A Narrative Review on Maternal Choline Intake and Liver Function of the Fetus and the Infant; Implications for Research, Policy, and Practice.

Dietary choline is needed to maintain normal health, including normal liver function in adults. Fatty liver induced by a choline-deficient diet has been consistently observed in human and animal studies. The effect of insufficient choline intake on hepatic fat accumulation is specific and reversible when choline is added to the diet. Choline requirements are higher in women during pregnancy and lactation than in young non-pregnant women. We reviewed the evidence on whether choline derived from the maternal diet is necessary for maintaining normal liver function in the fetus and breastfed infants. Studies have shown that choline from the maternal diet is actively transferred to the placenta, fetal liver, and human milk. This maternal-to-child gradient can cause depletion of maternal choline stores and increase the susceptibility of the mother to fatty liver. Removing choline from the diet of pregnant rats causes fatty liver both in the mother and the fetus. The severity of fatty liver in the offspring was found to correspond to the severity of fatty liver in the respective mothers and to the duration of feeding the choline-deficient diet to the mother. The contribution of maternal choline intake in normal liver function of the offspring can be explained by the role of phosphatidylcholine in lipid transport and as a component of cell membranes and the function of choline as a methyl donor that enables synthesis of phosphatidylcholine in the liver. Additional evidence is needed on the effect of choline intake during pregnancy and lactation on health outcomes in the fetus and infant. Most pregnant and lactating women are currently not achieving the adequate intake level of choline through the diet. Therefore, public health policies are needed to ensure sufficient choline intake through adding choline to maternal multivitamin supplements.

High maternal choline consumption during pregnancy and nursing alleviates deficits in social interaction and improves anxiety-like behaviors in the BTBR T+Itpr3tf/J mouse model of autism.

Autism is a neurodevelopmental disorder with multiple genetic and environmental risk factors. Choline is a fundamental nutrient for brain development and high choline intake during prenatal and/or early postnatal periods is neuroprotective. We examined the effects of perinatal choline supplementation on social behavior, anxiety, and repetitive behaviors in the BTBR T+Itpr3tf/J (BTBR) mouse model of autism. The BTBR or the more "sociable" C57BL/6J (B6) strain females were fed a control or choline-supplemented diet from mating, throughout pregnancy and lactation. After weaning to a control diet, all offspring were evaluated at one or two ages [postnatal days 33-36 and 89-91] using open field (OF), elevated plus maze (EPM), marble burying (MB), and three-chamber social interaction tests. As expected, control-diet BTBR mice displayed higher OF locomotor activity, impaired social preference, and increased digging behavior during the MB test compared to control-diet B6 mice. Choline supplementation significantly decreased digging behavior, elevated the percentage of open arm entries and time spent in open arms in the EPM by BTBR mice, but had no effect on locomotion. Choline supplementation did not alter social interaction in B6 mice but remarkably improved impairments in social interaction in BTBR mice at both ages, indicating that the benefits of supplementation persist long after dietary choline returns to control levels. In conclusion, our results suggest that high choline intake during early development can prevent or dramatically reduce deficits in social behavior and anxiety in an autistic mouse model, revealing a novel strategy for the treatment/prevention of autism spectrum disorders.

Gestational choline supply regulates methylation of histone H3, expression of histone methyltransferases G9a (Kmt1c) and Suv39h1 (Kmt1a), and DNA methylation of their genes in rat fetal liver and brain.

Choline is an essential nutrient that, via its metabolite betaine, serves as a donor of methyl groups used in fetal development to establish the epigenetic DNA and histone methylation patterns. Supplementation with choline during embryonic days (E) 11-17 in rats improves memory performance in adulthood and protects against age-related memory decline, whereas choline deficiency impairs certain cognitive functions. We previously reported that global and gene-specific DNA methylation increased in choline-deficient fetal brain and liver, and these changes in DNA methylation correlated with an apparently compensatory up-regulation of the expression of DNA methyltransferase Dnmt1. In the current study, pregnant rats were fed a diet containing varying amounts of choline (mmol/kg: 0 (deficient), 8 (control), or 36 (supplemented)) during E11-17, and indices of histone methylation were assessed in liver and frontal cortex on E17. The mRNA and protein expression of histone methyltransferases G9a and Suv39h1 were directly related to the availability of choline. DNA methylation of the G9a and Suv39h1 genes was up-regulated by choline deficiency, suggesting that the expression of these enzymes is under negative control by methylation of their genes. The levels of H3K9Me2 and H3K27Me3, tags of transcriptionally repressed chromatin, were up-regulated by choline supplementation, whereas the levels of H3K4Me2, associated with active promoters, were highest in choline-deficient rats. These data show that maternal choline supply during pregnancy modifies fetal histone and DNA methylation, suggesting that a concerted epigenomic mechanism contributes to the long term developmental effects of varied choline intake in utero.

