Phosphoenolpyruvate Carboxykinase Is Necessary for the

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/00/$04.0010

Sept. 2000, p. 6508–6517 Vol. 20, No. 17

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Phosphoenolpyruvate Carboxykinase Is Necessary for theIntegration of Hepatic Energy Metabolism

PENGXIANG SHE, MASAKAZU SHIOTA, KATHY D. SHELTON, ROGER CHALKLEY,CATHERINE POSTIC, AND MARK A. MAGNUSON*

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine,Nashville, Tennessee 37232

Received 29 March 2000/Accepted 5 May 2000

We used an allelogenic Cre/loxP gene targeting strategy in mice to determine the role of cytosolic phos-phoenolpyruvate carboxykinase (PEPCK) in hepatic energy metabolism. Mice that lack this enzyme die within3 days of birth, while mice with at least a 90% global reduction of PEPCK, or a liver-specific knockout ofPEPCK, are viable. Surprisingly, in both cases these animals remain euglycemic after a 24-h fast. However,mice without hepatic PEPCK develop hepatic steatosis after fasting despite up-regulation of a variety of genesencoding free fatty acid-oxidizing enzymes. Also, marked alterations in the expression of hepatic genes involvedin energy metabolism occur in the absence of any changes in plasma hormone concentrations. Given that aninefold elevation of the hepatic malate concentration occurs in the liver-specific PEPCK knockout mice, wesuggest that one or more intermediary metabolites may directly regulate expression of the affected genes. Thus,hepatic PEPCK may function more as an integrator of hepatic energy metabolism than as a determinant ofgluconeogenesis.

Gluconeogenesis is the process whereby glucose is formedfrom noncarbohydrate metabolic substrates such as lactate andalanine. This metabolic pathway occurs predominately in theliver and kidney and is essential for the production of glucoseduring extended fasting when glycogen stores have been de-pleted (18, 23). A key step in gluconeogenesis is the formationof phosphoenolpyruvate from oxaloacetate, which is catalyzedby phosphoenolpyruvate carboxykinase (PEPCK). This reac-tion has long been thought to be essential for gluconeogenesissince it bypasses the thermodynamically unfavorable conver-sion of pyruvate to phosphoenolpyruvate by pyruvate kinase(20). Although both a cytosolic and a mitochondrial isoform ofPEPCK are expressed in rodents, the cytosolic isoform ac-counts for over 95% of the activity in the liver and kidney (30,45).

Several lines of evidence have led to the widely held notionthat PEPCK is the rate-determining step in hepatic and renalgluconeogenesis. First, the cytosolic isoform of PEPCK isadaptively regulated at the transcriptional level in a mannerthat correlates with alterations in gluconeogenic flux (5, 12, 25,47). Second, treatment of fasted animals with 3-mercaptopico-linic acid, a PEPCK inhibitor, causes hypoglycemia (8). Third,overexpression of PEPCK in both cell lines and transgenicmice causes either increased gluconeogenesis or hyperglycemia(37, 44). However, other data suggest that gluconeogenic fluxis determined by alterations in activities of multiple enzymes,not just PEPCK. Indeed, in one study both pyruvate carboxy-lase and pyruvate kinase were suggested to play a greater rolein determining gluconeogenic flux in isolated rat hepatocytesthan PEPCK (13). Also, results of a perfused rat liver studyindicated that pyruvate carboxylase rather than PEPCK is aprimary rate-determining step for gluconeogenesis (10).

Energy metabolism in the liver involves the interconversion

of carbohydrates, lipids, and amino acids. The pathways in-volved are regulated by multiple mechanisms. First, allostericeffectors or covalent modifications affect the activity of certainenzymes. Second, substrate availability itself may play an es-sential role. Third, hormones and other extrahepatic effectorsregulate the expression of genes encoding various enzymes.However, the mechanisms that regulate flux between differentpathways have not been thoroughly elucidated. Since PEPCKcatalyzes a reaction near the intersection of several fundamen-tally important pathways, the absence of this enzyme mighthave unpredictable effects on the accumulation of specific me-tabolites. Moreover, excess hepatic glucose production is amajor factor contributing to fasting hyperglycemia in both type1 and type 2 diabetes mellitus (6, 7). Thus, knowledge of howgluconeogenesis is regulated is of both fundamental and clin-ical importance.

To gain greater insights into the role of PEPCK in bothgluconeogenesis and hepatic energy metabolism, we used anallelogenic Cre/loxP gene targeting strategy to generate micethat had variable degrees of impaired PEPCK gene expression.In addition, by intercrossing mice with a conditional PEPCKgene locus with a line of albumin (Alb)-cre transgenic animals,we were also able to produce mice with a liver-specific PEPCKgene knockout. Studies of these mice indicate that whilePEPCK has a much lower than expected effect on gluconeo-genesis, the enzyme plays an unexpected role in integratingmetabolic pathways in the liver.

MATERIALS AND METHODS

Targeting vector. The targeting vector shown in Fig. 1A contains a phospho-glycerol kinase (pgk)-neomycin resistance (neo) gene cassette, a pgk-herpes sim-plex virus type 1 thymidine kinase (tk) gene cassette, and three loxP sites. Thevector was assembled in pNTK(A) using loxP sites from pBS246 (39) and mousePEPCK gene fragments isolated from a 129/SvJ Bac genomic DNA clone (46).The long arm of the targeting vector, a 6-kb KpnI fragment, was ligated into theClaI site of pBS.LP, thereby forming BSLP.PEPCK.LA. A 1-kb excisable KpnIDNA fragment containing exons 4 and 5 was ligated into the ClaI site ofpNTK(A), thereby forming mPEP.B3. The short arm of the targeting vector wasa 2.1-kb KpnI/XbaI fragment that was ligated into the BamHI site of mPEP.B3,forming mPEP.K2. After destroying a SalI site in mPEP.K2, the 6-kb SalI

* Corresponding author. Mailing address: 702 Light Hall, Vander-bilt University School of Medicine, Nashville, TN 37232-0615. Phone:(615) 322-7006. Fax: (615) 322-7236. E-mail: [email protected].

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fragment of BSLP.PEPCK.LA was cloned into the remaining SalI site ofmPEP.K2, creating the final construct, mPEPCK.KO2. The correct assembly ofmPEPCK.KO2 was confirmed by DNA sequencing.

