Disruption of the Murine Protein Kinase C� Gene PromotesGallstone Formation and Alters Biliary Lipid and HepaticCholesterol Metabolism*

Received for publication, April 12, 2011, and in revised form, May 4, 2011 Published, JBC Papers in Press, May 5, 2011, DOI 10.1074/jbc.M111.250282

Wei Huang‡1, Rishipal R. Bansode‡1, Yan Xie§, Leslie Rowland‡, Madhu Mehta¶, Nicholas O. Davidson§,and Kamal D. Mehta‡2

From the ‡Department of Molecular and Cellular Biochemistry, The Dorothy M. Davis Heart and Lung Research Institute, and the¶Department of Medicine, The Ohio State University College of Medicine, Columbus, Ohio 43210 and the §Department of Medicine,Washington University School of Medicine, St. Louis, Missouri 63110

The protein kinase C (PKC) family of Ca2� and/or lipid-acti-vated serine-threonine protein kinases is implicated in thepathogenesis of obesity and insulin resistance. We recentlyreported that protein kinase C� (PKC�), a calcium-, diacylglyc-erol-, and phospholipid-dependent kinase, is critical for main-taining whole body triglyceride homeostasis. We now reportthat PKC� deficiency has profound effects on murine hepaticcholesterol metabolism, including hypersensitivity to diet-in-duced gallstone formation. The incidence of gallstones in-creased from 9% in control mice to 95% in PKC��/� mice. Gall-stone formation in the mutant mice was accompanied byhyposecretion of bile acids with no alteration in fecal bile acidexcretion, increased biliary cholesterol saturation and hydro-phobicity indices, as well as hepatic p42/44MAPK activation, allof which enhance susceptibility to gallstone formation. Litho-genic diet-fed PKC��/� mice also displayed decreased expres-sion of hepatic cholesterol-7�-hydroxylase (CYP7A1) and sterol12�-hydroxylase (CYP8b1). Finally, feeding a modified litho-genic diet supplemented with milk fat, instead of cocoa butter,both increased the severity of and shortened the interval forgallstone formation in PKC��/� mice and was associated withdramatic increases in cholesterol saturation andhydrophobicityindices. Taken together, the findings reveal a hitherto unrecog-nized role of PKC� in fine tuning diet-induced cholesteroland bile acid homeostasis, thus identifying PKC� as a majorphysiological regulator of both triglyceride and cholesterolhomeostasis.

Gallstone disease is a major public health problem in alldeveloped countries. Approximately 12% of individuals in theUnited States are affected by gallstones withmore than 750,000cholecystectomies being performed each year (1, 2). The patho-genesis of gallstone disease is multifactorial, involving interac-tions between genetic and environmental factors that result in

bile supersaturation, gallbladder hypomotility, and precipita-tion/nucleation of cholesterol microcrystals (3, 4). Obesity,aging, estrogen treatment, and diabetes are consistently associ-atedwith a higher risk of gallstones (5, 6). Interestingly, individ-uals undergoing weight loss also have a high risk of developinggallstones (7–9). The signalingmechanisms that link changes inthe triglyceride (TG)3 content of the body and impaired glucosemetabolism to cholesterol gallstone formation are unknown,but our knowledge is rapidly expanding. Systemic genome-wide scans in mice from experimental crosses of inbred mousestrains have mapped the “Lith” loci to increased gallstone sus-ceptibility (3, 4). A growing number of genetic variants in someof these genes have been linked to human gallstone disease (10).Protein kinase C (PKC) family members are lipid-dependent

serine/threonine protein kinases that play key roles in a widespectrum of signal transduction pathways and are of majorinterest as therapeutic targets for human diseases (11, 12).There are 12 family members that can be classified on the basisof their structures into three groups: classic (�, �I, �II, and �),novel (�, �, �, and �), and atypical (� and ). The exact functionof the different PKC isoforms is not yet established. However,there is evidence that individual PKC isoforms show diffe-rent substrate specificities, subcellular localization, cofactorrequirements, and sensitivity to agonist-induced down-regula-tion (13). Previous studies have firmly established that diabeticconditions activate the diacylglycerol-PKC signaling pathway(14–16). The induction appears to be restricted to a few “dia-betes-related” isoforms in a tissue-specific manner. PKC� isone of these isoforms and has been most directly linked toimportant aspects of hyperglycemia in vivo and in vitro (15).Interestingly, PKC�was one of the earliest isoforms recognizedin insulin signaling and appears to play dual roles in insulinsignaling (17). PKC� is involved in the transduction of specificinsulin signals but also contributes to the generation of insulinresistance. For instance, PKC� is activated by insulin and actsupstream of PI 3-kinase and mitogen-activated protein kinase(18). In contrast, activation of PKC� results in increased serine/* This work was supported, in whole or in part, by National Institutes of Health

Grants HL79091 (to K. D. M.) and DK56260, HL-38180, and DK52574 (toN. O. D.).

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Dept. of Molecular and

Cellular Biochemistry, The Dorothy M. Davis Heart and Lung Research Insti-tute, The Ohio State University College of Medicine, 464 Hamilton Hall,1645 Neil Ave., Columbus, OH 43210. Tel.: 614-688-8451; Fax: 614-292-4118; E-mail: Mehta.80@osu.edu.

