The FASEB Journal • Research Communication

Maternal choline supplementation programs greateractivity of the phosphatidylethanolamineN-methyltransferase (PEMT) pathway in adultTs65Dn trisomic mice

Jian Yan,* Stephen D. Ginsberg,†,‡,§ Brian Powers,* Melissa J. Alldred,†,‡

Arthur Saltzman,† Barbara J. Strupp,* and Marie A. Caudill*,1

*Division of Nutritional Sciences, Cornell University, Ithaca, New York, USA; †Center for DementiaResearch, Nathan Kline Institute, Orangeburg, New York, USA; and ‡Department of Psychiatry and§Department of Physiology and Neuroscience, New York University Langone Medical Center, NewYork, New York, USA

ABSTRACT Maternal choline supplementation (MCS)induces lifelong cognitive benefits in the Ts65Dn mouse,a trisomic mouse model of Down syndrome and Alzhei-mer’s disease. To gain insight into the mechanisms under-lying these beneficial effects, we conducted a study to testthe hypothesis that MCS alters choline metabolism inadult Ts65Dn offspring. Deuterium-labeled methyl-d9-choline was administered to adult Ts65Dn and disomic(2N) female littermates born to choline-unsupplementedor choline-supplemented Ts65Dn dams. Enrichment ofd9-choline metabolites (derived from intact choline) andd3 � d6-choline metabolites [produced when choline-derived methyl groups are used by phosphatidylethano-lamine N-methyltransferase (PEMT)] was measured inharvested tissues. Adult offspring (both Ts65Dn and 2N)of choline-supplemented (vs. choline-unsupplemented)dams exhibited 60% greater (P<0.007) activity of hepaticPEMT, which functions in de novo choline synthesis andproduces phosphatidylcholine (PC) enriched in docosa-hexaenoic acid. Higher (P<0.001) enrichment of PEMT-derived d3 and d6 metabolites was detected in liver,plasma, and brain in both genotypes but to a greaterextent in the Ts65Dn adult offspring. MCS also yieldedhigher (P<0.05) d9 metabolite enrichments in liver,plasma, and brain. These data demonstrate that MCSexerts lasting effects on offspring choline metabolism,including up-regulation of the hepatic PEMT pathway andenhanced provision of choline and PEMT-PC to thebrain.—Yan, J., Ginsberg, S. D., Powers, B., Alldred, M. J.,

Saltzman, A., Strupp, B. J., Caudill, M. A. Maternalcholine supplementation programs greater activity of thephosphatidylethanolamine N-methyltransferase (PEMT)pathway in adult Ts65Dn trisomic mice. FASEB J. 28,4312–4323 (2014). www.fasebj.org

Key Words: Alzheimer’s disease � docosahexaenoic acid � Downsyndrome � fetal programming � phosphatidylcholine

Down syndrome (DS) is the most common geneticcause of intellectual disability and is often accompaniedby behavioral disorders and attention deficits (1). Af-fecting �1 of 690 live births in the United States (2), DSis caused by the triplication of human chromosome 21(HSA21), which arises from nondisjunction duringmeiosis (3). In addition to intellectual disability, indi-viduals with DS develop Alzheimer’s disease (AD) pa-thology, including amyloid plaques and neurofibrillarytangles, usually by the fourth decade of life (4–7).Because no specific treatment is available for the intel-lectual disability or dementia in individuals with DS (8),prognosis is generally poor.

The Ts65Dn mouse model has been developed toreplicate the human DS condition via triplication ofthe distal portion of murine chromosome 16(MMU16) orthologous to HSA21 (9). Ts65Dn micemanifest many of the biochemical, neuronal, andcognitive abnormalities observed in humans with DS(10 –14). Specifically, these animals exhibit degener-ation of the basal forebrain cholinergic neurons(BFCNs), with significant age-related reductions intotal BFCN numbers and a reduction in cholineacetyltransferase activity in the neocortex and hip-

1 Correspondence: Division of Nutritional Sciences, Cor-nell University, 228 Savage Hall, Ithaca, NY 14853, USA.E-mail: mac379@cornell.edu

doi: 10.1096/fj.14-251736This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

Abbreviations: AD, Alzheimer’s disease; BFCNs, basal fore-brain cholinergic neurons; CDP-choline, cytidine diphos-phate-choline; DS, Down syndrome; FDR, false discovery rate;GPC, glycerophosphocholine; HSA21, human chromosome21; LC-MS/MS, liquid chromatography-tandem mass spec-trometry; LPC, lysophosphatidylcholine; MCS, maternal cho-line supplementation; MMU16, murine chromosome 16; PC,phosphatidylcholine; PCho, phosphocholine; PE, phosphati-dylethanolamine; PEMT, phosphatidylethanolamine N-meth-yltransferase; SAM, S-adenosylmethionine; SM, sphingomy-elin

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pocampus (11, 15–20), suggesting that some of thecognitive deficits observed in DS arise from impair-ments in cholinergic functioning.

Using the Ts65Dn mouse model of DS and AD,Strupp and colleagues (21, 22) have shown that supple-menting the mother’s diet with additional cholineduring pregnancy and lactation substantially lessens theneurocognitive dysfunction seen in this genetic disor-der. Adult trisomic offspring born to mothers whoconsumed extra choline during the perinatal periodperformed significantly better on tasks assessing atten-tion, spatial cognition, or emotional regulation com-pared to unsupplemented Ts65Dn mice and on sometests performed similarly to normal disomic (2N) litter-mate controls (21, 22). Comparable cognitive benefitshave been reported in normal animals (23–27) and inanimal models of seizure disorders (28, 29) and prena-tal alcohol exposure (30, 31).

Although much remains to be learned about themechanisms by which maternal choline supplementa-tion (MCS) produces these beneficial effects in this DSmouse model, it is very likely that the basic mechanismsare the same as those that provide cognitive benefit innormal animals and other disease models. In normalrodents, pre- and/or early postnatal MCS has beenshown to produce lasting changes in the cholinergicsignaling system, hippocampal neurogenesis, and hip-pocampal long-term potentiation, all of which maycontribute to the observed improvements in spatialmemory and attention seen in these animals (reviewedin ref. 32). Similarly, studies with the Ts65Dn mouse

model of DS have shown that MCS significantly in-creases hippocampal neurogenesis in the trisomic off-spring and offers protection against the age-relateddegeneration of cholinergic neurons in the medialseptal nucleus (22). Moreover, hippocampal neurogen-esis and medial septal cholinergic neuron density eachcorrelated significantly with the spatial cognition abili-ties of the offspring, suggesting functional relationships(21, 22). In addition, the persistent effects of MCS oncognition in adult offspring imply that an epigeneticmechanism mediated by the role of choline as a methyldonor may be involved (32).

To gain further insight into the mechanisms under-lying MCS-induced benefits, the present study sought totest the hypotheses that choline metabolism differsbetween Ts65Dn mice and their 2N littermates and thatMCS exerts lasting effects on choline metabolism in theadult Ts65Dn and 2N offspring. Isotopically labeledcholine (i.e., methyl-d9-choline) was administered as atracer in the drinking water of adult (16 mo) femaletrisomic (Ts65Dn) offspring and their 2N littermatesborn to dams who consumed the control or cholinesupplemented diet. As shown in Fig. 1, methyl-d9-choline is labeled with deuterium on all 3 methylgroups, which enables the tracing of the intact cholinemolecule (d9 metabolites) as well as the tracing of itsmethyl groups (d3 and d6 metabolites) (33, 34) inharvested tissues including liver, plasma, and discretebrain regions relevant to the neuropathology of DSand AD.