Gene targeting and production of chimeras. Fifty micrograms of the targetingvector was linearized with NotI and then electroporated into 5 3 107 TL-1embryonic cells (ES) cells, which were derived from 129/SvEvTac mice (22).Southern blot analysis of several clones resistant to both G418 and ganciclovirrevealed one clone, 2D7, that had undergone the desired recombination event(data not shown). The ES cell clone was microinjected into C57BL/6 blastocysts,which were then implanted into pseudopregnant female recipients to producechimeras. Germ line transmission of the targeted pcklox1neo allele was confirmedby Southern blotting and PCR analysis (data not shown).

Conversion of the pcklox1neo allele to a pcklox or pckdel allele. The pcklox1neo

allele was converted to both a pcklox and a pckdel allele by pronuclear microin-jection of supercoiled pBS185 (39), a cytomegalovirus-Cre expression plasmid,

into single-cell mouse embryos derived from mating pcklox1neo males with su-perovulated B6D2 F1 hybrid female (1, 35).

Genotype analysis. Four different pck alleles (pckw, pcklox1neo, pcklox, andpckdel [w and del denote wild type and deletion, respectively]) were routinely

FIG. 1. pck alleles generated by gene targeting and Cre-mediated recombi-nation. (A) Top, partial map of the pckw allele. Exons are indicated as solidrectangles. The location of the DNA fragment used as the Southern hybridiza-tion probe is shown. Middle, map of the PEPCK gene targeting vector. Thevector contains a pgk-neo cassette, a pgk-tk cassette, and three loxP sites (trian-gles). Two of the loxP sites flank neo, and the third is located between exons 4 and5 in the PEPCK gene. The pcklox1neo allele was generated by homologousrecombination (HR) in ES cells. Bottom, the pcklox and pckdel alleles, derivedfrom pcklox1neo by Cre-mediated recombination. Exons 4 and 5 and neo wereexcised by Cre microinjection of single-cell pcklox1neo/w embryos. (B and C) PCRgenotype analysis. Tail DNA was amplified by both primer pair W (59-TCTGTCAGTTCAATACCAATCT-39), 59-AATGTTCTCTGCAAGTCCTGGTG-39)and primer pair D (59-ATCAGCTTTAGTCGTCTCTGGT-39, 59-AATGTTCTCTGCAAGTCCTGGTG-39) or primer pair F (59-TCTGTCAGTTCAATACCAATCT-39, 59-AGCCTCTGTTCCACATACACTTCA-39. The amplified allelesand their sizes are shown on the right and left, respectively. (D) Western blotanalysis of liver homogenates from pckw/w (lane 1), pckdel/w (lane 2), and pckdel/del

(lane 3) mice.

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distinguished by PCR analysis (Fig. 1B and C). pckw and pcklox were detectedusing primers 59-TCTGTCAGTTCAATACCAATCT-39 and 59-AATGTTCTCTGCAAGTCCTGGTG-39. A 518-bp fragment was generated from pckw, and a620-bp fragment was generated from pcklox. For the pcklox1neo allele, a 360-bpfragment was amplified using 59-TCTGTCAGTTCAATACCAATCT-39 and 59-AGCCTCTGTTCCACATACACTTCA-39. The pckdel allele was detected as a815-bp fragment using primers 59-ATCAGCTTTAGTCGTCTCTGGT-39 and59-AATGTTCTCTGCAAGTCCTGGTG-39. The Alb-cre transgene (35) was de-tected by PCR with primers 59-ACCTGAAGATGTTCGCGATTATCT-39 and59-ACCGTCAGTACGTGAGATATCTT-39, which yielded a 370-bp fragment.

Animals. All mice were specific pathogen free, maintained on a 12-h light-darkcycle, and fed a standard rodent chow (Purina Mills, Inc., St. Louis, Mo.).Treatment and housing of animals met guidelines of the American Associationfor the Accreditation of Laboratory Animal Care, and the protocols were ap-proved by the Vanderbilt Institutional Animal Care and Use Committee.

Northern blot analysis. Total RNA was isolated using TRIzol (Life Technol-ogies, Inc., Grand Island, N.Y.). Northern blot analysis was performed as pre-viously described (28). The PEPCK probe was a 569-bp HindIII-EcoRI fragmentspanning exons 1 to 4 of the mouse cDNA. cDNA clones for rat CYP4A1, ratCYP4A1, and mouse medium-chain fatty acyl coenzyme A (acyl-CoA) dehydro-genase were provided D. Kelly (Department of Internal Medicine, WashingtonUniversity). Probes for pyruvate carboxylase, fructose-1,6-bisphosphatase, andglucose-6-phosphatase were prepared from human, pig, and rat cDNA clones,respectively. Probes (numbers in parentheses are those of the IMAGE consor-tium clones from which clones were prepared) for malonyl-CoA decarboxylase(692290), very long-chain fatty acyl-CoA dehydrogenase (2236538), long-chainfatty acyl-CoA dehydrogenase (2225775), acyl-CoA oxidase (1972335), car-nitine octanoyltransferase (1451465), carnitine acetyltransferase (1889414),and enoyl- CoA hydratase–L-3-hydroxyacyl-CoA dehydrogenase bifunctionalprotein (2076790), cytosolic (2064729) and mitochondrial (1972463) aspartateaminotransferase, cytosolic (329958) and mitochondrial (2192286) malate dehy-drogenase, citrate synthase (335882), isocitrate dehydrogenase (2136389), succinyl-CoA synthetase (2099765), lactate dehydrogenase (1451692), alanine aminotrans-

ferase (1886916), and glyceraldehyde-3-phosphate dehydrogenase (716832) wereobtained from Research Genetics, Inc., Huntsville, Ala., and verified by DNAsequencing prior to use. The relative abundance of each mRNA was correctedfor loading differences, using cyclophilin cDNA as a control probe.

PEPCK activity measurements and Western blot analysis. PEPCK activity wasmeasured using an NADH-coupled system to quantitate the conversion of phos-phoenolpyruvate into oxaloacetate and subsequent conversion to malate (45).All assays were performed within 3 h of removal of tissue from the animal.Activity was expressed as milliunits per milligram of protein in liver supernatant(1 mU 5 1 nmol of oxaloacetate produced/min). The protein content wasdetermined using a Bradford assay kit (Bio-Rad, Hercules, Calif.), with bovineserum albumin as a standard. Western blot analysis was performed as previouslydescribed (16). A sheep anti-PEPCK antiserum (a gift from D. K. Granner,Vanderbilt University) was used at a 1:5,000 dilution.

