to those of the controls (Fig. 2B), suggesting lowerPAP activity in the Pon3KO liver. We determined thatrecombinant PON3 does not exhibit PAP activity (data notshown), suggesting that the observed phosphatidate ac-cumulation may be related to altered activity of the lipinPAP enzymes. Lipins 1, 2, and 3 are known to exhibit PAPactivity that converts PA to diacylglycerol, the precursor oftriglyceride and phospholipid biosynthesis (41). We couldnot detect Lipin3mRNA in any liver samples. QuantitativePCR and Western blot analyses revealed no differences inhepatic Lipin1 or Lipin2 mRNA levels (Fig. 2C), or Lipin1protein levels (data not shown) between Pon3KO andcontrols. However, Lipin2 protein levels were elevated inthePon3KO liver (Fig. 2D). Liver PAP activity, on the otherhand,was similar betweenPon3KOandWTmice (Fig. 2E).

In mice that were fed the CC diet for 10 wk, we alsoobserved decreased expression of Cyp7a1, Cyp8b1 (cyto-chrome P450, family 8, subfamily B, polypeptide 1), andNtcp and increased expression of Shp in the livers ofPon3KOmice compared to those of theWTmice (Fig. 2C).Expression of these genes is known to be regulated by

farnesoidXreceptor (FXR). Inaddition, thePon3KOmiceexhibited decreased expression of hepatic enzymes in-volved in lipogenesis, including Acc, Me1, Scd1 (stearoyl-CoAdesaturase-1), andAgpat6 (1-acylglycerol-3-phosphateO-acyltransferase 6) (Fig. 2C). Furthermore, the expressionof genes involved in cholesterol homeostasis, includingLdlrand Hmgcr (3-hydroxy-3-methylglutaryl-CoA reductase),was significantly decreased in the Pon3KO livers (Fig. 2C).

Altered bile composition and increased gallstoneformation in Pon3KO mice

Gallbladderbile compositionwas analyzed in Pon3KOandWTmice on theCCdiet for 10wk.The decreased synthesisof bile acids in the liver (Fig. 2C) was accompanied byincreasedcholesterol andphospholipid levels in the bile ofPon3KO mice (Fig. 3A). In addition, we observed a signif-icant 39% increase in cholesterol levels in the hepatic bilesamples collected from the Pon3KO mice (Fig. 3B). Asshown in Fig. 3C, Pon3KO mice fed the CC diet exhibita 58% increase in gallstone weight. Presumably, the de-creased bile acid synthesis and increased cholesterol ex-cretion in the Pon3KOmice resulted in an increased ratioof cholesterol to bile acids in the gall bladder, favoring theformation of gallstones.

We performed mass spectrometry analysis to examinebile acid composition of plasma, liver, bile, and small in-testine samples of Pon3KO and WT mice on the CC diet(Fig. 4). Since the CC diet contains 0.5% cholic acid (CA),the major bile acid found in the samples of these mice wasCA (Fig. 4). The rest of the bile acids in these samples aredeoxycholic acid, the secondary bile acid generated by thegut bacteria using CA as the substrate, and other endoge-nously synthesized bile acids including muricholic acid(MCA), chenodeoxycholic acid (CDCA), and ursodeox-ycholic acid (UDCA) (Fig. 4). In contrast to humans,UDCA is a primary bile acid in rodents (42). In the plasma,there are significantly increased levels of all of the bile acidspecies in the Pon3KOmice compared to those of theWTmice (Fig. 4A). In the liver, the total bile acid content in the

TABLE 1. Plasma lipid, glucose, and bile acid levels of Pon3KO and WT mice on a CC diet for 16 wk

Genotype n TriglyceridesTotal

cholesterolHDL

cholesterolVLDL/IDL/LDL

cholesterolUnesterifiedcholesterol

Free fattyacids Glucose Bile acids

WT 16 6 (0.5) 250 (13) 74 (5) 176 (9) 68 (6) 29 (1) 128 (6) 73 (10)Pon3KO 6 22 (9.8)* 1051 (335)* 55 (16) 996 (349)* 677 (280)* 29 (6) 194 (42) 272 (90)*

All values shown are means (SE) in mg/dl except bile acids values, which are shown in mM. *P , 0.05, Pon3KO vs. WT, using log2 transformed data.