Figure 1. Schematic of the metabolism of the orally consumed d9-choline tracer. The intact d9-choline tracer can be used toproduce the d9-choline metabolites: d9-acetylcholine (d9-ACho), d9-betaine (d9-Bet), d9-phosphocholine (d9-PCho), d9-glycerophosphocholine (d9-GPC), and d9-phosphatidylcholine (d9-PC). Alternatively, the d9-choline tracer can be oxidized tod9-betaine, and one of its 3 methyl groups can be donated to homocysteine (Hcy), forming d3-methionine (d3-Met) andsubsequently d3-S-adenosylmethionine (d3-SAM), the universal methyl donor. A main consumer of SAM is the phosphatidyle-thanolamine N-methyltransferase (PEMT) pathway, which mediates the sequential methylation of phosphatidylethanolamine(PE) to PC. Under the current labeling strategy, the PEMT pathway generated d3-PC and d6-PC. These PEMT-PC labeledmetabolites can then undergo hydrolysis to release free choline (e.g., d3-Cho), which can be used to synthesize other metabolites(e.g., d3-ACho, d3-Bet, d3-PCho, and d3-GPC). Theoretically, d9-PC could also be generated by the PEMT pathway. However,the estimated amount of PEMT-derived d9-PC was negligible in the current study (as indicated by the very low enrichment ofPEMT-derived d6-PC). Therefore, d9-PC production via the PEMT pathway is not depicted in the figure.

4313PERINATAL CHOLINE PROGRAMS OFFSPRING PEMT ACTIVITY

MATERIALS AND METHODS

Mice and diets

Breeder pairs (Ts65Dn female and C57Bl/6J Eicher � C3H/HeSnJ F1 male) were purchased from Jackson Laboratories(Bar Harbor, ME, USA) and mated at Cornell University(Ithaca, NY, USA). All animals were housed in microisolatorcages (Ancare, Bellmore, NY, USA) in a temperature-con-trolled room (22–25°C and 70% humidity) with a 12-hlight-dark cycle. At the time of mating, breeder pairs wererandomized to either a diet containing standard cholinelevels (1.1 g choline chloride/kg diet; Dyets no.110098; Dyets,Bethlehem, PA, USA) or the same diet containing 5.0 gcholine chloride/kg diet, which has been shown to producelasting cognitive benefits in the offspring (22). These twolevels of maternal choline intake continued until the pupswere weaned at postnatal d 21. All pups were subsequently feda standard chow diet and genotyped for the presence of theextra chromosome by Jackson Laboratories (35, 36).

These breeding pairs produce litters containing both tri-somic (Ts65Dn) and disomic (2N) offspring. The presentstudy used only female offspring because the male littermateswere used for behavioral and cognitive testing (data notpublished). The study cohort included 8 disomic control(2N-C) and 7 trisomic control (Ts65Dn-C) offspring of 15dams fed a control diet with standard choline levels, as well as25 disomic MCS (2N-MCS) and 8 trisomic MCS (Ts65Dn-MCS) offspring of 15 choline-supplemented dams. For the2N-C, Ts65Dn-C, and Ts65Dn-MCS groups (7–8 subjects/group), subjects were from different litters; for the 2N-MCSgroup with 25 subjects, �2 subjects were from the samebreeder pairs.

At 16 mo of age, the animals were shipped to the NathanKline Institute for the tracer experiments. Methyl-d9-choline(1.6 mM; Cambridge Isotopes Laboratory, Andover, MA,USA) was provided as a tracer in the drinking water and wasadministered for 8 wk to the entire cohort as describedpreviously (37). On completion of the tracer dosing, mice (18mo of age) were overdosed with ketamine (13 mg/kg)/xylazine (83 mg/kg), blood was collected by cardiac punc-ture, mice were perfused with 1� PO4 buffer (38), and tissueswere collected and processed. In brief, blood was collectedinto vacutainer tubes containing 3.6 mg K2-EDTA (BectonDickinson, Franklin Lakes, NJ, USA), placed immediately onice, processed for plasma within 4 h, and stored at �80°C(37). Liver was removed, cut into pieces, immediately frozenon dry ice, and stored at �80°C. Brain was harvested anddissected according to established protocols (39, 40) toextract basal forebrain, hippocampus, neocortex, cerebellum,and the rest of the brain, frozen on dry ice, and stored at�80°C. The samples (liver, plasma, and brain regions) weresubsequently shipped (on dry ice) to the M.A.C. laboratory atCornell University and stored at �80°C until analyzed forcholine metabolite enrichments.

All protocols for the animal procedures were approved bythe Institutional Animal Care and Use Committees of CornellUniversity, the Nathan Kline Institute, and the New YorkUniversity Langone Medical Center and were conducted inaccordance with the National Institutes of Health Guide forthe Care and Use of Laboratory Animals.

Measurement of plasma and tissue metabolite enrichment

Choline metabolites were extracted from the biological sam-ples and quantified by liquid chromatography-tandem massspectrometry (LC-MS/MS) according to Koc et al. (41) withmodifications based on our instrumentation. The aqueous

extract contained the water-soluble choline metabolites [ace-tylcholine, choline, betaine, glycerophosphocholine (GPC),and phosphocholine (PCho)], while the organic extractcontained the lipid-soluble choline metabolites [phosphati-dylcholine (PC), lysophosphatidylcholine (LPC), and sphin-gomyelin (SM)]. The LC-MS/MS system consisted of anAccela pump with degasser (Thermo, San Jose, CA, USA), arefrigerated Accela autosampler (Thermo), and a TSQ Quan-tum mass spectrometer (Thermo), which was equipped withan electrospray ionization source and operated in positive ionmode. Aqueous extract (10 �l) was injected onto a Prevailsilica column (150�2.1 mm, 5 �m; Grace, Deerfield, IL,USA) with matching guard column (7.5�2.1 mm, 5 �m) forseparation of the water-soluble choline metabolites. Mobilephases included solvent A (acetonitrile/water/ethanol/1 Mammonium acetate/glacial acetic acid, 800/127/68/3/2,v/v) and solvent B (acetonitrile/water/ethanol/1 M ammo-nium acetate/glacial acetic acid, 500/500/85/27/18, v/v). A30 min gradient was used to achieve metabolite separation:t � 0 min, 100% solvent A; t � 2 min, 100% solvent A; t � 11min, 55% solvent A; t � 14 min, 55% solvent A; t � 18 min,40% solvent A; t � 19 min, 0% solvent A; t � 21.5 min, 0%solvent A; t � 25.5 min, 100% solvent A; t � 30 min, 100%solvent A. The metabolites of interest were detected by selectedreaction monitoring of the following m/z ratio transitions:d0-aceytlcholine, 146/87; d3-acetylcholine, 149/87; d6-acetyl-choline, 152/87; d9-acetylcholine, 155/87; d0-choline, 104/60; d3-choline, 107/63; d6-choline, 110/66; d9-choline, 113/69; d0-betaine, 118/59; d3-betaine, 121/62; d6-betaine, 124/65; d9-betaine, 127/68; d0-GPC, 258/104; d3-GPC, 261/107;d6-GPC, 264/110; d9-GPC, 267/113; d0-PCho, 184/125; d3-PCho, 187/125; d6-PCho, 190/125; d9-PCho, 193/125. Or-ganic extract (10 �l) was injected onto an Adsorbosphere XLSI column (150�2.1 mm, 5 �m; Grace) with matching guardcolumn (7.5�4.6 mm, 5 �m) for separation of the lipid-soluble choline metabolites. A 21 min gradient of the samesolvents A and B was used: t � 0 min, 95% solvent A; t � 3min, 100% solvent A; t � 8 min, 70% solvent A; t � 14 min,40% solvent A; t � 16 min, 0% solvent A; t � 18 min, 0%solvent A; t � 20 min, 95% solvent A; t � 21 min, 95% solventA. Because insource fragmentation of PC, SM, and LPCproduces PCho as a common ion (regardless of fatty acidcomposition), PC, LPC, and SM were identified by selectedion monitoring of the PCho fragment, with m/z 184 forunlabeled d0 metabolites, m/z 187 for d3 metabolites, m/z 190for d6 metabolites, and m/z 193 for d9 metabolites. Themonitoring of the PCho fragment and the use of normalphase chromatography on the Adsorbosphere XL SI columnprovided the greatest sensitivity for quantifying isotopic en-richments but precluded the quantification of the molecularspecies of PC, SM, and LPC. No internal standards were usedbecause only relative intensities of unlabeled and labeledmetabolites (e.g., d0-, d3-, d6-, and d9-PC) are needed forcalculations of fractional enrichments. Instruments were op-erated using Xcalibur 2.0.7 SP1 (Thermo), and data wereprocessed with the Xcalibur Quan Browser to obtain peakareas under the chromatography curves for labeled andunlabeled choline metabolites.