In vivo glucose kinetics. Glucose turnover rates and gluconeogenic index in theform of plasma [14C]glucose specific activity derived from [14C]lactate weremeasured as previously described (29), using mice in which both jugular andcarotid catheters had been surgically implanted 5 days prior to study. The ex-perimental protocol involved depriving animals of food for 26 h, followed by anexperimental period of 220 min, which consisted both of a 100-min equilibrationperiod (2100 to 0 min) and a 120-min experiment period. Basal glucose turnoverand gluconeogenic index were determined by 2-mCi bolus injection and constantinfusion at 0.5 mCi/min of high-pressure liquid chromatography-purified [3-3H]glucose (NEN Life Science Products, Boston, Mass.) for 220 min and constantinfusion at 0.1 mCi/min of [U-14C]lactate (NEN Life Science Products) for 120min beginning at 0 min. Blood samples were withdrawn from a carotid arterycatheter at 230, 0, 30, 60, 90, and 120 min. Liquid chromatography was used topurify radioactive glucose in plasma. The samples were deproteinized by passagethrough a 9- by 190-mm column filled with 14-mm sulfonate polystyrene cation-exchange resin (Benson BH-4, Reno, Nev.) at a flow rate 2.2 ml/min.

Endurance exercise study. Male mice that had been fasted for 24 h wereacclimated to a treadmill for 2 min each at 13.4 and 16.1 meters/min and then runto exhaustion at 18.8 meters/min. Blood glucose concentration was measured

FIG. 2. Analysis of PEPCK expression in mice that are homozygous for the pcklox1neo and pcklox alleles and that are compound heterozygotes of pcklox1neo andpckdel alleles. (A) Hepatic and renal PEPCK activity in pcklox1neo/lox1neo and pcklox/lox mice fasted for 24 h. ppp, P , 0.001, n 5 4. (B) Western blot analysis of liverand kidney tissues from 24-h-fasted mice. Lanes 1, 2, and 3 represent pckw/w, pcklox1neo/lox1neo, and pcklox/lox mice, respectively. (C) Plasma glucose concentration inpcklox1neo/lox1neo and pcklox1neo/del mice at fed and 24-fasted states (n 5 7 to 9). (D) Northern blot analysis of liver, kidney, and brown and white adipose tissues (BATand WAT) from 24-h-fasted mice. Lanes 1 and 2 represent pckw/w and pcklox1neo/del mice, respectively. cyclo., cyclophilin.

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with a Hemocue (Mission Viejo, Calif.) glucose meter immediately before, at 30min, and at the end of the exercise.

Analytical procedures. Plasma insulin, glucagon, and corticosterone concen-trations were determined by radioimmunoassay using a rat insulin kit (LincoResearch, St. Louis, Mo.), a rat glucagon kit (Linco Research), and a Coat-A-Count rat corticosterone kit (DPC, Los Angeles, Calif.), respectively. Plasmafree fatty acid (FFA) concentrations were measured with a NEFA C kit (WakoPure Chemical Industries, Osaka, Japan). Plasma triglyceride concentrationswere measured using a colorimetric kit (Sigma, St. Louis, Mo.). Plasma glucose,lactate, glycerol, b-hydroxybutyrate (BHBA), and hepatic malate concentrationswere determined by enzymatic assays using microassay procedures (3). Hepaticglycogen and triglyceride contents were analyzed as described elsewhere (3, 40).All results are presented as the mean 6 standard error of the mean. Statisticalsignificance was determined by one-way analysis of variance. P values of less than0.05 were considered statistically significant.

Nucleotide sequence accession number. The sequences altered in the pcklox

allele were deposited in GenBank (accession number AF220498).

RESULTS

Targeting of the mouse pck gene. We designed a Cre/loxPgene targeting strategy that allowed the creation of three dif-ferent alleles of the pck gene from a single gene targeting eventin ES cells (Fig. 1A). The parental pcklox1neo allele containsthree loxP sites and a neo cassette. Two of the loxP sites flanka pgk-neo cassette which is located between exons 3 and 4,whereas a third loxP site lies downstream of exon 5. After itsintroduction into mice, the pcklox1neo allele was further mod-ified by Cre-mediated recombination in single-cell pcklox1neo

embryos. Partial recombination yielded a conditional allele(pcklox) that lacks neo but retains two loxP sites flanking exons4 and 5. Complete recombination produced a null pck allele(pckdel) that contains a single loxP site and lacks exons 4 and 5.The generation of each allele was confirmed by both PCR (Fig.1B and C) and Southern blot analysis (data not shown).

Interbreeding of animals that were heterozygous for eitherthe pcklox1neo or pcklox allele yielded mice that were homozy-gous for each of these two alleles. In both cases the mice were

viable, thereby enabling PEPCK activity and protein content tobe determined from both the liver and kidney after a 24-h fast.PEPCK activity and protein amount in the pcklox/lox mice didnot differ significantly from those of wild-type mice (Fig. 2Aand B). In contrast, PEPCK activity and protein mass inpcklox1neo/lox1neo mice, which contains a neo cassette betweenexons 3 and 4, was reduced to about ;10 and 20% of normalin the liver and kidney, respectively (Fig. 2A and B). Thus, thepcklox1neo allele is functionally attenuated, presumably due tointerference with normal RNA processing as been observed inother genes that retain a neo cassette within an intron (27).Plasma glucose concentrations in fed and 24-h-fasted mice thatwere homozygous for the pcklox1neo allele did not differ fromthose in mice with two pckw alleles (Fig. 2C). However, therelative liver mass in pcklox1neo/lox1neo mice after a 24-h fastwas increased by 30% (5.6% 6 0.4% versus 4.3% 6 0.1% ofbody weight for wild-type mice, P , 0.004, n 5 8 to 10).Content of malate, a tricarboxylic acid (TCA) cycle interme-diate, in fasting mice was 5.4-fold greater in the liver (1.57 60.21 versus 0.29 6 0.01 mM/g, P , 0.001, n 5 6 to 7) and2.4-fold greater in the kidney (0.36 6 0.04 versus 0.15 6 0.01mM/g, P , 0.001, n 5 6 to 7) of pcklox1neo/lox1neo mice than inwild-type mice. Plasma FFA concentrations in 24-h-fastedpcklox1neo/lox1neo mice were not different from that in controlmice (1,345 6 123 versus 1161 6 74 mEq/liter n 5 7 to 9).

