a maternal high-fat diet is accompanied by alterations in the fetal primate metabolome

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maternal high-fat diet is accompanied by alterationsn the fetal primate metabolomeames Cox, PhD; Sarah Williams, MSc; Kevin Grove, PhD; Robert H. Lane, MD; Kjersti M. Aagaard-Tillery, MD, PhD

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BJECTIVE: To characterize the serum metabolome of a primate modelf in utero high-fat exposure.

TUDY DESIGN: Serum from maternal and fetal (e130) macaque mon-eys exposed to either a high-fat or control diet were analyzed by gashromatography-mass spectrometry. Multivariate data analysis waserformed to reduce the generated data set. Candidate metabolitesere further analyzed for significance by using the analysis of variancend comparative t tests.

ESULTS: Approximately 1300 chromatographic features were de-

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oi: 10.1016/j.ajog.2009.06.041

0 possible metabolites. With the use of comparative t tests, 22 me-abolites had statistical significance (P � .05) over the entire study. Byirtue of maternal high-fat diet alone, fetal phenotypic differences areccompanied by altered metabolite concentrations of 7 metabolites (P

.05).

ONCLUSION: In utero high-fat diet exposure is associated with anltered fetal epigenome and parlays a characteristic modification in theetal metabolite profile.

ey words: epigenetics, fetal programming, metabolomics, nutrition,
ected. Through multivariate data analysis this number was reduced to obesity, pregnancy
ite this article as: Cox J, Williams S, Grove K, et al. A maternal high-fat diet is accompanied by alterations in the fetal primate metabolome. Am J Obstet Gynecol

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he current global health epidemic ofobesity is considered a major con-

ributor to the increasing prevalence ofype 2 diabetes, as well as atherosclerotic,ardiovascular, and hypertensive mor-idity and mortality.1-6 In association

rom the Metabolomics Core Researchacility (Dr Cox) and the Division ofeonatology, Department of Pediatrics (Drsane and Aagaard-Tillery), University oftah Health Sciences, Salt Lake City, UT;regon National Primate Research Center

Ms Williams and Dr Grove), Oregon HealthScience University, Beaverton, OR; and

he Division of Maternal-Fetal Medicine,epartment of Obstetrics and Gynecology

Dr Aagaard-Tillery), Baylor College ofedicine, Houston, TX.

resented at the 29th Annual Meeting of theociety for Maternal-Fetal Medicine, Saniego, CA, Jan. 26-31, 2009.

eceived Feb. 21, 2009; revised April 22,009; accepted June 16, 2009.

eprints not available from the authors.

upport for this work was provided by theational Institutes of Health (NIH) Directorew Innovator Pioneer AwardP21DP2OD001500-01 (K.A.T.), and NIHrants 1R01DK080558 (R.H.L. and K.A.T.),K60685-0351 (K.L.G.), 1R01DK079194

K.L.G.), and RR00163 (K.L.G.-ONPRC).

002-9378/$36.002009 Published by Mosby, Inc.

ith this rise in adult obesity throughouthe developed and developing world,here is occurring a disproportionatearlier onset of obesity among childreninfants to adolescents).7-9 Given suchnticipation (eg, tendency of disease toppear at an earlier age of onset and withncreasing severity in successive genera-ions), the increased prevalence of child-ood obesity cannot be attributed to ei-her environment or genetics alone.

According to Barker’s fetal origins ofdult disease hypothesis, perturbationsn the gestational milieu influence theevelopment of diseases later in life.10,11

his supposition gains support frompidemiologic studies10-12 as well as ani-al models of nutritional constraint13-15

nd uteroplacental insufficiency-in-uced intrauterine growth restrictionIUGR).16-18 These outcomes have beenuggested to occur through the static re-rogramming of gene expression via al-erations in chromatin structure (epige-etic regulation). We and others havereviously demonstrated that uteropla-ental insufficiency and IUGR in the ratesults in covalent modifications ofhromatin structure16-18 or changes inNA methylation patterns,13-15 which

ead to persistent changes in postnatalene expression. In our initial studies es-ablishing our nonhuman primate

gh-fat diet (HFD) w

SEPTEMBER 2009 Americ

xposure in utero,19 we demonstratedhat a maternal HF/caloric-dense diet al-ered fetal hepatic chromatin structurend led to alterations in fetal and postna-al gene expression.20 These observa-ions supported our hypothesis that a

aternal HFD leading to obesity modi-es the gestational milieu and pro-

oundly influences the postnatal and fe-al phenotype in association with fetalistone (H3) covalent modifications.hese results led us to question whether

hese fetal epigenetic modificationsranslated into meaningful alterations inhe fetal and neonatal metabolome.

