the j b c © 2000 by the american society for biochemistry and … · 2000-08-31 · defect in...

Defect in Peroxisome Proliferator-activated Receptor a-inducibleFatty Acid Oxidation Determines the Severity of Hepatic Steatosisin Response to Fasting*

Received for publication, December 29, 1999, and in revised form, June 6, 2000Published, JBC Papers in Press, June 7, 2000, DOI 10.1074/jbc.M910350199

Takashi Hashimoto, William S. Cook, Chao Qi, Anjana V. Yeldandi, Janardan K. Reddy‡,and M. Sambasiva Rao

From the Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611-3008

Fasting causes lipolysis in adipose tissue leading tothe release of large quantities of free fatty acids intocirculation that reach the liver where they are metabo-lized to generate ketone bodies to serve as fuels forother tissues. Since fatty acid-metabolizing enzymes inthe liver are transcriptionally regulated by peroxisomeproliferator-activated receptor a (PPARa), we investi-gated the role of PPARa in the induction of these en-zymes in response to fasting and their relationship tothe development of hepatic steatosis in mice deficient inPPARa (PPARa2/2), peroxisomal fatty acyl-CoA oxidase(AOX2/2), and in both PPARa and AOX (double knock-out (DKO)). Fasting for 48–72 h caused profound impair-ment of fatty acid oxidation in both PPARa2/2 and DKOmice, and DKO mice revealed a greater degree of he-patic steatosis when compared with PPARa2/2 mice.The absence of PPARa in both PPARa2/2 and DKO miceimpairs the induction of mitochondrial b-oxidation inliver following fasting which contributes to hypoketone-mia and hepatic steatosis. Pronounced steatosis in DKOmouse livers is due to the added deficiency of peroxiso-mal b-oxidation system in these animals due to the ab-sence of AOX. In mice deficient in AOX alone, the sus-tained hyperactivation of PPARa and up-regulation ofmitochondrial b-oxidation and microsomal v-oxidationsystems as well as the regenerative nature of a majorityof hepatocytes containing numerous spontaneously pro-liferated peroxisomes, which appear refractory to storetriglycerides, blunt the steatotic response to fasting.Starvation for 72 h caused a decrease in PPARa hepaticmRNA levels in wild type mice, with no perceptible com-pensatory increases in PPARg and PPARd mRNA levels.PPARg and PPARd hepatic mRNA levels were lower infed PPARa2/2 and DKO mice when compared with wildtype mice, and fasting caused a slight increase only inPPARg levels and a decrease in PPARd levels. Fastingdid not change the PPAR isoform levels in AOX2/2

mouse liver. These observations point to the criticalimportance of PPARa in the transcriptional regulatoryresponses to fasting and in determining the severity ofhepatic steatosis.

Higher animals, under fed conditions, preferentially burncarbohydrate to generate ATP, and surplus carbohydrate isconverted into fatty acids, which are then stored as triacylglyc-erols (TG)1 in adipose tissue. When glucose availability is lowduring periods of starvation, the TG stored in adipose tissueare hydrolyzed to free fatty acids (FFA) and mobilized intoplasma to reach liver where they play a major role in energyproduction (1–3). In liver, the influxed fatty acids are oxidizedpredominantly by the mitochondrial b-oxidation system and toa lesser extent by the peroxisomal b-oxidation, as well as byCYP4A-catalyzed microsomal v-oxidation pathways (4–6).Partial oxidation of fatty acids by mitochondrial and peroxiso-mal b-oxidation systems in liver leads to the production ofacetyl coenzyme A (acetyl CoA), which then condenses withitself to form ketone bodies. Ketone bodies generated in liverare exported out of the liver to serve as fuels for other tissuessuch as the skeletal and cardiac muscle and brain duringstarvation. Thus, alternate use of carbohydrate and fatty acidsto produce ATP is well regulated, and this regulatory energyconsumption is referred to as “glucose fatty acid cycle” thatrequires the maintenance of efficient hepatic fatty acid oxida-tion. It is estimated that in the adult, FFA and their ketonebody derivatives provide ;80% of caloric requirements after24 h of fasting (7). Fasting causes a more dramatic depletion ofcarbohydrate energy source in infants and children, and as aconsequence they exhibit greater dependence on efficient FFA-dependent ketogenesis during starvation, underscoring the im-portance of fatty acid oxidation in energy metabolism (8). Atpresent, several genetically determined metabolic defects atthe individual enzyme level in mitochondrial and peroxisomalfatty acid b-oxidation pathways have been identified, and in-dividuals with these defects remain essentially asymptomaticas far as basal energy metabolism is concerned under normalfeeding conditions (8). However, during conditions that lead toshort term fasting, they manifest severe hypoketotic hypogly-

* This work was supported by National Institutes of Health GrantGM 23750 (to J. K. R), Veterans Administration Merit grants (toM. S. R. and A. V. Y.), and the Joseph L. Mayberry, Sr. EndowmentFund. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

‡ To whom correspondence and reprint requests should be addressed:Dept. of Pathology, Northwestern University Medical School, 303 EastChicago Ave., Chicago, IL 60611-3008. Fax: 312-503-8249; E-mail:[email protected].

1 The abbreviations used are: TG, triacylglycerols; AOX, straightchain fatty acyl-CoA oxidase; CAT, carnitine acetyltransferase; COT,carnitine octanoyltransferase; CYP4A1 and CY4A3, encode microsomalcytochrome P450 fatty acid v-hydroxylases; DKO, double knock-outnullizygous for both PPARa and AOX; FFA, free fatty acids; HADH,3-hydroxyacyl-CoA dehydrogenase; 3-HB, 3-hydroxybutyrate; HS, 3-hy-droxy-3 methylglutaryl-CoA synthase; MCAD, medium chain acyl-CoAdehydrogenase; MTL1, mitochondrial 3-ketoacyl-CoA thiolase; TFP,mitochondrial trifunctional protein; MTL2, mitochondrial acetoacetyl-CoA-specific thiolase; D-PBE, peroxisomal D-3-hydroxyacyl-CoA dehy-dratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein;L-PBE, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydro-genase bifunctional protein; PPAR, peroxisome proliferator-activatedreceptor a, b/d or g; PTL, peroxisomal 3-ketoacyl-CoA thiolase; SCAD,short chain acyl-CoA dehydrogenase; SCOT, succinyl-CoA: 3-oxoacidtransferase; VLACS, very long chain acyl-CoA synthetase; VLCAD,very long chain acyl-CoA dehydrogenase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 37, Issue of September 15, pp. 28918–28928, 2000© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org28918

by guest on February 21, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

cemia, increased plasma FFA, variable degree of hepatic stea-tosis, and sudden death in early life because of their inability tooxidize FFAs in liver due to enzymatic defects (8). These met-abolic diseases have provided valuable insights pertaining tothe role of individual enzymes in fatty acid oxidation and en-ergy utilization.

It is now well recognized that fasting causes a rapid tran-scriptional activation of genes encoding mitochondrial, peroxi-somal, and microsomal fatty acid oxidation in liver in healthyindividuals (9, 10). These observations point to the importancealso of regulatory step(s) controlling the levels of inducible fattyacid oxidation enzymes. Any defect in the inducibility of theseenzymes can also impact on the energy metabolism and degreeof hepatic steatosis in response to fasting similar to thoseencountered with metabolic defects at the enzymatic level.Fatty acid oxidation occurs in mitochondria, peroxisomes, andmicrosomes, and some of the critical enzymes of these oxidationsystems are transcriptionally controlled by peroxisome prolif-erator-activated receptor a (PPARa), a member of the nuclearhormone receptor superfamily (11). PPARs, which derive thedesignation by virtue of their ability to mediate predictablepleiotropic effects in response to peroxisome proliferators (12),consist of three isotypes, namely PPARa, PPARd (also calledPPARb), and PPARg which are products of separate genes (11,13, 14). Peroxisome proliferators are structurally diverseagents which, when administered to rats and mice, induce notonly a marked peroxisome proliferation and increase in theenzyme proteins of the peroxisomal fatty acid oxidation butalso induce changes in carbohydrate and lipid metabolisms (4,12). The induction of mitochondrial, peroxisomal, and microso-mal CYP4A genes involved in fatty acid oxidation requires theformation of PPARa heterodimerization with retinoid X recep-tor, and this PPARazretinoid X receptor complex binds to PPARresponse element, a region consisting of a degenerate directrepeat of the canonical AGGTCA sequence separated by 1 basepair (DR1), present in the 5 9-flanking region of target genes(15). The generation of PPARa2/2 mice established thatPPARa is critical for peroxisome proliferation and the coordi-nate transcriptional activation of fatty acid oxidation enzymesin liver (16). Furthermore, PPARa2/2 mice have provided val-uable information on the constitutive levels of expression ofmitochondrial and peroxisomal fatty acid-metabolizing en-zymes in liver (17) and the response of these mice to dietaryoverload as well as short term fasting (18–20). Mice deficient inperoxisomal fatty acyl-CoA oxidase (AOX2/2) exhibited sus-tained PPARa hyperfunction presumably caused by accumula-tion of endogenous ligand(s) due to the impairment of theperoxisomal fatty acid oxidation pathway (21). Mice nullizy-gous for both PPARa and AOX (PPARa2/2 AOX2/2 doublenulls (DKO)) have also served as valuable tools to explore therole of PPARa and fatty acid oxidation in constitutive lipidmetabolism and hepatic fatty liver phenotype under fed state(22). The availability of these genetically altered PPARa2/2

(16), PPARa2/2 AOX2/2 (DKO) (22), and AOX2/2 (21) miceprovides an opportunity to examine the comparative responsesto changes in energy metabolism imposed by fasting. We dem-onstrate the critical importance of PPARa-dependent inductionof fatty acid oxidation in determining the degree of hepaticsteatosis.