Characterization of pck null pups. Mice that were homozy-gous null for PEPCK (i.e., pckdel/del) were obtained by inter-breeding of pckdel/w mice. Genotype analysis of 97 1-day-oldpups revealed 22 pckw/w (22.7%), 49 pckdel/w (50.5%), and 26pckdel/del mice (26.8%), consistent with Mendelian inheritance.Western blot analysis of liver extracts confirmed the absence ofPEPCK in pckdel/del mice. PEPCK protein content was de-creased by approximately one-half in pckdel/w pups, indicatingthe lack of any significant compensatory changes in gene tran-

TABLE 1. Plasma and hepatic metabolites in 1-day-old pckdel/del and pckdel/w pupsa

Pup genotype

Concn (mean 6 SE)

Plasma Hepatic

Glucose (mg/dl) FFA (meq/liter) Lactate (mg/dl) Glycogen (mM/g) Malate (mM/g)

pckw/w 90.3 6 7.8 (9) 830 6 89 (9) 37.5 6 1.9 (13) 210.0 6 34.4 (8) 0.68 6 0.07 (6)pckdel/w 93.1 6 6.9 (20) 925 6 92 (14) 37.9 6 2.0 (16) 189.3 6 14.0 (16) 1.27 6 0.14 (7)*pckdel/del 31.8 6 7.2 (8)*** 1,610 6 210 (10)** 41.6 6 1.6 (13) 79.1 6 15.6 (7)** 6.59 6 0.22 (6)***

a *, P , 0.05, **, P , 0.01, and ***, P , 0.001 versus pckw/w; n is indicated in parentheses.

TABLE 2. Plasma insulin concentration, plasma and hepatic metabolite concentrations, and hepatic PEPCK activityin fed and 24-h-fasted pckdel/w and pckw/w micea

Pupgenotype

Concn (mean 6 SE)

Plasma Hepatic

Insulin(pg/ml)

Glucose(mg/dl)

FFA(meq/liter)

Glycerol(mg/dl)

BHBA(mg/dl)

Lactate(mg/dl)

Glycogen(mM/g)

Malate(mM/g)

PEPCK activity(mU/mg of protein)

Fedpckw/w 908 6 183 192.2 6 4.7 649 6 76 5.7 6 0.6 3.5 6 0.3 34.0 6 3.2 402.4 6 32.9 0.47 6 0.03 6.5 6 0.6pckdel/w 874 6 104 179.5 6 6.9 648 6 62 6.4 6 0.8 3.3 6 0.3 32.2 6 1.8 369.8 6 18.3 0.56 6 0.07 3.9 6 0.3**

24 h fastedpckw/w 178 6 56 137.0 6 7.1 1,199 6 78 6.2 6 0.8 16.6 6 1.6 35.4 6 23 51.3 6 17.0 0.35 6 0.03 16.2 6 2.3pckdel/w 235 6 50 150.0 6 6.9 1,052 6 61 7.0 6 0.8 13.5 6 1.2 36 6 2.3 58.5 6 13.6 0.35 6 0.03 8.5 6 1.0*

a Age- and sex-matched mice 8 to 10 weeks old were sacrificed by cervical dislocation at 8 to 10 a.m., and blood was collected from neck vessels. *, P , 0.05, and**, P , 0.01 versus pckw/w; n 5 8 except for PEPCK activity (n 5 4).

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scription (Fig. 1D). All pckdel/del pups died within 3 days ofbirth, mostly at days 2 and 3. Prior to becoming moribund,pckdel/del pups became lethargic and pale and were smallerthan heterozygous null or normal littermates. Necropsy of thedead pckdel/del pups revealed empty stomachs and pale livers.

One-day-old pckdel/del mice were markedly hypoglycemic,with plasma glucose concentrations of 32 6 7 mg/dl comparedto control values of 90.3 6 7.9 mg/dl. FFA concentrations were;2-fold greater in pckdel/del pups, but plasma lactate concen-trations were unchanged (Table 1). Hepatic glycogen contentin pckdel/del pups was only 38% of that in wild-type pups,although hepatic glycogen content of fetuses removed at em-bryonic day 20 did not differ among the three genotypes (553 611 mM/g in pckw/w pups, 594 6 26 in pckdel/w pups, and 551 624 in pckdel/del pups, n 5 3 to 6) and, in all cases, was greaterthan in postnatal day 1 (P1) pups (P , 0.001). Hepatic malateconcentration was 10-fold greater in livers of the pckdel/del pupsthan in those of wild-type pups (Table 1). While the malateconcentration was increased in the pckdel/w pups, although to alesser degree, there were no differences in the plasma glucose,FFA, lactate, and hepatic glycogen concentrations of theseanimals compared to pckw/w pups (Table 1).

PEPCK mRNA abundance was decreased by 42 and 33% inliver and brown adipose tissue of pckdel/w pups (P , 0.01, n 54), respectively, compared to wild-type pups. Consistent withprior studies of the developmental expression of PEPCK (41),PEPCK mRNA in kidneys of 1-day-old pups was not detected

by Northern blot analysis for either wild-type or knockout pups(data not shown). The body weight of pckdel/del pups was 6.8%less than that of pckw/w pups at P1 (P , 0.05).

Adult heterozygous pck null mice. To assess the impact ofthe loss of one functional pck allele, we performed furtherstudies on 8- to 10-week-old animals that were heterozygousnull for PEPCK (pckdel/w). Hepatic PEPCK activities of fedand 24-h-fasted pckdel/w mice were 60 and 52%, respectively, ofthat of control animals (Table 2), indicating that a single func-tional pck allele in liver was only minimally capable of com-pensating for the loss of the second allele. Similarly, renalPEPCK activity in pckdel/w mice was approximately two-thirdsof control level at both fed (7.4 6 1.0 versus 10.6 6 0.8 mU/mgof protein, P , 0.04, n 5 4) and 24-h-fasted (23.2 6 0.9 versus39.4 6 4.1 mU/mg of protein, P , 0.01, n 5 4) states. Mito-chondrial PEPCK activity in the liver was less than 2% of thetotal PEPCK activity and did not differ between pckdel/w andpckw/w mice (data not shown). PEPCK mRNA abundance wasdecreased by 35% in liver and by 33% in kidney in 24-h-fastedmice (data not shown). However, plasma glucose concentra-tions did not differ at either fed or fasted states (Table 2). Bothhepatic (Table 2) and muscle (data not shown) glycogen con-tents were also similar. No significant differences were found inplasma FFA, BHBA, lactate, insulin, and hepatic malate con-centrations between the two genotypes at both fed and fastedstates (Table 2).