TABLE 2. Plasma ALT, AST, and direct bilirubin levels of Pon3KOand WT mice on a CC diet for 16 wk

Genotype n ALT (U/L) AST (U/L) Direct bilirubin (mg/dl)

WT 11 262 (37) 216 (17) 0.19 (0.02)Pon3KO 6 853 (281)* 935 (312)* 8.65 (3.99)*

All values shown are means (SE). ALT, alanine aminotransferase;AST, aspartate aminotransferase. *P , 0.05, Pon3KO vs. WT, usinglog2 transformed data.

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Pon3KO mice was not different from the WT mice (Fig.4B).However, the levels of several unconjugated bile acids,including b-MCA, a-MCA, CDCA, and UDCA, were sig-nificantly decreased in the Pon3KO compared to those oftheWTmice (Fig. 4C).Thismight have been caused by thedecreased expression of bile acid synthesis genes observedin the Pon3KO liver (Fig. 1D), or by decreased uptake of

bile acid returning from the small intestine as a result ofdecreased expression of the bile acid transporter, Ntcp. Inthe bile samples we observed a significant decrease indeoxycholic acid levels and a trend of decreased levels ofother bile acids in thePon3KOcompared toWTmice (Fig.4D). Finally, in the small intestine, while the levels of CAand deoxycholic acid, presumably derived from the diet,

TABLE 3. Plasma lipid, glucose, and bile acid levels of Pon3KO and WT mice on a CC diet for 10 wk

Genotype n Triglycerides Total cholesterol HDLVLDL/IDL/

LDL Free fatty acids Bile acids

WT 11 9.9 (1.9) 193 (11) 62 (3) 131 (10) 31 (2) 48 (5)Pon3KO 12 7.6 (1.3) 240 (11)* 50 (3)* 190 (10)* 29 (2) 74 (9)*

All values shown are means (SE) in mg/dl, except bile acids values are shown in mM. *P , 0.01,Pon3KO vs. WT.

Figure 2. Altered plasma lipoprotein and liver lipid levels in Pon3KO mice fed the CC diet for 10 wk. Pon3KO and WT mice weremaintained on the CC diet for 10 wk before tissue/plasma collection. A) Plasma lipoprotein profiles of Pon3KO and WT mice asfractionated by FPLC. B) Liver lipid concentration as determined by mass spectrometry. C) Liver gene expression as determinedby qPCR. D) Immunoblotting of mouse lipin-2 protein in liver protein lysate. E) Liver PAP activity was determined as described inMaterials and Methods. #P = 0.09, *P , 0.05, **P , 0.01, ***P , 0.001 between the 2 genotype groups.

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were not different between the 2 groups ofmice, the levelsof endogenously synthesized bile acids, MCA, CDCA, andUDCA, were significantly decreased in the Pon3KO micecompared to those of the WT mice (Fig. 4E).

Pon3KO mice exhibit altered gene expression in smallintestine and kidney when maintained on the CC diet

The mRNA levels of FXR target genes, Mrp2 (multidrugresistance protein 2) and Abcg8 (ATP-binding cassettesubfamily Gmember 8) were significantly decreased in theileum samples of Pon3KOmice fed the CC diet comparedto those of the WTmice (Supplemental Fig. S2A). On theother hand, the expression of other FXR-regulated genesincluding Shp,Osta andOstb (organic solute transporter aand b), I-babp (ileal bile acid-binding protein), Fgf15 (fi-broblast growth factor 15), Abcg5 (ATP-binding cassettesubfamily G member 5), and Asbt (apical sodium-dependent bile salt transporter) was not significantly dif-ferent between these 2 groups of mice (Supplemental Fig.S2A). In kidney, the expression of FXR target genes, Shpand Mrp2, was significantly increased in the Pon3KOcompared to WTmice (Supplemental Fig. S2B), probablyresulted from theelevatedcirculatingbile acid levels foundin the Pon3KOmice (Fig. 4A). However, the expression ofother FXR target genes, includingOsta,Ostb, and Asbt wasnot different between the 2 genotype groups (Supple-mental Fig. S2B).

Pon3KO mice exhibit elevated plasma total bile acidlevels when maintained on a Western diet

Wealsodeterminedplasma total bile acid levelsofPon3KOand WT mice maintained on either low fat chow diet oraWestern diet.While therewas no significant difference inplasma total bile acid levels betweenPon3KOandWTmiceon chow diet (Supplemental Fig. S3A), Pon3KO exhibitedsignificantly elevated levels of plasma total bile acid com-pared to WT mice when fed the Western diet (Supple-mental Fig. S3A). Hepatic expression levels of genesinvolved in bile acid homeostasis, including Bsep (bile saltexport pump), Cyp7a1, and Ntcp, were not different be-tweenPon3KOandWTmice,whereas ShpmRNA level was

significantly elevated in the Pon3KO mice when main-tained on the Western diet (Supplemental Fig. S3B).

