Endogenous Production of Lipoic Acid Is Essential for Mouse Development

${alpha}$ -Lipoic acid (LA) is a cofactor for mitochondrial ${alpha}$ -ketoaciddehydrogenase complexes and is one of the most potent, naturalantioxidants. Reduction of oxidative stress by LA supplementationhas been demonstrated in patients with diabetic neuropathy andin animal models. To determine how normal development or pathologicalconditions are affected by genetic alterations in the abilityof mammalian cells to synthesize LA and whether dietary LA cancircumvent its endogenous absence, we have generated mice deficientin lipoic acid synthase (Lias). Mice heterozygous for disruptionof the Lias gene develop normally, and their plasma levels ofthiobarbituric acid-reactive substances do not differ from thoseof wild-type mice. However, the heterozygotes have significantlyreduced erythrocyte glutathione levels, indicating that theirendogenous antioxidant capacity is lower than those of wild-typemice. Homozygous embryos lacking Lias appear healthy at theblastocyst stage, but their development is retarded globallyby 7.5 days postcoitum (dpc), and all the null embryos die before9.5 dpc. Supplementing the diet of heterozygous mothers withLA (1.65 g/kg of body weight) during pregnancy fails to preventthe prenatal deaths of homozygous embryos. Thus, endogenousLA synthesis is essential for developmental survival and cannotbe replaced by LA in maternal tissues and blood.

INTRODUCTION

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Introduction

R- ${alpha}$ -Lipoic acid (6,8-thioctic acid) (LA) is a crucial cofactorthat is required for the activity of the multienzyme complexesinvolved in the decarboxylation of ${alpha}$ -ketoacids, important stepsin energy metabolism. These complexes include pyruvate dehydrogenasecomplex (PDC), ${alpha}$ -ketoglutarate dehydrogenase complex, branchedchain ${alpha}$ -ketoacid dehydrogenase complex (32), and a glycine cleavagesystem (11). LA is covalently attached via an amide bond tospecific lysine residues of the E2 subunits of these complexes.LA is universally present in prokaryotes and eukaryotes (28),and previous investigations have shown that it is synthesizedby lipoic acid synthase from octanoic acid and a sulfur source(23, 25). Mouse lipoic acid synthase is encoded by the nucleargene Lias.

LA, one of the most potent natural antioxidants, has receivedconsiderable attention as a general dietary supplement for thepast 15 years (3). Many lines of evidence have shown that bothLA and its reduced form, dihydrolipoic acid (DHLA), have multifunctionalantioxidant activities with the following characteristics (29).First, LA/DHLA are amphiphilic and readily cross the blood-brainbarrier and cell membranes. Second, LA/DHLA possess metal-chelatingactivity. Third, LA is reduced to DHLA by several antioxidantenzymes that are expressed constitutively in most types of cells.In addition, because of its strong negative redox potential,DHLA can recycle other antioxidants, such as vitamin C, vitaminE, glutathione, coenzyme Q10, and ubiquinone (14, 15, 21, 29,33).

A growing body of evidence implicates oxidative stress as animportant pathogenic element in a variety of diseases, includingatherosclerosis and diabetes (34, 36). Previous investigationssuggest that supplements of LA reduce oxidative stress in bothdiabetic patients and animal models (9, 24) and that it is particularlysuitable for prevention and/or treatment of diabetic complications(28). Indeed, LA has been used in Germany by patients with diabeticneuropathy for more than 30 years (44). However, treatmentswith LA at doses that produce a significant reduction of oxidativestress have not been clearly shown to affect the pathologicalprocesses resulting in diabetes and atherosclerosis. In addition,nothing is known about how endogenous LA synthesis is modifiedunder pathological conditions. Furthermore, it is not knownwhether genetic alterations that reduce LA synthesis will modifydisease development. To explore the physiological effects ofreduced endogenous LA synthesis and to assess to what degreethe exogenous LA can circumvent the effects of reduction inits synthesis, we have inactivated the Lias gene in mouse embryonicstem (ES) cells using homologous recombination. We show herethat homozygous Lias null embryos die in utero. Heterozygotessurvive but show evidence of increased oxidative stress comparedto their wild-type littermates.