3 The abbreviations used are: TG, triglyceride(s); CSI, cholesterol saturationindex; CYP7A1, cholesterol 7�-hydroxylase; CYP8b1, sterol 12�-hydroxyl-ase; MMLD, modified milk fat-containing lithogenic diet; PLD, purifiedlithogenic diet; WAT, white adipose tissue; LXR, liver X receptor; FXR, farne-soid X receptor; SHP, small heterodimer protein; PPAR�, peroxisome pro-liferator-activated receptor �.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 26, pp. 22795–22805, July 1, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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threonine phosphorylation of insulin receptor substrate-1 (19),and its excessive phosphorylation has been proposed to be oneof the mechanisms whereby dietary lipids and tumor necrosisfactor induce insulin resistance (20, 21).We recently reported that PKC�-deficient (PKC��/�) mice

are lean and exhibit resistance to high fat diet-induced obesity,hepatic steatosis, and insulin resistance (22, 23). Interestingly,these mice show hypocholesterolemia in response to a high fatdiet, suggesting an influence of PKC� in the regulation of cho-lesterol homeostasis. In addition, several previous studies havealso indicated a role for PKC isoforms in bile acid homeostasis.For example, cholesterol sulfate has been shown to activatespecific PKC isoforms, including PKC� (24, 25). In addition,tauroursodeoxycholic acid, which is used in the treatment ofcholestatic liver disease, is known to stimulate biliary secretionof bile acids throughCa2�-dependent PKC activation (26). Fur-thermore, PKC� (chromosome 7, 117.5 Mb, 61.0 centimor-gans) is a positional candidate for the Lith22 gene that wasdetected on chromosome 7 with peak linkage at 60 centimor-gans (27, 28). The potential effects of PKC� on insulin signal-ing, obesity, cholesterol, and TG homeostasis led us to hypoth-esize that PKC� may modify the pathogenesis of diet-inducedgallstone formation.In the present study, we explored the in vivo role of PKC� in

diet-induced gallstone formation using PKC��/� mice. Ourdata show that PKC�-deficient mice have greatly enhancedsensitivity to diet-induced gallstones despite their apparentprotection from high fat diet-induced obesity and insulin resis-tance. We propose that PKC� plays a pivotal role in the meta-bolic adaptation to lithogenic diet to prevent gallstoneformation.

EXPERIMENTAL PROCEDURES

Animals and Lithogenic Diet—PKC�-deficient C57BL/6Jmice have been described earlier (22, 23). Eight-week-old malewild-type (WT) and PKC��/� mice were fed ad libitum for7–8 weeks either a purified lithogenic diet (PLD) (TD-04428,Harlan Teklad, Madison, WI) containing 19% fat (cocoa butteris the primary fat source), 1% cholesterol, 0.5% cholic acid, andessential minerals and vitamins or a standard chow diet con-taining 8% fat (7912 rodent chow; Harlan Teklad). To studyeffects related to the substitution of cocoa butter with fat ofanimal origin, lithogenic diet composition was the same exceptthat cocoa butter was replaced with 15% anhydrous milk fat asthe primary fat source (modified milk fat-containing lithogenicdiet (MMLD)) (TD-10014, Harlan Teklad), and animals werefed this diet for 4 weeks. WT and PKC��/� mice were housedunder controlled temperature (23 °C) and lighting (12-h light/dark cycle) conditions with free access to water. C57BL/6J WTmice were obtained from The Jackson Laboratory (Bar Harbor,ME). Unless otherwise indicated, all experiments were per-formed on male animals that were fasted for �16 h. At theindicated period, animals were killed, cholecystectomies wereperformed, and gallbladders were examined for cholesterolgallstones. The presence of gallstones was determined by grossexamination of the gallbladder with the naked eye. All experi-ments were approved by the Institutional Animal Care and

Research Advisory Committee of the Ohio State UniversityCollege of Medicine.Lipid and Lipoprotein Analyses—Plasma TG, tissue TG, and

cholesterol concentrations were measured by colorimetricassays (Roche Diagnostics). Serum leptin and adiponectin lev-els weremeasured by enzyme-linked immunosorbent assay kits(Linco Inc.) according to the manufacturer’s protocol. Biliary(10 l) and hepatic (0.2 g of liver) lipids were extracted accord-ing to the Folchmethod (29). For comparative analysis of tissuelipids, lipids were extracted from liver, separated with ALSILGsilica gel TLC plates (Whatman) using hexane/ethyl ether/ace-tic acid (83:16:1), and stained with iodine vapor (30). Phospho-lipids were measured as inorganic phosphorus, and bile saltswere enzymatically quantified using the 3�-hydroxysteroiddehydrogenase method (Diazyme Laboratories, Poway, CA)(31). Analysis of lipids in lipoprotein fractions was performedafter separating pooled plasma samples by fast-performanceliquid chromatography (FPLC) with a Superose 6 10/300 GLhigh performance column (GE Healthcare Lifescience) by per-sonnel in the Mouse Metabolic Phenotyping Center facility atthe University of Cincinnati College of Medicine. Fractionswere assayed for total cholesterol and triglycerides. Plasma ala-nine aminotransferase and aspartate aminotransferase activi-ties were measured with the International Federation of Clini-cal Chemistry reference method without pyridoxal phosphateactivation by personnel in the chemistry laboratories at theVet-erinary Hospital of the Ohio State University.Histological Analysis of Tissues—Liver, white adipose tissue