Isotopic enrichment percentages [labeled metabolite/(la-beled � unlabeled metabolites) � 100%] of the cholinemetabolites were calculated in liver, plasma, and discretebrain regions by using the peak area under the chromatog-raphy curve of the labeled metabolite divided by the total areaof all isotopomers of the metabolite (37). These enrichmentpercentages were used to calculate 2 composite parameters torepresent the overall metabolic use of choline-associatedmethyl groups by the phosphatidylethanolamine N-methyl-transferase (PEMT) pathway [d3 � d6 enrichment � (totalenrichment of d3- and d6-choline metabolites)/(number of

4314 Vol. 28 October 2014 YAN ET AL.The FASEB Journal � www.fasebj.org

TABLE 1. Effects of genotype on choline metabolite enrichment in liver, plasma, and brain regions of adult disomic control (2N-C)and trisomic control (Ts65Dn-C) offspring born to unsupplemented control dams

Metabolite 2N-C, n � 8 Ts65Dn-C, n � 7 P Metabolite 2N-C, n � 8 Ts65Dn-C, n � 7 P

Hepatic d3- and d6-choline metabolite enrichment (%) Hepatic d9-choline metabolite enrichment (%)

d3-Betaine 1.1 (0.98–1.23) 0.6 (0.56–0.73) �0.016 d9-Betaine 0.4 (0.32–0.41) 0.1 (0.12–0.16) �0.016d3-Choline 1.6 (1.39–1.82) 0.9 (0.79–1.09) �0.016 d9-Choline 0.9 (0.81–1.06) 0.5 (0.40–0.55) �0.016d3-GPC 1.1 (0.97–1.27) 0.7 (0.57–0.77) �0.016 d9-GPC 0.8 (0.65–0.88) 0.3 (0.25–0.36) �0.016d6-GPC 0.007 (0.004–0.013) 0.002 (0.001–0.004) 0.006 d9-PCho 0.5 (0.34–0.64) 0.2 (0.13–0.27) 0.001d3-PCho 0.8 (0.70–1.01) 0.5 (0.36–0.56) 0.002 d9-LPC 0.6 (0.47–0.70) 0.2 (0.19–0.30) �0.016d3-PC 1.0 (0.90–1.13) 0.6 (0.55–0.71) �0.016 d9-PC 0.5 (0.43–0.58) 0.2 (0.15–0.22) �0.016d6-PC 0.015 (0.011–0.021) 0.007 (0.005–0.011) 0.006 d9-SM 0.7 (0.57–0.90) 0.4 (0.31–0.52) 0.004

Plasma d3- and d6-choline metabolite enrichment (%) Plasma d9-choline metabolite enrichment (%)

d3-Betaine 1.0 (0.88–1.20) 0.6 (0.51–0.71) �0.01 d9-Betaine 0.3 (0.23–0.51) 0.2 (0.10–0.24) 0.012d3-Choline 1.6 (1.47–1.82) 1.0 (0.88–1.11) �0.01 d9-Choline 3.0 (2.58–3.38) 1.8 (1.59–2.13) �0.01d3-GPC 1.2 (1.04–1.29) 0.7 (0.62–0.79) �0.01 d9-GPC 0.7 (0.63–0.82) 0.3 (0.25–0.33) �0.01d6-GPC ND ND d9-PCho ND NDd3-PCho ND ND d9-LPC 0.6 (0.54–0.70) 0.2 (0.20–0.26) �0.01d3-PC 0.9 (0.83–1.06) 0.6 (0.50–0.65) �0.01 d9-PC 0.5 (0.47–0.60) 0.2 (0.15–0.20) �0.01d6-PC ND ND d9-SM 0.7 (0.59–0.72) 0.4 (0.35–0.43) �0.01

Basal forebrain d3- and d6-choline metabolite enrichment (%) Basal forebrain d9-choline metabolite enrichment (%)

d3-ACho 1.3 (1.20–1.50) 1.2 (1.07–1.36) 0.6 d9-ACho 0.7 (0.61–0.83) 0.7 (0.63–0.88) 0.9d3-Betaine 1.3 (1.20–1.37) 1.1 (1.05–1.21) 0.3 d9-Betaine 0.4 (0.35–0.44) 0.3 (0.30–0.38) 0.3d3-Choline 2.5 (2.30–2.75) 2.3 (2.06–2.49) 0.5 d9-Choline 1.4 (1.26–1.59) 1.4 (1.24–1.60) 0.9d3-GPC 1.5 (1.36–1.60) 1.4 (1.28–1.52) 0.7 d9-GPC 1.1 (0.99–1.25) 1.2 (1.05–1.36) 0.7d6-GPC 0.025 (0.020–0.031) 0.024 (0.019–0.030) 0.9 d9-PCho 0.6 (0.54–0.73) 0.7 (0.56–0.77) 0.9d3-PCho 0.7 (0.36–1.20) 0.6 (0.30–1.08) 0.9 d9-LPC ND NDd3-PC 1.2 (1.09–1.33) 1.2 (1.07–1.32) 0.9 d9-PC 0.9 (0.82–1.04) 1.0 (0.92–1.19) 0.5d6-PC 0.023 (0.020–0.027) 0.021 (0.018–0.025) 0.7 d9-SM 0.7 (0.63–0.84) 0.9 (0.80–1.09) 0.2

Hippocampus d3- and d6-choline metabolite enrichment (%) Hippocampus d9-choline metabolite enrichment (%)

d3-ACho 1.2 (1.03–1.39) 1.1 (0.97–1.34) 0.9 d9-ACho 0.7 (0.57–0.84) 0.8 (0.63–0.96) 0.8d3-Betaine 1.3 (1.12–1.40) 1.1 (0.97–1.23) 0.8 d9-Betaine 0.3 (0.28–0.40) 0.3 (0.26–0.39) 0.9d3-Choline 2.3 (2.01–2.52) 2.0 (1.80–2.30) 0.9 d9-Choline 1.4 (1.24–1.61) 1.4 (1.19–1.57) 0.9d3-GPC 1.4 (1.33–1.57) 1.4 (1.25–1.50) 0.8 d9-GPC 1.1 (0.98–1.26) 1.2 (1.08–1.41) 0.7d6-GPC 0.020 (0.014–0.029) 0.023 (0.016–0.034) 0.9 d9-PCho 0.7 (0.61–0.81) 0.7 (0.61–0.83) 0.9d3-PCho 1.1 (0.97–1.22) 1.0 (0.87–1.11) 0.8 d9-LPC ND NDd3-PC 1.2 (1.07–1.30) 1.2 (1.05–1.29) 0.9 d9-PC 0.9 (0.79–1.01) 1.0 (0.91–1.17) 0.6d6-PC 0.022 (0.018–0.027) 0.021 (0.017–0.026) 0.9 d9-SM 0.7 (0.63–0.81) 0.9 (0.78–1.02) 0.4