The metabolome is broadly defined ashe complete set of the small moleculeshat contribute to metabolism.21,22

etabolomics has emerged over the pastear decade as the third major path of

unctional genomics, alongside its re-ated “omics” of messenger RNA profil-ng (transcriptomics) and proteomics. Its a comparative analysis describing met-bolic responses of living systems toathophysiologic stimuli, genetic, orpigenetic modification.22 Comprehen-ive analyses of the metabolome in a bi-logic system occurs by the combinedse of analytic methods and multivariatetatistical analyses: metabolic finger-rints are acquired with an analytic plat-

orm (ie, gas or liquid chromatography
ith mass spectroscopy), processed with
an Journal of Obstetrics & Gynecology 281.e1

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ultivariate analyses to allow for data vi-ualization and discrimination (ie, prin-ipal component analysis [PCA] andartial least squares discriminate analy-is [PLS-DA]). The mass spectrum issed to determine the identity of dis-riminate metabolites by automatedomparison to known metabolites in aatabases.22 The synergistic integrationf the metabolome with altered gene ex-ression profiling allows for a compre-ensive overview of living systemsiology.As metabolomics is unique from other

omics” in its capacity to efficiently de-ipher systems biology as a reflection ofts close proximity to phenotype, weought to use it to establish the maternalnd fetal metabolic profile under condi-ions of a maternal HFD. With respect tohe maternal phenotypic profile, we havereviously described19,20 that over timeeg, 3-5 years) 2 distinct maternal phe-otypes become apparent among ourams. These dams can be considered asensitive (HFD-S) or resistant (HFD-R)o our HFD. HFD-S dams display anique metabolic phenotype in both theonpregnant and pregnant state.19 By

he fourth year on the HFD, the HFD-Sroup demonstrates a �35% increase inody weight, �3-fold increase in insulinrea under the curve in response to a glu-ose tolerance test, �5-fold increase ineptin levels (leptin/body weight ratio),nd a near doubling in body fat.19 How-ver, fasting glucose levels did not signif-cantly differ in either the HFD-S or

FD-R groups, despite significant alter-tions (control, 2.53 � 0.50; HFD-R,.81 � 0.70; HFD-S, 7.87 � 1.50; P � .05or HFD-S vs control) in the homeostasis

odel assessment of insulin resistanceor HOMAIR, which serves as a generaleasure of insulin resistance in refer-

nce to basal glucose and insulin). Byomparison, HFD-R animals retainedimilar body weight and metabolic phe-otype as the controls throughout thentire 4 years.

With respect to the fetus, we have sim-larly characterized the effect of in uteroxposure to a maternal HFD.19,20 Weave observed that although the mater-al phenotype is relatively mild, regard-

ess of maternal HFD-R or HFD-S status, g

81.e2 American Journal of Obstetrics & Gynecolo

he fetus has nonalcoholic fatty liver de-elop and displays an altered fetal he-atic epigenomic profile. Because theffspring are delivered in the early latterrimester of gestation (term � 167 days),ppreciable differences in overall fetaleight are avoided.19,20 Of interest, weave shown that reversing the maternaliet in year 5 from HF to control (“dieteversal”) bears appreciable differencesn the fetal phenotype with partial rever-ion trending toward to control values.19

owever, it is unknown whether thesehenotypic and epigonomic characteris-ics translate into observable alterationsn the maternal and fetal metabolome.iven that we have observed significant

lterations in the maternal and fetal phe-otype and fetal epigenome and tran-criptome after in utero exposure to a

aternal HFD, we aimed to characterizehe full spectrum of metabolite differ-nces in our model system.

Leveraging these collective observa-ions, we set out to first study the serum

etabolome of HFD-R and HFD-Sams and to test whether these observedhenotypes were transferred in utero tohe developing fetus. We then character-zed the fetal metabolome from dams fedontrol, HFD, or HFD-reversal. We hy-othesized that this approach would notnly decipher the fetal and maternal se-um metabolome under maternal HFDonditions leading to maternal obesity,ut would potentially identify candidateiomarker(s) associated with the devel-pment of obesity.

