Folic acid supplementation in a mouse model of diabetes in pregnancy alters insulin sensitivity in female mice and beta cell mass in offspring.

Epidemiological studies have reported discrepant findings on the relationship between folic acid intake during pregnancy and risk for gestational diabetes mellitus (GDM). To begin to understand how folic acid impacts metabolic health during pregnancy, we determined the effects of excess folic acid supplementation (5× recommendation) on maternal and fetal offspring metabolic health. Using a mouse (female C57BL/6J) model of diet-induced diabetes in pregnancy (western diet) and control mice, we show that folic acid supplementation improved insulin sensitivity in the female mice fed the western diet and worsened insulin sensitivity in control mice. We found no unmetabolized folic acid in liver from supplemented mice suggesting the metabolic effects of folic acid supplementation are not due to unmetabolized folic acid. Male fetal (gestational day 18.5) offspring from folic acid supplemented dams (western and control) had greater beta cell mass and density than those from unsupplemented dams; this was not observed in female offspring. Differential sex-specific hepatic gene expression profiles were observed in the fetal offspring from supplemented dams but this differed between western and controls. Our findings suggest that folic acid supplementation affects insulin sensitivity in female mice, but is dependent on their metabolic phenotype and has sex-specific effects on offspring pancreas and liver.

Homocysteine-a retrospective and prospective appraisal.

The biologically important amino acid homocysteine links sulfur, methionine, and one-carbon metabolism. This review describes its initial discovery, the identification of the clinical condition of "homocystinuria" and the recognition of its close relationship to folate and vitamin B12 metabolism. It discusses the history behind its current association with diverse diseases including neural tube defects, cardio- and cerebrovascular disease and, more recently, dementia and Alzheimer's Disease. It also explores current controversies and considers potential future research directions. It is intended to give a general overview of homocysteine in relation to health and disease.

Vitamin B12 deficiency.

Of the water-soluble vitamins, vitamin B12 (B12) has the lowest daily requirement. It also has several unique properties including a complex pathway for its absorption and assimilation requiring intact gastric and terminal small intestinal function, an enterohepatic pathway, and several dedicated binding proteins and chaperons. The many causes of B12 deficiency include malabsorption and defects in cellular delivery and uptake, as well as limited dietary intake. B12 is required as a cofactor for only two reactions in humans, the cytosolic methionine synthase reaction and the mitochondrial methymalonyl CoA mutase reaction. Disruption of either of these reactions gives rise to B12 deficiency. Although more common with advancing age, because of the higher prevalence of malabsorptive disorders in the elderly, B12 deficiency is widely distributed across all age groups particularly where food insecurity occurs. The consequences and severity of B12 deficiency are variable depending on the degree of deficiency and its duration. Major organ systems affected include the blood, bone marrow and nervous system. Megaloblastic anemia results from a defect in thymidine and therefore DNA synthesis in rapidly dividing cells. Nervous system involvement is varied, some of which results from defective myelin synthesis and repair. Cognitive impairment and psychosis may also occur. Diagnosis of B12 deficiency rests on clinical suspicion followed by laboratory testing, which consists of a panel of tests, that together provide clinically reliable predictive indices. B12 metabolism and deficiency is closely intertwined with folate, another B-vitamin. This chapter explores the various aspects of a unique and fascinating micronutrient.

Associations between Genetic Variants and Blood Biomarkers of One-Carbon Metabolism in Postmenopausal Women from the Women's Health Initiative Observational Study.

BACKGROUND: Genetic variation in one-carbon metabolism may affect nutrient concentrations and biological functions. However, data on genetic variants associated with blood biomarkers of one-carbon metabolism in US postmenopausal women are limited, and whether these associations were affected by the nationwide folic acid (FA) fortification program is unclear. OBJECTIVES: We investigated associations between genetic variants and biomarkers of one-carbon metabolism using data from the Women's Health Initiative Observational Study. METHODS: In 1573 non-Hispanic White (NHW) and 282 Black/African American, American Indian/Alaska Native, Asian/Pacific Islander, and Hispanic/Latino women aged 50-79 y, 288 nonsynonymous and tagging single-nucleotide variants (SNVs) were genotyped. RBC folate, plasma folate, pyridoxal-5'-phosphate (PLP), vitamin B-12, homocysteine, and cysteine concentrations were determined in 12-h fasting blood. Multivariable linear regression tested associations per variant allele and for an aggregated genetic risk score. Effect modifications before, during, and after nationwide FA fortification were examined. RESULTS: After correction for multiple comparisons, among NHW women, 5,10-methylenetetrahydrofolate reductase (MTHFR) rs1801133 (677C→T) variant T was associated with lower plasma folate (-13.0%; 95% CI: -17.3%, -8.6%) and higher plasma homocysteine (3.5%; 95% CI: 1.7%, 5.3%) concentrations. Other associations for nonsynonymous SNVs included DNMT3A rs11695471 (T→A) with plasma PLP; EHMT2 rs535586 (G→A), TCN2 rs1131603 (L349S A→G), and TCN2 rs35838082 (R188W G→A) with plasma vitamin B-12; CBS rs2851391 (G→A) with plasma homocysteine; and MTHFD1 rs2236224 (G→A) and rs2236225 (R653Q G→A) with plasma cysteine. The influence of FA fortification on the associations was limited. Highest compared with lowest quartiles of aggregated genetic risk scores from SNVs in MTHFR and MTRR were associated with 14.8% to 18.9% lower RBC folate concentrations. Gene-biomarker associations were similar in women of other races/ethnicities. CONCLUSIONS: Our findings on genetic variants associated with several one-carbon metabolism biomarkers may help elucidate mechanisms of maintaining B vitamin status in postmenopausal women.

