Low Neonatal Plasma n-6/n-3 PUFA Ratios RegulateOffspring Adipogenic Potential and Condition AdultObesity ResistanceMichael C. Rudolph,1 Matthew R. Jackman,1 David M. Presby,1 Julie A. Houck,1 Patricia G. Webb,1

Ginger C. Johnson,1 Taylor K. Soderborg,2 Becky A. de la Houssaye,2 Ivana V. Yang,3 Jacob E. Friedman,1,2

and Paul S. MacLean1

Diabetes 2018;67:651–661 | https://doi.org/10.2337/db17-0890

Adipose tissue expansion progresses rapidly during post-natal life, influenced by both prenatal maternal factors andpostnatal developmental cues. The ratio of omega-6 (n-6)relative to n-3 polyunsaturated fatty acids (PUFAs) is be-lieved to regulate perinatal adipogenesis, but the cellularmechanisms and long-term effects are not well under-stood. We lowered the fetal and postnatal n-6/n-3 PUFAratio exposure in wild-type offspring under standard ma-ternal dietary fat amounts to test the effects of low n-6/n-3ratios on offspring adipogenesis and adipogenic potential.Relative to wild-type pups receiving high perinatal n-6/n-3ratios, subcutaneous adipose tissue in 14-day-old wild-type pups receiving low n-6/n-3 ratios had more adipocytesthat were smaller in size; decreased Pparg2, Fabp4, andPlin1; several lipid metabolism mRNAs; coincident hyper-methylation of the PPARg2 proximal promoter; and ele-vated circulating adiponectin. As adults, offspring thatreceived low perinatal n-6/n-3 ratios were diet-induced obe-sity (DIO) resistant and had a lower positive energy balanceand energy intake, greater lipid fuel preference and non–resting energy expenditure, one-half the body fat, and betterglucose clearance. Together, the findings support a modelin which low early-life n-6/n-3 ratios remodel adipose mor-phology to increase circulating adiponectin, resulting in apersistent adult phenotype with improved metabolic flexi-bility that prevents DIO.

The possibility that nutrient composition early in life haspermanent metabolic effects was recognized .40 years ago

(1), a phenomenon later called developmental origins ofmetabolic disease (2,3). Strong evidence indicates the exis-tence of long-term consequences of early-life nutrients onadult metabolic health, linking perinatal nutrients with pre-disposition for obesity (4–7). Obesity has doubled in chil-dren and quadrupled in adolescents over the past 30 years(8), coincident with a marked increase in maternal intake ofrefined vegetable oils containing high amounts of omega-6(n-6) polyunsaturated fatty acids (PUFAs) (4). Infant n-6PUFA exposures introduced through maternal diet duringthe perinatal window are believed to stimulate adipogenesisduring pre- and postnatal development on the basis of find-ings in rodent models (4,9–11). In the U.S., n-6 PUFAs inhuman milk have been threefold greater since the 1950s,whereas n-3 PUFAs have remained constant, thus increasingthe infant’s n-6/n-3 PUFA ratio exposure (9,10,12). Infantexposure to a high n-6/n-3 PUFA ratio during gestation andbreastfeeding has been associated with increased pediatricadiposity out to 3 years of age (13). We found that the higherthe n-6/n-3 PUFA ratio in human milk, the greater theinfant adipose deposition by 4 months of age independentof maternal prepregnancy BMI (14). Accordingly, perinatalexposure to a high n-6/n-3 PUFA ratio has been postulatedto contribute to the obesity epidemic in developed countries(4,10).

Obesity is defined by excessive adiposity, and regulationof adipose tissue expansion (ATE) occurs through prolifer-ation of adipocyte precursor (AP) cells to increase adipocytenumber (hyperplasia) and by adipocyte filling (hypertrophy)

1Division of Endocrinology, Metabolism and Diabetes, University of ColoradoSchool of Medicine, Aurora, CO2Department of Pediatrics, Section of Neonatology, University of Colorado Schoolof Medicine, Aurora, CO3Division of Biomedical Informatics and Personalized Medicine, University ofColorado School of Medicine, Aurora, CO

Corresponding author: Michael C. Rudolph, michael.rudolph@ucdenver.edu.

Received 31 July 2017 and accepted 6 November 2017.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0890/-/DC1.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

See accompanying article, p. 548.