Neocortex d3- and d6-choline metabolite enrichment (%) Neocortex d9-choline metabolite enrichment (%)

d3-ACho 0.8 (0.69–1.04) 0.7 (0.59–0.92) 0.6 d9-ACho 0.5 (0.35–0.64) 0.4 (0.29–0.55) 0.6d3-Betaine 1.2 (1.05–1.27) 1.0 (0.88–1.08) 0.2 d9-Betaine 0.3 (0.26–0.36) 0.2 (0.20–0.28) 0.2d3-Choline 2.2 (2.03–2.43) 2.0 (1.85–2.24) 0.4 d9-Choline 1.4 (1.26–1.61) 1.5 (1.33–1.73) 0.6d3-GPC 1.5 (1.33–1.62) 1.4 (1.23–1.52) 0.6 d9-GPC 1.2 (1.03–1.30) 1.2 (1.09–1.40) 0.7d6-GPC 0.021 (0.013–0.036) 0.011 (0.006–0.019) 0.3 d9-PCho 0.6 (0.46–0.86) 0.5 (0.32–0.63) 0.4d3-PCho 1.1 (0.88–1.29) 0.7 (0.61–0.91) 0.3 d9-LPC ND NDd3-PC 1.2 (1.13–1.35) 1.2 (1.10–1.33) 0.9 d9-PC 0.9 (0.81–1.04) 1.0 (0.91–1.19) 0.4d6-PC 0.023 (0.019–0.028) 0.022 (0.018–0.027) 0.9 d9-SM 0.7 (0.63–0.82) 0.9 (0.77–1.03) 0.1

Cerebellum d3- and d6-choline metabolite enrichment (%) Cerebellum d9-choline metabolite enrichment (%)

d3-ACho 0.8 (0.58–1.14) 0.6 (0.45–0.91) 0.5 d9-ACho 0.3 (0.20–0.51) 0.3 (0.16–0.42) 0.7d3-Betaine 1.3 (1.19–1.39) 1.0 (0.96–1.14) 0.034 d9-Betaine 0.4 (0.32–0.42) 0.3 (0.26–0.35) 0.1d3-Choline 2.3 (2.13–2.56) 2.0 (1.84–2.24) 0.1 d9-Choline 1.4 (1.27–1.61) 1.4 (1.22–1.58) 0.8d3-GPC 1.5 (1.37–1.61) 1.3 (1.24–1.47) 0.2 d9-GPC 1.2 (1.03–1.30) 1.2 (1.03–1.33) 0.9d6-GPC 0.027 (0.022–0.034) 0.020 (0.016–0.025) 0.1 d9-PCho 0.8 (0.68–0.89) 0.6 (0.54–0.73) 0.2d3-PCho 1.2 (1.08–1.34) 0.9 (0.85–1.06) 0.043 d9-LPC ND NDd3-PC 1.2 (1.10–1.32) 1.1 (1.03–1.24) 0.5 d9-PC 0.9 (0.80–1.01) 1.0 (0.86–1.12) 0.5d6-PC 0.024 (0.020–0.028) 0.022 (0.018–0.026) 0.7 d9-SM 0.5 (0.45–0.57) 0.6 (0.55–0.70) 0.1

Rest of brain d3- and d6-choline metabolite enrichment (%) Rest of brain d9-choline metabolite enrichment (%)

d3-ACho 1.4 (1.20–1.60) 1.3 (1.12–1.52) 0.9 d9-ACho 0.8 (0.67–0.92) 0.8 (0.64–0.91) 0.9d3-Betaine 1.3 (1.21–1.43) 1.2 (1.06–1.27) 0.2 d9-Betaine 0.4 (0.32–0.45) 0.4 (0.30–0.43) 0.9d3-Choline 2.3 (2.13–2.54) 2.2 (1.97–2.37) 0.8 d9-Choline 1.4 (1.22–1.52) 1.4 (1.25–1.59) 0.9d3-GPC 1.4 (1.32–1.56) 1.3 (1.23–1.48) 0.9 d9-GPC 1.1 (0.95–1.22) 1.2 (1.01–1.31) 0.9d6-GPC 0.026 (0.020–0.033) 0.027 (0.020–0.035) 0.9 d9-PCho 0.8 (0.71–0.89) 0.8 (0.68–0.87) 0.9d3-PCho 1.2 (1.08–1.28) 1.0 (0.93–1.12) 0.3 d9-LPC ND NDd3-PC 1.2 (1.12–1.32) 1.2 (1.10–1.31) 0.9 d9-PC 0.9 (0.83–1.05) 1.1 (1.05–1.22) 0.4d6-PC 0.024 (0.020–0.029) 0.022 (0.018–0.027) 0.9 d9-SM 0.7 (0.60–0.77) 0.9 (0.78–1.03) 0.1

Data were analyzed with 1-factor ANOVA and are presented as estimated marginal means with 95% confidence intervals. ND, not detected.

4315PERINATAL CHOLINE PROGRAMS OFFSPRING PEMT ACTIVITY

d3- and d6-choline metabolites)] and the overall metabolicuse of intact choline [d9 enrichment � (total enrichment ofd9-choline metabolites)/(number of d9-choline metabo-lites)] for each tissue type. Because of the very low enrich-ment of d6-PC (a PEMT derivative), all of the d9-PC wasassumed to have been derived from the cytidine diphosphate-choline (CDP-choline) pathway. Hepatic S-adenosylmethio-nine (d3-SAM) enrichment and PEMT activity were alsocalculated according to the principles of mass isotopomerdistribution analysis (42, 43) using these equations: d3-SAMenrichment � (enrichment of d6-PC)/(enrichment of d3-PC � d6-PC); PEMT activity � (enrichment of d3-PC �d6-PC)/(enrichment of d3-SAM). Because d3- and d6-PC canbe produced by the CDP-choline pathway using d3- andd6-choline released from PEMT-derived d3- and d6-PC (i.e.,recycling of PEMT-PC), our calculated PEMT activity mayoverestimate actual activity. Nonetheless, this overestimationwould transpire across all experimental groups, thereby en-abling comparisons between genotypes and between treat-ment groups.

Statistical analysis

Data were logarithmically transformed to achieve normalityand equal variance of residuals for all dependent variables;transformed data were used in subsequent analyses. Theeffect of genotype on the dependent variables (i.e., individualmetabolite enrichments and composite parameters) was ex-amined in the adult offspring of the unsupplemented damsusing 1-factor ANOVA with genotype as the independentvariable. The effect of MCS on the dependent variables (i.e.,individual metabolite enrichments and composite parame-ters) was examined in all adult offspring using 2-factorANOVA with MCS, genotype, and the interaction term(MCS�genotype) as the independent variables. If a signifi-cant interaction was detected, data were stratified by geno-type, and the effect of MCS was tested using 1-factor ANOVAwithin the 2N and Ts65Dn offspring separately. Because bodyweight is a main determinant of water intake (44) (and thustracer consumption), body weight was considered in theinitial statistical analyses but was not retained in the finalmodels because it was not a significant predictor.

Statistical analyses were conducted using SPSS 20 (IBMCorp., Armonk, NY, USA), and the Benjamini-Hochbergprocedure was employed for false discovery rate (FDR)control at a level of 0.05 for the number of comparisons ineach tissue type. Data are presented as estimated marginalmeans with 95% confidence intervals. FDR-adjusted Pvalues are reported. Differences were considered statisti-cally significant at values of P � 0.05, and 0.05 � P � 0.10was indicative of trends.

RESULTS

Body weight at the end of tracer administration

A main effect of genotype (P�0.004) was detected forbody weight with 22% greater weight observed in 2N(331 g) vs. Ts65Dn (271 g) mice. No effect of MCS(P�0.17) was detected for body weight, nor was aninteraction between MCS and genotype detected(P�0.25).

Effect of genotype on individual enrichments of d3-,d6-, or d9-choline metabolites in adult offspring ofunsupplemented dams

Liver

Ts65Dn (vs. 2N) offspring of unsupplemented damsexhibited significantly lower (P�0.016) hepatic enrich-ments of all the labeled choline metabolites, includingd3-betaine (�42%), d3-choline (�42%), d3-GPC(�40%,), d6-GPC (�74%), d3-PCho (�46%), d3-PC(�38%), d6-PC (�52%), d9-betaine (�63%), d9-cho-line (�50%), d9-GPC (�60%), d9-PCho (�60%), d9-LPC (�59%), d9-PC (�64%), and d9-SM (�44%)(Table 1), suggesting that hepatic choline uptake isreduced in Ts65Dn adult offspring. No differences(P�0.16) in hepatic PEMT activity were detected be-tween genotypes with activities of 0.55% (0.43–0.68) vs.0.67% (0.55–0.82) of total PC in Ts65Dn vs. 2N adultoffspring, respectively. In addition, the activity of thehepatic CDP-choline pathway appeared to be unaf-fected by genotype, because the enrichment level ofboth its product (d9-PC) and its precursors (d9-cholineand d9-PCho) were similarly reduced in Ts65Dn (vs.2N) offspring.