ATERIALS AND METHODSonhuman primate modelf maternal HFD exposureur model is described in detail else-here.19,20 Briefly, all animal proceduresere in accordance with the guidelines of

he Institutional Animal Care and Useommittee of the Oregon National Pri-ate Research Center (ONPRC). Age-

nd weight-matched young (5-6 years ofge at the start of the studies) adult fe-ale Japanese macaque monkeys were

ocially housed in indoor/outdoor en-losures in groups of 5-9 with 1-2 maleser group. Animals were separated into 2
roups: The control group was fed stan- s
gy SEPTEMBER 2009

ard monkey chow that provides 14% ofalories from fat (Monkey Diet 5052; Labiet, Richmond, IN) and the HFD groupas fed a diet that supplied 32% of calo-

ies from fat (Custom Diet 5A1F, Testiet) and included calorically dense

reats.19,20 Both diets are sufficient in vi-amin, mineral, and protein content forormal growth. Before this study, all an-

mals were maintained on standardonkey chow in large outdoor enclo-

ures and were naive to any experimentalrotocols. During the fifth year, a cohortf HFD-fed animals were switched backo the standard monkey chow specifi-ally during the breeding/pregnancy sea-on (“HFD-reversal”).

Dams were allowed to breed naturally.oth groups consisted of primiparousnd multiparous gravid monkeys, and allregnancies were singleton. Pregnanciesere terminated by cesarean delivery,erformed by ONPRC veterinarians, atestational day 130 by sonographic fetaliometry. For the experiments involvingostnatal (eg, juvenile) offspring, theregnancies were allowed to progress toerm and vaginal birth occurred, and off-pring were subsequently maintained onhe maternal diet after weaning.

Immediately after cesarean delivery ort the time of death, the fetuses/juvenilenimals were delivered to necropsyhere they were weighed and measured.he fetuses were deeply anesthetizedith sodium pentobarbital (�30 mg/kg,

ntravenously) and then exsanguinatedor attainment of serum and necropsyissue. For metabolomic profiling, wetudied serum samples from 47 damsnd 40 fetuses collected in heparinizedapillary tubes from the fetal aorta andentrifuged at 2400g rpm. All samplesere flash frozen and stored at – 80°C in

liquots such that freeze-thawing wasinimized.

etabolite profilingrotein removal from serum samplesas performed by using the method of

iye et al23 with the addition of 20 uni-ormly labeled stable isotope amino ac-ds of known concentration as an inter-al standard.To ensure metabolite volatility each

ample underwent a 2-step derivatiza-

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ion process. The dried extracts were sus-ended in 50 �L of pyridine containing0 mg/mL O-methylhydroxylamine hy-rochloride, sonicated for 5 minutes,hen incubated at 60°C for 30 minutes.his was followed by the addition of 50L MSTFA �1% TMCS (Pierce, Rock-

ord, IL) and incubated at 37°C for 30inutes. A retention index standardixture consisting of fatty acid methyl

sters was added and each sample imme-iately analyzed.All samples were analyzed in a random

rder by gas chromatography-masspectrometry (GC-MS). With an au-osampler, 1 �L of each sample was in-ected into an Agilent 6890 gas chro-

atograph (Agilent, Santa Clara, CA).he injector was set to a 2:1 split rationd the temperature was held at 220°C. A0-m Rtx-5MS with Integra guard col-mn (Restek, Bellefonte, PA) was used

or separation. Helium was used as thearrier gas at a 1-mL/min flow rate. Theransfer line into the mass spectrometeras held at 250°C. The temperature pro-ram was as follows: an initial 2-minuteold at 70°C, followed by an 8°C perinute ramp to 250°C, then a second

0°C per minute ramp to 330°C that waseld for 2 minutes. A MicroMass GCTremier (Waters, Beverly, MA) was used

or mass spectrometric analysis. Normallectron impact ionization conditionsere used. Data were recorded by usingassLynx (Waters).

odel development andetabolite identificationhromatographic peak detection andrea analysis for each chromatogram waserformed by using MarkerLynx (Wa-ers). To find possible variation in eachample group, the data were transferredo SIMCA-P version 12.0 (Umetrics,innelon, NJ), in which PCA andLS-DA were performed. This producedpossible list of metabolites that had

hanged because of diet or sensitivity tohe HFD. Each metabolite from this listas investigated further by extracting

he area under the chromatographicurve and analyzing it with analysis ofariance (ANOVA) and comparative tests. Metabolites were identified with ei-
her an inhouse database or the NIST MS o
earch 2.0. Unknown (un) metabolitesere identified by using their retention

ndex (ri) and characteristic mass-to-harge ratio (m/z) in the following for-at: un(ri_m/z).

tatistical analysisLS-DA was performed by usingIMCA-P (Umetrics). This multivariatetatistical analysis technique is similar toCA with the addition of a second dataatrix identifying specific classes, thus it

s a supervised method when comparedith PCA that is unsupervised. ANOVA

nd comparative t tests were performedor each metabolite by using the JMP 7.0tatistics package (SAS; SAS Institute,ary, NC). Excel (Microsoft, Redmond,A) was used for spread sheet develop-ent and simple statistics.