EXPERIMENTAL PROCEDURES

Animals—Wild type (C57BL/6J), AOX-null (AOX2/2) (21), PPARa-null (PPARa2/2) (16), and AOX2/2 PPARa2/2 double knock-out (DKO)(22) mice were housed in a controlled environment with a 12-h light/dark cycle with free access to water and standard laboratory chow asdescribed (22). All experiments were performed using mice ranging inage from 16 to 20 weeks. Starvation was commenced by removing foodat 8:00 a.m., and groups of mice were fasted up to 96 h. Control mice

were fed ad libitum. After mice were anesthetized, blood was collectedin heparinized tubes and centrifuged, and the plasma was frozen untiluse. Organs were removed and frozen in liquid nitrogen and stored at280 °C. All animal procedures used in this study were reviewed andpreapproved by the Institutional Review Boards for Animal Research ofthe Northwestern University.

Morphological Studies—For light microscopy, pieces of liver werefixed in 10% neutral buffered formalin, embedded in paraffin, and4-mm-thick sections stained with hematoxylin and eosin. Frozen sec-tions of formalin-fixed liver (5 mm) were stained with Oil Red O andcounterstained with Giemsa. For cell proliferation analysis, mice weregiven bromodeoxyuridine (0.5 mg/ml) in drinking water, and their liverswere processed for immunohistochemical localization as described pre-viously (23), using antibodies raised against bromodeoxyuridine (Bec-ton Dickinson). Histological analysis and image processing were carriedout using Leica DMRE microscope equipped with a Spot digital camera.Images were taken at 3 20 and 40 magnification and captured at1315 3 1033 pixels. Montages of images were prepared with the use ofPhotoshop 5.0 (Adobe, Mountain View, CA).

Determination of Metabolites—Plasma glucose (24), lactate (25), and3-hydroxybutyrate (3-HB) (26) were determined by the cited proce-dures. Plasma FFA and TG were determined by the use of reagent kits(NEFA C-Test Wako and Triglyceride E-Test Wako, respectively, fromWako Pure Chemical Industries, Ltd. Osaka, Japan). Liver glycogenwas determined by the use of coupling reactions of amyloglucosidaseand glucose oxidase (27). Total carnitines were determined using car-nitine acetyltransferase (CAT) (28).

Western Blot Analysis and Quantification of Proteins—Protein con-centrations were determined using a protein assay kit (Bio-Rad) usingbovine serum albumin as standard. Liver, kidney, and heart extractswere subjected to 10% SDS-polyacrylamide gel electrophoresis andtransferred to nitrocellulose membranes. The membranes were incu-bated with the primary antibody (see Refs. 17 and 22 for the source ofvarious primary antibodies used in this study) followed by alkalinephosphatase-conjugated goat anti-rabbit IgG. Antibodies against 3-hy-droxy-3-methylglutaryl-CoA synthase (HS), 3-hydroxy-3-methylglu-taryl-CoA lyase (29), and succinyl-CoA:oxoacid transferase (SCOT) (30)were provided by Dr. G. A. Mitchell and Dr. T. Fukao, respectively. TheWestern blot signals were quantified by scanning densitometry, and thevalues from mice fed control diets were assigned the number 1.0.Results are expressed as the means 6 S.D. of three determinations.

Northern Blot Analysis and RNase Protection Assay—Total RNA wasisolated from liver using the acid guanidinium thiocyanate/phenol/chlo-roform extraction method. RNA was glyoxylated, electrophoresed,transferred to a nylon membrane, and then hybridized at 42o in 50%formamide hybridization solution using 32P-labeled cDNA probes asdescribed previously (21, 22). Equal loading was verified by the inten-sity of methylene blue-stained 18 S and 28 S RNA or by probing theblots with 18 S RNA probe. Changes in mRNA levels were estimated bydensitometric scanning of autoradiograms.

RNase protection assay was performed using the following gene-specific probes: PPARa, nucleotides 1186–1565 (GenBankTM accessionnumber X57638) (16); PPARg, nucleotides 1597–1914 (GenBankTM ac-cession number U01841) (39); PPARd, nucleotides 1004–1268 (Gen-BankTM accession number U10375) (22); and CYP4A1, nucleotides1421–1555 (referred to as CYP4A10, GenBankTM accession numberABO18421). Antisense RNA probes were transcribed in the presence of[32P]UTP (20 mCi/ml, 800 Ci/mmol, Amersham Pharmacia Biotech)using the MAXIscript in vitro transcription kit (Ambion, Austin, TX).After transcription, the labeled riboprobes were purified in a 5% TBE/urea polyacrylamide Ready Gel (Bio-Rad). Probes were eluted from thepolyacrylamide gel fragments, and their activity was measured in ascintillation counter. Total RNA isolated from liver was hybridized withlabeled probes overnight and then digested for 30 min with RNaseA/RNase T1 mix at 37 °C. Protected fragments were precipitated andresuspended in 3 ml of gel loading buffer. The samples were loaded ontoa 6% polyacrylamide sequencing gel 0.4 mm in thickness (Bio-Rad).After electrophoresis, the gel was dried and exposed to film or a Phos-phorImager plate (Molecular Dynamics, Amersham Pharmacia Bio-tech) overnight at room temperature without intensification. Quantita-tion was with a Molecular Dynamics Storm 860 PhosphorImager.

Statistical Analysis—Statistical comparisons were made by usingStudent’s t test or two-way analysis of variance. A statistically signifi-cant difference was defined as p , 0.05.

PPARa and Fatty Liver 28919


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

RESULTS

Fatty Liver in Fasted Mice—Since fasting increases the ca-pacity for fatty acid oxidation in liver under normal conditions,we subjected wild type, PPARa2/2, DKO, and AOX2/2 mice tofasting for up to 96 h for a comparative analysis of liver mor-phology. After 48 h starvation, the livers of PPARa2/2 andDKO mice were paler compared with those of fasted wild typemice, and this difference in pallor indicative of severe steatosiswas grossly exaggerated with prolonged fasting. A representa-tive example of a typical gross appearance of liver of a fed and66-h fasted DKO mouse is illustrated in Fig. 1 A. Comparativehistologic appearance of liver of fed and fasted wild type,PPARa2/2, and DKO mice, as revealed by Oil Red O staining(to visualize neutral lipid) of frozen sections, is illustrated inFig. 1. In fed wild type mice there is no detectable fatty changein hepatocytes other than the presence of Oil Red O-positivedroplets in stellate cells (Fig. lB). When fasted for 48–72 h,these wild type mice exhibited subtle steatosis in centrizonalhepatocytes (Fig. 1C, arrows). As reported elsewhere (22), un-der fed state, only a few centrilobular hepatocytes in PPARa2/2

mice and few scattered periportal hepatocytes in DKO micerevealed fatty change (Fig. 1, D and F). When these mice werefasted for 48–72 h, steatosis was extensive involving the entire

liver lobule (Fig. l, E and G). At 48, 66, or 72 h of fasting, the OilRed O staining clearly showed marked differences in the degreeof hepatic steatosis between PPARa2/2 and DKO mice in thatfatty change appeared more prominent in DKO livers. Oil RedO-stained liver sections of 48–72 h fasted AOX2/2 mice re-vealed fatty change in centrizonal hepatocytes, but a majorityof hepatocytes with intense eosinophilic cytoplasm indicative ofregeneration did not accumulate fat (Oil Red O stain not illus-trated but see hematoxylin and eosin staining pattern in Fig.2G). Examination of hematoxylin and eosin-stained histologicsections of livers from 66-h fasted wild type, PPARa2/2, andDKO mice demonstrated clear-cut differences in steatosis (Fig.2, A–C). In wild type animals fasted for 66 or 72 h the fattychange was minimal with few microvesicular lipid droplets(Fig. 2A). In contrast, microvesicular fatty change appearedprominent in PPARa2/2 livers (Fig. 2B), and this change wasgreatly exaggerated in DKO livers in which many hepatocytesalso had macrovesicular lipid droplets (Fig. 2C). When fastedfor 96-h, wild type mice exhibited no fatty change, and in factthe liver cells revealed features of cytoplasmic atrophy (Fig.2D). On the other hand, the fatty change persisted and pro-gressed predominantly to macrovesicular type in the midzonaland centrilobular areas of liver lobules, while maintaining the