PON3 is localized in MAM and influencesmitochondrial respiration and hepatic inflammation

Previous studies had suggested that PON2 may influencemitochondrial functions (6, 43). Therefore, we sought totest whether the effects of PON3 deficiency on bile acidmetabolism might involve various mitochondrial func-tions. We began by examining the subcellular localizationof PON3. WT and Pon3KO livers were subjected to sub-cellular fractionation on density gradients to producefractions enriched in ER, crude mitochondria (CM),highly purified mitochondria (PM), and MAM. We usedWestern blot analysis to examine the presence in thesefractions of PON3, calnexin (MAM and ER marker),VDAC, and cytochrome c (mitochondrialmarkers). As canbe seen, PON3 is present in crudemitochondrial fractionsbut not in highly purified fractions, and it colocalizes withcalnexin inERandMAM,withhigher abundanceof PON3in MAM than ER (Fig. 5A). The PON3 antibody we usedexhibits no reactivity with any of the fractions in Pon3KOmice, indicating its specificity (Fig. 5A).

To examine whether the Pon3KO mice exhibitedalterations inmitochondrial functions, we used a SeahorseBioscience (Lowell, MA, USA) XF24-3 instrument tomeasure OCR, an indicator of mitochondrial respiration.In the presence of succinate and rotenone (for measure-ment of respiration driven by complex II–IV activity),Pon3KO mitochondria exhibited significantly lower OCRin response to ADP (state 3) and FCCP (state 3u), in-dicating decreasedmitochondrial respiration (Fig. 5B, C).

Mitochondria are a major source of superoxide (44),and we investigated whether superoxide might contributeto the increased inflammationobserved inmice fed theCCdiet. As shown in Fig. 5D, Pon3KO mice exhibited abouttwice the level of superoxide compared to WT mice.Complexes II–III are essential parts of mitochondrialelectron transport chain. As shown in Fig. 5E, Pon3KOmice exhibited a significantly decreased complex II–IIIactivity compared to WT mice when fed the CC diet, sug-gesting impaired mitochondrial respiration. Because the

Figure 3. Gallstone formation is increased in Pon3KO mice fed the CC diet. WT and Pon3KO mice were maintained on the CCdiet for 10 wk (A, C) or 2 wk (B) before (A) gallbladder bile composition (8 WT and 10 Pon3KO mice), (B) hepatic bilecomposition (6 WT and 6 Pon3KO mice), and (C) gallstone weight were determined. Symbols as in Fig. 1.

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Pon3KO mice on the CC diet exhibited increased super-oxide and inflammatory gene expression, we wonderedwhether theremight be an increase in apoptosis. As shownin Fig. 5F, Pon3KO mice exhibited a 66% increase inTUNEL-positive cells compared to WT mice.

Increased obesity in Pon3KO mice due to impairedmitochondrial respiration and decreased fattyacid oxidation

After a 10 wk feeding of a Western diet, the Pon3KOmiceexhibited a significant 37% increase in body weight com-pared to WT mice (Fig. 6A), despite similar daily foodconsumption between the 2 groups (data not shown).Furthermore, the weights of gonadal, retroperitoneal, andsubcutaneous fat pads of the Pon3KO mice were signifi-cantly increased compared to those of the WT mice (Fig.

6B). The average size of adipocytes of the gonadal fat padwas also significantly increased by 25% in Pon3KO com-pared toWTmice (Fig. 6C). Thus, PON3deficiency resultsin increased obesity.

We thenexaminedmitochondrial respirationof fat padsisolated from the 2 groups of mice. Tissues from the go-nadal and subcutaneous fat pads were assessed for theiroxygen consumption rate after injection of the uncouplerFCCP. By revealing the maximal respiration capacity,FCCP can be used to uncover disrupted electron transportchain activity. Pon3KO mice showed decreased OCR inresponse to FCCP in the fat depots compared to those ofthe WT mice (Fig. 6D). The fact that the Pon3KO tissuescannot increase respiration under stress to the same de-gree as WT mice suggests that the Pon3KO mice haveimpaired mitochondrial respiration. This defect was con-firmed when tissues were challenged with palmitate to in-duce fatty acid oxidation. The OCR response to palmitatewas substantially decreased in the fat depots from Pon3KOmice (Fig. 6E).