Characterization of compound heterozygotes. Given thelack of any effect of fasting on the plasma glucose concentra-tion in either the pcklox1neo/lox1neo or pckdel/w mice, we pro-ceeded to intercross pcklox1neo/lox1neo mice with pckdel/w mice.By doing so, we produced compound heterozygotes(pcklox1neo/del) that had 95, 90, and almost 100% reductions ofPEPCK gene expression in liver, kidney, and adipose tissues,respectively, as judged Northern blot analysis (Fig. 2D).Plasma glucose concentrations in both fed and 24-h-fastedmice were no different from those in wild-type mice (Fig. 2C).However, in this case the relative liver mass in pcklox1neo/del

mice after a 24-h fast was increased by 65% (7.1% 6 0.1% ofbody weight) and their livers were pale at necropsy.

Liver-specific knockout of PEPCK. Since liver is the majorsite of gluconeogenesis, we proceeded to generate liver-specificPEPCK knockout (i.e., pcklox/lox1Alb-cre) mice by matingpcklox/lox with animals that express cre in the liver under controlof the albumin promoter. Genotype analysis of 126 mice atweaning revealed frequencies of 72 pcklox/lox mice and 54pcklox/lox1Alb-cre mice (P , 0.11 according to chi-square test).

FIG. 3. Liver-specific recombination in pcklox/lox1Alb-cre (lanes 1) and pck-lox/lox (lanes 2) mice. (A) Northern blot analysis of overnight-fasted 5- to 6-week-old mice; BAT, brown adipose tissue; cyclo., cyclophilin. (B) Western blot anal-ysis of liver tissue from overnight-fasted mice.

TABLE 3. Body and liver weight, plasma hormone concentrations, and plasma and hepatic metaboliteconcentrations in fed and 24-h-fasted pcklox/lox and pcklox/lox1Alb-cre micea

Pup genotype Body wt (g) Relative liver wt(% of body wt)

Concn (mean 6 SE)

Plasma

Insulin(pg/ml)

Glucagon(pg/ml)

Corticosterone(ng/ml)

Glucose(mg/dl)

Glycerol(mg/dl)

Fedpcklox/lox 22.2 6 0.6 5.7 6 0.1 412 6 61 NDb ND 195.9 6 8.3 5.4 6 0.9pcklox/lox1Alb-cre 21.9 6 0.9 6.7 6 0.2*** 357 6 86 ND ND 185.4 6 45.2 5.3 6 0.7

24 h fastedpcklox/lox 17.2 6 0.6 4.5 6 0.1 253 6 76 50.0 6 8.0 367.5 6 45.2 131.7 6 10.7 6.5 6 1.1pcklox/lox1Alb-cre 17.8 6 1.5 7.7 6 0.1*** 206 6 34 52.1 6 4.6 426 6 38.9 127.9 6 6.1 6.1 6 0.5

a Age- and sex-matched mice 8 to 9 weeks old were sacrificed by cervical dislocation at 8 to 10 a.m., and blood was collected from neck vessels. *, P , 0.05, **, P ,0.01, and ***, P , 0.001 versus pcklox/lox; n 5 6 to 11.

b ND, not determined.



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Southern blot analysis of tissue DNA from 6-week-oldpcklox/lox1Alb-cre mice showed over 80% conversion of thepcklox to pckdel allele in the liver, with no sign of recombinationin other tissues (data not shown), consistent with previousstudies (35). To further assess the efficiency of recombinationwithin hepatocytes, both Western and Northern blot analyseswere performed. PEPCK mRNA and protein amounts in6-week-old pcklox/lox1Alb-cre mice were markedly reduced(Fig. 3), cytosolic PEPCK activity was undetectable, and mito-chondrial PEPCK activity was not altered (data not shown).PEPCK mRNA abundance in kidney and brown adipose tis-sues of pcklox/lox1Alb-cre mice was unaffected (Fig. 3A). Thesmall amount of residual PEPCK protein and mRNA detectedin the livers of adult animals by both of these assays is likelydue to a nonhepatocyte source, since a previous study of theAlb-cre mice showed them to confer complete recombinationby 6 to 8 weeks of age (35). However, recombination using thistransgene was age dependent since PEPCK protein levels de-creased only by ;70 and ;80% at 1 and 7 days after birth,respectively, in pcklox/lox1Alb-cre mice (data not shown).

Euglycemia in liver-specific PEPCK knockout mice at rest.Plasma glucose concentrations in pcklox/lox1Alb-cre mice atboth fed and fasted states were not different from those inpcklox/lox control mice (Table 3). However, fed (postabsorp-tive) hepatic glycogen content in the livers of PEPCK knockoutmice was only 56% of the level in control mice, and glycogenwas depleted completely in 24-h-fasted animals (Table 3).Plasma concentrations of the gluconeogenic substrates, lactateand glycerol, did not differ between the two genotypes, whilefasting plasma alanine concentration was increased by 25% inpcklox/lox1Alb-cre mice.

Given the normal plasma glucose concentrations in pcklox/lox1Alb-cre mice after fasting, we measured concentrations ofplasma hormones involving regulation of energy metabolism(Table 3). Both fed and fasting insulin concentrations were notdifferent between the two genotypes, nor were the fasting glu-cagon and corticosterone concentrations, despite hepaticmalate concentrations that were increased 5.5- and 9-fold atthe fed and fasted states, respectively, in pcklox/lox1Alb-cremice (Table 3).

The plasma glucose concentration in pcklox/lox1Alb-cre pupswas less than that in pcklox/lox pups at P1 (31.7 6 4.6 versus62.3 6 3.1 mg/dl, n 5 8 to 14, P , 0.0001). However, plasmaglucose concentrations did not differ between the two geno-types at P3 and P7 (data not shown). Hepatic glycogen content

in pcklox/lox1Alb-cre pups was decreased by 50% at P1 and by85% at P3 (data not shown).