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OBESITYSTUDIES

(15). In a rat model of maternal obesity and high-fat diet(HFD), cross-fostering techniques identified an offspring’searly-life exposure tomaternalHFD-inducedobesity as a crit-ical window that conditions adipogenic potential throughaltered DNA methylation patterning (5,11). In mice, mater-nal pre- and postnatal consumption of an HFD containingexcessive n-6 PUFAs increases the offspring’s n-6/n-3 PUFAratio exposure and stimulates ATE pathways, resultingin greater offspring adiposity (12,16–18). Moreover, a ma-ternal diet rich in n-6 relative to n-3 PUFAs conditionsadipogenic potential in offspring across generations (19),which is believed to occur by regulating expression of masteradipogenic transcription factors C/EBPa and peroxisomeproliferator–activated receptor-g (PPARg) (5,20,21). How-ever, identification of specific molecular targets of n-3PUFAs, independent of maternal obesity and HFD feeding,is less well characterized.

By using an established transgenic model that over-expresses an n-3 fatty acid desaturase (fat-1) in combinationwith maternal HFD-induced obesity, we reported that low-ering the endogenous maternal n-6/n-3 PUFA ratio reducesplacental inflammation and protects adult wild-type (WT)offspring against excessive body fat accumulation and insulinresistance (22). In the current study, we control for con-founding maternal variables (HFD and obesity) by compar-ing offspring born to WT (high n-6/n-3) and fat-1 (lown-6/n-3) mothers provided a standard amount of dietaryfat to study outcomes during early-life development inde-pendent of maternal obesity or HFD. We tested the hy-pothesis that lowering n-6/n-3 PUFA ratios in offspringcirculation during the preweaning window tempers adipo-genesis at the molecular and cellular levels. Of note, a lowearly-life PUFA ratio imparts long-term metabolic benefitto the adult offspring.

RESEARCH DESIGN AND METHODS

MiceAnimal procedures were approved by the institutional ani-mal care and use committee and housed at the Universityof Colorado Anschutz Campus vivarium. WT C57Bl/6J micewere purchased from The Jackson Laboratory (Bar Harbor,ME). Fat-1 mice were provided by J.X. Kang and genotypedaccording to his protocol (23). Heterozygous fat-1 femaleswere bred with WT males, generating 50% WT offspring aspreviously described (22). The study design (Fig. 1) used WT(high n-6/n-3 PUFA ratio) and fat-1 (low n-6/n-3 PUFAratio) mothers to test PUFA ratio exposures in offspringdevelopment. Female mice were bred at 10 weeks old. Damswere provided chow and underwent normal gestation andlactation. At postnatal day 14 (PND14), both dams and theirnative litters were assessed for body composition, and pupswere sacrificed for adipose morphology, cellularity, gene ex-pression, protein levels, and circulating hormone, glucose,and fatty acid composition. Only WT offspring were usedfor downstream assays, and litters were standardizedto six to eight pups per dam. In study 2, long-term effects

of early-life PUFA exposure were tested by provision of awestern-style diet (high fat, high sucrose [HF/HS]) (TekladTD.88137; Envigo RMS, Indianapolis, IN). WT offspringwith high or low n-6/n-3 PUFA ratio exposure were weanedto chow until 17 weeks old, when adult offspring wereassessed by indirect calorimetry during the lead-in pe-riod and challenge with HF/HS diet for 1 week. Animalswere then maintained on an HF/HS diet for 3 more weeks(Fig. 1).

Adipocyte CellularityContralateral subcutaneous fat pads from one pup werecombined and processed for cellularity as described previously(24). Briefly, adipose tissue was enzymatically digested, iso-lated adipocytes were stained with 0.2% methylene bluefor cell integrity, and samples were imaged by using a0.01-mm stage micrometer. Eight to 12 fields of each sam-ple were quantified by using AdCount software (Mayo Clinic,Rochester, MN) to obtain diameters and cell size frequencydistributions.

Gas Chromatography–Mass SpectrometryTotal lipids were extracted and fatty acid profiles quantifiedby gas chromatography–mass spectrometry as previouslydescribed (14,25). Data are expressed in micromoles of fattyacid per milliliter of milk or milligram of total plasma protein.The total n-6/n-3 PUFA ratio is the sum of n-6 divided by thesum of n-3 PUFAs; the arachidonic acid/docosahexaenoicacid + eicosapentaenoic acid (AA/DHA + EPA) ratio isthe micromoles of 20:4n-6 divided by 22:6n-3 + 20:5n-3;the linoleic/a-linoleic (LA/LNA ratio) is the micromoles of18:2n-6 (LA) divided by 18:3n-3 (LNA).

ELISAsPND14 pups were anesthetized by isofluorane and de-capitated, and trunk blood was collected in K2EDTA tubes(BD Microtainer, Franklin Lakes, NJ). Samples were storedon wet ice for 30 min and then centrifuged at 2,000g for20 min at 4°C. Insulin (#90080; Crystal Chem, DownersGrove, IL), leptin (#22-LEPMS-E01; ALPCO, Salem, NH),and adiponectin (#47-ADPMS-E01; ALPCO) were processedaccording to manufacturer protocols.