MATERIALS AND METHODS

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Materials and Methods

Construction of a targeting vector. A ${lambda}$ phage clone containing exons 4 to 9 of the endogenous Liasgene was isolated from a mouse genomic phage library. A 1.3-kbNheI/XhoI fragment containing exon 3 and its flanking intronicsequences and an approximately 6-kb BamHI/NotI fragment containingexons 7 to 9 were isolated from the phage clone and used as5' and 3' homologous arms, respectively, flanking the neomycinphosphotransferase gene (Neo [see Fig. 1]). The construct wasdesigned so that homologous recombination between the endogenousLias gene and the targeting construct would delete 3.7 kb ofDNA, including exons 4 through 6 of the Lias gene, and replacethem with the Neo gene. The targeting construct was linearizedat a NotI site and electroporated into W4/129S6 cells (Taconic,Germantown, NY), which are derived from 129S6 strain mice. Coloniessurviving after selection with G418 and ganciclovir were firstscreened by PCR with a primer, p1, from introns 2 to 3 (5'-GACTGG TGT ACA GCG TAG TCT-3') and a primer, p2, from the 3' endof the Neo gene (5'-GCT TCC TCG TGC TTT ACG GTA T-3'). TargetedES cells were identified by the presence of 1.3-kb PCR productand were confirmed by Southern blot analysis of EcoRI-digestedES cell genomic DNA with a probe specific to exon 2.

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FIG. 1. Targeted modification of the mouse Lias gene. (A) Schematic representation of the endogenous mouse Lias allele, targeting vector, and the mutant allele. The thick horizontal black line indicates genomic DNA containing exons 3 to 9. The NheI/XhoI and BamHI/NotI fragments flank the Neo gene in the targeting construct. Small arrowheads labeled P1 and P2 represent the locations of the P1 and P2 PCR primers used to identify the targeted allele in embryonic stem cells. The probe used for Southern blot analysis is shown below the mutant allele. Eco, EcoRI; Nhe, NheI; Xho, XhoI; Bam, BamHI; Not, NotI. (B) Confirmation of the targeted allele by Southern blot analysis. Genomic DNA from Lias^+/^– (+/–) or Lias^+/+ (+/+) mice was digested with EcoRI and hybridized with the probe shown in panel A. The probe detects an 11.0-kb targeted fragment in addition to a 5.4-kb endogenous fragment in the correctly modified ES cells. (C) Detection of mouse genotype by PCR. Genomic DNA from F₁ offspring of chimeric mice was amplified with the primers described in Materials and Methods. A 415-bp fragment indicates the presence of the targeted locus, while a 560-bp fragment indicates the endogenous locus.

Lias-deficient mice. Two ES cell lines with a correctly targeted Lias locus wereidentified and injected into C57BL/6J recipient blastocysts,which were transferred to the uteri of CD1 pseudopregnant females.The chimeric male mice were bred with C57BL/6J females (TheJackson Laboratory, Bar Harbor, ME) to produce F₁ progeny carryingthe mutant allele. F₂ offspring were obtained by crossing theF₁ heterozygotes. Transmitting chimeras were also mated with129S6 females (Taconic, Germantown, NY) to establish an inbredline of mutants. Mice were maintained according to the Guidefor the Care and Use of Laboratory Animals (26a), and all experimentswere carried out in accordance with protocols approved by theInstitutional Animal Care and Use Committee of the Universityof North Carolina at Chapel Hill. Mice were on regular chowexcept that the lipoic acid-supplemented diet contained 45%(wt/wt) fat, 0.05% cholesterol, 20% sucrose, and 1.65 g of LA(Research Diets, Inc., New Brunswick, NJ) per kg of body weight.