Plasma

Comparable to the labeling pattern observed in theliver, Ts65Dn (vs. 2N) offspring of unsupplementeddams exhibited significantly lower (P�0.012) plasmaenrichment of all the labeled metabolites, includingd3-betaine (�41%), d3-choline (�39%), d3-GPC(�39%), d3-PC (�40%), d9-betaine (�54%), d9-cho-line (�37%), d9-GPC (�60%), d9-LPC (�63%), d9-PC(�67%), and d9-SM (�40%) (Table 1). d3-PCho,d6-GPC, d6-PC, and d9-PCho were not detected inplasma in either genotype.

Brain

In contrast to the labeling pattern observed in liver andplasma, enrichment of the choline metabolites did notdiffer (P0.1) between genotypes in the examinedbrain regions, with the exception of the cerebellum,where d3-betaine and d3-PCho were �20% lower(P�0.043) in the Ts65Dn (vs. 2N) adult offspring ofunsupplemented dams (Table 1).

Effect of genotype on the overall enrichment of d3-and d6- or d9-choline metabolites in adult offspringof unsupplemented dams

Ts65Dn (vs. 2N) offspring of unsupplemented damsexhibited significantly lower (P�0.001) d3 and d6 enrich-ment and significantly lower (P�0.001) d9 enrichment inliver (�41 and �56%, respectively) and plasma (�40 and�46%, respectively) (Fig. 2). In contrast, d3 and d6enrichment and d9 enrichment did not differ (P�0.07)between genotypes in the examined brain regions, with

4316 Vol. 28 October 2014 YAN ET AL.The FASEB Journal � www.fasebj.org

the exception of cerebellum d3 and d6 enrichment,which was 14% lower (P�0.025) in the Ts65Dn (vs. 2N)adult offspring of unsupplemented dams (Fig. 2).

Effect of MCS during the perinatal period onindividual enrichment of d3-, d6-, or d9-cholinemetabolites in the adult offspring

Liver

A main effect of MCS on d9-choline metabolite enrich-ment (except d9-betaine) was detected (P�0.007) withhigher enrichments of d9-choline (�43%), d9-GPC(�58%), d9-PCho (�91%), d9-LPC (�48%), d9-PC(�58%), and d9-SM (�41%) in the adult offspring ofsupplemented (vs. unsupplemented) dams (Table 2).Hepatic PEMT activity was also 60% higher (P�0.007,main effect) in the adult offspring of supplemented vs.unsupplemented dams. For the d3- and d6-cholinemetabolites, as well as d9-betaine, MCS interacted withgenotype (P�0.041) to affect hepatic enrichments(Table 3). Ts65Dn adult offspring of supplemented (vs.unsupplemented) dams exhibited a markedly signifi-cant increase (P�0.009) in enrichment of d3-betaine(�140%), d3-choline (�161%), d3-GPC (�155%), d6-GPC (�640%), d3-PCho (�201%), d3-PC (�159%),

d6-PC (�249%), and d9-betaine (�148%); whereas 2Nadult offspring of supplemented (vs. unsupplemented)dams exhibited a less robust but still significantlyhigher (P�0.018) enrichment in select metabolites,including d3-choline (�29%), d3-GPC (�30%), d3-PC(�30%), and d9-betaine (�42%).

Because the Ts65Dn genotype perturbed hepaticcholine metabolism, we examined whether these ab-normalities were normalized by MCS. Compared to the2N adult offspring of unsupplemented dams, Ts65Dnadult offspring of supplemented dams exhibited similar(P�0.2) or higher (P�0.043) choline metabolite en-richment, as well as significantly higher PEMT activity(�57%, P�0.016) (Supplemental Table S1).

Plasma

The enrichment patterns of choline metabolites inplasma mirrored those observed in liver. A main effectof MCS on d9-choline metabolite enrichment (exceptd9-betaine) was detected (P�0.021) with higher en-richment of d9-choline (�24%), d9-GPC (�38%), d9-LPC (�48%), d9-PC (�49%), and d9-SM (�43%) inthe adult offspring of supplemented (vs. unsupple-mented) dams (Table 2). For the d3-choline metabo-lites, MCS interacted with genotype (P�0.01) to affect

Figure 2. Effects of genotype on overall d3 and d6enrichment (A) and overall d9 enrichment (B) in liver, plasma, and brainregions of adult disomic control (2N-C) and trisomic control (Ts65Dn-C) offspring born to unsupplemented control dams. Dataare estimated marginal means with 95% confidence intervals. *P � 0.05, ***P � 0.001.

4317PERINATAL CHOLINE PROGRAMS OFFSPRING PEMT ACTIVITY

plasma enrichment (Table 3). Ts65Dn adult offspringof supplemented (vs. unsupplemented) dams exhib-ited substantially significantly higher (P�0.004) enrich-ment of d3-betaine (�147%), d3-choline (�143%),d3-GPC (�138%), and d3-PC (�155%), whereas 2Nadult offspring of supplemented (vs. unsupplemented)

dams tended to exhibit higher (P�0.056) enrichmentof d3-PC (�26%) only. Finally, MCS normalized cho-line metabolism in the trisomic offspring. Specifically,Ts65Dn adult offspring of supplemented dams exhib-ited similar (P�0.068) or higher (P�0.05) plasmacholine metabolite enrichments than the unsupple-

TABLE 2. Effects of MCS on choline metabolite enrichment in liver, plasma, and brain regions of adult disomic control (2N-C) andtrisomic control (Ts65Dn-C) offspring born to unsupplemented control dams vs. 2N-MCS and Ts65Dn-MCS offspring born to MCS dams

Metabolite2N-C � Ts65Dn-C,

n � 152N-MCS � Ts65Dn-MCS,

n � 33 P Metatolite2N-C � Ts65Dn-C,

n � 152N-MCS � Ts65Dn-MCS,

n � 33 P

Hepatic d9-choline metabolite enrichment (%) Plasma d9-choline metabolite enrichment (%)

d9-Betaine See Table 3 See Table 3 d9-Betaine 0.2 (0.17–0.33) 0.3 (0.25–0.43) 0.1d9-Choline 0.7 (0.56–0.78) 0.9 (0.84–1.06) 0.001 d9-Choline 2.3 (2.04–2.68) 2.9 (2.60–3.21) 0.019d9-GPC 0.5 (0.40–0.58) 0.8 (0.67–0.87) �0.007 d9-GPC 0.5 (0.37–0.56) 0.6 (0.54–0.73) 0.021d9-PCho 0.3 (0.24–0.38) 0.6 (0.48–0.68) �0.007 d9-PCho ND NDd9-LPC 0.4 (0.31–0.44) 0.6 (0.49–0.63) 0.001 d9-LPC 0.4 (0.31–0.45) 0.6 (0.48–0.64) 0.003d9-PC 0.3 (0.26–0.37) 0.5 (0.43–0.56) �0.007 d9-PC 0.3 (0.26–0.37) 0.5 (0.40–0.53) 0.002d9-SM 0.5 (0.44–0.65) 0.8 (0.65–0.87) 0.006 d9-SM 0.5 (0.44–0.58) 0.7 (0.64–0.80) 0.006