Oral Glucose Tolerance Tests and Blood GlucoseMeasurementsBlood glucose was measured in fasted adults (4 h) by using aContour blood glucose meter (Ascensia Diabetes Care, Parsippany,NJ) after oral gavage of 50% dextrose (Vet One, Boise, ID)as previously described (26). Pup blood glucose was measuredfrom trunk blood immediately after decapitation.

Global and Targeted Gene ExpressionTotal RNA was isolated by using the RNAqueous-MicroTotal RNA Isolation Kit (Thermo Fisher Scientific, Waltham,MA), including DNAse digestion, according to the manufac-turer’s protocol. Two hundred fifty nanograms of total RNAwas used in the Whole-Transcript Expression RNA kit (LifeTechnologies, Carlsbad, CA), and samples were hybridizedto mouse 1.1 ST Gene Arrays (Affymetrix, Santa Clara, CA),washed, stained, and imaged with the Affymetrix GeneAtlas

652 Enriched n-3 PUFAs Protect Against Adult Obesity Diabetes Volume 67, April 2018

Personal Microarray System. Raw data were GC-RMA (ro-bust multiarray averaging) normalized, log2 transformed,and analyzed by using the Partek Genomic Suite as pre-viously described (27). Quantitative RT-PCR was performedby using mRNA copy number and normalized to PolR2bcopies with TaqMan gene expression assays (ThermoFisher Scientific) as previously described (28).

Semiquantitative Protein AnalysisSubcutaneous white adipose tissue (sWAT) was homogenizedin 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 2 mmol/LEDTA, 50 mmol/L NaF, 1% Triton X-100, 1% sodiumdeoxycholate, 0.1% SDS, and 5 mmol/L sodium vanadatesupplemented with Halt Protease and Phosphatase Inhibi-tor cocktail (#1861281; Thermo Fisher Scientific). Totalprotein concentration was determined by bicinchoninic acidanalysis (Pierce BCA Protein Assay; Thermo Fisher Scientific,Rockford, IL). Immunoblots were run on the Western capillaryelectrophoresis system, and data were analyzed by Compasssoftware (ProteinSimple, San Jose, CA) as in Checkley et al.(29). Primary antibodies included PPARg (#2435S; Cell Sig-naling, Danvers, CT), fatty acid synthase (FASN) (#3180S;Cell Signaling), and perilipin 1 (PLIN1) (#70R-1297; FitzgeraldIndustries International, Acton, MA) and were normalized tocyclophilin A (CypA) (#2175; Cell Signaling).

PPARg2 DNA Methylation Analysis by PyrosequencingTotal DNA was isolated by using the AllPrep DNA/RNAMini Kit (QIAGEN, Germantown, MD) according to themanufacturer’s protocol, and DNA was quantified by using

the Qubit Fluorometric Quantitation assay (Thermo FisherScientific). Bisulfite-converted DNA was PCR amplified byusing primers designed in the PyroMark Assay Design Soft-ware (QIAGEN) as in Yang et al. (30). Pyrosequencing wasperformed on the PyroMark Q96 MD (QIAGEN), with re-agents and protocols supplied by the manufacturer. Eachplate contained 0%, 50%, and 100% methylated controls.Percent methylation was calculated from the peak heightsof C and T by Pyro Q-CpG software (QIAGEN) in duplicatefor each sample, and averaged values were used in thestatistical analysis (30).

Body Composition and Indirect CalorimetryWhole-body composition was quantified by magnetic reso-nance (EchoMRI; Echo Medical Systems, Houston, TX) onPND14 for dams and litters or for adult offspring before,during, and after calorimetry. Energy balance of adults wasconducted at 17 weeks old as previously described (24,31).Mice were housed individually at 27°C in an eight-chambersystem (Oxymax Comprehensive Lab Animal MonitoringSystem; Columbus Instruments, Columbus, OH) with a 14-hlight, 10-h dark cycle. Mice were acclimated for 1 week onchow and then challenged with an HF/HS diet for 5 days.Food and water intake were measured during the entire timewhile in calorimetry. VO2, VCO2, respiratory quotient, activ-ity measurements, and energy expenditure were calculatedas previously described (31). After calorimetry, mice werereturned to individual housing for an additional 2 weeksand then sacrificed, and samples were collected.

Figure 1—Experimental study designs for perinatal programming and adult obesogenic diet. A: Study 1 was of WT (high n-6/n-3 PUFA ratio) or n-3fat-1 (low n-6/n-3 PUFA ratio) mothers maintained on chow and bred at 10 weeks old. Dams had normal gestation and reared their biologicallitters during lactation, and litters were standardized with six to eight pups per dam. Pups and mothers were assessed for body compositionbefore sacrifice at PND14. Offspring outcomes were adipose morphology and cellularity, adipose gene and protein expression, and circulatinghormone, glucose, and fatty acid composition. B: In study 2, litters born to and reared by WT or fat-1 dams (high and low n-6/n-3 ratioexposures, respectively) were weaned and maintained on chow until 17 weeks old. At 17 weeks, adults were assessed for body composition,placed into the indirect calorimeter for 1 week, assessed for body composition and switched to an HFD for 1 week, and assessed for bodycomposition at week 19. Adults were maintained on an HFD for an additional 3 weeks, when a final body composition and oral glucose tolerancetest (OGTT) were taken before sacrifice (Sac).