Genotyping. Genotypes of mice were determined by PCR of tail DNA with threeprimers, a primer (p2) from Neo, p3 (5'-GGA GAC ATA GGC AGTGGA TA-3') from 5' to the XhoI site in intron 3, and p4 (5'-GAAGCA GAA GCA GGC AGA TA-3') from 3' to the XhoI site in intron3. A 560-bp fragment produced with p2 and p3 detects the endogenouslocus, and a 415-bp fragment generated with p3 and p4 detectsthe targeted locus, allowing homozygous, heterozygous, and wild-typemice to be distinguished.

Laser capture microdissection. The genotype of 7.5 days postcoitum (dpc) embryos was determinedas follows. Decidual swellings were dissected at 7.5 dpc fromheterozygous mothers, fixed in 4% formaldehyde in phosphate-bufferedsaline overnight, and embedded in paraffin. The embryos withtheir decidua were serially sectioned and deparaffinized throughxylene and graded alcohols, stained with hematoxylin and eosin,dehydrated in graded ethanol, and cleared in xylene. Embryonictissues were captured under direct microscopic visualization,collected by laser capture microdissection (Leica Microsystems,Wetzlar, Germany) following the manufacturer's protocol, andlysed in 10 mM Tris-HCl (pH 8.0) containing 0.1% Tween 20. Aftertreatment with proteinase K (100 ng/ml) at 55°C for 4 hfollowed by inactivation of proteinase K at 95°C for 5 min,DNA was subjected to 40 cycles of PCR.

mRNA analysis. Tissue RNA was extracted with TRIzol reagent (Carlsbad, CA)according to the manufacturer's protocol. The amount of LiasmRNA was determined by real-time quantitative reverse transcription-PCR,with ß-actin as the reference gene in each reactionmixture (20). Primers for Lias gene amplification were 5'-CCCGTC GGC CAC ATC ATC TCG AT-3' and 5'-GGG TCT GGA TTA TGT TGTCC-3'. The probe for Lias detection was 5'-FAM-ATC GAG ATC ATGTGG CCG ACG GG-TAMRA-3' (FAM, 6-carboxyfluorescine; TAMRA, 6-carboxytetramethylrhodamine).The primers for ß-actin detection were 5'-GAA GCGGCA TCC ATT GCT-3' and 5'-GAA GCG GCA TCC ATT GCT-3', and theprobe was 5'-tetrachloro-fluorescein phosphoramidite-CAC TATTGG CAA CGA GCG GTT CCG-TAMRA-3'.

Biochemical analysis. Blood was collected from the retro-orbital sinus under anesthesiaand was anticoagulated with heparin or EDTA. Oxidative stressin plasma was assessed by measuring thiobarbituric acid-reactivesubstances (TBARS), a common indicator of oxidative stress (22).In erythrocytes, reduced levels of erythrocyte glutathione (GSH),an endogenous antioxidant marker, were determined using a commerciallyavailable kit (Cayman, Ann Arbor, MI). Plasma glucose, totalcholesterol, and triglycerides were measured using the respectivekits (Sigma, St. Louis, MO) following the manufacturers' protocols.Lactic acid concentrations in tissues were determined as describedpreviously (41). Protein content was determined by the Bio-Radprotein assay kit using bovine serum albumin as a standard.PDC activity in liver was determined by spectrophotometric assayas described previously (35)

Histological analysis. Embryos (7.5 dpc) from timed mating between heterozygotes wereremoved from uteri and fixed together with their surroundingdeciduas in 4% paraformaldehyde in phosphate-buffered saline(pH 7.4), dehydrated, and embedded in paraffin. Five-micronsections were cut, mounted on slides, and stained with hematoxylinand eosin.