To confirm the mitochondrial respiration defect in thePon3KO fat pads, we isolated stromal vascular cells fromthe subcutaneous fat pads. Cells were differentiated for7 d andwere found toharbor a white adipocyte phenotypewith large lipid droplet–filled cells (data not shown). OCRwas monitored during a mitochondrial assay where oligo-mycin, FCCP, and amixof rotenone andmyxothiazol weresequentially injected after the baseline measures. Thisallowed for an estimation of the contribution of ATP-linked mitochondrial oxygen consumption, non-mitochondrial respiration, and maximal mitochondrialrespiratory capacity. OCR response from the Pon3KOadipocyte were not different from the WT cells after oli-gomycin or rotenone/myxotiazol (Fig. 6F), suggestingno difference in ATPase activity or nonmitochondrialrespiration. In contrast, the response to FCCP was sub-stantially lower in Pon3KO compared to WT cells (Fig. 6F),confirming thedefect inmitochondrial respirationcapacity.

DISCUSSION

We report the generation of Pon3KO mice and theircharacterization to further understand the functions ofthis enzyme. Several novel and unexpected findingsemerged. First, PON3 deficiency clearly influences bileacid metabolism by an as yet unknown mechanism. Theincreased gallstone formation observed in the Pon3KOmice is probably caused by the decreased bile acid secre-tion and increased cholesterol excretion into bile. Second,PON3 is enriched in the mitochondria-associated mem-brane fraction of the hepatocytes, and PON3 deficiencyhasaclear impactonmitochondrial functionandoxidativestress. The increased diet-induced obesity observed in thePon3KO mice may be explained in part by impaired mi-tochondrial function and fatty acid oxidation observed inthe fat pads isolated from the Pon3KOmice. Third, PON3deficiency results in increased superoxide levels andapoptosis in liver as well as increased expression of in-flammatory genes. PON3 deficiency also leads to a proa-therogenic lipoprotein profile, including elevated IDL/LDL and decreasedHDL cholesterol levels, when fed theCC diet. The proatherogenic lipoprotein profile and

Figure 4. Bile acid composition analysis. WT and Pon3KOmice were maintained on the CC diet for 16 wk before varioustissues were collected for bile acid composition analysis.Shown are data from (A) plasma, (B, C) liver, (D) bile, and(E) small intestine. Data are the sum of both conjugated andunconjugated bile acids, except for (C), where only levels ofunconjugated bile acids are shown. Symbols as in Fig. 1.

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increased inflammation in the Pon3KO mice likely leadto the increased atherosclerotic lesion formation com-pared toWTmice.On the other hand, we cannot rule outthe notion that PON3 deficiency in the artery wall mayalso contribute to atherogenesis because mouse Pon3 isexpressed in themacrophages (45). However, there is noevidence that mouse Pon3 is expressed by endothelial orsmooth muscle cells.

A recent publication described generation of anotherline of PON3nullmice independently; Kempster et al. (46)concluded that PON3deficiency led to embryonic lethalityinmice. In the present study, we showed a complete loss ofPON3 at the mRNA, protein, and activity (based on lova-statinase activity) levels, yet we did not observe any

embryonic lethality of Pon3KOmice (data not shown). Infact, PON3null allelewas inherited in aMendelian fashionwith a 1:2:1 genotype ratio of WT, heterozygous, and ho-mozygous PON3 mutant mice being observed in the off-spring of a cross between PON3 heterozygous mice (datanot shown). There are 2 plausible explanations for thedifference in lethality observed between these 2 lines ofPon3KO mice. First, different genetic backgrounds havebeen shown to influence the phenotype and lethality ofmice harboring null mutations (47, 48). Our PON3 nullmutation was generated using an embryonic stem cell linederived from the 129X1/SvJ background. The null muta-tion was then introduced onto the C57BL/6J geneticbackground by 10 generations of backcrossing. Kempster

Figure 5. PON3 is localized in MAM and influences mitochondrial function. A) Distribution of PON3 protein in variousorganelles isolated from the WT and Pon3KO livers. CM, crude mitochondria; ER, endoplasmic reticulum; MAM, mitochondria-associated membrane; PM, pure mitochondria; T, total lysate. Distribution patterns of calnexin, VDAC, and cytochrome c are alsoshown. B, C) Complex II–IV-dependent respiration of mitochondria isolated from the liver of WT and Pon3KO mice fed a chowdiet. D–F) Mice were maintained on the CC diet for 10 wk before liver samples were collected for determination of (D)mitochondrial superoxide levels, (E) mitochondrial complex II + III activity, and (F) extent of apoptosis as measured by TUNELassay. *P , 0.05, ***P , 0.001 between the 2 genotype groups.