Perspective: The High-Folate-Low-Vitamin B-12 Interaction Is a Novel Cause of Vitamin B-12 Depletion with a Specific Etiology-A Hypothesis.

Vitamin B-12 is a water-soluble vitamin that plays important roles in intermediary metabolism. Vitamin B-12 deficiency has many identifiable causes, including autoimmune and other gastrointestinal malabsorption disorders, dietary deficiency, and congenital defects in genes that are involved in vitamin B-12 trafficking and functions. Another putative cause of vitamin B-12 deficiency is the high-folate-low vitamin B-12 interaction, first suspected as the cause for observed relapse and exacerbation of the neurological symptoms in patients with pernicious anemia who were prescribed high oral doses of folic acid. We propose that this interaction is real and represents a novel cause of vitamin B-12 depletion with specific etiology. We hypothesize that excessive intake of folic acid depletes serum holotranscobalamin (holoTC), thereby decreasing active vitamin B-12 in the circulation and limiting its availability for tissues. This effect is specific for holoTC and does not affect holohaptocorrin, the inert form of serum vitamin B-12. Depletion of holoTC by folic acid in individuals with already low vitamin B-12 status further compromises the availability of vitamin B-12 coenzymes to their respective enzymes, and consequently a more pronounced state of biochemical deficiency. This hypothesis is drawn from evidence of observational and intervention studies of vitamin B-12-deficient patients and epidemiological cohorts. The evidence also suggests that, in a depleted state, vitamin B-12 is diverted to the hematopoietic system or the kidney. This most likely reflects a selective response of tissues expressing folate receptors with high affinity for unmetabolized folic acid (UMFA; e.g., hematopoietic progenitors and renal tubules) compared with those tissues (e.g., liver) that only express the reduced folate carrier, which is universally expressed but has poor affinity for UMFA. The biochemical and physiological mechanisms underlying this interaction require elucidation to clarify its potential public health significance.

Sex-specific effects of neonatal oral sucrose treatment on growth and liver choline and glucocorticoid metabolism in adulthood.

Hospitalized preterm infants experience painful medical procedures. Oral sucrose is the nonpharmacological standard of care for minor procedural pain relief. Infants are treated with numerous doses of sucrose, raising concerns about potential long-term effects. The objective of this study was to determine the long-term effects of neonatal oral sucrose treatment on growth and liver metabolism in a mouse model. Neonatal female and male mice were randomly assigned to one of two oral treatments (n = 7-10 mice/group/sex): sterile water or sucrose. Pups were treated 10 times/day for the first 6 days of life with 0.2 mg/g body wt of respective treatments (24% solution; 1-4 µL/dose) to mimic what is given to preterm infants. Mice were weaned at age 3 wk onto a control diet and fed until age 16 wk. Sucrose-treated female and male mice gained less weight during the treatment period and were smaller at weaning than water-treated mice (P ≤ 0.05); no effect of sucrose treatment on body weight was observed at adulthood. However, adult sucrose-treated female mice had smaller tibias and lower serum insulin-like growth factor-1 than adult water-treated female mice (P ≤ 0.05); these effects were not observed in males. Lower liver S-adenosylmethionine, phosphocholine, and glycerophosphocholine were observed in adult sucrose-treated compared with water-treated female and male mice (P ≤ 0.05). Sucrose-treated female, but not male, mice had lower liver free choline and higher liver betaine compared with water-treated female mice (P < 0.01). Our findings suggest that repeated neonatal sucrose treatment has long-term sex-specific effects on growth and liver methionine and choline metabolism.

Perspective: Planning and Conducting Statistical Analyses for Human Nutrition Randomized Controlled Trials: Ensuring Data Quality and Integrity.