(WAT), and gallbladder were isolated fromWT and PKC��/�

mice. Tissues were fixed with 4% formaldehyde in phosphate-buffered saline, processed, and stainedwith hematoxylin-eosin.Bile Flow, Biliary Lipid Output, and Bile Acid Species

Determination—Mice were anesthetized after a 4-h fast. Anexternal bile fistula was established surgically via the gallblad-der fundus. Hepatic bile was collected for up to 60 min whilemaintaining body temperature at 37 °C. Hepatic bile volumewas determined gravimetrically, assuming a density of 1 g/ml.Bile samples were stored at�80 °C until analyzed. Biliary phos-pholipids and cholesterol were determined enzymatically usinga phospholipids B kit and a cholesterol E kit fromWako Chem-icals (32, 33). Cholesterol saturation indices in hepatic bile werecalculated using published parameters (34). Total bile acid con-tent and individual bile acid concentrations were measured viahigh performance liquid chromatography (32, 33). In brief, 100l of diluted hepatic bile (1:20 dilution prepared in methanol)was injected onto a Pursuit C18 reverse phase column (Varian,Lake Forest, CA) using a Rainin HPXL HPLC system (Varian).Bile acid standards were purchased from Steraloids Inc. (New-port, RI) or from Calbiochem. The hydrophobic index ofhepatic bile samples was calculated as described earlier (32, 33).Bile Acid Pool Size and Fecal Bile Acid Output Determina-

tion—Bile acid pool size was determined as the sum total bileacid content of the entire small intestine, gallbladder, and liver,which were homogenized and extracted together in ethanol(32). Total bile acid mass was determined enzymatically. Inaddition, fecal bile acid output was determined from stoolquantitatively collected from individually housedmice for peri-ods up to 72 h. For each determination, 100-mg duplicate ali-

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quots of dried feces were extracted into ethanol as described(32), and total bile acidmasswas againmeasured enzymatically.RNA Extraction and Gene Expression Analyses—We ob-

tained liver samples from lithogenic diet-fed mice (16 h afterstarvation). Total RNAwas extracted with TRIzol (Invitrogen),and samples were pooled (n � 5) for analysis as described ear-lier (22, 23).Microsome Preparation—Livers from mice of the indicated

genotype were quickly removed and homogenized at 4 °C inbuffer I (40 mM Tris-HCl, 1 mM EDTA, 5 mM dithiothreitol, 50mMKCl, 50mMKF, 300mM sucrose, and 1� protease inhibitormixture. The homogenate was centrifuged at 20,000 � g for 20min, and the supernatant was further centrifuged at 108,000 �g for 60min. The pelletwaswashed and resuspended in buffer II(buffer I without sucrose), and aliquots representing 80 g ofmicrosomal protein were separated by 10% SDS-PAGE. West-ern blotting was performed with the anti-mouse CYP7A1 anti-body (Cosmo Bio USA, Inc., Carlsbad, CA).Western Blotting—Tissue samples were snap-frozen, pulver-

ized, and dispersed in lysis buffer. Samples were loaded onto12% acrylamide gels and blotted. Blots were probed using anti-phospho-p42/44MAPK or phosphorylation-independent anti-bodies. Immunoreactive proteins were visualized via enhancedchemiluminescence (ECL Plus; Amersham Biosciences) andquantified via densitometry using the Molecular Analyst soft-ware (Bio-Rad).

Statistical Analysis—Statistical comparisons were per-formed using unpaired Student’s t tests with p � 0.05 beingconsidered statistically significant.

RESULTS

PKC� Deficiency Sensitizes Mice to Lithogenic Diet-inducedGallstone Formation—To study the susceptibility of PKC��/�

mice to gallstone formation, we fed a PLD for 8 weeks andmeasured bodyweight, food intake, and gallstone formation.Asexpected from our previous work,WTmice were on an average2.5 g heavier than PKC��/� mice, although the food intake ofPKC��/� mice was �20% higher than that ofWTmice duringthe feeding study (Fig. 1, A and B). There were no discernablegallstones in the gallbladders from either genotype when fed achow diet for 8 weeks (Table 1). However, in WT mice fed alithogenic diet, 9% of gallbladders exhibited grossly visible gall-stones. In marked contrast, almost all gallbladders (95%) fromPKC��/� mice fed this diet were engorged with gallstones tovarying degrees (Fig. 1C). Table 1 shows semiquantitative anal-ysis of cholesterol gallstone formation in WT and PKC��/�

FIGURE 1. PKC� deficiency sensitizes mice to gallstone formation following PLD feeding. A and B, body weight (A) and food intake (B) of WT and PKC��/�

mice before and after feeding PLD diet for 8 weeks. *, p � 0.05. Each value represents the mean � S.D. (n � 5–10 mice/group). C, gallbladder of WT mouseappeared transparent, whereas the gallbladder from PKC��/� mouse contained visible gallstones. Gallstone formation was visually scored with the naked eye.