To further determine the effect of the lack of hepaticPEPCK on whole body glucose metabolism, we performed astudy of basal glucose kinetics in 26-h-fasted pcklox/lox1Alb-cremice. During the entire 220-min experimental period, plasmaglucose concentrations in the liver-specific PEPCK knockoutmice did not differ from the control values. The [3-3H]glucose-measured glucose turnover rate and specific activity of plasma[14C]glucose were also not different (Table 4).

Since pcklox/lox1Alb-cre mice are able to maintain normalglucose metabolism at rest, we tested their response to exer-cise, a maneuver that increases peripheral glucose utilization.At 30 min and at the end of the exercise, blood glucose con-centrations in the knockout mice fell to approximately half ofthe level in controls (Fig. 4). While the exercise endurancetime between the two genotypes did not reach the definedstatistically significant level (50.6 6 7.3 versus 71.4 6 6.8, P ,0.07, n 5 5), the animals lacking hepatic PEPCK appeared tobe less tolerant of exercise than the pcklox/lox controls.

Impaired lipid metabolism and up-regulation of genes en-coding enzymes of energy metabolism in mice lacking hepaticPEPCK. The liver weights in pcklox/lox1Alb-cre mice were in-creased by 18 and 71% in the fed and 24-fasted states, respec-tively, compared to pcklox/lox mice (Table 3 and Fig. 5B). Oilred O and hematoxylin-eosin staining of liver sections frompcklox/lox1Alb-cre mice showed larger lipid droplets and vacu-oles, respectively (Fig. 5D and F). Although not different in fedmice, hepatic triglyceride content in the 24-h-fasted pcklox/lox1Alb-cre mice was increased by 94% compared with controls.Fasting plasma FFA and triglyceride concentrations were alsogreater in the knockout mice than in the controls. In contrast,

TABLE 4. Basal glucose kinetics in pcklox/lox pcklox/lox1Alb-cremice after a 26-h fasta

Pup genotype

Mean 6 SE

Plasmaglucose(mg/dl)

Glucoseturnover rate(mg/min/kg)

Plasma[14C]glucose

sp act (cpm/mg)

pcklox/lox 119.3 6 7.0 13.6 6 0.7 42,820 6 3,492pcklox/lox1Alb-cre 139.6 6 4.8 12.4 6 0.6 35,049 6 2,457

a Male mice approximately 7 months old were used; n 5 4.

TABLE 3—Continued

Concn (mean 6 SE)

Plasma Hepatic

BHBA(mg/dl)

Lactate(mg/dl)

Alanine(mM)

FFA(meq/liter)

Triglycerides(mg/dl)

Glycogen(mM/g)

Triglycerides(mg/g)

Malate(mM/g)

Malonyl-CoA(nM/g)

3.6 6 0.3 32.3 6 2.8 ND 558 6 38 116.2 6 4.6 356.7 6 19.4 17.5 6 3.0 0.56 6 0.07 ND3.2 6 0.3 33.4 6 3.4 ND 649 6 63 109.8 6 8.7 198 6 26.1*** 19.2 6 3.7 3.64 6 0.40*** ND

17.6 6 1.0 31.5 6 5.5 350 6 21 1,050 6 113 61.3 6 5.3 36.7 6 9.3 66.1 6 7.9 0.35 6 0.02 7.2 6 0.612.5 6 1.7* 37.9 6 3.5 439 6 14** 1,613 6 140** 82 6 8* 1.0 6 0.2*** 127.8 6 9.3*** 3.48 6 0.24*** 7.4 6 0.4



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the plasma BHBA concentration was less in the knockout mice(Table 3), suggesting a possible decrease in FFA b oxidation inlivers of the knockout mice. Hepatic malonyl-CoA concentra-tions were not different between the two genotypes at thefasted state (Table 3).

To begin to determine the basis for abnormalities in theregulation of lipid metabolism in these mice, malonyl-CoAdecarboxylase mRNA abundance was assessed and found to beincreased by 71% in livers of the knockout mice (Fig. 6A). The

mRNA levels for the mitochondrial and peroxisomal FFAb-oxidation enzymes, acyl-CoA oxidase, very long-chain fattyacyl-CoA dehydrogenase, and long-chain fatty acyl-CoA dehy-drogenase were increased by 551, 320, and 214%, respectively.The mRNA levels for medium-chain fatty acyl-CoA dehydro-genase, carnitine acetyltransferase, and enoyl-CoA hydratase–L-3-hydroxyacyl-CoA dehydrogenase bifunctional protein werealso increased, but to smaller extents (Fig. 6A). There was nochange in the mRNA abundance for microsomal FFA v-oxi-dation enzyme, CYP4A1, and CYP4A4 (data not shown).Palmitate b-oxidation activity in liver homogenates was alsoincreased by 16% (P , 0.01 [data not shown]), consistent withelevated mRNA abundance for FFA oxidation enzymes.

DISCUSSION

We used an allelogenic Cre/loxP gene targeting strategy todetermine role of cytosolic PEPCK in hepatic gluconeogenesisand energy metabolism. This strategy enabled the creation of aseries of three functionally distinct pck alleles from a single EScell line. In addition, since one of the alleles created wasconditional, by intercrossing these animals with a line of Alb-cre transgenic mice that we have previously described (35), wewere also able to generate mice with a liver-specific knockoutof PEPCK.

PEPCK is essential for life. The initiation of PEPCK geneexpression and gluconeogenesis occur at birth since sucklingrodent pups are thought to depend on gluconeogenesis forglucose production (2, 11). Thus, the facts that pckdel/del micedie within 3 days of birth and that 1-day-old pck-null pups aremarkedly hypoglycemic suggest impaired gluconeogenesis.Moreover, the lack of differences in fetal liver glycogen content

FIG. 4. Changes of blood glucose concentrations during exercise in pcklox/

lox1Alb-cre mice. ppp, P , 0.001 versus pcklox/lox at each time point; n 5 5.

FIG. 5. Hepatic steatosis in 24-h-fasted pcklox/lox1Alb-cre mice. (A and B) Necropsy indicates increased liver size with pale color in ;9-week-old pcklox/lox1Alb-cremice compared to pcklox/lox mice. (C and D) Oil red O histochemistry for neutral lipids in liver sections. Larger lipid droplets (red) are shown in livers ofpcklox/lox1Alb-cre mice than in those of pcklox/lox mice. (E and F) Hematoxylin-eosin histological staining for liver sections. Open circles in liver sections ofpcklox/lox1Alb-cre mice suggest lipid vacuoles. Scale bars in panels C to F represent 50 mm.