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StatisticsPND14 WT offspring from litters of four to five indepen-dent dams per genotype were used in analyses. One-way ortwo-way ANOVA followed by Bonferroni multiple compar-isons test was performed in GraphPad Prism version 7.0afor Macintosh software (GraphPad Software, La Jolla, CA).For microarray analysis, one-way ANOVA with a Bonferronifalse discovery rate of 0.1 was performed by using PartekGenomics Suite version 6.6 software (Partek, St. Louis, MO)followed by Bonferroni multiple comparisons test.

RESULTS

Offspring Plasma Has Decreased n-6 and Enriched n-3PUFAs Independent of Maternal ObesityWhen provided a low-fat diet, maternal fat-1 transgeneexpression did not influence body composition of eitherdams or their biological litters, and differences were notobserved in the macronutrient composition of the milk(data not shown). Maternal fat-1 expression reduced the

n-6/n-3 long-chain (LC) PUFA and AA/DHA + EPA ratiosin milk (Fig. 2A) without affecting quantitative amounts (inmicromoles per milliliter) of saturated fatty acids (SFAs),monounsaturated fatty acids (MUFAs), PUFAs, or the ratioof essential dietary fatty acids (LA [18:2n-6]/LNA [18:3n-3]). The plasma of PND14 pups qualitatively mimicked thecomposition of the milk with decreased LC-PUFA and AA/DHA + EPA ratios (Fig. 2B). Despite maternal provision ofthe same diet, WT offspring exposed to fat-1 mother’s milkhad significantly reduced plasma amounts (in micromoles permilligrams of protein) of 20:2n-6 and 20:4n-6 (P # 0.01),with enriched amounts of 22:6n-3 (P = 0.038) (Fig. 2B).

Low n-6/n-3 PUFA Ratios Increase Pup AdipocyteCellularity and Increase Systemic AdiponectinIn PND14 pups exposed to low n-6/n-3 PUFA ratios, adi-pocyte cellularity from digested sWAT revealed small-sizedadipocytes (10–20 mmol/L) had a significantly greaterpercentage of total adipocytes, whereas the percentage ofmedium-sized adipocytes (30–60 mmol/L) was significantly

Figure 2—Offspring reared by fat-1 dams have lower n-6 PUFAs and greater n-3 PUFAs in circulation. A: Milk fatty acid composition quantified bylipid mass spectrometry indicates that fat-1 dams synthesize milk containing significantly lower n-6/n-3 ratios for both LC-PUFA and AA/DHA +EPA (P, 0.001) without significantly changing amounts of essential dietary fatty acids (LA/LNA) or levels of de novo fatty acids, SFAs, MUFAs, orPUFAs (n = 5 dams/genotype). B: Offspring serum lipid composition quantified by lipid mass spectrometry mirrored the milk composition with sig-nificantly lower LC-PUFA and AA/DHA + EPA ratios (P, 0.001) and identified significantly reduced n-6 PUFAs (20:2 and 20:4;P, 0.01) with significantlyenriched n-3 PUFAs (22:6; P = 0.038) (n = 5 pups/condition from five independent dams). *P, 0.05; **P, 0.01; ***P, 0.001. n/s, not significant.

654 Enriched n-3 PUFAs Protect Against Adult Obesity Diabetes Volume 67, April 2018

Figure 3—Reduced n-6/n-3 PUFA ratio exposure leads to smaller and more numerous subcutaneous adipocytes and higher systemic adipo-nectin. A: Quantification of enzymatically digested sWAT from PND14 offspring. Exposure to a low n-6/n-3 PUFA ratio results in an adipocytecellularity profile with a greater percentage of small-sized and fewer large-sized adipocytes (n = 29 WT pups from five WT dams; n = 18WT pupsfrom five fat-1 dams). B: Total adipocyte number per gram of sWAT and overall sWAT weight were both greater in low n-6/n-3 PUFA ratiooffspring (P = 0.01 and 0.005, respectively; same number of pups and dams as in A). C: Systemic adiponectin, specifically the low-molecular-weight (LMW) isoforms, was significantly increased in response to a low n-6/n-3 PUFA ratio in PND14 pups (P , 0.001), whereas systemicglucose, insulin, and leptin levels were equivalent (n = 8 WT pups from four WT dams; n = 8 WT pups from five fat-1 dams). *P , 0.05. HMW,high molecular weight.