FIG. 1. Liver morphology in fed andfasted wild type mice and mice lack-ing PPARa and mice nullizygous forboth PPARa and AOX (DKO). A, rep-resentative gross photograph of liver offed (left) and 66-h fasted (right) DKOmouse. Note the marked pallor of liver offasted DKO mouse. B–G represent OilRed O-stained frozen sections of liverfrom fed (B, D, and F) and 48-h fasted (C,E, and G) wild type (B and C), PPARa2/2

(D and E), and DKO (F and G) mice. Thered color represents Oil Red O staining ofneutral lipid. C, mild centrilobular stea-tosis in wild type fasted mouse (arrows)and arrows in F point to scattered hepa-tocytes in the periportal region showinglipid deposition in fed DKO mouse. Fattychange (intensity of Oil Red O staining) isgreater in fasted DKO liver (G) whencompared with that seen in fastedPPARa2/2 mouse liver (E).

PPARa and Fatty Liver28920


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

microvesicular steatotic pattern in periportal areas of bothPPARa2/2 and DKO mice (Fig. 2, E and F). The degree ofhepatic steatosis appeared slightly more prominent in femalesduring the first 48 h of starvation but with prolonged starva-tion (66–96 h); the differences in fatty change between malesand females were not apparent. We also subjected AOX2/2

mice to 48- and 72-h starvation and found that regeneratedhepatocytes with eosinophilic cytoplasm are resistant to lipidaccumulation, whereas cells already steatotic appeared nearlythe same or only slightly more steatotic (Fig. 2G). In order todemonstrate that the eosinophilic hepatocytes that do not ex-hibit steatosis in response to fasting are indeed cells that haveregenerated and therefore resistant, we administered bromode-oxyuridine for 4 days in drinking water and assessed its incor-poration in hepatocyte nuclei by immunoperoxidase staining(Fig. 2H). During the 4-day labeling period, several hepatocyteshave incorporated this precursor indicating DNA synthesis andcell proliferation in cells that are at the interface betweensteatotic cells and cells with abundant eosinophilic cytoplasmthat are resistant to fatty change. In contrast, an occasional cellshowed bromodeoxyuridine incorporation in the livers of eitherfed or starved wild type, PPARa2/2, and DKO mice (Fig. 2I)indicating minimal cell proliferation.

Changes in Major Metabolic Fuels upon Starvation—Plasma

glucose levels were not much different among the four groups ofmice under fed conditions, and these levels were decreased byabout 50% at 48 h of starvation, and similar levels were main-tained at 72 h in all groups (Fig. 3A). Plasma lactate levels inall groups were nearly the same under non-starved conditions,and on fasting gradual decreases in lactate concentrations wereobserved in all four groups of animals (data not shown). Lac-tate, together with alanine, serves as a major precursor forgluconeogenesis in liver under starvation. Glycogen in liver, areservoir of glucose, was largely depleted within 48 h of star-vation, and this reduction was sustained at 72 h of fasting inwild type, PPARa2/2, and DKO mice (Fig. 3B). The hepaticglycogen content in AOX2/2 mice was lower than that found inwild type mice even under the non-starved state. Similar de-crease in hepatic glycogen content was noted in non-fasted wildtype mice maintained on a diet containing a peroxisome prolif-erator such as ciprofibrate suggesting that PPARa activationleads to reduction in hepatic glycogen content.2 Hepatic glyco-gen content of PPARa2/2 and DKO mice maintained on controldiet was similar to that of wild type mice, but the glycogencontent in liver of these mice did not decrease following cipro-

2 T. Hashimoto, unpublished data.

FIG. 2. Comparative liver morphology as revealed in sections stained with hematoxylin and eosin. A–C (all taken at the samemagnification) represent livers of wild type (A), PPARa2/2 (B), and DKO (C) mice fasted for 66 h. The inset in B depicts an enlarged version of cellswith microvesicular fatty change in PPARa2/2 mouse liver. D–F (all taken the same magnification) represent livers of wild type (D), PPARa2/2 (E),and DKO (F) mice fasted for 96 h. In D–F the insets represent higher magnification of areas marked with asterisks to show the absence of fattychange in wild type mouse liver starved for 96 h (D), the presence of steatosis in all cells in PPARa2/2 (E), and DKO (F) liver. G, liver of AOX2/2

mouse starved for 72 h. Note the absence of fatty starvation-induced steatosis in regenerated hepatocytes that contain eosinophilic cytoplasm;hepatocytes in the centrizonal area show microvesicular fatty change similar to that seen in fed state. H, liver of AOX2/2 mouse showing a leadingedge of hepatocellular proliferation, at the steatotic cell interface, as evidenced by bromodeoxyuridine labeling. No significant liver cell proliferationwas noted in wild type, PPAR, and DKO (I) mice. The insets in H and I show immunoperoxidase-stained nuclei representing bromodeoxyuridinelabeling in proliferated cells. Eventually all steatotic cells will be replaced by regenerated liver cells in AOX2/2 mouse liver.



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

fibrate, further suggesting that the basal glycogen content isaffected by the PPARa function.2

In the wild type mice, fasting caused a nearly 2-fold increasein plasma FFA levels at 48 h, and by 72 h the FFA returned tonear normal levels (Fig. 3C). Plasma FFA levels in other groupswere nearly the same as those of wild type mice at 48 h fasting.Unlike in wild type mice, high levels of FFA were maintainedat 72 h in PPARa2/2, DKO, and AOX2/2 mice. A large part ofthe product of hepatic fatty acid oxidation, acetyl CoA, is con-verted into ketone bodies, acetoacetate, and 3-HB. These areexported by the liver to other organs where they are utilized asenergy substrates. The plasma 3-HB concentration was dra-matically increased in fasted wild type and AOX2/2 mice, sug-gesting enhanced hepatic mitochondrial fatty acid b-oxidation.In both PPARa2/2 and DKO mice, increases in plasma ketonebody levels were not as marked (Fig. 3D), suggesting thatfasting in these animals does not lead to increased mitochon-drial oxidation.

Under fed conditions, plasma TG levels were slightly higherin both PPARa2/2 and DKO mice as compared with wild typemice (Fig. 3E). Plasma TG levels decreased following fasting inwild type, PPARa2/2, and DKO mice (Fig. 3E). Interestingly,plasma TG levels in AOX2/2 mice were lower even under non-starved conditions, and there appeared to be a slight increasein TG levels as a result of starvation. As expected TG accumu-lated in liver when starved (Fig. 3F). This is in part to in-creased TG synthesis using an excess amount of FFA fromadipose tissue, and this synthetic capacity exceeds that of he-

patic TG secretion. As shown in Fig. 3F, the TG content in wildtype mouse increased about 10-fold by starvation, reachingabout 150 mg/g liver. TG accumulation in livers of mutantmice (i.e. in mice lacking PPARa) was more than 300 mg/gliver. In contrast, the TG content in AOX2/2 mouse liver washigh under fed conditions, but TG accumulation by starvationin this mouse was lower than that in the wild type mouse (Fig.3F). This is in most part due to the predominance of regener-ated hepatocytes in AOX2/2 livers that are resistant to lipidaccumulation (Fig. 2G). Dramatic increases in hepatic TG/protein ratio are evident in 72-h fasted PPARa2/2 when com-pared with similarly fasted wild type mice, but the increasewas more pronounced in fasted DKO livers (Fig. 4A). In con-trast, the TG/protein ratio was increased only modestly in 72-hfasted AOX2/2 mouse liver, which is consistent with the pres-ence in liver of hepatocytes that are regenerated and resistantto steatosis (Fig. 2G). Hepatic carnitine levels were high in thelivers of AOX2/2 mice under fed state when compared withother groups (Fig. 4B). Fasting for 48 and 72 h produced dra-matic increases in hepatic carnitine levels in the wild typemice, but mice deficient in PPARa and those nullizygous forboth PPARa and AOX showed no changes in carnitine content.No significant increases in TG levels were observed in kidneyand heart of starved wild type, PPARa2/2, DKO, and AOXa2/2

mice (data not shown). This may be due to controlled uptake ofFFA by these extrahepatic organs and that uptake does notexceed the energy demand of these organs.