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et al. (46) obtained their Pon3KO mice from Texas A&MInstitute for Genomic Medicine (TIGM). According toinformation obtained from the TIGM website, theirPon3KO mice were on a mixed background of 129S5/SvEvBrd and C57BL/6. This difference in genetic back-ground between the 2 Pon3KO mice may have causeddifference in lethality, but the rather closely related back-grounds suggest that this is unlikely. Second, a spontane-ous null mutation of a passenger gene near the Pon3 locusmay have occurred during embryonic stem cell manipu-lation, and this mutation of the passenger gene could bethe cause of embryonic lethality observed in Pon3KOmiceobtained from the TIGM. An example of this has beenpreviously reported (49), and this seems the more likelyexplanation. A detailed expression analysis of passengergenes near the Pon3 locus of the TIGM Pon3KO micecould help to determine whether these mice harbor ad-ditional null mutations that could be the cause of embry-onic lethality.

Pon3KOmice exhibited increased plasma total bile acidlevels when maintained on a CC diet containing choles-terol and cholic acid (Tables 1 and 3). This increase ofplasma bile acids observed in the Pon3KO mice was likelydue to the deceased Ntcp expression (Figs. 1D and 2C),a transporter that mediates bile acid portal uptake. Livergene expression pattern showed increased activation ofFXR in the Pon3KO mice, as evidenced by increased ex-pression of Shp and Bsep and decreased expression ofCyp7a1, compared to WTmice when fed the CC diet (Fig.1D). However, we failed to observe higher total concen-trations of bile acids in the livers of Pon3KO mice com-pared to WTmice (Fig. 4B). This could be because we didnot perfuse the livers before tissue collection, which couldobscure the difference between the 2 groups of liver sam-ples. The significantly decreasedexpressionofCyp7a in thelivers of Pon3KO mice could be due to the actions of theFXR-SHP-dependent pathway (50) and of FXR-independentpathways such as the inhibitory effects of inflammatory

Figure 6. Increased obesity and impaired mitochondrial function in the white adipose tissues of Pon3KO mice. The 5.5-mo-oldPon3KO and WT mice were fed a Western diet for 10 wk before (A) body weight and (B) fat pad weight (expressed as percentageof body weight) were determined (n = 13 for each genotype). Retro, retroperitoneal. C) The average size of adipocytes of thegonadal fat pad is shown. Gonadal fat pads from 6 mice of each group were examined. The oxygen consumption rate of gonadaland subcutaneous fat pads was examined in the presence of (D) FCCP and (E) palmitate. F) OCR of differentiated whiteadipocytes derived from the Pon3KO and WT mice was examined at baseline and after the sequential injection of oligomycin,FCCP, and rotenone/myxothiazol. Symbols as in Fig. 1.

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cytokines on the expression of Cyp7a1 (51). Becausethere was increased expression of inflammatory genes inthe livers of Pon3KOmice (Fig. 1D), this could contributeto further down-regulation of Cyp7a1 gene expression inthese mice. The elevated plasma total bile acid levels andsignificantly increased hepatic expression of Shp in thePon3KO mice fed the Western diet suggest that alteredbile acid metabolism in these mice can occur without thefeeding of cholic acid, a bile acid known to cause in-flammation in the liver (52). The Western diet, which ishigh in fat and contains a moderate amount of choles-terol (0.15%), is known to induce hepatic inflammatorygene expression as well (53). However, we did not ob-serve significant differences in the hepatic expression ofinflammatory genes, including Mcp-1, between Pon3KOand WT mice fed the Western diet (data not shown),suggesting the preferentially increased inflammation inthe livers of Pon3KOmice only occurs in the presence ofcholic acid feeding.

We observed increased mortality in the Pon3KO micewhen maintained on the CC diet for 16 wk (Fig. 1B). Thecause of death in thesemicewas likely cholestasis causedbyincreasedgallstone formation (Fig. 3C) that led tobileductobstruction. In fact, we observed jaundice in critically illPon3KO mice (data not shown). Significantly elevatedplasmabilirubin, total bile acids, and total cholesterol levelswere also observed in the Pon3KO mice that survived the16 wk feeding of the CC diet (Tables 1 and 2), consistentwith the notion that these mice might have cholestasis.