TABLE 1Comparison of gallstone formation between genotypes

Genotype Diet Gallstones

WT Chow 0% (0/22)PKC��/� Chow 0% (0/22)WT PLD 9% (2/22)PKC��/� PLD 95% (21/22)

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mice fed PLD. The low incidence of gallstone formation inWTmice observed in our study ismainly attributed to the feeding ofa semisynthetic and refined PLD as compared with lithogenicdiets used in other studies (35, 36). Because the fat content isderived primarily from cocoa butter, this purified diet has beenshown to consistently reduce liver damage and gallstone forma-tion in this strain (37, 38).There was a 28% increase in plasma cholesterol concentra-

tions and a 39% decrease in plasma TG concentrations inPKC��/� mice as compared with WT mice fed PLD (Fig. 2A).Fig. 2B shows the lipoprotein profile of the fresh plasma sam-ples obtained from each experimental group of mice. VLDL,intermediate density lipoprotein, and HDL TG were signifi-cantly low in PKC��/� mice; however, these mice exhibited alipoprotein cholesterol distribution that had a slight increasein intermediate density lipoprotein/LDL fractions, possiblyreflecting the replacement ofTG in the core of apoB-containinglipoproteins with cholesterol esters. Marked differences inhepatic TG contents were also observed between the WT andPKC��/�mice (Fig. 2C). PKC��/�mice exhibited significantlylower hepatic TG content, whereas hepatic total cholesterolcontent was 29% higher (Fig. 2C).We also examined liver and fat histology and measured adi-

pokine levels. The PLDdid not cause any significant differencesin liver weights betweenWT and PKC��/� mice (Fig. 3A), andno obvious histological liver damage was observed in PKC��/�

mice (Fig. 3B). Neither alanine aminotransferase nor aspartateaminotransferase activities were significantly different betweenWT and PKC��/� mice (data not shown). In contrast, epidid-

ymal WAT weights of PKC��/� mice were significantly lower(�2.5-fold) than those in controlmice (Fig. 3A), but theweightsof other organs, such as the kidney and muscles, did not differ(results not shown). Histological analysis of WAT showed thatthe size of lipid droplets in adipocytes was generally smaller inPKC��/� mice than in controls (Fig. 3B), suggesting that thedecreased cell size partly contributed to the fat mass decrease.Consistentwith adipose tissuemass, PKC��/�mice fed a litho-genic diet exhibited significantly lower serum leptin levels,whereas serum adiponectin levels were unchanged (Fig. 3C). Inaddition, gallbladder volume and turbidity were increased inPKC��/� mice versus WT mice (Fig. 3D). The gallbladdersfrom PKC��/� mice showed wall thickening and submucosalvasodilation (Fig. 3D).Alterations in Biliary Lipid Profiles in PKC��/� Mice on

Chow or Lithogenic Diet—The lithogenic phenotype promptedus to analyze the biliary lipid rates and lipid composition. Com-parable bile flow rates were observed in chow-fed mice of bothgenotypes with comparable secretion rates of cholesterol (Fig.4,A andD). By contrast, there was a trend to reduced phospho-lipid secretion but a significant decrease in bile acid secretionrates in PKC��/� mice (Fig. 4B), which was accompanied by asignificant increase in cholesterol saturation index (CSI) (Fig.4C). On the other hand, biliary concentrations of cholesterol,phospholipids, and bile salt were significantly higher in PLD-fed PKC��/� mice (Fig. 4E), but no significant difference wasobserved in biliary CSI (Fig. 4F). Individual bile acid speciesdetermination by HPLC revealed that the primary bile acids�-muricholate and taurocholate together represent the domi-

FIGURE 2. Comparison of metabolic profiles of WT and PKC��/� mice fed PLD. A, plasma cholesterol and TG levels. B, cholesterol- and TG-lipoproteindistributions are altered by PKC� deficiency in C57BL/6J mice on the PLD. Lipoprotein cholesterol and TG distributions were determined in pooled serum fromfive individual mice/group. Open shapes, WT mice; closed shapes, PKC��/� mice. IDL, intermediate density lipoprotein. C, relative amounts of liver TG andcholesterol (CE) contents following PLD feeding for 8 weeks. Each value represents the mean � S.D. (n � 5–10 mice/group). Thin layer chromatography of totallipid extract from the livers of WT and PKC��/� mice is also shown. Each lane represents lipids from the livers of individual mice. *, p � 0.05.

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nant species in chow-fed animals of both genotypes (Fig. 5A),without alterations to the hydrophobicity index of bile (Fig. 5C).In response to lithogenic feeding, there was a shift in the bileacid species, with taurocholate representing the major specieswith a corresponding decrease in �-muricholate for both gen-otypes (Fig. 5A). In addition, there was a further increase in therelative abundance of taurodeoxycholate in PKC��/� mice

(Fig. 5B). There was a significant increase in hydrophobicity inPKC��/� bile upon lithogenic feeding (Fig. 5D). Increases ineither CSI or hydrophobicity are known to facilitate cholesterolprecipitation (39), providing a plausible biochemical explana-tion for the lithogenic phenotype of PKC��/� mice.BileAcidPoolSizeandSyntheticRates inPKC��/�Mice—Tobet-

ter understand the general bile acid homeostasis, we also eval-

FIGURE 3. Feeding PLD did not cause any major hepatotoxicity. A, weights of liver and WAT for WT and PKC��/� mice fed PLD diet for 8 weeks (n � 5 for eachgenotype). BW, body weight. B, liver and WAT histology (hematoxylin-eosin staining) of the same group of mice. Pictures are representative of 8 –10 imagesobtained per animal in groups of 3– 4 animals. This experiment was performed 2–3 times. Magnification: �20. C, serum leptin and adiponectin levels in thesemice. Each value represents the mean � S.D. (n � 5–10 mice/group). *, p � 0.05; **, p � 0.01. D, gallbladder volumes and histology from each genotype.