RESULTS

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Results

Generation of Lias-deficient mice. Homologous recombination between the endogenous Lias locus andthe targeting DNA construct deleted DNA containing exons 4 through6 and replaced it with the Neo gene (Fig. 1A). This deletionresulted in a null allele because these exons encode the functionallyconserved Cys motifs of the enzyme that are important for insertinga sulfur atom into the hydrocarbon chain of octanoic acid (23).Two correctly targeted ES cells carrying a disrupted Lias genewere identified by PCR and confirmed by Southern blot analysis,which showed an 11.0-kb band from the disrupted Lias gene, togetherwith a 5.4-kb fragment band from the unmodified Lias gene (Fig.1B).

The Lias gene is essential for the development of mouse embryos. Fifteen chimeric mice were obtained from the two targeted EScell lines. Six transmitted the modified Lias gene to the nextgeneration. F₁ Lias^+/^– mice showed no overt differencesfrom their Lias^+/+ littermates in gross appearance, behavior,growth curve, and fertility. However, intercrossing the Lias^+/^–F₁ mice failed to produce any homozygous Lias^–^/^–offspring (Table 1). The numbers of wild-type and heterozygouspups, genotyped by PCR (Fig. 1C), were as expected. This suggeststhat the complete lack of Lias is prenatally lethal.

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TABLE 1. Genotypes of embryos from Lias^+/– x Lias^+/– mating

In order to determine the stage of embryonic death, embryosat different dpc were collected from the uteri of pregnant damsfrom Lias^+/^– x Lias^+/^– mating. As shown in Table1, homozygous Lias^–^/^– embryos were present at the3.5 dpc blastocyst stage. They were indistinguishable in appearancefrom Lias^+/+ and Lias^+/^– blastocysts. However, after 9.5dpc Lias^–^/^– embryos were not recovered. Three outof the four resorbed embryos from pregnancies at 9.5 dpc weregenotyped as Lias^–^/^–; the other was wild type. Thelast is not unexpected, since a small percentage of wild-typeembryos are naturally resorbed. Lias^–^/^– embryoswere recovered at 7.5 dpc but were noticeably smaller than Lias^+/+or Lias^+/^– embryos (Fig. 2). The genotypes of the embryoswere determined using laser capture microdissection (Fig. 2F).Histological examinations revealed that the homozygous 7.5 dpcembryos had no ectoplacental cavities and were developmentallyretarded (Fig. 2C). This suggests that the Lias^–^/^–embryos do not reach the early primitive streak stage or latestreak stage; they appear to stop growing at the egg cylinderstage (6.5 dpc). In addition, hemorrhage surrounding the homozygous7.5 dpc embryos suggests that they were being resorbed. Theseresults indicate that the Lias^–^/^– embryos die atan early implantation stage between 7.5 dpc and 9.5 dpc andthat LA in maternal blood and tissues cannot support their development.

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FIG. 2. (A) Developmental retardation of homozygous embryos. Embryos (7.5 dpc) from timed Lias^+/^– x Lias^+/^– mating were dissected from the uteri with the removal of deciduas, along with extraembryonic membrane and ectoplacental cone. This panel was photographed at an original objective lens magnification of x15. Lias^–^/^– null embryos (–/–) are smaller than heterozygous (+/–) or wild-type embryo littermates. Embryos (7.5 dpc) were subjected to histological analysis and genotyping by microdissection. Sections were stained with hematoxylin and eosin and photographed at an original objective lens magnification of x10. (B) A Lias^+/^– embryo shows postgastrulation with distinct germ layers, ectoderm, and visceral endoderm. ve, visceral endoderm; ee, embryonic ectoderm; al, allantois; ac, amniotic cavity; exc, exocoelomic cavity; am, amnion; ch, chorion; rm, Reichert's membrane. (C) A Lias^–^/^– embryo shows obvious lack of organization. pc, proamniotic canal. (D and E) Embryonic tissues were collected by laser capture microdissection, and the remaining sections are shown in panels D and E. The genotypes of the embryos were determined by PCR of the captured tissues (F). The 415-bp band indicates the null allele, and the 560-bp band indicates the wild-type alleles.