Basal forebrain d3- and d6-choline metabolite enrichment (%) Basal forebrain d9-choline metabolite enrichment (%)

d3-ACho 1.3 (1.11–1.48) 1.6 (1.41–1.74) 0.049 d9-ACho 0.7 (0.61–0.87) 0.8 (0.69–0.89) 0.5d3-Betaine 1.2 (1.09–1.33) 1.6 (1.50–1.74) �0.017 d9-Betaine 0.4 (0.32–0.42) 0.4 (0.40–0.49) 0.058d3-Choline 2.4 (2.17–2.65) 3.1 (2.85–3.31) �0.017 d9-Choline 1.4 (1.23–1.60) 1.7 (1.53–1.86) 0.057d3-GPC 1.4 (1.31–1.57) 1.9 (1.75–2.00) �0.017 d9-GPC 1.1 (1.00–1.32) 1.3 (1.22–1.49) 0.088d6-GPC 0.025 (0.019–0.032) 0.028 (0.023–0.034) 0.5 d9-PCho 0.6 (0.54–0.76) 0.7 (0.65–0.84) 0.2d3-PCho 0.6 (0.48–0.79) 1.3 (1.09–1.57) �0.017 d9-LPC ND NDd3-PC 1.2 (1.09–1.32) 1.6 (1.45–1.67) �0.017 d9-PC 1.0 (0.86–1.12) 1.1 (1.04–1.26) 0.093d6-PC 0.022 (0.019–0.026) 0.031 (0.028–0.035) 0.003 d9-SM 0.8 (0.72–0.94) 0.9 (0.84–1.02) 0.2

Hippocampus d3- and d6-choline metabolite enrichment (%) Hippocampus d9-choline metabolite enrichment (%)

d3-ACho 1.2 (1.04–1.32) 1.6 (1.46–1.74) �0.017 d9-ACho 0.7 (0.62–0.87) 0.9 (0.77–0.99) 0.1d3-Betaine 1.2 (1.06–1.30) 1.5 (1.43–1.66) �0.017 d9-Betaine 0.3 (0.28–0.38) 0.4 (0.33–0.42) 0.2d3-Choline 2.2 (1.95–2.38) 2.9 (2.67–3.10) �0.017 d9-Choline 1.4 (1.21–1.59) 1.7 (1.53–1.87) 0.039d3-GPC 1.4 (1.28–1.55) 1.9 (1.75–2.01) �0.017 d9-GPC 1.2 (1.02–1.33) 1.4 (1.25–1.52) 0.061d6-GPC 0.022 (0.018–0.028) 0.031 (0.026–0.037) 0.03 d9-PCho 0.7 (0.60–0.82) 0.9 (0.79–1.00) 0.03d3-PCho 1.0 (0.94–1.15) 1.4 (1.32–1.53) �0.017 d9-LPC ND NDd3-PC 1.2 (1.07–1.29) 1.5 (1.40–1.61) �0.017 d9-PC 1.0 (0.84–1.09) 1.1 (1.01–1.23) 0.081d6-PC 0.022 (0.018–0.025) 0.031 (0.028–0.035) 0.002 d9-SM 0.8 (0.70–0.91) 0.9 (0.79–0.97) 0.2

Neocortex d3- and d6-choline metabolite enrichment (%) Neocortex d9-choline metabolite enrichment (%)

d3-ACho 0.8 (0.69–0.92) 1.2 (1.07–1.34) �0.015 d9-ACho 0.4 (0.29–0.63) 0.5 (0.40–0.72) 0.3d3-Betaine 1.1 (0.97–1.18) 1.4 (1.34–1.55) �0.015 d9-Betaine 0.3 (0.23–0.32) 0.4 (0.35–0.43) �0.015d3-Choline 2.1 (1.93–2.36) 2.9 (2.69–3.11) �0.015 d9-Choline 1.5 (1.28–1.68) 1.7 (1.57–1.93) 0.066d3-GPC 1.4 (1.30–1.56) 1.9 (1.77–2.03) �0.015 d9-GPC 1.2 (1.04–1.36) 1.4 (1.27–1.55) 0.074d6-GPC See Table 3 See Table 3 d9-PCho 0.5 (0.43–0.67) 0.7 (0.63–0.87) 0.035d3-PCho See Table 3 See Table 3 d9-LPC ND NDd3-PC 1.2 (1.11–1.34) 1.6 (1.47–1.69) �0.015 d9-PC 1.0 (0.85–1.11) 1.1 (1.04–1.26) 0.07d6-PC 0.023 (0.019–0.027) 0.032 (0.025–0.034) 0.002 d9-SM 0.8 (0.70–0.91) 0.9 (0.83–1.00) 0.1

Cerebellum d3- and d6-choline metabolite enrichment (%) Cerebellum d9-choline metabolite enrichment (%)

d3-ACho 0.7 (0.55–0.95) 1.0 (0.81–1.22) 0.089 d9-ACho 0.3 (0.22–0.38) 0.4 (0.35–0.54) 0.043d3-Betaine 1.2 (1.05–1.30) 1.6 (1.46–1.72) �0.017 d9-Betaine 0.3 (0.29–0.39) 0.4 (0.36–0.45) 0.085d3-Choline 2.2 (2.00–2.40) 2.9 (2.75–3.16) �0.017 d9-Choline 1.4 (1.23–1.61) 1.7 (1.55–1.90) 0.037d3-GPC 1.4 (1.30–1.56) 1.9 (1.79–2.05) �0.017 d9-GPC 1.2 (1.01–1.34) 1.4 (1.29–1.59) 0.04d6-GPC 0.023 (0.017–0.032) 0.031 (0.024–0.039) 0.2 d9-PCho 0.7 (0.57–0.86) 0.8 (0.71–0.97) 0.2d3-PCho 1.1 (0.95–1.22) 1.5 (1.32–1.60) �0.017 d9-LPC ND NDd3-PC 1.2 (1.06–1.29) 1.5 (1.43–1.65) �0.017 d9-PC 0.9 (0.81–1.08) 1.1 (0.48–0.65) 0.085d6-PC 0.023 (0.019–0.027) 0.032 (0.029–0.036) 0.002 d9-SM 0.6 (0.48–0.65) 0.6 (0.55–0.68) 0.3

Rest of brain d3- and d6-choline metabolite enrichment (%) Rest of brain d9-choline metabolite enrichment (%)

d3-ACho 1.3 (1.22–1.50) 2.0 (1.85–2.15) �0.017 d9-ACho 0.8 (0.66–0.89) 1.0 (0.90–1.12) 0.009d3-Betaine 1.2 (1.13–1.36) 1.7 (1.61–1.85) �0.017 d9-Betaine 0.4 (0.32–0.42) 0.5 (0.44–0.54) 0.004d3-Choline 2.2 (2.04–2.48) 3.0 (2.77–3.20) �0.017 d9-Choline 1.4 (1.21–1.57) 1.6 (1.47–1.79) 0.059d3-GPC 1.4 (1.27–1.53) 1.8 (1.71–1.96) �0.017 d9-GPC 1.1 (0.97–1.27) 1.3 (1.19–1.45) 0.062d6-GPC 0.026 (0.022–0.032) 0.039 (0.034–0.045) 0.002 d9-PCho 0.8 (0.67–0.89) 0.9 (0.84–1.04) 0.05d3-PCho 1.1 (1.00–1.21) 1.5 (1.40–1.62) �0.017 d9-LPC ND NDd3-PC 1.2 (1.10–1.32) 1.6 (1.47–1.67) �0.017 d9-PC 1.0 (0.87–1.14) 1.1 (1.04–1.26) 0.1d6-PC 0.023 (0.020–0.027) 0.034 (0.030–0.038) �0.017 d9-SM 0.8 (0.67–0.89) 0.9 (0.79–0.96) 0.2

Data were analyzed with 2-factor ANOVA (MCS, genotype, MCS�genotype). MCS did not interact with genotype (P0.13) to affect thesedependent variables; thus, the main effect of MCS is presented. Data are estimated marginal means with 95% confidence intervals. ND, notdetected.

4318 Vol. 28 October 2014 YAN ET AL.The FASEB Journal � www.fasebj.org

mented 2N offspring, with the exception of d9-PC,which remained lower (�40%; P�0.048) than theunsupplemented 2N mice (Supplemental Table S1).