Changes in Quantities of Fatty Acid b-Oxidation Enzymes—Systematic quantification of fatty acid b-oxidation enzymes inliver was conducted by immunoblot analysis. As shown in Ta-ble I, hepatic very long chain acyl-CoA synthetase (VLACS)was higher in AOX2/2 mice under non-starved conditions andincreased by starvation in wild type but not in PPARa2/2 andDKO, suggesting that the increase of this enzyme is dependenton the presence of PPARa. In AOX2/2 mice, VLCAS amountdid not increase any further because of sustained PPARa acti-vation in these mice and attendant spontaneous peroxisomeproliferation (21). The levels of VLACS do not relate to themitochondrial b-oxidation activity, because of its presence onlyin microsomes and peroxisomes and not in mitochondria.

The mitochondrial fatty acid b-oxidation enzymes that in-creased by starvation in wild type mouse liver consisted ofCAT, very long chain acyl-CoA dehydrogenase (VLCAD), andthe mitochondrial trifunctional protein (TFP), an enzyme com-plex exhibiting the activities of enoyl-CoA hydratase/3-hy-droxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase. Theseenzymes were higher in AOX2/2 mice than in wild type mice. Itis noteworthy that VLCAD and TFP are inner mitochondrialmembrane-associated proteins and are responsible for the ini-tial b-oxidation spiral of long chain fatty acids before thesefatty acids are oxidized by the classical matrix enzymes. Al-though the levels of MCAD and SCAD were elevated in theliver of fed AOX2/2 mouse, and these high levels were sus-tained following fasting, no increases in MCAD and SCADprotein content were found in the liver of wild type mousefollowing 48 and 72 h of fasting (Table I).

Mitochondrial acetoacetyl-CoA-specific thiolase (MTL2) andHS are the ketolytic enzymes. Ketogenesis is a final phase offatty acid oxidation in liver, forms a single pathway utilizingacetyl CoA, and is regulated by the supply of acetyl CoA and bythe activity of rate-limiting reaction catalyzed by HS. It wasdescribed that the half-life of the enzyme protein was shorter(31), and its expression was related to the PPARa function (32)and that the enzyme catalytic activity was reduced by succiny-lation (33). The HS content in AOXa2/2 mouse was higher thanthat in the wild type mouse, and the contents in both groups

FIG. 3. Changes in the levels of glucose, FFA, TG, and HB inplasma and the glycogen and TG content in liver in fed andfasted wild type, PPARa2/2, DKO, and AOX2/2. Mice fed (0) orfasted for 48 or 72 h were killed, and plasma was collected for biochem-ical determinations as described under “Experimental Procedures.”Frozen liver samples were analyzed for glycogen and TG.



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

increased by starvation (Table I). Most of the peroxisomalenzymes listed here increased by starvation in the liver of thewild type mouse. The protein content of these enzymes waslower in PPARa2/2 and DKO mice and higher in AOX2/2 mice.

Changes in the content of kidney and heart mitochondrialand peroxisomal enzyme proteins were less responsive to star-vation (Table II). None of the mitochondrial enzymes, except forcarnitine palmitoyltransferase II, changed in the kidney of wildtype mice following fasting. The amounts of several enzymes inkidney under fed state were lower in PPARa2/2 mice (Table II).The contents of octanoyl-CoA synthetase, a mitochondrial ma-trix enzyme activating medium chain fatty acids, and carnitinepalmitoyltransferase II were higher in AOX2/2 mouse kidney.The changes in heart enzymes were much less remarkable(data not shown). No increase in the enzyme content by star-vation was observed. The content of a few enzymes such asVLCAD, SCAD, and carnitine palmitoyltransferase were foundto be lower in PPARa2/2 and DKO mice than those of the wildtype mice under fed conditions. Ketolysis takes place in themitochondria of extrahepatic cells. Acetoacetate is activated tothe CoA ester by SCOT and is then converted to acetyl CoA byMTL2. Ketone body utilization is proportional to the circulat-ing level (34), and the ketolytic capacity varies directly with theSCOT activity. Kidney and heart have high SCOT activityamong the extrahepatic tissues. The SCOT content in theseorgans was not much different among the animal groups andwas not changed by starvation.

Liver mRNA Levels—The expression levels of a select subsetof genes encoding peroxisomal and microsomal fatty acid oxi-dation enzymes have been ascertained by Northern analysis(Fig. 5). In fasted wild type mouse liver, the mRNA levels ofL-PBE, PTL, CY4A1, and CYP4A3 increased 5–20-fold,whereas no induction occurred in the livers of starved

PPARa2/2 and DKO mice (Fig. 5). The mRNA levels of all thesegenes were already elevated in AOX2/2 mouse liver, and fast-ing did not cause additional increases. Catalase mRNA levelsremained unchanged in all groups. We determined the mRNAlevels of PPARa, PPARd, and PPARg in liver of wild type andmutant mice following 72 h of starvation by Northern analysisand RNase protection assay (Fig. 6). PPARa mRNA contentwas slightly reduced in fasted wild type mouse liver, and asexpected no PPARa mRNA was detected in PPARa2/2 andDKO mouse livers (Fig. 6A). In AOX2/2 livers, the PPARamRNA level was somewhat lower than that seen in fed wildtype mouse (Fig. 6, A and B). PPARg mRNA concentration waslower in fed PPARa2/2 and DKO mouse livers as comparedwith wild type. Fasting caused an up-regulation in PPARgmRNA level in PPARa2/2 and DKO mouse liver, whereas thePPARg mRNA level decreased in wild type mouse liver. Thelevel of PPARg mRNA in fasted PPARa2/2 and DKO mouseliver was similar to that seen in the liver of fed wild type mouse(Fig. 6B). PPARd mRNA levels in the liver of all mutant micewere lower than that found in wild type mice, and fastingcaused a reduction in all animals. We also examined thePPARa mRNA level in wild type mice fasted for 24, 48, and72 h and found that fasting caused a progressive decrease inmRNA concentrations at 48 and 72 h (data not shown).

DISCUSSION

Fatty acids are metabolized via the mitochondrial and per-oxisomal b-oxidation enzyme systems with partly overlappingsubstrate spectra (4). Oxidation of the major portion of mediumand long chain fatty acids occurs in mitochondria and that ofthe very long chain fatty acids takes place preferentially inperoxisomes (4). Long chain and very long chain fatty acids arealso metabolized by the microsomal CYP4A1 and CYP4A3 fattyacid v-oxidases, resulting in the formation of dicarboxylic acidsthat are further degraded by peroxisomal b-oxidation system(5, 6). Under normal physiological conditions, mitochondrialb-oxidation is the dominant metabolic pathway, whereas theextramitochondrial fatty acid oxidation occurring within per-oxisomes and endoplasmic reticulum plays a minor role (4).During starvation, fatty acids entering into the liver constitutethe major source of energy, and they require efficient hepaticoxidation to generate ketone bodies to serve as fuels for othertissues (1, 2, 7). The availability of mice (i) deficient in perox-isomal AOX (21), (ii) deficient in PPARa (16), and (iii) thosenullizygous for both PPARa and peroxisomal AOX (22) enabledus to explore the effect of genotype on energy utilization duringfasting and on hepatic phenotype. In this paper, we show thatfasted PPARa2/2 and DKO mice exhibit profound impairmentof fatty acid oxidation and that DKO mice reveal a greaterdegree of hepatic steatosis when compared with PPARa2/2

mice (Figs. 1 and 2). Following 48 and 72 h of fasting, hepaticTG/protein ratio was substantially higher in DKO mice thanthat observed in PPARa2/2 mice. Both DKO and PPARa2/2

mice manifested hypoglycemia, hypoketonemia, increased se-rum FFA, and increased serum TG, all indicative of impairedhepatic fatty acid oxidation (Fig. 3). The reductions in hepaticglycogen level and increase in hepatic TG concentration result-ing from fasting further attest to disturbed fatty acid oxidationin these DKO and PPARa2/2 animals. On the other hand, asdiscussed below, in AOX2/2 mice starvation did not substan-tially affect total hepatic TG levels suggesting that regeneratedhepatocytes with massive spontaneous peroxisome prolifera-tion are resistant to steatosis. Fasting did induce hypoglycemiaand an increase in plasma FFA in AOX2/2 mice. Nevertheless,they exhibited ketogenesis similar to that occurring in wildtype mice as evidenced by increased levels of serum 3-HBreflecting increased mitochondrial and microsomal fatty acid

FIG. 4. Changes in liver TG/protein ratio and carnitine contentfollowing fasting. Wild type, PPARa2/2DKO, and AOX2/2 mice fed (0)or fasted for 48 or 72 h were analyzed for hepatic TG, carnitine, andprotein content. A represents TG/protein ratios, and B represents livercarnitine levels in various groups under fed and fasted conditions.