Supplementing the diet of Lias^+/^– mothers with LA fails to rescue Lias^–^/^– embryos. We attempted to rescue the Lias^–^/^– embryos by providingtheir heterozygous mothers with extra lipoic acid (1.65 g/kg)in their diet. This dose is commonly used in rodents for therapeuticpurposes, and a 50% effective dose of LA for correcting bloodflow deficits in diabetic rats has been estimated at approximately38 mg kg^–1 day^–1 (5). Supplementation of LA in thediet for 1 month increased GSH in erythrocytes and decreasedplasma TBARS significantly (Fig. 3), which demonstrates thatdietary LA reaches maternal circulation. However, no live-bornLias^–^/^– mice were found among 34 pups of LA-supplementeddams, and the supplementation did not affect the average littersize. Thus, high-dose LA supplementation, which sufficientlyaffects the levels of TBARS and GSH in the maternal blood, failedto rescue pups that completely lack endogenous production ofLA.

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FIG. 3. Plasma TBARS and erythrocyte GSH levels before (black bars) and after (open bars) Lias^–^/^– mothers were fed a diet containing LA for 4 weeks. Data are the means ± standard errors of the means (error bars) for seven mice. Values that are significantly different from the values of mice before they were fed a diet containing LA are indicated (P < 0.01 [**]; P < 0.001 [***]).

The Lias^+/^– mice have reduced Lias mRNA expression. Quantitative reverse transcription-PCR revealed that the Liasgene is highly expressed in the testes, kidney, liver, heart,skeletal muscle, and aorta of wild-type C57BL/6J mice (Fig.4A). Northern blot analysis showed similar results (data notshown). The levels of expression of Lias mRNA in the kidney,liver, and heart of the heterozygous Lias^+/^– mice are45 to 60% of the levels of their wild-type littermates (Fig.4B), which does not differ significantly from the 50% levelexpected in a heterozygote for a gene that is not subject tohomeostatic compensation.

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FIG. 4. (A). Expression of the Lias gene. (A) The mRNA expression levels of the Lias gene in aorta (A), skeletal muscle (M), heart (H), liver (L), kidney (K), and testis (t) in eight wild-type C57BL/6J mice. Lias mRNA levels relative to ß-actin mRNA in each tissue were determined by the real-time quantitative PCR as described in Materials and Methods. Expression levels in tissues are presented relative to the level in the aorta, which was set at 1.0. Data are shown as means ± standard errors of the means (error bars). (B) Lias mRNA expression in the heterozygotes. The amounts of Lias^+/^– mRNA in the heart, kidney, and liver are expressed relative to those in Lias^+/+ mice. Values are the means ± standard errors of the means (error bars) of nine animals in each group of Lias^+/+ or Lias^–^/^– mice.

Metabolic status of Lias^+/^– mice. To test whether the 50% reduction in the level of expressionof Lias alters the metabolic and antioxidant status of the heterozygousmice, we assayed a series of biochemical parameters (Table 2).The Lias^+/^– mice had normal levels of plasma glucose,triglyceride, and cholesterol. They exhibited normal glucosetolerance (data not shown). Lactic acid in the liver was higher,and the activity of liver pyruvate dehydrogenase complex waslower in Lias^+/^– mice than in wild-type mice, althoughneither reached significance (P = 0.08 and 0.09, respectively).The level of GSH in the erythrocytes of Lias^+/^– mice wassignificantly lower than that of wild-type mice, although theplasma TBARS levels in the heterozygotes were not differentfrom the levels in wild-type mice. Thus, these data show thathalf the normal level of Lias mRNA is not sufficient to maintainthe normal antioxidant capacity of mice even under the unstressedcondition of this experiment.