FIGURE 4. Biliary flow, lipid secretion rates, and hepatic bile cholesterol saturation indices on chow or PLD. Hepatic bile was collected as described for 60min. A and D, biliary flow rates (l/min/kg of body weight). B and E, biliary bile salt secretion (mol/min/kg of body weight). C and F, hepatic bile CSIs werecalculated from the parameters in panels B and E using Carey’s critical tables. Bar graphs show the mean � S.E. (n � 4 – 8 for each genotype). *, p � 0.05.

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uated the total bile acid pool size and fecal excretion. Weobserved comparable total bile acid pool size in chow-fed WTand PKC��/� mice (Fig. 6A), and fecal bile acid excretion rates(at steady state, a surrogate for bile acid synthesis rates)revealed no statistically significant difference between the gen-otypes. By contrast, upon feeding the PLD, we observed a 1.4-fold increase in the bile acid pool size in PKC��/� mice (Fig.6A), which was not accompanied by any alteration in fecal bileacid output (Fig. 6B). The expanded bile acid pool (largely theresult of dietary cholate supplementation) in the setting of anyalteration in fecal losses suggests the possibility that enterohepaticbile acid recycling is increased inPKC��/�mice.Thispossibility is

also supported by the aforementioned changes in chow-fedPKC��/�mice (Fig. 4B), where decreased bile acid secretion ratesin the setting of unchanged bile acid pool size or fecal bile acidexcretion (Fig. 6, A and B) again imply that alterations in entero-hepatic cycling may contribute to this phenotype.Substitution of Cocoa Butter to Milk Fat Shortened Time

Required for Gallstone Formation in PKC��/� Mice—To beginto understand the influence of individual dietary componentson increased lithogenicity of PKC��/� mice, we turned to anMMLD in which cocoa butter was replaced with a fat of animalorigin (anhydrous milk fat). Cocoa butter (57.8% saturated,34.6%monounsaturated, and 7.6% polyunsaturated fatty acids)

FIGURE 5. Bile acid species distribution and hydrophobicity index. A and B, individual bile acid species were examined in newly secreted bile samples frommice fed chow (A) (n � 4/genotype) or PLD (B) (n � 8/genotype) by HPLC. Bar graphs represent the mean � S.E. GCDC, Glycochenodeoxycholate; T�MC,tauro-�-muricholate; TUDC, tauroursodeoxycholate; TC, taurocholate; GC, glycocholate; TCDC, taurochenodeoxycholate; TDC, taurodeoxycholate; TLC, tauro-lithocholate. C and D, bile salt hydrophobicity index (HI) was comparable on chow diet in both genotypes (C) but increased in PKC��/� mice versus WT mice (D)fed PLD for 7 weeks. *, p � 0.05.

FIGURE 6. Bile acid pool size and fecal bile acid excretion. A, bile acid pool size (mol/100 g of body weight (BW)) tended to be lower (but never reachedstatistical significance) in PKC��/� mice on chow diet and was higher in PKC��/� mice on PLD diet for 7 weeks. Liver, gallbladder, and intestine were pooledand subjected to ethanolic bile acid extraction. Total bile acid mass was measured enzymatically. B, fecal bile acid excretion rates were comparable in bothgenotypes on both diets. Mice were individually housed, and feces were collected up to 72 h. Fecal bile acids were extracted and quantitated enzymatically. Datawere from five mice/genotype fed chow diet and seven mice/genotype on PLD. Each value represents the mean � S.D. (n � 5–10 mice/group). **, p � 0.01.

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is comparable in fatty acid composition with milk fat (55.2%saturated, 29.8% monounsaturated, and 15.1% polyunsaturatedfatty acids). However, the actual percentage of specific types offatty acids differed between these diets. For example, 31% of satu-rated fatty acids in cocoa butter came from stearic acid, whereas itaccounted for only 10% of saturated fatty acids in milk fat.WT and PKC��/� mice were fed MMLD that contained 1%

cholesterol, 0.5% cholate, and 15% fat (anhydrousmilk fat) for 4weeks before being analyzed for gallstone formation. Notably,there were visible stones in 100% of PKC��/� mice as com-pared with 10% ofWT controls when fedMMLD. Importantly,this increased susceptibility to gallstones in PKC��/� mice wasaccompanied by dramatic increases in both CSIs and hydro-phobicity indices (Fig. 7). Comparison of gallstone formationbetween genotypes is shown in Table 2.PKC��/� Mice Exhibit Lower Expression of Hepatic Choles-

terol-7�-hydroxylase (CYP7A1) Gene and Increased HepaticPhospho-p42/44MAPK Levels—A reduction in biliary bile acidsin relation to cholesterol and phospholipids can result fromhyposecretion of bile acids. Accordingly, there are many candi-date enzymes, transporters, or regulators of hepatic cholesterolmetabolism that could theoretically influence the formation ofcholesterol gallstones. To understand the molecular mecha-nism by which PKC� deficiency promotes lithogenesis, mRNAexpression of candidate genes was examined in the livers frommice of both genotypes fed a PLD diet for 8 weeks. Hepatic