ESULTSnalytic analysis and chromatographicata deconvolution produced more than300 chromatographic features. Throughultivariate statistical analysis this num-

er was reduced to 60 possible metaboliteshat contributed to PLS-DA model devel-pment. Integration of the chromato-raphic area of these individual metaboliteeaks, followed by statistical analysis pro-uced 22 metabolites that had statisticalignificance (P� .05) over the entire study.

aternal metabolic profilesur first study aim was to characterize

he metabolomic footprint from damsed a 13% fat by calories (control) dietnd a 35% fat by calories (HFD). Chro-atographic data from 6 control and 8FD animals was processed to give the

nitial PCA plot (not shown). To there-fter quantitate and identify metabolitehanges a PLS-DA was performed (Fig-re 1, A). The loadings plot from thisodel was used to find chromatographic

eatures that differ between the controlnd HF animals. We found 10 metabo-ites that differed significantly (P � .05)etween control and HF animals (Ta-le 1).As anticipated by virtue of their pro-

ression to obesity on the maternal HFD,holesterol levels were elevated 1.5-foldigher in HF-fed dams relative to those
n control diet, whereas no significant c

ifferences could be detected in the levelsf palmitic, stearic, lauric, oleic, andlaidic free-fatty acids; higher lipid spe-ies are not detectable by using ourC-MS methods. We similarly observed

levated level of 2 primary constituent ofhe vitamin E complex: �- and �-to-opherol in the HFD dams. This is con-istent with obesity physiology in re-ponse to a HFD as these lipid solubleompounds act as antioxidants, protect-ng the cell membrane by reacting withipid radicals24 or triggering intracellularignaling cascades.25 Glycine, aminoma-onic acid, and 4 unknowns, un(2252_03), un(2281_131), un(2298_144), andn(2682_116) were also elevated in theFD dams, whereas citric acid was

ecreased.Based on the previously described

henotypic differences, we sought toompare the maternal sensitive (ie, obesend insulin resistant; n � 4) and resistantie, nonobese and insulin sensitive; n �) metabolomic profiles (Figure 1, B). Bysing a combination of PLS-DA andtandard statistical analysis from the pre-iously identified metabolites, HFD-Snimals contributed the bulk of changeetween HFD and control animals.leven metabolites were significantly al-

ered as shown in Table 1. Nine of theseetabolites are shared between the HFD

s control data set and 2 additional un-nowns were also found. The fat-solubleetabolites cholesterol, �-tocopherol,

nd �-tocopherol are increased in theFD-S vs the control animals. Signifi-

ant metabolite alterations as aminoma-onic acid, citric acid, and 6 unknowns;n(2252_203), un(2281_131), un(2298_44), un(2682_116), un(823_200), andn(1346_104). Only cholesterol (Table 1)as found to be significant in the HFD-R

nimals vs the control.

etal metabolic profilesur second objective was to characterize

he fetal serum metabolome to deter-ine its association to both maternal

iet and phenotype. A PLS-DA scatterlot was generated comparing fetal se-um from HFD-fed dams (n � 16) vshose on control diet (n � 6; Figure 2, A).ight metabolites were found to have
hanged in control vs HFD-exposed off-

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pring (Table 2), with cholesterol and-tocopherol being found in both ma-

ernal and fetal serum but with inverseoncentration profiles (ie, lower fetalholesterol under maternal HFD condi-ions). Of note, our findings have beenimilarly observed with attained mater-al and fetal serum cholesterol values aseasured in conventional clinical assays

n a limited cohort (n � 4 control vs n �HFD; maternal mean serum choles-

erol 81 � 5 vs 118 � 17 mg/dL, P � .002;etal 73 � 10 vs 63 � 7, P � .143; Dr D.

arks, unpublished observations). Alsonteresting was the 8-fold decrease of

FIGURE 1PLS-DA scatter plots of serum

LS-DA scatter plots of serum from dams mainifferences as quantitated by bar graphs (loweretabolites from HFD animals was stratified into

sed to effectively search for metabolites that cignificance are as summarized in Table 1. PLSox. Altered fetal epigenome associated with in utero HFD. A

scorbic acid in HFD fetal serum when g


ompared with control (P � .0069). Itas been proposed that ascorbic acid actss part of the antioxidant network inhich vitamin E participates.24

As an initial step in our effort to differ-ntiate the effects of maternal obesity vsFD exposure on the fetal metabolome,e characterized fetal serum profiles

mong a subgroup of dams whose HFDas reverted to control (reversal) in year(eg, control diet in an obese maternal

nvironment). We used a PLS-DA scat-er plot (Figure 2, B) and the underlyingoading profile to separate both maternaliet and obesity (ie, horizontal plot

ed for 2-3 years on either the high-fat diet (HFDel). B, In accordance with their phenotype as

ther high-fat diet resistant (HFD-R) or sensitiveribute to differences between groups based on, partial least squares discriminate analysis.