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Figure 4—PPARg2 proximal promoter is hypermethylated in sWAT under low n-6/n-3 PUFA ratios. A: Heat map of significant differentiallyexpressed genes by high and low n-6/n-3 PUFA ratio exposure in PND14 pup sWAT for lipid metabolism, signal transduction, and adipocyte-specific categories (P, 0.05 by using one-way ANOVA with a Bonferroni false discovery rate of 0.1; n = 4 pups/condition from four independentdams). B: Quantitative levels of PPARg2 mRNA and downstream target genes by quantitative RT-PCR by using a different subset of offspring

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lower (Fig. 3A). Exposure to low n-6/n-3 PUFA ratios yielded1.48-fold more total adipocytes per gram of sWAT (P =0.01), and sWAT depots were 1.33-fold heavier (Fig. 3B). ByPND14, total adiponectin was significantly greater in thelow n-6/n-3 PUFA ratio pups (P , 0.001) (Fig. 3C), drivenby the low- (trimer) and medium- (hexamer) molecular-weight forms (total/high-molecular-weight adiponectinratio P , 0.001). Other systemic factors, including bloodglucose, insulin, and leptin, were unchanged (Fig. 3C). To-gether, these data indicate that low n-6/n-3 PUFA ratioperinatal exposures establish an early adipose cellularityassociated with increased insulin sensitivity, producingmore adiponectin.

Adipogenic and Lipid Metabolism Gene ExpressionIs SuppressedMicroarray gene expression identified multiple differentiallyexpressed pathways in sWAT in PND14 pups with lowplasma n-6/n-3 PUFA ratios (Fig. 4A). Several lipid metab-olism genes were downregulated in pups with low plasman-6/n-3 ratios, including actyl-CoA carboxylase, ATP citratelyase, fatty acid transporter (CD36), elongase 5, fatty aciddesaturase 1, FASN, lipoprotein lipase (LPL), malic enzyme,prostaglandin E synthase, prostaglandin reductase, stearoyl-CoA desaturase 2, and SREBP1. Conversely, acyl-CoA thioes-terase 12, acyl-CoA synthetase medium-chain family member4, fatty acid desaturase 6, lipoyltransferase 1, phospholipaseA2, and retinol dehydrogenase 7 were upregulated in the lowplasma n-6/n-3 ratio offspring. Signal transduction geneswere downregulated in the low plasma n-6/n-3 ratio pups,including the adiponectin receptor 2, frizzled family mem-bers (Fzd-1, -5, -7), leptin receptor, LDL receptor–relatedprotein 8 and 10, lysophosphatidic acid receptor 4, prosta-glandin F2-a receptor, tumor necrosis factor (TNF) receptorsuperfamily member 11b, TNF receptor–associated factor 7,and TNF receptor–associated protein 1, as well as secretedligands angiopoietin-related protein 1, C-C motif chemo-kines (2, 27a, 27b, and 7), and Wnt10a. Conversely, IGF-Iand Wnt7b were both induced in low plasma n-6/n-3 PUFApups (Fig. 4A). Regulatory and adipogenic markers de-creased by low n-6/n-3 PUFA ratios were PPARg, PPARgcoactivator 1 and estrogen-related receptor–induced regula-tor in muscle protein 1 (Perm1), Berardinelli-Seip congeni-tal lipodystrophy gene (Bscl2, Seipin), fatty acid bindingprotein 4 (Fabp4), and Plin1. Significantly regulated genesare presented in Supplementary Table 1. Together, expres-sion data suggest that low plasma n-6/n-3 ratio conditionsduring offspring adipogenesis exerts broad control overlipid uptake and metabolism, surface receptors, chemokinelevels, protein hormones, and adipocyte regulatory factors.

Low n-6/n-3 PUFA Ratios Increase DNA Methylationof PPARg2 in sWATPrincipal adipogenic transcription factors and downstreamtarget levels were verified by quantitative RT-PCR by usingan independent set of PND14 offspring (Fig. 4B). Pparg2was significantly decreased (P = 0.024) in sWAT of lown-6/n-3 PUFA ratio offspring, but mRNA level of C/ebpawas unchanged. In addition, Pparg2 transcriptional targetswere either significantly decreased (Fabp4, Fasn, Plin1, andAdipoQ) (P # 0.04) or tended to be decreased (Cidea, Lpl,and leptin) (P # 0.075) (Fig. 4B). The mRNA level for theBscl2 gene, which regulates lipid droplet formation and sizein adipocytes (32,33), was significantly decreased by lown-6/n-3 PUFA ratio exposure (Fig. 4B). Reduction ofPPARg2 mRNA was consistent with its decreased proteinlevels (P = 0.007) and for PPARg2 targets FASN and PLIN1(P = 0.04 and 0.01, respectively) (Fig. 4C). Consistent withthe decreased mRNA and protein levels, DNA methylationof the PPARg2 proximal promoter at 23,195 and 2322upstream of the transcriptional start site was increased by6.4% and 4.5%, respectively (P , 0.01) (Fig. 4D).