mRNA expression levels of the bile salt export pump ABCB11,the organic cation transporter SLC22A1, and the canalicularcholesterol transporters ABCG5 and ABCG8 were similar inboth groups of mice (Fig. 8A), whereas the ATP-binding cas-sette transporter A1 (ABCA1) showed slightly elevated levels inPKC��/� mice as compared with WT mice. Interestingly,PKC� deficiency down-regulated the classic and alternativepathways of bile salt synthesis by dramatically decreasinghepatic mRNA expression of the CYP7A1 gene (classic path-way) with no change in CYP27A1 mRNA expression (alterna-tive pathway) (Fig. 8A), a finding that is consistent with theincreased gallstone formation in PKC��/� mice. Additionally,significantly reduced expression of the sterol 12�-hydroxylase(CYP8b1) gene is expected to increase the bile hydrophobicityof PKC��/� mice (40). The reduction in CYP7A1 expressionwas not accompanied by any alteration in expression of tran-scription factors (LXR�, FXR, and SREBP-1c), which areknown to regulate its expression (41–43). Interestingly, feedingMMLD caused a greater suppression of both CYP7A1 andCYP8b1 genes in the livers of PKC��/� mice as compared withcontrols (Fig. 8B). We next asked whether the decreased

FIGURE 7. Biliary lipid secretion rates, cholesterol saturation indices, bile acid species distribution, and hydrophobicity indices in mice fed MMLD for4 weeks. A and B, WT and PKC��/� (n � 6/genotype) mice were fed MMLD containing milk fat for 4 weeks. A total of 10 mice/genotype were analyzed forgallstone formation, and a subset (6/genotype) was used to determine lipid secretion rates and bile acid species. C and D, CSIs and hydrophobicity indices werecalculated as described earlier (32, 33). Each value represents the mean � S.D. (n � 5–10 mice/group). *, p � 0.05.

TABLE 2Comparison of gallstone formation

Genotype Diet Gallstones

WT MMLD 10% (1/10)PKC��/� MMLD 100% (10/10)

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CYP7A1mRNA expression was accompanied by a correspond-ing decrease in protein expression and found decreased micro-somal CYP7A1 protein in PKC��/� livers as compared withWT mice fed PLD (Fig. 8C) or in livers of mice fed MMLD(results not shown). It is likely that higher liver cholesterol con-centration in PKC��/�mice is due to reducedmetabolic flux ofliver cholesterol into bile acids, possibly as a result of reducedhepatic CYP7A1 levels.Because hepatic p42/44MAPK activation has recently been

linked to suppression of CYP7A1 expression (44–47), weexamined activation levels of this kinase in WT and PKC��/�

mice by immunoblotting with antibodies that recognize theactivated phosphorylated form. PKC��/� mice fed PLD, butnot the standard chow diet, exhibited significantly increasedhepatic phospho-p42/44MAPK levels as compared with WTmice with apparently little or no effect on total p42/44MAPK

protein levels (Fig. 9). The increase in kinase phosphorylation

was accompanied by a corresponding increase in phosphoryla-tion of immediate upstream kinase, namely MEK-1/2. How-ever, the abundance of p38MAPKwas similar between genotypesfed chow or PLD. These findings together point to an unantic-ipated adaptation in PKC��/� mice in which CYP7A1 expres-sion is markedly attenuated in chow-fed animals and furthersuppressed following a lithogenic diet feeding.

DISCUSSIONThe present study defines, for the first time, a component of

the hepatic signaling pathway that is required for adaptation tolithogenic diet feeding and implicates PKC� as a critical kinasein mediating the metabolic response to this diet. Our observa-tion that PKC� deficiency stimulates diet-induced gallstoneformation provides direct evidence that this signaling kinase isimportant in maintaining cholesterol and bile acid homeosta-sis. This makes PKC� an excellent positional candidate for the

FIGURE 8. PKC�-dependent modulation of expression of CYP7A1 and CYP8b1 genes. A, the expression levels of each gene in WT and PKC��/� mice fedPLD for 8 weeks, which were normalized to the expression level in WT mice (n � 5 mice/group). Levels for each gene in WT mice were assigned a value of 1.B, expression levels of indicated genes in the livers of MMLD-fed WT and PKC��/� mice. C, Western blot analysis of CYP7A1 protein expression in livermicrosomes prepared as described from chow or PLD-fed animals (n � 4 mice of each genotype). Eighty micrograms of protein was subjected to SDS-PAGE andWestern blotting with anti-CYP7A1 IgG and quantitated by imaging with Bip used as a loading control (results not included). Each value represents the mean �S.D. (n � 5–10 mice/group). *, p � 0.05; **, p � 0.01.

FIGURE 9. Increased hepatic phospho-p42/44MAPK abundance in PKC��/� mice as compared with WT mice fed PLD. Equal amounts of the whole liverlysates from WT and PKC��/� mice fed chow or PLD for 8 weeks were separated by SDS-PAGE, and phosphorylation levels of p42/44MAPK, MEK-1/2, andp38MAPK were analyzed by Western blotting using phospho-specific or phosphorylation-independent p42/44MAPK antibodies following transfer of totalproteins onto nitrocellulose. The relative phosphorylation levels were determined by densitometry and are shown in the right panel. Phosphorylation levels forWT were assigned a value of 100% as shown in the graph. Each value represents the mean � S.D. (n � 5–10 mice/group). *, p � 0.05.