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

TA

BL

EI

Qu

anti

fica

tion

ofm

itoc

hon

dri

alan

dpe

roxi

som

alfa

tty

acid

oxid

atio

nen

zym

esin

live

rT

otal

live

rpr

otei

ns

wer

esu

bjec

ted

toim

mu

nob

lot

anal

ysis

,an

dth

esi

gnal

sw

ere

quan

tifi

edby

scan

nin

gde

nsi

tom

etry

.Th

eva

lues

wer

en

orm

aliz

ed(5

1.0)

toth

esi

gnal

inte

nsi

ties

obta

ined

wit

hth

ew

ild

type

mic

efe

dth

eco

ntr

oldi

et.

DK

O,

dou

ble

knoc

k-ou

tm

ice

nu

lliz

ygou

sfo

rbo

thP

PA

Ra

and

AO

X.

Gro

up

star

vati

onW

ild

type

PP

AR

a2

/2D

KO

AO

X2

/2

048

720

4872

048

720

4872

VL

AC

S1.

006

0.16

3.2

60.

7a4.

06

0.7a

0.77

60.

10.

776

0.13

0.71

60.

280.

736

0.05

1.1

60.

11.

16

0.1

3.1

60.

73.

56

0.5

2.8

60.

6P

CS

b1.

006

0.19

1.1

60.

10.

996

0.2

0.88

60.

150.

566

0.09

0.41

60.

120.

746

0.10

0.35

60.

050.

316

0.04

1.4

60.

21.

26

0.3

1.0

60.

1O

CS

1.00

60.

121.

26

0.2

1.4

60.

20.

826

0.19

0.86

60.

180.

726

0.16

0.80

60.

060.

906

0.24

0.89

60.

200.

996

0.2

0.98

60.

100.

886

0.10

CP

TII

1.00

60.

061.

16

0.3

1.1

60.

30.

476

0.01

0.55

60.

090.

506

0.16

0.46

60.

030.

676

0.06

0.84

60.

160.

956

0.30

0.98

60.

180.

946

0.05

CA

T1.

006

0.11

1.3

60.

31.

76

0.3

0.80

60.

110.

966

0.04

0.83

60.

130.

866

0.19

0.90

60.

031.

06

0.1

3.6

60.

33.

96

0.6

3.2

60.

3V

LC

AD

1.00

60.

061.

36

0.1

1.8

60.

1a0.

426

0.05

0.49

60.

150.

506

0.01

0.24

60.

010.

246

0.01

0.24

60.

112.

46

0.2

2.4

60.

32.

46

0.1

LC

AD

1.00

60.

061.

06

0.2

1.4

60.

10.

366

0.01

0.44

60.

060.

476

0.06

0.51

60.

081.

16

0.19

1.3

60.

31a

1.5

60.

41.

66

0.4

1.0

60.

1M

CA

D1.

006

0.14

1.0

60.

11.

26

0.1

1.1

60.

10.

926

0.14

0.97

60.

110.

746

0.10

1.0

60.

11.

26

0.1

1.7

60.

22.

06

0.1

1.9

60.

4S

CA

D1.

006

0.14

0.77

60.

140.

646

0.2

0.58

60.

050.

496

0.06

0.47

60.

120.

546

0.05

0.47

60.

070.

516

0.04

1.4

60.

21.

86

0.1

2.3

60.

5E

TF

1.00

60.

180.

956

0.05

0.96

60.

10.

926

0.03

0.93

60.

090.

886

0.16

0.97

60.

070.

506

0.02

0.54

60.

030.

846

0.04

1.0

60.

11.

46

0.1a

MH

1.00

60.

281.

06

0.1

1.2

60.

41.

36

0.2

1.2

60.

11.

26

0.1

1.3

60.

21.

26

0.3

1.2

60.

10.

916

0.07

1.1

60.

11.

16

0.1

HA

DH

1.00

60.

040.

866

0.04

0.98

60.

20.

926

0.09

0.89

60.

020.

826

0.12

0.93

60.

020.

896

0.14

0.72

60.

010.

976

0.11

0.84

60.

150.

946

0.03

MT

L1

1.00

60.

040.

816

0.05

0.91

60.

050.

616

0.06

0.59

60.

030.

776

0.24

0.47

60.

060.

586

0.14

0.80

60.

080.

886

0.16

0.68

60.

080.

946

0.20

TF

P1.

006

0.06

1.3

60.

41.

76

0.3

0.94

60.

10.

836

0.19

0.96

60.

351.

16

0.2

0.78

60.

250.

916

0.10

2.8

60.

12.

56

0.1

2.0

60.

1M

TL

21.

006

0.09

1.3

60.

071.

66

0.3

1.0

60.

10.

706

0.02

1.1

60.

31.

16

0.1

1.0

60.

11.

26

0.2

0.98

60.

190.

956

0.21

1.1

60.

1H

S1.

006

0.04

1.8

60.

2a3.

56

0.6a

0.54

60.

030.

626

0.05

0.68

60.

070.

506

0.01

0.66

60.

040.

706

0.04

1.6

60.

12.

06

0.1

3.2

60.

5a

CO

T1.

006

0.06

1.5

60.

1a1.

86

0.2a

0.65

60.

040.

656

0.13

0.89

60.

212.

36

0.3

2.4

60.

22.

76

0.3

3.2

60.

63.

16

0.3

3.2

60.

2A

OX

1.00

60.

051.

26

0.2

1.9

60.

4a1.

06

0.1

0.89

60.

050.

856

0.16

L-P

BE

1.00

60.

221.

76

0.3

3.1

60.

7a0.

396

0.11

0.20

60.

020.

116

0.05

1.9

60.

11.

56

0.1

1.4

60.

12.

16

0.6

2.1

60.

52.

36

0.5

PT

L1.

006

0.10

1.2

60.

11.

56

0.2

0.55

60.

020.

256

0.01

0.10

60.

011.

16

0.1

1.1

60.

11.

96

0.2

2.2

60.

52.

36

0.3

2.4

60.

3D

-PB

E1.

006

0.03

1.6

60.

2a2.

26

0.3a

0.57

60.

090.

546

0.04

0.47

60.

082.

06

0.2

2.9

60.

11.

66

0.5

2.5

60.

72.

26

0.6

2.7

60.

1S

CP

x1.

006

0.10

1.8

60.

3a2.

26

0.3a

0.55

60.

130.

806

0.11

0.50

60.

111.

06

0.2

1.2

60.

11.

26

0.2

3.4

60.

43.

16

0.5

3.4

60.

8a

Sig

nif

ican

tin

crea

ses

offa

sted

sam

ples

rela

tive

tofe

dsa

mpl

es(p

,0.

05by

one-

tail

edS

tude

nt’s

tte

st)

are

show

n.

bT

he

abbr

evia

tion

su

sed

are:

CO

T,

carn

itin

eoc

tan

oylt

ran

sfer

ase;

CP

T,

carn

itin

epa

lmit

oyl-

tran

sfer

ase;

ET

F,

elec

tron

tran

sfer

flav

opro

tein

;H

AD

H,

3-h

ydro

xyac

yl-C

oAde

hyd

roge

nas

e;L

CA

D,

lon

gch

ain

acyl

-CoA

deh

ydro

gen

ase;

MH

,m

itoc

hon

dria

len

oyl-

CoA

hyd

rata

se;

MT

L1,

mit

och

ondr

ial

3-ke

toac

yl-C

oAth

iola

se;

MT

L2,

mit

och

ondr

ial

acet

oace

tyl-

CoA

-spe

cifi

cth

iola

se;

OC

S,

octa

noy

l-C

oAsy

nth

etas

e;P

CS

,pa

lmit

oyl-

CoA

syn

thet

ase;

SC

Px,

ster

olca

rrie

rpr

otei

nx

or3-

keto

acyl

-CoA

thio

lase

/ste

rol

carr

ier

prot

ein

2.