Obstet Gynecol 2009.

roupings among HFD and reversal off- t

gy SEPTEMBER 2009

pring distinct from control offspring).etabolites from the loadings plot that

ontributed to this model were furthernalyzed and are detailed in Table 2. Fe-al profiles could not be significantly dis-inguished by virtue of maternal diet (eg,FD relative to reversal) but could by

irtue of maternal obesity (eg, diet rever-al relative to control) for all metabolites,xcept cholesterol.

As a second step in differentiating theffects of maternal obesity vs HFD expo-ure on the fetal metabolome, we againtratified our analysis by virtue of mater-al HFD sensitivity (Figure 2, C). Six me-

r control (con) diet A, (upper panel) with meaniously described,19 PLS-DA analysis of serumD-S). The loadings plot from these graphs wasass spectrometry. Those metabolites achieving

tain ) opan prevei (HF

ont m-DAm J

abolites were found to be significantly

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ltered in the HFD-S serum vs control,holesterol, �-tocopherol, hypoxan-hine, myoinositol, un(698_103), andn(2298_144). When HFD-R is com-ared with control diet serum only �-to-opherol was found to be significantly al-ered. Three fetal metabolites were foundo be significantly elevated when com-aring HFD-S with HFD-R: hypoxan-hine, myoinositol, and un(2298_144).

It bears mentioning that 3-hydroxybu-yrate, initially identified as un(922_605)nd later confirmed to be 3-hydroxybu-yrate by mass spectrometry of purchasedtandard, is unique among the signifi-antly distinct fetal metabolites by virtue ofts elevations in association with maternalbesity (Figure 3). Thus, fetal levels of-hydroxybutyrate can be distinguishedith a maternal HFD and obesity (HFD vs

ontrol 2.3-fold, P � .006) or obesity alonereversal vs control 2.8-fold, P � .004). Inontrast, fetal levels are not distinguishedn maternal HFD relative to reversal con-itions (0.82-fold, P � .33).

OMMENTaternal obesity is thought to increase a

TABLE 1Analysis of maternal metabolites c

HFD vs con

Metabolite P value

Cholesterol .0093...................................................................................................................

�-tocopherol .023...................................................................................................................

�-tocopherol .039...................................................................................................................

Glycine .037...................................................................................................................

Aminomalonic acid .024...................................................................................................................

Citric acid .015...................................................................................................................

un(2252_203) .016...................................................................................................................

un(2281_131) .047...................................................................................................................

un(2298_144) .038...................................................................................................................

un(2682_116) .0028...................................................................................................................

un(823_200) .065...................................................................................................................

un(1346_104) .086...................................................................................................................

Con, control; HFD, high-fat diet; HFD-S, high-fat diet-sensitMetabolites from maternal serum that differ significantly betwresistance to the diet (eg, do not develop obesity despite muspectra data as presented in Figure 1.Control (n � 6); HFD (n � 8).

Cox. Altered fetal epigenome associated with in utero HFD

hild’s risk of juvenile obesity and meta- d

olic diseases; however, the mecha-ism(s) whereby excess maternal nutri-

ion impacts fetal development remainsoorly understood. According to the de-elopmental origins of adult disease hy-othesis, perturbations in the gestationalilieu influence the development of dis-

ases later in life through the static repro-ramming of genes. Phenotypic charac-erization in our primate model stronglymplicate in utero exposure to excess

aternal lipids during early and midfetalevelopment as an independent risk fac-or for the development of nonalcoholicatty liver disease (NAFLD) and meta-olic diseases during postnatal life as ev-

denced by alterations in the epigenomend transcriptome.19,20 Whether thesepigenomic alterations would parlaynto meaningful alterations in the fetalunctional genome (eg, the fetal metabo-ome) had not yet been addressed.

To begin to address this issue, we usednonhuman primate model to deter-ine the effect of chronic maternal con-

umption of a HF/high calorie diet onhe development of metabolic systems inhe fetal offspring. We have previously

ributing to the PLS-DA modelHFD-S vs con

ld changeigh-fat/con) P value

Fold c(HFD-S

5 .0038 1.6.........................................................................................................................