Our study showed that Pon3KOmice have a proathero-genic lipoprotein profile with elevated IDL/LDL and de-creased HDL levels when maintained on the CC diet (Fig.2A).Wealsoobserved increasedcholesterol anddecreasedtriglyceride levels in the livers of the Pon3KOmice fed theCC diet (Fig. 1C). These changes are likely caused by thealtered bile acid metabolism observed in the Pon3KOmice. Decreased Cyp7a1 protein levels observed in thelivers of Pon3KO mice lead to decreased conversion ofcholesterol to bile acid, causing increased cholesterol ac-cumulation in the liver. Increased cholesterol accumula-tion in the livers of the Pon3KO mice may then result indecreased hepatic LDL receptor expression (Fig. 2C) thatleads todecreasedclearanceof IDL/LDLfromcirculation.Bile acids are known to lower liver triglyceride accumula-tion through inhibitionof expressionof lipogenic genes bya pathway involving FXR, SHP, and SREBP-1c (54). Like-wise, we observed decreased expression of lipogenic genes(Fig. 2C) and decreased triglyceride accumulation (Fig.1C) in the livers of Pon3KO mice fed the CC diet.

To our knowledge, ours is the first report to localizePON3 to MAM (Fig. 5A). MAM is the physical associationlocation between ER and mitochondria. The functions ofMAM include phospholipid synthesis, lipid transport, cal-cium homeostasis, and control of apoptosis (55, 56). Itremains unclear how PON3 influences MAM functions.However, we demonstrated that PON3 deficiency leads toimpaired respiratory function and increased superoxidelevels in the mitochondria isolated from the livers ofPon3KOmice (Fig. 5). Hydrophobic bile acids are knownto induce reactive oxygen species generation, and releaseof cytochrome c and apoptosis-inducing factors by the mi-tochondria (57). Our previous findings demonstrated therole of PON3 in protecting against superoxide formation

in mitochondria and superoxide-induced apoptosis incultured cells (13). We reason that the increased hepaticbile acid levels caused by cholic acid feeding lead to thesignificantly increased mitochondrial superoxide levels(Fig. 5D) and increased apoptosis (Fig. 5F) observed in thePon3KO mice. Furthermore, the accumulation of choles-terol has been shown to increase ER stress, inflammation,oxidative stress, and apoptosis in various cell types (58–64).The increased accumulation of cholesterol in the livers ofCC diet–fed Pon3KOmice (Fig. 1C) could also contributeto increased oxidative stress, inflammation, and apoptosisobserved in these mice.

PON3 deficiency also leads to impaired mitochondrialrespiration and fatty acid oxidation in the fat pads andadipocyte cultures derived from thePon3KOmice (Fig. 6).Themitochondrial dysfunction in thewhite adipose tissuesof Pon3KO mice could in part contribute to increasedadiposity observed in the Pon3KO mice compared to WTmice (Fig. 6). However, our study could not exclude thenotion that lack of PON3 in other tissues might contributeto the increased adiposity observed in Pon3KO mice. Inaddition, inflammation is known to contribute toobesity aswell. For example, mice that are deficient in TNF-a orprotease-activated receptor 2, a substantial contributor toinflammation, are both protected against obesity (65, 66).Thus, the increased inflammation associated with PON3deficiency could also contribute to obesity.

In summary, our data demonstrate that PON3 de-ficiency leads to impaired hepaticmitochondrial function,increased oxidative stress, inflammation, and cell deathwhen the Pon3KOmicewere challengedwith aCCdiet. Inaddition, our results suggest a role for PON3 in bile acidmetabolism. However, because Pon3KOmice on a low-fatchow diet did not exhibit altered bile acid levels, it is alsopossible that thePON3effectonbile acidmetabolism is theresult of liver injury caused by the CC diet. This studydemonstrates a protective role of PON3 against athero-sclerosis, gallstone disease, and obesity.

The authors thank Yu-Rong Xia, Yi-Shou Shi, YonghongMeng, Judy Wu, Xu-Ping Wang, Zhiqiang Zhou, and SaradaCharugundla for excellent technical support. This study wassupported by the following grants: U.S. National Institutes ofHealth (NIH) HL030568-26A1 (to A.J.L. and D.M.S.), andR01 HL074214 and RR019232 (to D.A.F.); National Center forResearch Resources S10RR026744 and NIH P01 HL028481(to K.R.), and 2 RO1 HL71776 (to S.T.R. and D.M.S.).

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Received for publication July 29, 2014.Accepted for publication November 7, 2014.

PON3 KNOCKOUT MICE 1197