Adult Diet-Induced Obesity Resistance Is Conditionedby Early-Life Low n-6/n-3 PUFA RatiosThe long-term effects of early-life PUFA exposures weretested when adult offspring were challenged with an HF/HSdiet. Between perinatal exposure groups, body fat percentage,lean mass, and body weight of adult offspring at 17, 18, and19 weeks were not significantly different (Fig. 5A); however,the low n-6/n-3 PUFA ratio group had one-half the body fatrelative to the high n-6/n-3 PUFA ratio group by week 22.Of note, the lean body mass was not affected after 4 weeksof HF/HS diet, so the significantly increased body weight(P # 0.001) was due to greater adiposity. Metabolic phe-notyping revealed a lower energy balance by21.04 kcal/dayduring the lead-in period (P = 0.001) in adults from the lowplasma n-6/n-3 PUFA perinatal exposure group, and thesemice exhibited an attenuation of the positive energy imbal-ance in response to the HF/HS diet (Fig. 5B). Energy intakewas significantly decreased in the low perinatal n-6/n-3ratio group during the lead-in period (P = 0.046) as wellas at days 2 (P = 0.003) and 5 (P = 0.04) of the HF/HS diet.Although the total energy expenditure was significantlygreater in the lead-in period (P = 0.006), it did not differsignificantly during the HF/HS diet challenge. The non–resting energy expenditure was significantly increased dur-ing the lead-in period and on day 3 of the HF/HS dietrelative to the high perinatal n-6/n-3 ratio group (P ,0.05). The respiratory exchange ratio was significantly lowerin the low n-6/n-3 ratio group during the lead-in period

than for the microarray analysis, indicating significant decreases and decreasing trends as a result of low n-6/n-3 PUFA ratio exposure in sWATof PND14 offspring (P , 0.05, n = 6 pups/condition from five independent dams). C: Significant reductions in protein levels of PPARg2, FASN,and PLIN1 in the sWAT of PND14 offspring relative to CypA loading control (n = 6 pups/condition from five independent dams). D: The proximalpromoter of PPARg2 is hypermethylated at positions 23,195 and 2322 upstream from the transcriptional start site in sWAT of PND14 pupsexposed to a low n-6/n-3 PUFA ratio (n = 8 pups/condition from four independent dams). *P , 0.01. DMR, differentially methylated region; Kb,kilobase.

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Figure 5—Adult DIO is programmed by early-life n-6/n-3 PUFA ratio exposures. A: Body composition of adult animals exposed to high or lown-6/n-3 PUFA ratios during the perinatal window. Both groups initially underwent significantly increased adipose deposition in response to theHF/HS diet between weeks 18 and 19 (P , 0.05; n = 8/condition). The low n-6/n-3 PUFA exposure group was resistant to additional adiposedeposition during HF/HS diet maintenance (weeks 19–21), whereas the high n-6/n-3 PUFA exposure group continued to accumulate adiposetissue as reflected by the total mass increase by 21 weeks. B: Indirect calorimetry of adult offspring exposed to high or low n-6/n-3 PUFA ratios

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(P = 0.002) as well as at days 1 (P = 0.05) and 3 (P = 0.02).After 4 weeks of HF/HS diet feeding, adult offspring in the lowperinatal n-6/n-3 ratio group had significantly lower fastingglucose levels and reduced response during an oral glucosetolerance test (area under the curve P = 0.003) (Fig. 5C)indicative of a healthier metabolic phenotype.

DISCUSSION

The novel observation of this study is that enriching n-3and simultaneously reducing n-6 LC-PUFA exposure duringperinatal development establishes epigenetic, gene expres-sion, and morphological changes in adipose tissue as well asincreases circulating adiponectin in 14-day-old offspring. Ofnote, these early-life responses to the low n-6/n-3 PUFAratio were independent of confounding factors potentiallyintroduced by maternal obesity or the mother’s high-fatfeeding. Consequently, the low plasma n-6/n-3 ratio andhigh circulating adiponectin levels early in life, without sig-nificant differences in blood insulin, glucose, or leptin levels,set up a persistent metabolic phenotype that amelioratesthe predisposition to develop obesity later in life. Early-lifeexposure to a low perinatal n-6/n-3 PUFA ratio resulted inan adipose phenotype characterized by a greater numberof total adipocytes that were smaller in size, a broad-basedreduction in adipogenic gene expression, and hypermethy-lation of two regions in the proximal promoter of PPARg2.Cumulatively, the current observations indicate that perina-tal n-6/n-3 exposure affects fundamental molecular andcellular characteristics of sWAT in mice, and these effectson adipose tissue development may have functional conse-quences for metabolic disease predisposition later in life.