7 .00059 2.2.........................................................................................................................

2 .047 2.8.........................................................................................................................

3 .187 1.4.........................................................................................................................

5 .041 1.6.........................................................................................................................

3 .0089 1.3.........................................................................................................................

7 .015 2.1.........................................................................................................................

5 .023 1.9.........................................................................................................................

5 .015 1.9.........................................................................................................................

2 .017 2.1.........................................................................................................................

4 .018 1.5.........................................................................................................................

8 .012 2.4.........................................................................................................................

FD-R, high-fat diet-reversal; PLS-DA, partial least squares-discHFD and control diet-fed animals. The HFD-fed dams are furthe

years of feeding). Significantly discrete serum metabolites by

J Obstet Gynecol 2009.

emonstrated that only a portion of the s


dult female monkeys chronically con-uming a HFD become obese and insulinesistant.19 However, irrespective of ma-ernal obesity and insulin resistance, alletal offspring of dams fed a HFD dem-nstrated clinically and histologicallyvident NAFLD, including hepatic in-ammation, oxidative stress/damage,

riglyceride accumulation, and prema-ure gluconeogenic gene activation.19 Inhis study, we have extended our pheno-ypic and fetal epigenomic characteriza-ion with metabolomic analysis of ma-ernal and fetal serum. Altered serumoncentrations of cholesterol, membersf the vitamin E tocopherol complex,lycine, aminomalonic acid, citric acid,nd 3 unknowns was found betweenFD vs control maternal serum. Associ-

ted increases in maternal cholesterol areost likely attributable to the HFD itself,

s elevations were observed among bothFD-R and HFD-S dams (Figure 1, Ta-

le 1). By contrast, �- and �-tocopherolere not associated with HFD-fed damsith the resistant phenotype, suggestivef a reduced ability to metabolize fats inhe HFD-S animals that results in the ob-

HFD-R vs con

gen) P value

Fold change(HFD-R/con)

.043 1.4..................................................................................................................

.36 1.2..................................................................................................................

.45 1.6..................................................................................................................

.16 1.3..................................................................................................................

.27 1.4..................................................................................................................

.16 1.3..................................................................................................................

.26 1.3..................................................................................................................

.41 1.2..................................................................................................................

.41 1.1..................................................................................................................

.051 2.3..................................................................................................................

.24 1.2..................................................................................................................

.48 1.3..................................................................................................................

ate analysis; un, unknown.tified by sensitivity to the diet (eg, develop obesity) andspectroscopy are as listed and correspond to the mass

ont

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le compounds also act as antioxidants,rotecting the cell membrane by reactingith lipid radicals24 or triggering intra-

ellular signaling cascades.25 The inabil-ty of HFD-S animals to metabolize thisompound could be suggestive of the in-bility of these animals to cope with ox-dative stress.

To question the effects of maternalbesity and HFD exposure in utero, weharacterized the effects of diet, maternalensitivity to HFD, and diet reversal withersistent obesity on the fetal metabolic

FIGURE 2PLS-DA scatterplot of fetal serum

, PLS-DA scatterplot of fetal serum from damserum from dams fed a HFD and who were eithontrol diet. The loadings plot from these grappectrometry. Those metabolites achieving signLS-DA, partial least squares discriminate analysis.

ox. Altered fetal epigenome associated with in utero HFD. A

erum profile. When we extended our o


etabolic profiling of fetal serum to in-lude offspring from animals reverted tocontrol diet after 4 years (diet-reversal

ohort; Table 2) we observed that asso-iated fetal profiles could not be signifi-antly distinguished by virtue of mater-al diet (eg, HFD relative to dieteversal), but could be by virtue of ma-ernal obesity (eg, diet reversal relative toontrol) for all metabolites, except cho-esterol (Table 2).

The observed decrease in HFD ani-als of 3 metabolites that are involved in

a high-fat diet (HFD) vs B, fetal serum from dHFD-S or HFD-R. PLS-DA scatter plot of fetal swas used to identify metabolites that contribunce are as summarized in Tables 2 and 3.