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

TA

BL

EII

Qu

anti

fica

tion

ofm

itoc

hon

dri

alan

dpe

roxi

som

alfa

tty

acid

oxid

atio

nen

zym

esin

kid

ney

Tot

alki

dney

prot

ein

sw

ere

subj

ecte

dto

imm

un

oblo

tan

alys

is,

and

the

sign

als

wer

equ

anti

fied

.T

he

valu

esre

pres

ent

the

sign

alin

ten

siti

esob

tain

edw

ith

the

fed

wil

d-ty

pem

ice

(51.

0).

SC

Px

was

un

dete

ctab

le.

DK

O,

dou

ble

knoc

k-ou

tm

ice

nu

lliz

ygou

sfo

rbo

thP

PA

Ra

and

AO

X.

Gro

up

star

vati

onW

ild

type

PP

AR

a2

/2D

KO

AO

X2

/2

048

720

4872

048

720

4872

VL

AC

S1.

006

0.13

1.0

60.

10.

856

0.11

1.0

60.

10.

496

0.08

0.49

60.

060.

606

0.18

0.81

60.

110.

586

0.11

1.2

60.

10.

926

0.12

1.1

60.

11P

CS

b1.

006

0.23

1.2

60.

31.

26

0.1

0.51

60.

090.

416

0.09

0.40

60.

050.

596

0.05

0.40

60.

050.

386

0.05

2.3

60.

353.

26

0.4

2.5

60.

2O

CS

1.00

60.

060.

826

0.07

0.84

60.

090.

626

0.15

0.60

60.

220.

756

0.20

0.74

60.

050.

586

0.15

0.86

60.

274.

76

0.4

4.0

60.

42.

96

0.3

CP

TII

1.00

60.

131.

56

0.2

2.3

60.

2a0.

676

0.26

0.70

60.

120.

686

0.05

0.82

60.

140.

876

0.20

1.3

60.

32.

86

0.3

2.5

60.

22.

96

0.2

CA

T1.

006

0.25

1.1

60.

20.

956

0.29

1.0

60.

20.

946

0.13

1.0

60.

21.

06

0.3

0.86

60.

090.

766

0.11

1.3

60.

21.

26

0.1

1.2

60.

1V

LC

AD

1.00

60.

071.

16

0.1

1.2

60.

11.

16

0.2

0.80

60.

081.

06

0.2

0.88

60.

211.

16

0.2

1.0

60.

21.

16

0.2

0.98

60.

071.

06

0.1

LC

AD

1.00

60.

201.

36

0.2

1.2

60.

20.

346

0.08

0.26

60.

080.

206

0.05

0.26

60.

120.

306

0.08

0.35

60.

091.

96

0.3

1.7

60.

31.

56

0.2

MC

AD

1.00

60.

181.

06

0.1

0.92

60.

110.

876

0.07

0.83

60.

090.

896

0.09

0.93

60.

110.

816

0.26

0.90

60.

161.

16

0.1

1.2

60.

11.

26

0.1

SC

AD

1.00

60.

100.

996

0.22

0.76

60.

150.

356

0.07

0.35

60.

080.

456

0.07

0.49

60.

090.

536

0.07

0.49

60.

071.

56

0.3

1.3

60.

11.

46

0.1

ET

F1.

006

0.15

1.2

60.

11.

06

0.1

1.1

60.

20.

796

0.12

0.82

60.

130.

906

0.03

0.83

60.

040.

736

0.08

1.0

60.

10.

946

0.1

0.89

60.

1M

H1.

006

0.05

0.87

60.

090.

726

0.07

1.2

60.

20.

836

0.08

1.0

60.

10.

806

0.18

1.1

60.

21.

06

0.2

1.1

60.

10.

606

0.05

0.52

60.

05H

AD

H1.

006

0.06

0.85

60.

050.

796

0.11

0.83

60.

150.

766

0.08

0.82

60.

160.

946

0.03

0.97

60.

090.

866

0.14

1.2

60.

11.

36

0.1

1.4

60.

1M

TL

11.

006

0.08

1.1

60.

11.

06

0.1

0.88

60.

180.

566

0.05

0.63

60.

080.

716

0.20

0.74

60.

150.

766

0.08

1.4

60.

21.

56

0.1

1.1

60.

1T

FP

1.00

60.

240.

866

0.20

1.0

60.

20.

926

0.09

0.77

60.

210.

826

0.18

0.58

60.

040.

626

0.14

0.92

60.

090.

966

0.15

1.1

60.

10.

896

0.08

SC

OT

1.00

60.

170.

686

0.1

0.95

60.

11.

16

0.3

1.0

60.

20.

816

0.10

0.72

60.

080.

606

0.11

0.63

60.

111.

06

0.1

1.2

60.

11.

16

0.2

MT

L2

1.00

60.

080.

876

0.08

0.85

60.

10.

876

0.08

1.1

60.

21.

26

0.1

0.91

60.

160.

806

0.11

1.2

60.

11.

26

0.2

1.3

60.

21.

36

0.1

CO

T1.

006

0.12

1.6

60.

22.

06

0.2a

1.4

60.

31.

26

0.2

1.1

60.

21.

36

0.1

1.5

60.

31.

46

0.4

3.6

60.

32.

96

0.1

2.6

60.

1a

AO

X1.

006

0.14

1.5

60.

11.

86

0.2a

0.87

60.

060.

846

0.19

0.90

60.

06L-P

BE

1.00

60.

211.

56

0.3

1.8

60.

2a0.

966

0.1

0.74

60.

10.

876

0.1

0.90

60.

21.

26

0.3

1.4

60.

25.

86

0.2

5.7

60.

14.

96

1.0

PT

L1.

006

0.07

1.2

60.

21.

46

0.2

0.53

60.

10.

476

0.1

0.43

60.

11.

36

0.4

1.3

60.

31.

26

0.3

1.2

60.

32.

66

0.3

2.5

60.

1D

-PB

E1.

006

0.02

1.1

60.

21.

16

0.1

0.18

60.

050.

206

0.1

0.23

60.

11.

06

0.3

1.7

60.

12.

56

0.6a

8.1

61.

39.

76

1.0

7.9

61.

4a

Sig

nif

ican

tin

crea

ses

offa

sted

sam

ples

rela

tive

tofe

dsa

mpl

es(p

,0.

05by

one-

tail

edS

tude

nt’s

tte

st)

are

show

n.

bS

eeT

able

Ifo

rab

brev

iati

ons.



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

oxidation due to sustained activation of PPARa by endogenousligands (21).

In wild type mice, as well as in other groups of mice used inthis study, starvation caused marked increases in plasma FFAdue to excessive lipolysis in adipose tissue resulting from car-bohydrate deficit. If mobilization of FFA exceeds the demandfor lipid oxidation, re-esterification of surplus FFA to TG willoccur in liver. There is sufficient evidence to indicate thatcertain crucial enzymes involved in fatty acid metabolism inmitochondria, peroxisomes, and endoplasmic reticulum are up-regulated during starvation by PPARa (9, 18–20). In the pres-ent study, animals were starved for up to 72 h to determinewhether hepatic mitochondrial and peroxisomal fatty acid-me-tabolizing enzymes are increased by starvation and if suchincreases depend on the presence of PPARa. In wild type mouseliver, starvation produced increases in certain enzymes such asVLACS, CAT, VLCAD, TFP, MTL2, HS, AOX, L-PBE, D-PBE,and sterol carrier protein X or 3-ketoacyl-CoA thiolase/sterolcarrier protein 2 (Table I). Increases in hepatic MCAD andSCAD mRNA levels were reported in wild type mice following24 h of starvation (19, 20), whereas in the present study weobserved no significant increases in the levels of these twoproteins following 48 and 72 h of fasting. The observed differ-ences may be due to the fact that mRNA levels and not proteinlevels were determined, and the duration of fasting was 24 h inpreviously reported studies (19, 20) and not prolonged as in thepresent study. In AOX null mouse liver, the basal MCAD andSCAD protein levels were significantly higher than that of wildtype mice, and these levels remained high following fasting(Table I). Our studies also confirm increases in mRNA levels ofPPARa-regulated genes such as L-PBE, PTL, CYP4A1, andCY4A3 in livers of wild type mice following fasting. Thus,PPARa-dependent increases in fatty acid oxidation systemsobserved in fasted wild type mice appear to metabolize FFAentering the liver efficiently and minimize the development ofhepatic steatosis. Indeed, in wild type mice fasted for 96 h,there was no morphologically discernible hepatic steatosis.