Emerging evidence links perinatal fatty acid exposurewith DNA methylation changes in liver, skeletal muscle, andadipose tissue (11,34–38). Consistent with these reports,the current findings of increased PPARg2 proximal pro-moter DNA methylation, in combination with suppressedlipogenic and adipocyte markers, support the hypothesisthat low n-6/n-3 PUFA ratios modulate perinatal adipogen-esis epigenetically independent of maternal obesity. Othersfound that MUFA (trans 18:1n-9) provided in the maternaldiet is associated with DNA hypermethylation in offspringadipose tissue, although the exact genes were not identified(39), and that in vitro treatment of cells with n-6 PUFAcauses dose-dependent DNA methylation patterns consistentwith those observed in obese individuals and those with diabetes(40). In an elegant maternal DIO study in rats, Borengasseret al. (5) identified comprehensive DNA methylationmodifications in adipogenic genes present in the whiteadipose tissue of cross-fostered offspring, which enhancedin vitro differentiation of AP cells present in the stromal

vascular fraction. Together, enriching n-3 while decreasingn-6 LC-PUFA throughout gestation and lactation couldregulate a common genetic program in AP cells to establishimportant epigenetic modifications. Future investiga-tions should determine how DNA methylation, particularlyin AP cells, is changed during the reduction of n-6 PUFAs (20:2and 20:4) or enrichment of n-3 PUFAs (22:6) or whether thebalance of n-6/n-3 PUFAs is most critical in epigenetic controlof ATE potential.

That ATE occurs both by increasing total adipocytenumber (proliferation) and increasing mature adipocyte size(hypertrophy) is well established (15,41). In Zucker rats,LC-MUFA and LC-PUFA favor adipose hyperplasia, whereasLC-SFA favors adipose hypertrophy, indicating differentsaturation affects of adipocyte cellularity in vivo (42). InPND14 pup plasma, we observed no significant differencein absolute amounts of SFAs, MUFAs, or PUFAs (data notshown); however, offspring with a low plasma n-6/n-3 PUFAratio had more small adipocytes in their sWAT, a morphologyconsistent with the regulation of AP cell proliferation and/ordifferentiation (43). We identified significantly increased20:2n-6 and 20:4n-6 in PND14 high n-6/n-3 PUFA ratioplasma. Arachidonic acid (20:4n-6) and its downstream bio-synthetic product prostacyclin promote preadipocyte pro-liferation by stimulating G-coupled protein receptor PTGIRsignaling in a paracrine loop and by driving differentiationby activating PPARs (9,12,16). Conversely, decreasing en-dogenous n-6/n-3 PUFA ratios by transient fat-1 expressionin 3T3-L1 cells inhibits preadipocyte proliferation, suppressesPPARg and FABP4, and inhibits lipid accumulation (44)consistent with what we observed in vivo. In the mouse,HFD initiates extensive in vivo proliferation of CD24+ ad-ipocyte progenitors, which then lose CD24 expression andare rapidly committed to differentiate into lipid-laden ma-ture adipocytes (15,43). With respect to early-life adipogen-esis, the current findings support that low perinatal n-6/n-3PUFA ratios might act 1) on the CD24+ AP cells to regulateproliferation and adipocyte number or 2) on the CD242

preadipocytes to regulate differentiation genes. Conse-quently, the early-life n-6/n-3 ratio may regulate AP cells,establishing a sWAT morphology capable of synthesizinggreater adiponectin and ultimately influencing adult appe-tite regulation and metabolic fuel preference during an obe-sogenic diet.

The reduction of early-life n-6/n-3 PUFA ratio exposurethrough maternal fat-1 expression appears to have persis-tent, functional consequences because these adult offspringhave proven to be less susceptible to the developmentof obesity in response to HF/HS feeding. The attenuatedintake, enhanced metabolic requirements, and lower

during the perinatal window. The low n-6/n-3 exposure group had a lower overall energy balance, with reduced energy intake on days 2 and5 after challenge with the HF/HS diet. For the low n-6/n-3 exposure group, the respiratory exchange ratio was significantly lower during thelead-in period and days 1–3 of the HF/HS diet, total energy expenditure was significantly greater during the lead-in period, and non–restingenergy expenditure was significantly greater during the lead-in period and on day 3 of the HF/HS challenge. C: Oral glucosetolerance was improved in adult offspring in the low n-6/n-3 exposure group relative to the high n-6/n-3 exposure group (area under the curveP , 0.05). *P , 0.05; **P , 0.01; ***P , 0.001. Acc, acclimation.