Raising gestational choline intake alters gene expression in DMBA-evoked mammary tumors and prolongs survival.

Choline is an essential nutrient that serves as a donor of metabolic methyl groups used during gestation to establish the epigenetic DNA methylation patterns that modulate tissue-specific gene expression. Because the mammary gland begins its development prenatally, we hypothesized that choline availability in utero may affect the gland's susceptibility to cancer. During gestational days 11-17, pregnant rats were fed a control, choline-supplemented, or choline-deficient diet (8, 36, and 0 mmol/kg of choline, respectively). On postnatal day 65, the female offspring received 25 mg/kg of a carcinogen 7,12-dimethylbenz[alpha]anthracene. Approximately 70% of the rats developed mammary adenocarcinomas; prenatal diet did not affect tumor latency, incidence, size, and multiplicity. Tumor growth rate was inversely related to choline content in the prenatal diet, resulting in 50% longer survival until euthanasia, determined by tumor size, of the prenatally choline-supplemented rats compared with the prenatally choline-deficient rats. This was accompanied by distinct expression patterns of approximately 70 genes in tumors derived from the three dietary groups. Tumors from the prenatally choline-supplemented rats overexpressed genes that confer favorable prognosis in human cancers (Klf6, Klf9, Nid2, Ntn4, Per1, and Txnip) and underexpressed those associated with aggressive disease (Bcar3, Cldn12, Csf1, Jag1, Lgals3, Lypd3, Nme1, Ptges2, Ptgs1, and Smarcb1). DNA methylation within the tumor suppressor gene, stratifin (Sfn, 14-3-3sigma), was proportional to the prenatal choline supply and correlated inversely with the expression of its mRNA and protein in tumors, suggesting that an epigenetic mechanism may underlie the altered molecular phenotype and tumor growth. Our results suggest a role for adequate maternal choline nutrition during pregnancy in prevention/alleviation of breast cancer in daughters.

Prenatal choline supplementation in rats increases the expression of IGF2 and its receptor IGF2R and enhances IGF2-induced acetylcholine release in hippocampus and frontal cortex.

Choline is an essential nutrient whose availability during the second half of gestation produces long-lasting cognitive effects. Rats that obtain supplemental choline during embryonic day (E) 11-17 have enhanced depolarization-evoked acetylcholine (ACh) release from hippocampal slices, whereas choline deficiency during this time reduces this release. Previously we reported that rats whose mothers consumed a choline-supplemented diet during E11-17 have higher levels of insulin-like growth factor II (IGF2) mRNA and protein in the frontal cortex compared to control and prenatally choline-deficient animals. Since IGF2 has been shown to stimulate endogenous ACh release, we measured the release of ACh from hippocampal and frontal cortical slices from rats on postnatal day (P) 18, P24, P34 and P80 in response to a depolarizing concentration of potassium (45 mM or 25 mM) or to IGF2 treatment in the absence or presence of a depolarizing concentration of potassium (25 mM). On P18, IGF2/depolarization-evoked ACh release from hippocampal slices was enhanced by prenatal choline supplementation. In the frontal cortex on P80, prenatal choline supplementation dramatically potentiated ACh release induced by depolarization, IGF2 or the combination of the two. On P18 and P90 and in both brain regions, IGF2 mRNA and protein levels, as well as protein levels of the IGF2 receptor (IGF2R), were higher in prenatally choline-supplemented rats. Choline supplementation also increased IGF2R mRNA levels in the septum. In summary, prenatal choline supplementation produced alterations in IGF2 signaling, via increased levels of IGF2 and IGF2R, which may enhance cholinergic neurotransmission and confer neuroprotection against insult.

Prenatal choline deficiency increases choline transporter expression in the septum and hippocampus during postnatal development and in adulthood in rats.

Supplementation of maternal diet with the essential nutrient, choline, during the second half of pregnancy in rats causes long-lasting improvements in spatial memory in the offspring and protects them from the memory decline characteristic of old age. In contrast, prenatal choline deficiency is associated with poor performance in certain cognitive tasks. The mechanism by which choline influences learning and memory remains unclear; however, it may involve changes to the hippocampal cholinergic system. Previously, we showed that the hippocampi of prenatally [embryonic days (E) 11-17] choline-deficient animals have increased synthesis of acetylcholine (ACh) from choline transported by the high-affinity choline transporter (CHT) and reduced ACh content relative to the control and to the E11-17 choline-supplemented rats. In the current study, we found that, during postnatal period [postnatal days (P) 18-480], prenatal choline deficiency increased the expression of CHT mRNA in the septum and CHT mRNA and protein levels in the hippocampus and altered the pattern of CHT immunoreactivity in the dentate gyrus. CHT immunoreactivity was more prominent in the inner molecular layer in prenatally choline-deficient rats compared to controls and prenatally choline-supplemented animals. In addition, in all groups, we observed a population of hilar interneurons that were CHT-immunoreactive. These neurons are the likely source of the hippocampal CHT mRNA as their number correlated with the levels of this mRNA. The abundance of hippocampal CHT mRNA rose between P1 and P24 and then declined reaching 60% of the P1 value by P90. These data show that prenatal availability of choline alters its own metabolism (i.e., CHT expression). While the upregulated CHT expression during the period of prenatal choline deficiency may be considered as a compensatory mechanism that could enhance ACh synthesis when choline supply is low, the persistent upregulation of CHT expression subsequent to the brief period of prenatal deprivation of choline in utero might be beneficial during choline deficiency in adulthood.