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Lith22 gene due to its close proximity within the locus nearD7Mit12 on mouse chromosome 7.

Although clinical studies have suggested that obesity is animportant risk factor for gallstone disease, little is knownregarding the precise metabolic pathways through which obe-sity contributes to gallstone formation (48). Previous animalstudies showed that some but by nomeans allmurinemodels ofobesity are associated with increased gallstone susceptibility.Indeed, prior studies in L-Fabp�/� mice have demonstratedprotection against high fat diet-induced obesity in the setting ofa strikingly increased susceptibility to lithogenic diet-inducedgallstone formation (33). These paradoxical findings suggestthat obesity itself is not a gallstone risk but rather that specificalterations in key pathophysiologic pathways leading to obesitymay in turn augment susceptibility to both diseases (49). Con-sistentwith these results, our findings demonstrate that protec-tion against high fat diet-induced obesity in PKC��/� mice isnot accompanied by protection against gallstone formation.Onthe contrary, PKC��/�mice lostmoreweightwhen consumingthe lithogenic diet and exhibited lower hepatic TGcontent thandid WT controls, suggesting that increased gallstone suscepti-bility observed in PKC��/� mice cannot be attributed to aug-mented obesity, steatosis, or impaired glucose intolerance. Thechanges observed in hepatic cholesterol metabolism in litho-genic diet-fed PKC��/� mice, by contrast, suggest a plausiblemechanism for the increased susceptibility to gallstone forma-tion. In particular, we observed a 50% increase in hepatic cho-lesterol content, coupled with higher plasma cholesterol levelsin lithogenic diet-fed PKC��/� mice.The liver eliminates cholesterol from the body via bile as

unmodified cholesterol and through conversion of cholesterolinto bile salts (50). This second route represents a major path-way for cholesterol elimination, accounting for approximatelyhalf of daily excretion. CYP7A1 catalyzes the first and rate-limiting step in the major classic pathway of bile acid biosyn-thesis and plays a crucial role in cholesterol balance. In thepresent study, we found significantly reduced hepatic CYP7A1expression, coupled with decreased biliary bile salt concentra-tions, in PKC��/� mice as compared with WT mice. Thereduction in CYP7A1 gene expression in PKC��/� mice islikely responsible for increased susceptibility to gallstone for-mation as compared with WT mice. Human CYP7A1 defi-ciency is known to promote premature gallstone formation (51,52), whereas CYP7A1 transgenic mice provide resistance togallstone formation (53). Several human studies also suggestthat an A to C nucleotide substitution in position �204 of theCYP7A1 promoter is associated with variation in plasma LDLcholesterol concentrations (54, 55). These findings led us topropose that a change in hepatic CYP7A1 expression inPKC��/� mice is a critical determinant of gallstone formationin vivo and suggest a plausible mechanism for their increasedsusceptibility.Among the most intriguing questions posed by this study is

the mechanism whereby CYP7A1 expression is modulated inthe setting of PKC� deletion. The control of CYP7A1 level isknown to occur primarily at the transcriptional level and ismodulated by the relative ratio of cholesterol to bile acids in the

liver. The transcriptional regulation is highly complex andinvolves a multitude of recognized mechanisms and, mostlikely, many still elusive regulatory mechanisms (56–58). Inrodents, CYP7A1 transcription is up-regulated by sterolsthrough an LXR�-dependent pathway. Conversely, theCYP7A1 gene is down-regulated by FXR through a feedbackloop involving both small heterodimer protein (SHP)-depen-dent and SHP-independent pathways. The results presentedhere argue strongly in favor of a mechanistic link betweenPKC� deficiency and CYP7A1 suppression. The finding thatmRNA levels were decreased in PKC��/� mice suggests that atranscriptional mechanism is involved. There are many poten-tial mechanisms by which PKC� either indirectly or directlycan affect hepatic CYP7A1 transcription. First, p42/44MAPK

activation has been linked with CYP7A1 suppression by bothSHP-dependent and SHP-independent pathways (44, 45). Onesuch study has shown that p42/44MAPK-induced phosphoryla-tion of hepatic SHP at serine 26 stabilizes this protein by inhib-iting ubiquitin-proteasomal degradation (45). However, lack ofa significant change in hepatic SHP content between genotypesargues against involvement of SHP-dependent pathways inCYP7A1 repression.4 Alternately, PKC� directly phosphory-lates nuclear receptors (LXR, FXR, and PPAR�) and/or thetranscription factor SREBP-1c and suppresses CYP7A1 tran-scription, which is in line with earlier reports showing the abil-ity of PKCs to phosphorylate and modify the above transcrip-tion factors (59–63). The absence of changes in LXR(ABCG5/8) or FXR (ABCB11) target genes argues against amechanism involving these transcriptional regulators. PKC�has also been shown to regulate a molecular switch betweentransactivation and transrepression activity of the PPAR� (61);loss of PKC�-mediated phosphorylation is associated with aloss of function for ligand-induced transcriptional activity and again of function for transrepression activity. Given the role ofPPAR� in CYP7A1 repression (65), loss of PPAR� phosphoryl-ation in PKC��/� mice is expected to cause greater suppres-sion of CYP7A1 expression as compared with WT mice. Fur-thermore, fat-derived diacylglycerol, which is a strong PKC�activator, may provide the possible link between PKC� andmodulation of PPAR� function and thus may reconcile studiesthat have shown dietary fat requirement for cholesterol-depen-dent induction of CYP7A1 expression (66, 67). Future studieswill focus on underlying molecular mechanisms linking PKC�deficiency with CYP7A1 suppression.Another consideration is whether alteration in gallbladder