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TABLE 2. Comparison of biochemical parameters in Lias^+/+ and Lias^+/– mice^a

DISCUSSION

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Discussion

We have demonstrated that the absence of Lias gene expressionleads to early embryonic death and that this absence cannotbe rescued by maternal LA or by supplementing the diet of themothers with LA. Heterozygotes expressing Lias mRNA at 50% ofthe normal levels showed metabolic disturbances indicating asignificant reduction in erythrocyte GSH levels, even underunstressed conditions. Nevertheless, they develop normally andshow no overt abnormalities in 1 year.

The death of the Lias^–^/^– embryos shortly after implantationcoincides with the time when oocyte-derived proteins are nolonger able to support the rapidly growing embryos (42). Whilefurther studies are necessary to determine whether the absenceof LA synthesis damages preimplantation embryos, no visual abnormalitiesin the Lias^–^/^– blastocysts were observed. Publishedstudies have shown that the mouse embryo switches from oxidativemetabolism to glycolytic metabolism shortly prior to implantationbut reverts to oxidative metabolism soon after implantation(6, 39). Thus, dihydrolipoamide dehydrogenase (E3)-deficientembryos fail gastrulation and die before 7.5 dpc, and pyruvatedehydrogenase (E1)-deficient mice die around 9.5 dpc (18, 19).The similarities in timing of death of these and the Lias^–^/^–embryos indicates that LA synthesized by Lias, E1, and E3 areall essential components of the PDC enzyme complex so that inactivationof E1 or E3 activity and total loss of Lias would all lead toimpairment of glucose oxidative metabolism. Dihydrolipoamideacyltransferase (E2) has a central catalytic and structuralrole in the enzyme complex, because it provides a scaffold forthe binding and positioning of E1 and E3. The carboxyl groupof LA forms an amide bond with a lysine residue in E2 and generatesa "swinging arm" that permits the thiol/disulfide moieties ofthe lipoyl arm to rotate between the active sites of E1, E2,and E3 and facilitates electron transfer between them (31).Since E2 is shared in the multienzyme complexes that produceessential amino acids, pyrimidines, and porphyrins and ATP viathe citric acid cycle, cellular metabolism is likely to be compromisedmore broadly by the total absence of LA than it is by the absenceof individual E1 or E3.

Nevertheless, we were surprised to find that LA cannot be suppliedthrough maternal tissues, despite the fact that it is a smallamphiphilic molecule, readily able to cross the blood-brainbarrier and cell membranes (27, 28). Supplementing the maternaldiet with LA failed to rescue Lias^–^/^– mice fromembryonic lethality, although it is likely that LA can crossthe placenta efficiently, since Al Ghafli et al. have demonstratedthat LA supplementation (20 mg kg^–1, given intraperitoneally)improved the survival of embryos in rats with diabetes inducedby streptozotocin during the pregnancy (1).

The covalent attachment of exogenous LA to target apoenzymesoccurs in two steps. The first step is the formation of lipoyl-nucleotidemonophosphate, catalyzed by a lipoate-activating enzyme. Thesecond step is the transfer of the lipoyl moiety to the apoproteins,catalyzed by a lipoyl transferase. In Escherichia coli, a singleenzyme, LipA, catalyzes both of these steps (26). In mammals,a gene encoding a protein with the lipoate-activating enzymeactivity has been cloned in cows (13); its nucleotide sequencesuggests that it is a homolog of medium-chain acyl coenzymeA synthase. A different gene encoding lipoyl transferase activityhas also been cloned and characterized in cows and humans (12,13). Their presence suggests that mammalian cells may also becapable of utilizing exogenous LA to provide the LA moiety ofthe multienzyme complexes. However, the death of Lias^–^/^–embryos at an early implantation stage shows that the transferof exogenous LA to apoenzyme, even if it occurs in vivo, isnot sufficient to supplant the need for endogenous synthesisat least during mammalian development.