Obstet Gynecol 2009.

xidative stress (2-hydroxybutyrate, 3

gy SEPTEMBER 2009

scorbic acid, and �-tocopherol) withoncomitant increase in the gluconeo-enic metabolite 3-hydroxybutyrate de-erves further comment. The HFD se-um metabolomic phenotype of loweredntioxidant levels and the increased me-abolite 3-hydroxybutyrate persists inhe fetal metabolome even after diet re-ersal occurs. This is of particular inter-st, as other investigators have demon-trated in obese children that as insulinnd insulin resistance increases, plasmaevels of nonesterified fatty acids and

fed a control diet. PLS-DA scatter plot of fetalm whose dams were fed a HFD, HFD-R, or C,o differences between groups based on mass

fed amser eruhs te t

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-hydroxybutrate decrease.26 However,

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n biopsy-proven NAFLD adult subjectsith confirmed peripheral insulin resis-

ance, plasma 3-hydroxybutrate levelsncrease in concert with hypertriglyceri-emia independent of body mass in-ex.27 On the basis of these observationsnd others, it has been proposed thatAFLD may result from increased deliv-

ry of circulating lipids to the liver sec-ndary to insulin functioning as an anti-

ipolytic agent in adipose tissue. Withinhe liver, nonesterified fatty acids arehen oxidized and reesterified and circu-ate as triglyceride lipoproteins. Becausehis drives a shift in energy metabolismoward fatty acid �-oxidation and keto-enesis, the liver retains excessive lipidsnd steatosis (NAFLD) ensues. Thesendings are consistent with our findings

n the fetal metabolomic profile, as weimilarly observe NAFLD with elevatedlasma 3-hydroxybutyrate both by vir-ue of maternal diet and phenotype (Ta-le 2). Taken together, we speculate thatur model serves as the first evidencehat in primate fetal life maternal obesitynd in utero HFD exposure associateith elevations in 3-hydroxybutyrate

hat may serve as a marker for a correla-ion between fetal lipid oxidation, hyper-riglyceridemia, and NAFLD. If this wereo hold true in human studies, this serum

arker could be used to screen for

TABLE 2Analysis of fetal metabolites contristratified by in utero diet exposure

Metabolite

Maternal HFD v

P value

2-hydroxybutyrate .003...................................................................................................................

Ascorbic acid .0069...................................................................................................................

�-tocopherol .0031...................................................................................................................

Cholesterol .015...................................................................................................................

3-hydroxybutyrate .0055...................................................................................................................

un(1462_100) .014...................................................................................................................

un(1574_304) .071...................................................................................................................

un(1952_334) .029...................................................................................................................

Con, control; DR, diet reversal; HFD, high-fat diet; PLS-DA,Significantly discrete serum metabolites by mass spectroscoControl (n � 7); HFD (n � 15); reversal (n � 6).

Cox. Altered fetal epigenome associated with in utero HFD

AFLD and disposition toward obesity. f

We similarly find the inverse correla-ion of cholesterol (elevated in obeseams but diminished in the fetus) to bef further interest. Consistent with an as-ociation of an altered fetal metabolomeith maternal HFD intake as well asbese phenotype, we again observed sig-ificant differences among offspring ofFD-R vs HFD-S dams (Figure 2 andable 3). We speculate that fetal hypo-holesterolemia relative to maternal hy-ercholesterolemia may result from 3ossible (and nonmutually exclusive)echanisms. First, it is possible that

here is altered deposition in the fetus.herefore, circulating fetal cholesterolould be less but deposition could be in-reased. Others have previously ob-erved that maternal hypercholesterol-mia is associated with accelerated fetallaque formation26 and we have ob-erved fetal NAFLD, both supportinguch a mechanism.19,20 Second, it is pos-ible that maternal hypercholesterol-mia alters placental lipid and choles-erol metabolism to decrease fetal deovo synthesis resulting in relatively de-reased circulating fetal levels. Our ob-ervations pertaining to significantlyecreased fetal cholesterol levels prefer-ntially in HFD-S dams (Table 3) sup-orts this notion. Finally, it is possiblehat the maternal HFD and obesity dif-

ting to the PLS-DA modeld maternal phenotypen Reversal vs con

ld changeFD/con) P value

Fold ch(DR/co

59 .00098 0.48.........................................................................................................................

11 .0078 0.38.........................................................................................................................

53 .029 0.71.........................................................................................................................

77 .093 0.83.........................................................................................................................

3 .0036 2.8.........................................................................................................................

41 .0053 0.63.........................................................................................................................

71 .012 0.59.........................................................................................................................

77 .011 0.67.........................................................................................................................

al least squares-discriminate analysis; un, unknown.e as listed and correspond to the mass spectra data as present

J Obstet Gynecol 2009.

erentially alter fetal de novo synthesis of i


oth lipid and oxidative stress pathways.uch a “developmental plasticity” modelains support from others observationsn animal models as well as human stud-es.24-28 Consistent with such a “develop-

ental plasticity” model, the observedbsence of ascorbic acid in both HF andiet reversal cohorts is indicative of per-istent oxidative stress (Table 2).