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between the pckdel/del and pckw/w fetuses, but decreased glyco-gen content and plasma glucose concentrations in 1-day-oldpckdel/del pups, is also consistent with impaired gluconeogene-sis in these knockout pups. However, it is not entirely clearwhether the cause of death in these pups is actually hypogly-cemia. First, glucose-6-phosphatase knockout mice, whichhave impaired hepatic and renal gluconeogenesis as well assevere hypoglycemia, do not die until 5 weeks of age (24).Second, the liver-specific PEPCK knockout (pcklox/lox1Alb-cre) mice, which at day 1 exhibited hypoglycemia as severe asthat in the pckdel/del animals, survived and were euglycemic by3 days of age. Thus, it remains possible that other factorsbesides hypoglycemia contribute to the earlier death of

PEPCK knockout pups. Indeed, since PEPCK immunoreactiv-ity has been detected in a variety of cells and tissues (48), it ispossible that the animals die as a result of loss of PEPCK inone or more of these cell types.

Fasted mice with diminished PEPCK gene expression areeuglycemic at rest. The Cre/loxP strategy used in this studyinvolved placement of a neo cassette between exons 3 and 4 ofthe PEPCK gene and its subsequent removal by cre plasmidmicroinjections after germ line transmission. However, themice that retained the neo cassette have a functionally im-paired allele that proved to be valuable in assessing the controlstrength of PEPCK on gluconeogenesis. Interestingly, bothpcklox1neo/lox1neo and pcklox1neo/del mice were viable and didnot exhibit any abnormality of blood glucose concentrationsafter a 24-h fast. Indeed, the fact that mice with a 90% reduc-tion of PEPCK expression globally, as well as animals with atotal knockout of hepatic PEPCK, are able to maintain eugly-cemia after fasting clearly indicates that this enzyme exerts amuch weaker control of gluconeogenesis than previouslythought. Considering that the isotopic studies in pcklox/lox1Alb-cre mice showed that these animals had similar glucoseturnover rates and plasma [14C]glucose specific activities (de-rived from [14C]lactate) as wild-type mice, this conclusion isvirtually inescapable. However, while the isotopic studies indi-cate that resting mice without hepatic PEPCK remain capableof synthesizing almost the same amount of glucose as a normalanimal, how this is achieved remains unclear.

The liver and kidney are the only two gluconeogenic tissues.In humans, the contribution of renal glucose production duringthe postabsorptive state to systemic glucose appearance hasbeen reported to vary from 5 to 28% (9, 42). The kidney cancontribute more than 50% of total glucose production duringeither a 5- to 6-week starvation in humans (33) or insulin-induced hypoglycemia in dogs (4). Thus, we cannot rule out thepossibility that net glucose production in the kidney increasesto compensate for diminished hepatic glucose production inpcklox/lox1Alb-cre mice although PEPCK gene expression inthe kidney of pcklox/lox1Alb-cre mice is unchanged.

It is also possible that the liver in pcklox/lox1Alb-cre mice isstill capable of converting lactate to glucose, although there isno known pathway other than via PEPCK for the conversion ofoxaloacetate to phosphoenolpyruvate. Mitochondrial PEPCK,even though it accounts for only ;2% of total PEPCK activity,may be sufficient to maintain sufficient gluconeogenesis duringfasting, provided there is no additional stress such as exercise.There is little reason to think that the thermodynamically un-favorable conversion of pyruvate to phosphoenolpyruvate bypyruvate kinase occurs in these mice, although this possibilitymay need to be directly tested.

Hepatic gluconeogenesis may also be activated by enhancedpyruvate carboxylation, malate-aspartate shuttle, TCA cycle,and oxidative phosphorylation and ATP production (14, 19,32). The increased hepatic mRNA levels for pyruvate carbox-ylase, alanine aminotransferase, mitochondrial malate dehy-drogenase, cytosolic malate dehydrogenase, cytosolic aspartateaminotransferase, citrate synthase, and succinyl-CoA syn-thetase in pcklox/lox1Alb-cre mice (Fig. 6B) after a 24-h fast areall consistent with this possibility.

Glycerol is normally considered a minor gluconeogenic sub-strate since it accounts for only ;3% of overall glucose presentin the postabsorptive state (31). Plasma glycerol is derivedfrom adipose tissue as a result of lipolysis, which probably isenhanced in fasted pcklox/lox1Alb-cre mice as suggested byincreased plasma FFA concentration, thereby increasing glyc-erol availability. On the other hand, because of the increasedhepatic triglyceride content in the knockout mice, more plasma

FIG. 6. Altered gene expression for energy metabolism enzymes in 24-h-fasted pcklox/lox1Alb-cre mice. Northern blots were first probed with the specificcDNA; then the membranes were stripped and reprobed with cyclophilin cDNA.The relative abundance for each mRNA was normalized to the cyclophilinmRNA level. (A) Lipid-metabolizing enzymes. MCD, malonyl-CoA decarboxyl-ase; VLCAD, very long-chain fatty acyl-CoA dehydrogenase; LCAD, long-chainfatty acyl-CoA dehydrogenase; MCAD, medium-chain fatty acyl-CoA dehydro-genase; COT, carnitine octanoyltransferase; CAT, carnitine acetyltransferase;ACO, acyl-CoA oxidase; PBE, enoyl-CoA hydratase–L-3-hydroxyacyl-CoA de-hydrogenase bifunctional protein. (B) Gluconeogenic, TCA cycle, and otherenzymes. cAAT and mAAT, cytosolic and mitochondrial aspartate aminotrans-ferase, respectively; cMDH and mMDH, cytosolic and mitochondrial malatedehydrogenase, respectively; CS, citrate synthase; IDH, isocitrate dehydroge-nase; SCS, succinyl-CoA synthetase; LDH, lactate dehydrogenase; ALT, alanineaminotransferase; PC, pyruvate carboxylase; G6Pase, glucose-6-phosphatase;FBPase, fructose-1,6-bisphosphatase; GAPDH, glyceraldehyde-3-phosphate de-hydrogenase. p, P , 0.05, pp, P , 0.01, and ppp, P , 0.001; n 5 4.