The pathway for endogenous LA synthesis in mitochondria beginsin Saccharomyces cerevisiae with the transfer of octanoic acid,linked by a thioester bond to the 4'-phosphopantetheine sulfhydrylgroup of the acyl carrier protein (ACP), to a specific ${varepsilon}$ -aminogroup of a target apoenzyme with the formation of an amide bond(4, 43). This transfer is mediated by lipoyl (octanoyl)-transferase,encoded by yeast LIP2. Subsequently, two sulfur atoms are insertedinto the C-6 and C-8 positions of octanoic acid to form a lipoicacid moiety in situ on the enzyme. Lipoyl synthase, a metalloenzymeencoded by LIP5 in yeast, and the substrate S-adenosylmethionineare required for this reaction. In yeasts, the absence of endogenousLA synthesis in mutants lacking either ACP1 or LIP5 cannot becomplemented by LA in the medium (4, 38). The early embryonicdeath of Lias^–^/^– mice that we observe suggests thatfree LA cannot be used to form the lipoic acid moiety of E2-containingenzyme complexes or that LA cannot be transported into mitochondriain mice even though it can probably pass through the placenta.

Mammalian cells have numerous antioxidant systems that playcomplex roles in the cellular antioxidant system. These antioxidantgenes are expressed early in embryonic development but reachpeak levels only during the neonatal period (2). Low levelsof antioxidants predispose embryos to the risk of oxidativedamage (10), and the absence of some antioxidant gene products,such as phospholipid hydroperoxide glutathione peroxidase, manganese-dependentsuperoxide dismutase (MnSOD), and ${gamma}$ -glutamylcysteine synthetase,cause early embryonic death (17, 37, 40). On the other hand,other endogenous antioxidants are not essential for embryonicsurvival. Thus, the absence of catalase (16), glutathione peroxidase-1(7), or copper/zinc-dependent SOD (CuZnSOD) (30) does not affectembryonic survival. The corresponding null embryos show no pathologicalchanges under normal physiological conditions, although theyexhibit a pronounced susceptibility to oxidative stress. Thedeath of the Lias^–^/^– embryos at 7.5 dpc, becauseit is so sudden, is unlikely to be due to the lack of LA asan antioxidant but is readily understood if it is caused bythe inability of the developing embryos to obtain energy fromoxidative metabolism. Nevertheless, this does not exclude thepossibilities that antioxidant activity of endogenously producedLA could be crucial for controlling reactive oxidative speciesproduction in mitochondria. Such a failure to balance the increasedgeneration of reactive oxygen species that accompanies the embryo'sswitch to oxidative metabolism may further delay embryonic development(8).

In conclusion, our study shows that the endogenous productionof LA is essential for early embryonic development, mainly wesuggest because of its role as an enzyme cofactor but partlybecause it is an antioxidant. These functions cannot be replacedby exogenous LA synthesized by the Lias^+/^– mother or givenas a dietary supplement. We also show that the heterozygousLias^+/^– mice have reduced antioxidant capacities, andthese mice may therefore provide a useful model for studyingthe role of mitochondrial antioxidant function in the pathogenesisof various metabolic disorders and for defining the role ofantioxidant enzymes in cellular defense against oxidant-mediatedtissue injury.

ACKNOWLEDGMENTS

This work was supported by grants (HL42630 and HL70523) fromthe National Institutes of Health.

We thank Oliver Smithies, Kathleen Caron, Kathleen Sulik, Hyung-SukKim, Seigo Hatada, and Nobuyuki Takahashi for valuable advice.We also thank Sylvia Hiller, Angie Ponguta, John Hagaman, JenniferWilder, and John Cowhig for expert technical assistance andRaymond Givens and Lance Johnson for helpful comments on themanuscript.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, 701 Brinkhous-Bullitt Building, Chapel Hill, NC 27599-7525. Phone: (919) 966-6912. Fax: (919) 966-8800. E-mail: nobuyo{at}med.unc.edu.

REFERENCES

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