As reported elsewhere, the constitutive levels of expressionof several mitochondrial enzymes involved in lipid metabolismare substantially lower in the livers of PPARa2/2 and DKOmice as compared with wild type mice indicating defectivemitochondrial fatty acid catabolism (17, 22). Absence ofPPARa, in both PPARa2/2 and DKO mice, impairs the induc-tion of mitochondrial b-oxidation in liver following fasting

which contributes to hypoketonemia and hepatic steatosis.Fasting was associated with even greater increases in TG/protein ratio and more pronounced steatosis in the liver of DKOmice than that observed in PPARa2/2 mice. We attribute thisadded severity of hepatic steatosis in DKO mice to the absenceof peroxisomal b-oxidation system due to AOX deficiency. Fur-thermore, since peroxisomal b-oxidation system is required forthe metabolism of toxic dicarboxylic acids generated by consti-tutive levels of microsomal fatty acid v-oxidation enzymes,these metabolites can act synergistically to induce a greaterdegree of fatty change in fasted DKO mice than that encoun-tered in PPARa2/2 mice. We determined the hepatic mRNAlevels of PPARa, PPARg, and PPARd isoforms in all micefollowing 72 h of fasting using RNase protection assay, and wefound slight reductions in the levels of all three isoforms in wildtype mice. In PPARa2/2, and DKO mouse livers, fasting causeda less than 2-fold increase in PPARg mRNA levels, whereas thelevels of PPARd were reduced as seen in wild type mice. Noappreciable reductions in PPAR isoform levels occurred inAOX2/2 mice following fasting. In a previous study, increasesin PPARa mRNA content were noted in the livers of wild typemice fasted for 24 h (19). Since we found reduction in PPARa

mRNA level in mice starved for 72 h, we determined PPARa

levels in mice fasted for 24–72 h, and we noted a decrease inmRNA levels in animals fasted for 48 and 72 h, suggesting thatprolonged fasting results in an adaptive state.

In this paper we also present data on the effect of fasting inmice deficient in peroxisomal AOX. Although the basal contentof hepatic TG in AOX2/2 mice was somewhat higher than thatof other groups, reflecting the presence of preexisting microve-sicular steatosis in some centrizonal hepatocytes (Fig. 2G),fasting for 48 and 72 h induced only a minimal increase inTG/protein ratios. There are two possible explanations for theabsence of fasting-related amplification of hepatic steatosis inthese animals. First, PPARa is hyperactive in these animalsleading to sustained spontaneous peroxisome proliferation andincreases in the levels of mitochondrial and microsomal fattyacid oxidation systems in liver (21). PPARa-mediated sponta-neous increase in hepatic mitochondrial b-oxidation system inthese AOX2/2 animals, as evidenced by increases in VLCAS,CAT, VLCAD, MCAD, and SCAD, appears highly effective ingenerating ketone bodies. Increases in plasma 3-HB levelsoccurred in AOX2/2 mice similar to those observed in fastedwild type mice. In contrast, the plasma 3-HB levels in fastedPPARa2/2 and DKO mice indicate a significantly lower activityof fatty acid oxidation than that occurring in the liver of wildtype and AOX2/2 mice. Second, the reduced TG/protein ratio in16–20-week-old AOX2/2 mice subjected to starvation in thisstudy can be due to the fact that their livers consist of manyregenerated hepatocytes that progressively replace steatotichepatocytes, and this regenerative process extends toward cen-trizonal areas (Fig. 2G). We have presented evidence for hep-atocyte proliferation at the interface between steatotic andnon-steatotic hepatocytes. These regenerated hepatocytes withabundant cytoplasm and massive peroxisome proliferation areresistant to fatty change. This is analogous to the emergence ofhepatocytes in chronic alcoholic liver disease that no longerbecome steatotic (35). No liver cell proliferation occurred inwild type, PPARa2/2, and DKO mice either fed or fasted underthe conditions used in this study. The livers of fed AOX2/2 miceshow a remarkable increase in microsomal fatty acid oxidationenzymes due to sustained activation of PPARa by unmetabo-lized endogenous ligands (21), and fasting produced no furtherincrease in the levels of CYP4A1 and CYP4A3 mRNA levelsreflecting saturation of the receptor by ligands. Increased v-ox-idation generates dicarboxylic acids that cannot be metabolized

FIG. 5. Northern blot analysis of total RNA extracted from theliver of wild type (wild), PPARa2/2PPARa2/2/AOX2/2 (DKO), andAOX2/2 mice. Lanes 0, 48, and 72 in each group represent fed (0), 48-hfasting (48), and 72-h fasting (72). Twenty mg of total RNA was electro-phoresed on a 0.8% agarose gel, blotted onto a nylon membrane, andhybridized with different random-primed 32P-labeled L-PBE, peroxiso-mal 3-ketoacyl-CoA thiolase (PTL), CYP4A1 CYP4A3, and catalase(CTL) probed as shown. It should be noted that rat CYP4A1 andCYP4A3 probes used for hybridization may exhibit differences in thehybridization to mouse CYP4A mRNAs. The 18 S RNA is for loadingcontrol.



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

in the absence of peroxisomal b-oxidation. Although these toxicmetabolites seem to exert severe fatty change in the livers ofyounger AOX2/2 mice (21), the regenerated hepatocytes inolder mice appear immune to fatty change.

It has been known by in vitro experiments that maximaluptake of FFA by liver is not changed by fasting (36), that theuptake rate is dependent on the FFA concentration, and thatexogenously imported fatty acids are oxidized to ketone bodyand converted into TG and phospholipids in proportion to theuptake of FFA (37). However, a recent report (38) describesthat uptake of FFA in vivo is saturated at the level of about 1mM. Rate of FFA uptake was not determined in this study, butincreases in the plasma ketone body and the hepatic TG con-tent suggest that the FFA uptake did not limit the oxidationand esterification of the imported FFA. Studies on the relation-ship of PPARa2/2 expression to the proteins involved in fattyacid import (39, 40) suggest that proteins such as fatty-acidtranslocase, fatty acid transport protein, and L-fatty acid-bind-ing protein play a role in the uptake of fatty acids by liver. Butmarked increases in hepatic TG content in PPARa2/2 and DKOmice, irrespective of the PPARa function, suggest that a majormechanism of FFA uptake by liver is not limited by theseproteins under the starved conditions. Marked differences infasting-related increases in the levels of TG between liver andthese extrahepatic organs are evident. Differences in lipid me-tabolisms between liver and extrahepatic organs under starva-tion are attributed to rapid activation of PPARa-responsivegenes in liver and PPARa-inducible fatty acid oxidation sys-tems in liver play a vital role in energy metabolism and in theprevention of hepatic steatosis.

The mitochondrial b-oxidation is regulated by the carnitinecontent (4, 41). The hepatic carnitine content under fed condi-tions was slightly lower in mice without PPARa than that ofwild type mice. Carnitine content in wild type mice increasedabout 3-fold upon starvation for 48 h, whereas in PPARa2/2

and DKO mice carnitine levels remained unchanged, suggest-ing that absence of PPARa in these two genetically alteredmice influences the carnitine metabolism and its response tofasting. It is of considerable interest to note that hepatic car-nitine content in AOX2/2 mice, which are under PPARa hyper-function (21), is higher than that found in wild type mice, andthese high levels were maintained during starvation. We pro-pose that hepatic carnitine level is regulated by PPARa al-though the mechanism remains unknown.

Inborn errors in mitochondrial and peroxisomal b-oxidation

enzymes have received major attention thus far as causes ofhypoglycemia and hepatic dysfunction (1–3). In summary, ourresults with PPARa2/2, DKO, and AOX2/2 mice and data fromother laboratories with PPARa2/2 and other genetically al-tered mice also focus on the importance of transcriptional reg-ulation of genes involved in lipid metabolism in energy utiliza-tion (18–22, 42). In humans, the PPARa levels appear lowerthan that found in rats and mice (43), raising the issue of theeffectiveness of PPARa-inducible fatty acid oxidation systemsin different species in dealing with conditions of stress thatlead to reduced energy intake. A PPARa splice variant thatmay negatively interfere with wild type PPARa has been de-scribed recently (44), and this finding also raises the questionof countering the induction of PPARa-regulated genes leadingto abnormal energy utilization.

Acknowledgments—We are grateful to Dr. Frank J. Gonzalez for thegenerous gift of PPARa null mice and to V. Subbarao for technicalassistance.