contractility, based upon the differences observed in gallblad-der volumes in the two genotypes, contributes to the increasedsensitivity of PKC��/� mice to gallstones. Human studies haveshown that formation of cholesterol-supersaturated bile inpatients with gallstones is causally related to their decreasedgallbladder contractility (68, 69). Furthermore, it has beenshown in cholesterol-fed animals that the decrease in gallblad-der contractility is related to the impairment of cholecystokininsignaling-induced biliary alterations (70). These changes favorcholesterol crystal formation and may pathogenetically

4 W. Huang, R. R. Bansode, Y. Xie, L. Rowland, M. Mehta, N. O. Davidson, andK. D. Mehta, unpublished results.

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contribute to gallstone formation. Cholecystokinin-inducedgallbladder contraction via its receptor is mediated by the acti-vation of G-proteins and subsequent hydrolysis of phospha-tidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphateand diacylglycerol. Ultimately, these events lead to increasedsmoothmuscle contraction through the PKC, p42/44MAPK, andp38MAPK pathways (71–73). Thus, it is tempting to speculatethat enlarged gallbladder size and reduced p38MAPK activationin PKC��/� mice4 may reflect reduced gallbladder emptyingdue to a decrease in cholecystokinin signaling. Future studieswill focus on the potential alteration in the cholecystokinin-de-pendent signaling network and its physiological relevance toincreased lithogenesis in these mice.Dietary fats are known to modify cholesterol metabolism,

thereby affecting the whole body cholesterol homeostasis. Therole of dietary fat as an etiological factor for cholesterol gall-stone disease has received considerable attention but remainsunresolved (74, 75). Epidemiological studies have suggestedthat cholesterol gallstone disease development is positively cor-related with saturated fat intake and negatively related tomonounsaturated fat intake (76, 77). Subsequent studies inexperimental animal models have, in general, supported thissuggestion, but individual studies have shown variableresponses to diet-induced cholesterol gallstone disease (78–80). A significant difference in time required for gallstone for-mation was observed in PKC��/� mice despite the fact thatboth diets contained 1% cholesterol, 0.5% cholate, and similarfat content. The time required for gallstone formation was lon-gest in PKC��/� mice consuming cocoa butter diet, whereasmice consuming saturated fat of animal origin showedincreased severity of gallstone risk with gallstones visible after3–4 weeks. Results presented support the hypothesis that riskof diet-induced cholesterol gallstone disease is not only influ-enced by the degree of fatty acid unsaturation but also by thefatty acid composition.Finally, we also considered the possibility that alterations in

adipokine levels might also play a role in this phenotype (81).Previous studies have demonstrated that adipokines are indeedinvolved in gallstone development. For instance, increasedserum leptin has been shown to enhance biliary cholesterolconcentration, resulting in subsequent bile supersaturationwith cholesterol and hence an increased risk of gallstones (82).Data from leptin-deficient and leptin-resistant mice have addi-tionally suggested that leptin may decrease gallbladder activity(83) and shorten the formation time of cholesterol crystals (84).In contrast, a lower adiponectin level has been linked with gall-stone disease, obesity, and insulin resistance (85). In the presentstudy, we found a reduction in leptin concentration inPKC��/� mice, and such a change is expected to block diet-induced gallstone formation. With respect to PKC� deficiencypromoting gallstone formation, the negative effects describedabove likely supersede the potential beneficial effects offered byreduced leptin levels. The net result of these complex adapta-tions is a striking increase in gallstone susceptibility.In summary, we provide compelling evidence that PKC�

deficiency can disrupt the physiological balance between pro-and antilithogenic forces in favor of stone formation, at least inpart via repression of CYP7A1 expression. An increased super-

saturation of bile with cholesterol as a result of decreased bilesalts appears particularly relevant, although the contributionsof changes in gallbladder motility or enterohepatic cyclingremain to be explored. Our findings suggest that PKC� defi-ciency, either quantitative or qualitative, may contribute toalterations in diet-induced gallstone formation. A range ofpathophysiological conditions can affect PKC� expression oractivity in humans (64, 86). It will be of particular interest andimportance to define whether alterations in PKC� activity con-tribute to the pathogenesis of gallstone formation under thesedisease conditions.

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PKC� and Gallstone Prevention

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Davidson and Kamal D. MehtaWei Huang, Rishipal R. Bansode, Yan Xie, Leslie Rowland, Madhu Mehta, Nicholas O.

and Alters Biliary Lipid and Hepatic Cholesterol Metabolism Gene Promotes Gallstone FormationβDisruption of the Murine Protein Kinase C

doi: 10.1074/jbc.M111.250282 originally published online May 5, 20112011, 286:22795-22805.J. Biol. Chem. 

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