The strengths of our study our multi-old. We have used high throughput dis-overy “functional genomics” and char-cterized the metabolomic profile ofaternal and fetal serum under condi-

ions of a maternal HFD leading to obe-ity in nonhuman primates. Our modelystem has advantages over other animal

odels on the developmental origins ofuman disease in so much as we use pri-ates rather than phylogenetically dis-

imilar litter mate mammals. By stratifi-ation with respect to maternal dietensitivity and diet reversal we are able toistinguish effects of diet from the obeseaternal environment. Thus, our in-

ights are well characterized and repre-entative of human development.

There are inherent limitations to pri-ate research. First, the time and ex-

ense in establishing such a model sys-em prohibits large numbers of animalsnd may limit statistically significant dis-overy. Second, as with humans, the an-

Maternal HFD vs reversal

eP value

Fold change(HFD/DR)

.31 1.2..................................................................................................................

.66 0.29..................................................................................................................

.15 0.75..................................................................................................................

.51 0.93..................................................................................................................

.33 0.82..................................................................................................................

.38 1.3..................................................................................................................

.16 1.2..................................................................................................................

.39 1.1..................................................................................................................

Figure 2.

buan

s co

Fo(H

angn)

0.......... .........

0.......... .........

0.......... .........

0.......... .........

2.......... .........

0.......... .........

0.......... .........

0.......... .........

partipy ar ed in

mals behavioral and social hierarchy


WiC


2

FIGURE 3Identification of “un(922_165)” as 3-hydroxybutyrate

e found unknown (un) compound of 922_165 to be of particular interest. Subsequent analysis by mass spectrometry against known metabolic librariesdentified it as 3-hydroxybutyrate (�97% certainty).ox. Altered fetal epigenome associated with in utero HFD. Am J Obstet Gynecol 2009.

TABLE 3Fetal metabolites that differ between high-fat sensitive and resistant

HFD-S vs HFD-R HFD-S vs con HFD-R vs con

Metabolite P valueFold change(HFD-S/HFD-R) P value

Fold change(HFD-S/con) P value

Fold change(HFD-S/con)

Cholesterol .0099 0.73 .16 0.81 .45 0.89................................................................................................................................................................................................................................................................................................................................................................................

�-tocopherol .0030 0.53 .0038 0.53 .98 1.0................................................................................................................................................................................................................................................................................................................................................................................

Hypoxanthinea .025 1.6 .63 1.1 .016 1.4................................................................................................................................................................................................................................................................................................................................................................................

Myoinositola .040 1.9 .99 1 .042 1.9................................................................................................................................................................................................................................................................................................................................................................................

un(698_103) .0025 0.79 .15 .86 .44 1.1................................................................................................................................................................................................................................................................................................................................................................................

un(2298_144) .022 1.9 .98 0.99 .029 1.9................................................................................................................................................................................................................................................................................................................................................................................

Con, control, HFD-S, high-fat diet sensitive; HFD-R, high-fat diet resistant; un, unknown.HFD-S (n � 5); HFD-R (n � 3); Con (n � 6).Significantly discrete serum metabolites by mass spectroscopy are as listed and correspond to the mass spectra data as presented in Figure 2.a Metabolites found through the second round of gas chromatography-mass spectrometry.

Cox. Altered fetal epigenome associated with in utero HFD. Am J Obstet Gynecol 2009.

81.e8 American Journal of Obstetrics & Gynecology SEPTEMBER 2009

mmrtts

mtmmnonTHpsmcprm

R1c22l3ocI4nA5G6tt7SP

8Rmy39th21d1mnB1Sd1avn21Ppgpi21MdhonfmB1Uaa21Lnp1wi

HG1Stgm2ammd2rtssriC2JimJ2Eb72f12ls22Albn2Inm2aetp


ay confound our findings. Third,onitoring of metabolic parameters is

estrictive relative to other model sys-ems. Thus, we cannot provide a longi-udinal analysis as we cannot support aignificant cohort of fetuses over time.

The maternal and fetal serum metabolo-ic profiling presented herein gives fur-

her insight into our complex biologicodel. Moreover, because the fetaletabolome is proximal to the fetal phe-

otype our observations lend support tour prior findings on the effects of mater-al HFD exposure on the fetal epigenome.aken together, these data suggest thatFD exposure as well as maternal obese

henotype alters the fetal epigenome to re-ult in functional transcriptome and

etabolome variations. Further ongoingharacterization of our unique model willrovide insight and understanding of theole maternal diet and obesity play in fetaletabolism. f

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a maternal high-fat diet is accompanied by alterations in the fetal primate metabolome

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