Brain

A main effect of MCS was detected (P�0.05) on themajority of d3-choline metabolites and, to a lesserextent, on the d9-choline metabolites. Specifically,higher choline metabolite enrichment was detected in93% of the d3 metabolites (i.e., 28 of 30) and 29% ofthe d9 metabolites (i.e., 10 of 35) in the adult offspringof supplemented (vs. unsupplemented) dams (Table2). Notably, while the enrichment of all the d3-cholinemetabolites was higher in the basal forebrain, none ofthe d9-choline metabolite enrichment levels achievedstatistical significance (Table 2). MCS also tended tointeract with genotype (P�0.068) to influence d3-PChoand d6-GPC in the neocortex (Table 3). Specifically,Ts65Dn adult offspring of supplemented (vs. unsupple-mented) dams exhibited substantially significantlyhigher (P�0.004) enrichment of d6-GPC (�263%) andd3-PCho (�96%) whereas no differences (P0.17) inthe enrichments of these metabolites were detected in2N adult offspring of supplemented (vs. unsupple-mented) dams (Table 3). Finally, MCS normalizedcholine metabolism in the trisomic mice. Ts65Dn adult

offspring of supplemented dams exhibited similar(P�0.068) or higher (P�0.05) brain choline metabo-lite enrichments than the unsupplemented 2N mice(Supplemental Tables S2–S4).

Effect of MCS during the perinatal period on overallenrichment of d3- and d6- or d9-choline metabolitesin the adult offspring

A main effect of MCS on the metabolic use of d9-choline was detected in liver (�53%, P�0.001), plasma(�34%, P�0.001), and several brain regions (i.e., hip-pocampus, neocortex, cerebellum, and the rest of thebrain, �18 to �22%, P�0.05), reflecting the higher d9enrichment in the adult offspring of supplemented (vs.unsupplemented) dams (Fig. 3A). In addition, d9 en-richment in basal forebrain tended to be higher(�16%, P�0.07) among adult offspring born to sup-plemented (vs. unsupplemented) dams. MCS inter-acted with genotype to affect d3 and d6 enrichment inliver (P�0.001), plasma (P�0.001), and basal forebrain(P�0.042), with similar trends seen in hippocampus(P�0.1), neocortex (P�0.077), and cerebellum (P�0.075) (Fig. 3B). All of these interactions reflected asignificantly greater effect of MCS for the trisomicoffspring than their 2N littermates. Specifically,

TABLE 3. Effects of the interaction of genotype and MCS on choline metabolite enrichments in liver, plasma, and brain regions of adultdisomic control (2N-C) and trisomic control (Ts65Dn-C) offspring born to unsupplemented control dams vs. 2N-MCS and Ts65Dn-MCSoffspring born to MCS dams

Metabolite 2N-C, n � 8 2N-MCS, n � 25 P Ts65Dn-C, n � 7 Ts65Dn-MCS, n � 8 P

Hepatic d3- and d6-choline metabolite enrichment (%)

d3-Betaine 1.1 (0.94–1.29) 1.3 (1.16–1.39) 0.2 0.6 (0.53–0.77) 1.5 (1.30–1.81) �0.009d3-Choline 1.6 (1.37–1.85) 2.0 (1.88–2.23) 0.011 0.9 (0.75–1.15) 2.4 (2.02–2.92) �0.009d3-GPC 1.1 (0.97–1.28) 1.4 (1.33–1.56) 0.014 0.7 (0.53–0.84) 1.7 (1.39–2.07) �0.009d6-GPC 0.007 (0.004–0.013) 0.009 (0.006–0.013) 0.6 0.002 (0.001–0.004) 0.014 (0.008–0.026) �0.009d3-PCho 0.8 (0.71–0.99) 1.0 (0.94–1.14) 0.063 0.5 (0.35–0.59) 1.4 (1.09–1.71) �0.009d3-PC 1.0 (0.87–1.16) 1.3 (1.21–1.42) 0.018 0.6 (0.51–0.77) 1.6 (1.35–1.94) �0.009d6-PC 0.015 (0.012–0.020) 0.019 (0.016–0.021) 0.2 0.007 (0.005–0.011) 0.026 (0.018–0.038) 0.001Hepatic d9-choline metabolite enrichment (%)

d9-Betaine 0.4 (0.30–0.44) 0.5 (0.46–0.58) 0.012d3-Betaine 0.1 (0.10–0.19) 0.3 (0.25–0.45) 0.001Plasma d3- and d6-choline metabolite enrichment (%)

d3-Betaine 1.0 (0.81–1.31) 1.1 (0.95–1.26) 0.7 0.6 (0.49–0.73) 1.5 (1.22–1.82) �0.004d3-Choline 1.6 (1.35–1.98) 1.8 (1.57–1.97) 0.5 1.0 (0.81–1.21) 2.4 (1.97–2.94) �0.004d3-GPC 1.2 (0.99–1.35) 1.4 (1.24–1.50) 0.1 0.7 (0.57–0.86) 1.7 (1.36–2.04) �0.004d6-GPC ND ND ND NDd3-PCho ND ND ND NDd3-PC 0.9 (0.81–1.09) 1.2 (1.08–1.29) 0.056 0.6 (0.46–0.70) 1.4 (1.17–1.78) �0.004d6-PC ND ND ND NDNeocortex d3- and d6-choline metabolite enrichments (%)

d6-GPC 0.021 (0.015–0.031) 0.025 (0.020–0.030) 0.5 0.011 (0.006–0.020) 0.040 (0.023–0.068) 0.004d3-PCho 1.1 (0.90–1.26) 1.3 (1.14–1.39) 0.2 0.7 (0.59–0.94) 1.5 (1.17–1.82) 0.002

Data were analyzed with 2-factor ANOVA (MCS, genotype, MCS�genotype). Significant (P�0.041) and borderline significant (P�0.068)MCS and genotype interactions were detected for these dependent variables; thus, data were stratified by genotype, and the effect of MCS ispresented separately for each genotype (2N-C vs. 2N-MCS; Ts65Dn-C vs. Ts65Dn-MCS). Data are estimated marginal means with 95%confidence intervals. ND, not detected.

4319PERINATAL CHOLINE PROGRAMS OFFSPRING PEMT ACTIVITY

Ts65Dn adult offspring of supplemented (vs. unsupple-mented) dams exhibited significantly higher (P�0.001) d3 and d6 enrichment in liver (�159%), plasma(�147%), basal forebrain (�57%), hippocampus(�51%), neocortex (�57%), and cerebellum (�57%),whereas 2N adult offspring of supplemented (vs. un-supplemented) dams exhibited modestly higher (butsignificant; P�0.022) d3 and d6 enrichment in liver(�26%), basal forebrain (�21%), hippocampus(�23%), neocortex (�25%), and cerebellum (�23%)(Fig. 3B). For the metabolic use of endogenouslyproduced d3- and d6-choline in the rest of the brain, amain effect of MCS was detected, with a significantlyhigher (�36%, P�0.001) d3 and d6 enrichment in theadult offspring of supplemented (vs. unsupplemented)dams (Fig. 3C). Finally, MCS normalized d9-cholinemetabolite enrichment and improved the d3- and d6-choline metabolite enrichment in trisomic mice. Spe-cifically, in liver, plasma, and all examined brain re-gions, Ts65Dn adult offspring of supplemented damsexhibited d9-enrichment that was similar (P�0.08) tothe unsupplemented 2N mice, and d3 and d6 enrich-ment that was significantly higher (P�0.009) thanthese mice (Supplemental Tables S1–S4).

DISCUSSION

The study findings show that choline metabolism isaltered in trisomic mice, with evidence of preferentialpartitioning of choline toward the brain; and MCS haslasting effects on the metabolic use of choline in adultoffspring, including enhanced de novo production, andutilization, of PEMT-PC molecules in both genotypes,but to a great extent in trisomic offspring.