Prenatal choline availability modulates hippocampal and cerebral cortical gene expression.

An increased supply of the essential nutrient choline during fetal development [embryonic day (E) 11-17] in rats causes life-long improvements in memory performance, whereas choline deficiency during this time impairs certain aspects of memory. We analyzed mRNA expression in brains of prenatally choline-deficient, choline-supplemented, or control rats of various ages [postnatal days (P) 1 to 34 for hippocampus and E16 to P34 for cortex] using oligonucleotide microarrays and found alterations in gene expression levels evoked by prenatal choline intake that were, in most cases, transient occurring during the P15-P34 period. We selected a subset of genes, encoding signaling proteins, and verified the microarray data by reverse transcriptase-polymerase chain reaction analyses. Prenatally choline-supplemented rats had the highest expression of calcium/calmodulin (CaM)-dependent protein kinase (CaMK) I and insulin-like growth factor (IGF) II (Igf2) in the cortex and of the transcription factor Zif268/EGR1 in the cortex and hippocampus. Prenatally choline deficient rats had the highest expression of CaMKIIbeta, protein kinase Cbeta2, and GABA(B) receptor 1 isoforms c and d in the hippocampus. Similar changes in the expression of the proteins encoded by these genes were observed using immunoblot analyses. These data show that the prenatal supply of choline causes multiple modifications in the developmental patterns of expression of genes known to influence learning and memory and provide molecular correlates for the cognitive changes evoked by altered availability of choline in utero.

Developmental periods of choline sensitivity provide an ontogenetic mechanism for regulating memory capacity and age-related dementia.

In order to determine brain and behavioral sensitivity of nutrients that may serve as inductive signals during early development, we altered choline availability to rats during 7 time frames spanning embryonic day (ED) 6 through postnatal day (PD) 75 and examined spatial memory ability in the perinatally-treated adults. Two sensitive periods were identified, ED 12-17 and PD 16-30, during which choline supplementation facilitated spatial memory and produced increases in dendritic spine density in CA1 and dentate gyrus (DG) regions of the hippocampus while also changing the dendritic fields of DG granule cells. Moreover, choline supplementation during ED 12-17 only, prevented the memory decline normally observed in aged rats. These behavioral changes were strongly correlated with the acetylcholine (ACh) content of hippocampal slices following stimulated release. Our data demonstrate that the availability of choline during critical periods of brain development influences cognitive performance in adulthood and old age, and emphasize the importance of perinatal nutrition for successful cognitive aging.

Prenatal choline supplementation advances hippocampal development and enhances MAPK and CREB activation.

Choline is an essential nutrient for animals and humans. Previous studies showed that supplementing the maternal diet with choline during the second half of gestation in rats permanently enhances memory performance of the adult offspring. Here we show that prenatal choline supplementation causes a 3-day advancement in the ability of juvenile rats to use relational cues in a water maze task, indicating that the treatment accelerates hippocampal maturation. Moreover, phosphorylation and therefore activation of hippocampal mitogen-activated protein kinase (MAPK) and cAMP-response element binding protein (CREB) in response to stimulation by glutamate, N-methyl-D-aspartate, or depolarizing concentrations of K+ were increased by prenatal choline supplementation and reduced by prenatal choline deficiency. These data provide the first evidence that developmental plasticity of the hippocampal MAPK and CREB signaling pathways is controlled by the supply of a single essential nutrient, choline, during fetal development and point to these pathways as candidate mechanisms for the developmental and long-term cognitive enhancement induced by prenatal choline supplementation.

Prenatal choline supplementation protects against postnatal neurotoxicity.

Choline, a dietary compound present in many foods, has recently been classified as an essential nutrient for humans. There is evidence from animal models that the availability of choline during the prenatal period influences neural and cognitive development. Here we report that choline supplementation during a 6 d gestational period protects against neurodegeneration in the posterior cingulate and retrosplenial cortices of female adolescent rats produced by peripheral administration of the NMDA receptor antagonist dizocilpine (MK-801). These data show that availability of a single nutrient, choline, during a brief period of prenatal development diminishes vulnerability to neurotoxicity in adolescent offspring.