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respiratory exchange ratio in the perinatal low n-6/n-3 ratiooffspring generally is consistent with the diet-resistant phe-notype, which remains lean in response to this obesogenicdiet challenge (45). Diet-resistant rodents are able to sensenutrient excess, tend to burn more dietary fat, and respondby adjusting food intake and increasing energy expenditureaccordingly. Although speculating that the altered predispo-sition to develop obesity in the face of this HF/HS challengeresults from the molecular and cellular characteristics ob-served in adipose tissue is tempting, other key tissues in-volved in energy homeostasis (hypothalamus, muscle, liver,etc.) likely are affected in a concordant manner to affectappetite, satiety, nutrient trafficking, and/or metabolic reg-ulation. The combined milieu of PUFA and high adiponectincirculation in the low n-6/n-3 ratio pups could mediate pro-gramming of peripheral tissues. For example, no changes inbeige or brown adipocyte regulators (Ucp1, Prdm16, orZfp423 [46]) were observed in the sWAT of pups, so skeletalmuscle or alternative adipose depots could have increasedlipid oxidation to account for the elevated energy expendi-ture in adults. In addition, reduction of the n-6/n-3 ratiohas been shown to enhance beneficial gut bacteria and re-duce low-grade inflammation and metabolic disease in adultmice (47), providing another possible long-term effectorthat could be manipulated early in life by low n-6/n-3 ratioexposure. Together, metabolic phenotyping data suggestthat an early-life low n-6/n-3 ratio programs adult diet re-sistance by suppressing calorie intake and potentiallydiverting fat from storage to oxidative pathways, therebypreventing excessive adipose accumulation. In any case, morestudies are needed to place the contribution of early-life ad-ipose tissue programming into the context of the numerousother critical nodes of homeostatic regulation, which collec-tively explain the phenotype we observed in adult offspring.

This mouse model is potentially limited by the timing ofadipogenesis in rodents versus humans. In human infants,adipose tissue develops progressively by increasing bothadipocyte size and number until gestational week 23, whenin the weeks following, adipose growth is almost exclusivelyaccomplished through hypertrophy (48). Mice develop adi-pose tissue postnatally (49), but despite any temporal dif-ferences between infants and mouse pups, the mechanismsunderlying adipogenesis are believed to be equivalent(15,49). Moreover, human offspring are likely not to beweaned onto a singular dietary source as in our controlledmodel where weanlings were maintained on chow untilchallenge with an HF/HS diet. Rather, human childrenlikely have access to potentially obesogenic foods earlierthan in adulthood. Evaluation of an obesogenic diet pro-vided at weaning and during adolescence in the mouse isthe focus of future studies.

Our observations implicate enriched n-3 and reduced n-6PUFAs in the control of a genetic program that regulatesneonatal ATE in mice. With regard to the human mother/infant dyad, these findings strengthen support for enhancedmaternal dietary intake of n-3 PUFAs in combination with amore measured intake of n-6 PUFAs to lower the perinatal

n-6/n-3 PUFA ratio. The possibility that maternal-suppliedPUFAs conditions early-life adipogenic potential and appe-tite regulation in offspring, protecting against childhoodobesity, opens an opportunity for early intervention toreduce the incidence of obesity in future generations.

Acknowledgments. Instruments were provided by the Mass SpectrometryLipidomics Core Facility of the University of Colorado School of Medicine.Funding. M.C.R. is supported by National Institute of Diabetes and Digestive andKidney Diseases (NIDDK) grant K01-DK-109079, an Office of Research on Women’sHealth Building Interdisciplinary Research Careers in Women’s Health Scholar Award(K12-HD-057022), and a Eunice Kennedy Shriver National Institute of Child Healthand Human Development (NICHD) training grant (T32-HD-007186). M.C.R. and J.E.F.are supported by the Colorado NIDDK-Nutrition and Obesity Research Center(P30-DK-048520). J.E.F. is supported by NIDDK grant R24-DK-090964. P.S.M.is supported by NICHD grants P01-HD-038129 and R01-HD-075285.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. M.C.R. designed the experiments, generated andanalyzed the data, and wrote the manuscript. M.R.J., D.M.P., J.A.H., P.G.W., G.C.J., T.K.S.,and B.A.d.l.H. generated the data and reviewed and edited the manuscript. I.V.Y.assisted with DNA methylation/gene expression studies and reviewed and editedthe manuscript. J.E.F. and P.S.M. designed the experiments, contributed to thediscussion, and reviewed and edited the manuscript. M.C.R., J.E.F., and P.S.M.are the guarantors of this work and, as such, had full access to all the data in thestudy and take responsibility for the integrity of the data and the accuracy of thedata analysis.

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