Choline metabolism is altered in trisomic mice

Ts65Dn (vs. 2N) offspring of unsupplemented damsexhibited substantially lower enrichment of d3- and d6-and d9-choline metabolites in liver and plasma but notin brain tissue. Indeed, brain choline metabolite en-richment was remarkably unaffected by genotype, sug-gesting that choline is preferentially partitioned to thebrain at the expense of other organs in trisomic mice.Redistribution of choline from other organs (e.g., liver,plasma, kidney, and intestines) to the brain has alsobeen demonstrated in a mouse model of choline de-privation (45), suggesting that the trisomy seen in DSincreases choline requirements. Based on the present

Figure 3. Effects of MCS on overall d9-enrichment (A) and overall d3 and d6 enrichment (B, C) in liver, plasma, and brainregions of adult Ts65Dn and 2N offspring born to unsupplemented control vs. MCS dams. MCS did not interact with genotype(P0.14) for dependent variables shown in A and C; thus the main effect of MCS is presented. Significant (P�0.042) andborderline significant (P�0.1) MCS and genotype interactions were detected for the dependent variables shown in B; thus, datawere stratified by genotype, and the effect of MCS is presented separately for each genotype (2N-MCS vs. 2N-C; Ts65Dn-MCSvs. Ts65Dn-C). Data are estimated marginal means with 95% confidence intervals. *P � 0.05, **P � 0.01, ***P � 0.001.

4320 Vol. 28 October 2014 YAN ET AL.The FASEB Journal � www.fasebj.org

findings, it appears that increasing choline intake bypregnant dams with a trisomic fetus may help normal-ize their aberrant choline metabolism, which may, inturn, contribute to the observed improvements in cog-nitive functioning also produced by this maternal di-etary intervention.

MCS exerts lasting effects on adult offspring cholinemetabolism

Both Ts65Dn and 2N offspring of supplemented damsexhibited higher enrichment of d9-choline metabolitesin liver and several brain regions, indicating that expo-sure to higher choline levels during the perinatalperiod permanently enhances choline uptake and me-tabolism in these tissues. The programming effect ofMCS on d9-choline metabolism was most pronouncedin liver (�53% d9-choline metabolite enrichment inoffspring of supplemented vs. unsupplemented dams),although also evident in plasma (�34%) and variousbrain regions (��20%) (Fig. 3). The decreasing mag-nitude of the effects of MCS from liver to plasma tobrain suggests that hepatic choline metabolism is mostresponsive to the programming effect of perinatalcholine, and that changes in other organs likely arisefrom the primary alterations that occurred in liver. Thishypothesis is consistent with the central role of liver insupplying extrahepatic tissue with choline and othernutrients (46).

Notably, activity of the hepatic PEMT pathway was60% higher in Ts65Dn and 2N offspring of choline-supplemented dams. This pathway functions in the denovo biosynthesis of choline (i.e., PC) and the mobili-zation of hepatic DHA into circulation (43, 47, 48).Specifically, the PEMT pathway (vs. CDP-choline path-way) produces a PC molecule that is enriched in DHA(43, 47, 48) and other long-chain unsaturated fattyacids, which can be incorporated into very low densitylipoproteins, released into circulation, and made avail-able to extrahepatic organs (43, 47, 48). Althoughrecycling of PEMT-derived PC to produce PC via theCDP-choline pathway could alter the fatty acid profileof PC, PEMT-PC is mobilized to extrahepatic tissuebefore extensive recycling occurs (43). Thus, up-regu-lation of the hepatic PEMT pathway by MCS would beexpected to increase both choline and DHA supply tobrain and other tissues. Indeed, higher levels of thePEMT-derived d3- and d6-choline metabolites wereobserved in plasma and several brain regions amongboth genotypes but to a greater extent in Ts65Dnoffspring. The genotype-specific programming effectsof MCS may reflect a higher demand for PEMT prod-ucts (e.g., choline and DHA) among adult Ts65Dn (vs.2N) offspring, which is consistent with their highercholine requirement. Future attempts at employingd9-choline and stable isotope methodology to study thekinetics of DHA supply from liver to brain shouldemploy a more dynamic pulse-chase approach andinclude an analysis of the fatty acid composition of thenewly synthesized PEMT-PC before recycling of

PEMT-PC to produce PC via the CDP-choline pathwayoccurs.

Enhancement of the hepatic PEMT pathway maybeneficially influence cognitive functioning

The long-lasting enhanced supply of choline and DHAto the brain as a result of up-regulation of the hepaticPEMT pathway by MCS may contribute to the improvedcognitive functioning produced by this maternal inter-vention. DHA is abundant among the phospholipids inbrain membranes (49) and appears to be particularlyimportant in the development and maintenance ofbrain mechanisms underlying cognitive functions (50).Administration of nutritional regimens (i.e., supple-mental DHA, choline, and uridine) designed to in-crease PC synthesis and phospholipid-DHA content hasbeen shown to increase PC content in brain mem-branes, increase dendritic spines, and thus synapses, onhippocampal neurons in animals, enhance neurotrans-mitter release, and improve cognitive function (re-viewed in refs. 49, 51). In addition, the long-lastingelevated supply of choline and DHA as a result ofenhanced hepatic PEMT activity in the offspring ofcholine-supplemented dams may attenuate neurode-generation, a characteristic of normal aging that isaccelerated in DS and AD (7, 52). For example, admin-istering choline to adult mice improves memory in amouse model of dementia (53) while administeringDHA to adult mice reduces amyloid burden in a mousemodel of AD (54). Further, provision of choline- andDHA-containing nutritional supplements to olderadults with mild AD was shown to improve memory(55). These findings collectively suggest that the life-long increased activity of the hepatic PEMT pathwayproduced by MCS and consequent increased provisionof PEMT-PC (and its associated DHA) to the brain maymediate some of the cognitive benefits previously re-ported in adult Ts65Dn and 2N offspring born tocholine-supplemented dams (21, 22). However, a limi-tation of the present study was the lack of data on thefatty acid composition of the PC molecules and otherphospholipids (e.g., PE) in liver and brain tissue. Thesedata, along with measurements of PEMT activity (andtheir correlations with cognitive outcomes), are neededto demonstrate that up-regulation of hepatic PEMTwould enhance DHA supply to the brain and contrib-ute to the MCS-induced cognitive benefits. Alsorequiring clarification is the exact time period (e.g.,prenatal or postnatal) required to reap the benefitsof MCS delivery and whether the programming ef-fects of MCS on offspring choline metabolism arespecific to females.

CONCLUSIONS

Ts65Dn (vs. 2N) mice exhibit alterations in cholinemetabolism indicative of a higher requirement for thiscritical bioactive nutrient. MCS increases hepatic PEMT

4321PERINATAL CHOLINE PROGRAMS OFFSPRING PEMT ACTIVITY

activity in both trisomic and disomic adult offspringand results in higher enrichment of PEMT-derived d3and d6 metabolites in the brain for both genotypes, butwith a more pronounced effect in the Ts65Dn off-spring. The increased hepatic supply of PEMT-PC (andits associated fatty acids) to the brain may be onemechanism through which MCS induces lifelong cog-nitive benefits in the Ts65Dn mouse model of DS andAD, as well as the normal offspring. Translation of MCSto human populations is increasingly warranted basedon the collective behavioral, neurochemical, andmetabolomic evidence that is being accrued in thishighly relevant animal model. In the interim, thepresent data provide support for advising all pregnantwomen to consume recommended amounts of choline,especially those carrying a DS fetus.

This work was funded by the U.S. National Institutes ofHealth (HD057564, AG043375, AG017617, and AG014449)and the Alzheimer’s Association (IIRG-12-237253). The fund-ing sources had no role in the study design, interpretation ofthe data, and/or publication of the results. The authors thankOlga Malysheva for expert technical assistance.

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Received for publication March 11, 2014.Accepted for publication June 9, 2014.

4323PERINATAL CHOLINE PROGRAMS OFFSPRING PEMT ACTIVITY