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glycerol would be phosphorylated by hepatic glycerol kinasefor esterification of fatty acid in the liver of the knockout miceduring fasting. Moreover, plasma glycerol concentrations arenot changed in pcklox/lox1Alb-cre mice. Thus, it is unlikely thata compensatory increase in gluconeogenesis from glycerol oc-curs in these mice.

It should also be pointed out that futile cycling of substrates(e.g., glucose and fructose-6-phosphate cycling) might lead toan overestimation of the measurement of glucose turnover asdetermined by [3-3H]glucose, with glucose utilization in pe-ripheral tissues actually being decreased. Since the plasmaglucose concentration reflects the balance of glucose produc-tion and utilization, a decrease in glucose utilization wouldhelp maintain glucose homeostasis even if glucose productionwere diminished in pcklox/lox1Alb-cre mice. It is also possiblethat increased plasma FFA in pcklox/lox1Alb-cre mice competewith glucose for metabolism in tissues like skeleton muscle, amajor organ of glucose consumption, thereby impairing glu-cose utilization in this tissue (36).

Despite a normal fasting glucose concentration at rest, bloodglucose concentrations of pcklox/lox1Alb-cre mice decreasedwith exercise. The exercise response pattern of blood glucoseconcentrations in the control and knockout mice is similar tothat observed in a study of fed rats treated with 3-mercaptopi-colinic acid (17). Since the mice were fasted for 24 h beforeexercise, glycogen stores would not be expected to contributesubstantially to glucose production. Thus, the diminishedblood glucose concentration in pcklox/lox1Alb-cre mice after 30min of exercise probably reflects inadequate gluconeogenicflux in the face of increased peripheral glucose utilization. Ithas previously been shown that the glucose appearance rate incontrol rats is increased ;2-fold during submaximal exercisebut decreased in 3-mercaptopicolinic acid-treated rats (43).Therefore, the exercise study demonstrates that the liver-spe-cific PEPCK knockout mice have limited gluconeogenic capac-ity.

While additional studies may be required to confirm ourexperimental interpretations, the results we have obtained areconsistent with the conclusion that PEPCK is much less effec-tive in determining gluconeogenic flux than previously thought.Instead of a single dominant control point, these results sup-port the concept of multienzyme regulation of the gluconeo-genic pathway (13, 34). This difference may have significantimplications for the development of strategies to inhibit theexcess hepatic glucose production that occurs in type 2 diabe-tes mellitus.

Impaired lipid metabolism in liver-specific PEPCK knock-out mice. While these studies strongly suggest that PEPCKexerts much less control of gluconeogenesis than previouslythought, the finding of marked steatosis pcklox/lox1Alb-cremice during fasting indicates that the enzyme has a previouslyunsuspected role in regulating hepatic lipid metabolism. Theincreased hepatic triglyceride accumulation in pcklox/lox1Alb-cre mice may result from the 60% increased plasma FFA con-centration in these mice. The hepatic steatosis in pcklox/lox1Alb-cre mice may also be caused by a decrease in hepatic FFAoxidation, as suggested by lower plasma BHBA concentrationdespite increased plasma FFA concentration in these animals.Malonyl-CoA is a potent inhibitor of carnitine palmitoyltrans-ferase I, the key enzyme for transport of FFA into mitochon-dria for oxidation (26). Moreover, the concentration of malo-nyl-CoA in muscle is correlated positively with the sum ofcitrate and malate concentrations (38). However, the fastinghepatic malonyl-CoA concentration in pcklox/lox1Alb-cre micewas not increased, presumably due to increased malonyl-CoAdecarboxylase, as indicated by RNA analysis.

It is also surprising that the expression of genes encodingFFA-oxidizing enzymes both in mitochondria and in peroxi-somes is actually elevated in pcklox/lox1Alb-cre mice duringfasting. mRNA abundance for both acyl-CoA oxidase and fattyacyl-CoA dehydrogenases was elevated severalfold in the liver-specific knockout mice. Acyl-CoA oxidase is an important en-zyme for peroxisomal FFA oxidation, and acyl-CoA oxidaseknockout mice also have severely fatty livers (15). Fatty acyl-CoA dehydrogenases are also important enzymes for mito-chondrial FFA oxidation, as demonstrated by the mortalityrates of long-chain fatty acyl-CoA dehydrogenase knockoutmice (21). The mechanism(s) responsible for the increasedexpression of the genes encoding these enzymes is not known.The activation of peroxisome proliferation-activated receptoralpha (PPARa) signaling by long-chain acyl-CoA, as suggestedby studies of the acyl-CoA oxidase knockout mice (15), doesnot provide an explanation because the expression of othergenes known to be regulated by PPARa, such as the CYP4A1,CYP4A3, and carnitine octanoyltransferase genes, is not al-tered in pcklox/lox1Alb-cre mice.

This finding, as well as the lack of significant alterations inplasma hormone concentrations, suggests that alterations inthe concentrations of metabolic intermediates may affect ex-pression of numerous genes encoding various enzymes in-volved in energy metabolism. Indeed, the markedly elevatedexpression of genes encoding hepatic FFA oxidation enzymesstands in direct contradiction to the fatty liver phenotype inpcklox/lox1Alb-cre mice.

Concluding statements. This study provides new and unex-pected insights into the role of PEPCK in regulating hepaticenergy metabolism. While fasting plasma glucose concentra-tions are normal in mice with greatly diminished PEPCK geneexpression, the absence of PEPCK causes impaired lipid me-tabolism and marked alterations in the expression of a varietyof hepatic genes involved in energy metabolism. These changesoccur in the absence of alterations in plasma concentrations ofmajor hormones. Thus, PEPCK appears to play a vital role inthe integration of multiple pathways of energy metabolism, afunction that has not heretofore been attributed to this en-zyme.

ACKNOWLEDGMENTS

We thank D. Wasserman, P. Flakoll, Y. Fujimoto, E. P. Donahue,M.-Y. Zhu, and J. Lindner for help and advice in performing thesestudies, and we thank A. D. Cherrington and D. K. Granner forreading the manuscript and providing comments. We are also indebtedto A. Saha (Boston University) for measuring malonyl-CoA and to D.Kelly (Washington University) for providing cDNA for CYP4A3,CYP4A1, and MCAD.

This study was supported by funding from the National Institutes ofHealth (grant DK42502). P. She is a recipient of a JDFI postdoctoralfellowship.

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Phosphoenolpyruvate Carboxykinase Is Necessary for the