REFERENCES

1. Seitz, H. J., Mullerm M. J., Krone, E., and Tarnowski, W. (1977) Arch. Bio-chem. Biophys. 183, 647–663

2. Eaton, S., Bartlett, K., and Pourfarazam, M. (1996) Biochem. J. 320, 345–3573. Roe, C. R., and Coates, P. M. (1995) in Metabolic and Molecular Absence of

Inherited Disease (Scriver, C. R., Beaudet, A. I., Sly, W. S., and Valle, D.,eds) pp. 1501–1533, McGraw-Hill Inc., New York

4. Hashimoto, T. (1999) Neurochem. Res. 24, 551–5635. Aoyama, T., Hardwick, J. P., Imaoka, S., Funae, Y., Gelboin, H. V., and

Gonzalez, F. J. (1990) J. Lipid Res. 31, 1477–14826. Roman, L. J., Palmer, C. N., Clark, J. E., Muerhoff, A. S., Griffin, K. J.,

Johnson, E. F., and Masters, B. S. (1993) Arch. Biochem. Biophys. 307,57–65

7. Berk, P. D. (1999) Hepatology 29, 1607–16098. Taroni, F., and Uziel, G. (1996) Curr. Opin. Neurol. 9, 477–4859. Nagao, M., Parimoo, B., and Tanaka, K. (1993) J. Biol. Chem. 268,

24114–2412410. Hillgartner, F. B., Salati, L. M., and Goodridge, A. G. (1995) Physiol. Rev. 75,

583–59511. Issemann, I., and Green, S. (1990) Nature 347, 645–65012. Reddy, J. K., and Chu, R. (1996) Ann. N. Y. Acad. Sci. 804, 176–20113. Zhu, Y., Alvares, K., Huang, Q., Rao, M. S., and Reddy, J. K. (1993) J. Biol.

Chem. 268, 26817–2682014. Dryer, C., Krey, G., Keller, H., Givel, F., Helftenbein, G., and Wahli, W. (1992)

Cell 68, 879–88715. Kliewer, S. A., Umesono, K., Noonan, D. J., Heyman, R. A., and Evans, R. M.

(1992) Nature 358, 771–77416. Lee, S.-S.-T., Pineau, T., Drago, J., Lee, E. J., Owens, J. W., Kroetz, D. L.,

Fernandez-Salguero, P. M., Westpal, H., and Gonzalez, F. J. (1995) Mol.Cell. Biol. 15, 3012–3022

17. Aoyama, T., Peters, J. M., Iritani, N., Nakajima, T., Furihata, K., Hashimoto,T., and Gonzalez, F. J. (1998) J. Biol. Chem. 273, 5678–5684

18. Kroetz, D. L., Yook, P., Costet, P., Bianchi, P., and Pineau, T. (1998) J. Biol.Chem. 273, 31581–31589

19. Kersten, S., Seydoux, J., Peters, J. M., Gonzalez, F. J., Desvergne, B., andWahli, W. (1999) J. Clin. Invest. 103, 1489–1498

FIG. 6. The levels of PPARa, PPARg,and PPARd in mouse liver followingfasting. A and B, wild type (WT),PPARa2/2, DKO, and AOX2/2 mice wereeither fasted (1) or fed (2) for 72 h. TotalRNA was isolated and hybridized with anequal amount of radioactivity of antisenseriboprobes specific for each of the PPARgenes, PPARa, PPARg, and PPARd. Actinis used as a control for RNA. After RNasedigestion, the reaction mixtures were sep-arated on a 6% polyacrylamide gel. Asexpected, PPARa mRNA is absent inPPARa2/2 and DKO mouse liver. B, thedata are presented as average of threeanimals in each group and expressed asrelative light units and normalized to ac-tin mRNA content.



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

20. Leone, T. C., Weinheimer, C. J., and Kelly, D. P. (1999) Proc. Natl. Acad. Sci.U. S. A. 96, 7473–7478

21. Fan, C.-Y., Pan, J., Usuda, N., Yeldandi, A. V., Rao, M. S., and Reddy, J. K.(1998) J. Biol. Chem. 273, 15639–15645

22. Hashimoto, T., Fujita, T., Usuda, N., Cook, W., Qi, C., Peters, J. M., Gonzalez,F. J., Yeldandi, A. V., Rao, M. S., and Reddy, J. K. (1999) J. Biol. Chem. 274,19228–19236

23. Qi, C., Zhu, Y., Pan, J., Yeldandi, A. V., Rao, M. S., Maeda, N., Subbarao, V.,Pulikuri, S., Hashimoto, T., and Reddy, J. K. (1999) Proc. Natl. Acad. Sci.U. S. A. 96, 1585–1590

24. Bergmeyer, H. U., and Bernt, E. (1974) in Methods of Enzymatic Analysis(Bergmeyer, H. U., ed) 2nd English Ed., pp. 1205–1215, Academic Press,Inc., New York

25. Gutmann, I., and Wahlefeld, A. W. (1974) in Methods of Enzymatic Analysis(Bergmeyer, H. U., ed) 2nd English Ed., pp. 1464–1468, Academic Press,Inc., New York

26. Williamson, D. H., and Mellanby, J. (1974) in Methods of Enzymatic Analysis(Bergmeyer, H. U., ed) 2nd English Ed., pp. 1837–1839, Academic Press,Inc., New York

27. Roehrig, K. L., and Allred, J. B. (1974) Anal. Biochem. 58, 414–42128. Pearson, D. J., Tubbs, P. K., and Chase, J. F. A. (1974) in Methods of Enzy-

matic Analysis (Bergmeyer, H. U., ed) 2nd English Ed., pp. 1758–1771,Academic Press, Inc., New York.

29. Boukaftane, Y., and Mitchell, G. A. (1997) Gene (Amst.) 195, 121–12630. Song, X. Q., Fukao, T., Mitchell, G. A., Kassouska-Bratinova, S., Ugarte, M.,

Wanders, R. J., Hirayama, M. K., Shintaku, H., Churchill, P., Watanabe,

H., Orii, T., and Kondo, N. (1997) Biochim. Biophys. Acta 12, 151–15631. Serra, D., Casals, N., Asins, G., Royo, T., Ciudad, C. J., and Hegardt, F. G.

(1993) Arch. Biochem. Biophys. 307, 40–4532. Rodriguez, J. C., Gil-Gomez, G., Hegardt, F. G., and Haro, D. (1994) J. Biol.

Chem. 269, 18767–1877233. Lowe, D. M., and Tubbs, P. K. (1985) Biochem. J. 232, 37–4234. Williamson, D. H., Bates, M. W., Page, M. A., and Krebs, H. A. (1971) Biochem.

J. 121, 41–4735. Lands, W. E. M. (1995) Alcohol Clin. Exp. Res. 19, 928–93836. Sorrentino, R., Stump, R. D., Zhou, S. L., Van Kess, K., Isola, L. M., and Berk,

P. R. (1994) Gastroenterology 107, 1415–142537. Soler-Argilaga, C., and Heimberg, M. (1976) J. Lipid Res. 17, 605–61538. Huang, M. (1997) Life Sci. 60, 1915–192239. Martin, G, Schoonjans, K., Lefebvre, A. M., Staels, B., and Auwerx, J. (1997)

J. Biol. Chem. 272, 28210–2821740. Motojima, K., Passilly, P., Peters, J. M., Gonzalez, F. J., and Latruffe, N. (1998)

J. Biol. Chem. 273, 16710- 1671441. Kerner, J., and Hoppel, C. (1998) Annu. Rev. Nutr. 18, 179–20642. Kim, J. B., Sarraf, P., Right, M., Yao, K. M., Mueller, E., Solanes, G., Lowell,

B. B., and Spiegelman, B. M. (1998) J. Clin. Invest. 101, 1–943. Palmer, C. N. A., Hsu, M.-H., Griffin, K. J., Raucy, J. L., and Johnson, E. F.

(1998) Mol. Pharmacol. 53, 14–2244. Gervois, P., Torra, I. P., Chinetti, G., Grotzinger, T., Dubois, G., Fruchart,

J.-C., Fruchart-Najib, J., Leitersdorf, E., and Staels, B. (1999) Endocrinol-ogy 13, 1535–1549



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

and M. Sambasiva RaoTakashi Hashimoto, William S. Cook, Chao Qi, Anjana V. Yeldandi, Janardan K. Reddy

Oxidation Determines the Severity of Hepatic Steatosis in Response to Fasting-inducible Fatty AcidαDefect in Peroxisome Proliferator-activated Receptor

doi: 10.1074/jbc.M910350199 originally published online June 7, 20002000, 275:28918-28928.J. Biol. Chem.

10.1074/jbc.M910350199Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/275/37/28918.full.html#ref-list-1

This article cites 39 references, 18 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M910350199

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;275/37/28918&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/275/37/28918

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=275/37/28918&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/275/37/28918

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/275/37/28918.full.html#ref-list-1

http://www.jbc.org/

the j b c © 2000 by the american society for biochemistry and … · 2000-08-31 · defect in...

Documents