Appropriate planning, execution, and reporting of statistical methods and results is critical for research transparency, validity, and reproducibility. This paper provides an overview of best practices for developing a statistical analysis plan a priori, conducting statistical analyses, and reporting statistical methods and results for human nutrition randomized controlled trials (RCTs). Readers are referred to the other NURISH (NUtrition inteRventIon reSearcH) publications for detailed information about the preparation and conduct of human nutrition RCTs. Collectively, the NURISH series outlines best practices for conducting human nutrition research.

Letrozole-Induced Polycystic Ovary Syndrome Attenuates Cystathionine-ß Synthase mRNA and Protein Abundance in the Ovaries of Female Sprague Dawley Rats.

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects 10% of reproductive-aged women and leads to hyperandrogenism, anovulation, and infertility. PCOS has been associated with elevated serum homocysteine as well as altered methylation status; however, characterization of one-carbon metabolism (OCM) in PCOS remains incomplete. OBJECTIVES: The aim of our research was to assess OCM in a letrozole-induced Sprague Dawley rat model of PCOS. METHODS: Five-week-old female rats (n = 36) were randomly assigned to letrozole [0.9 mg/kg body weight (BW)] treatment or vehicle (carboxymethylcellulose) control that was administered via subcutaneously implanted slow-release pellets every 30 d. For both treatment groups, 12 rats were randomly assigned to be euthanized during proestrus at one of the following time points: 8, 16, or 24 wk of age. Daily BW was measured and estrous cyclicity was monitored during the last 30 d of the experimental period. Ovaries were collected to assess mRNA and protein abundance of OCM enzymes. RESULTS: Letrozole-induced rats exhibited 1.9-fold higher cumulative BW gain compared with control rats across all age groups (P < 0.0001). Letrozole reduced the time spent at proestrus (P = 0.0001) and increased time in metestrus (P < 0.0001) of the estrous cycle. Cystathionine ß-synthase (Cbs) mRNA abundance was reduced in the letrozole-induced rats at 16 (59%; P < 0.05) and 24 (77%; P < 0.01) wk of age. In addition, CBS protein abundance was 32% lower in 8-wk-old letrozole-induced rats (P = 0.02). Interestingly, betaine-homocysteine S-methyltransferase mRNA abundance increased as a function of age in letrozole-induced rats (P = 0.03). CONCLUSION: These data demonstrate that letrozole-induced PCOS Sprague Dawley rats temporally decrease the ovarian abundance of Cbs mRNA and protein in the early stages of PCOS.

Physiologic Responses to Dietary Sulfur Amino Acid Restriction in Mice Are Influenced by Atf4 Status and Biological Sex.

BACKGROUND: Dietary sulfur amino acid restriction (SAAR) improves body composition and metabolic health across several model organisms in part through induction of the integrated stress response (ISR). OBJECTIVE: We investigate the hypothesis that activating transcription factor 4 (ATF4) acts as a converging point in the ISR during SAAR. METHODS: Using liver-specific or global gene ablation strategies, in both female and male mice, we address the role of ATF4 during dietary SAAR. RESULTS: We show that ATF4 is dispensable in the chronic induction of the hepatokine fibroblast growth factor 21 while being essential for the sustained production of endogenous hydrogen sulfide. We also affirm that biological sex, independent of ATF4 status, is a determinant of the response to dietary SAAR. CONCLUSIONS: Our results suggest that auxiliary components of the ISR, which are independent of ATF4, are critical for SAAR-mediated improvements in metabolic health in mice.

Perspective: Laboratory Considerations and Clinical Data Management for Human Nutrition Randomized Controlled Trials: Guidance for Ensuring Quality and Integrity.

In human nutrition randomized controlled trials (RCTs), planning, and careful execution of clinical data collection and management are vital for producing valid and reliable results. In this article, we provide an overview of best practices for biospecimen collection and analyses, and for the fundamentals of clinical data management, including preparation and study startup; data collection, entry, cleaning, and authentication; and database lock. The reader is also referred to additional resources for information to assist in the planning and conduct of human RCTs. The tools and strategies described are expected to improve the quality of data produced in human nutrition research that can, therefore, be used to support food and nutrition policies.

Expression Changes in Epigenetic Gene Pathways Associated With One-Carbon Nutritional Metabolites in Maternal Blood From Pregnancies Resulting in Autism and Non-Typical Neurodevelopment.

The prenatal period is a critical window for the development of autism spectrum disorder (ASD). The relationship between prenatal nutrients and gestational gene expression in mothers of children later diagnosed with ASD or non-typical development (Non-TD) is poorly understood. Maternal blood collected prospectively during pregnancy provides insights into the effects of nutrition, particularly one-carbon metabolites, on gene pathways and neurodevelopment. Genome-wide transcriptomes were measured with microarrays in 300 maternal blood samples in Markers of Autism Risk in Babies-Learning Early Signs. Sixteen different one-carbon metabolites, including folic acid, betaine, 5'-methyltretrahydrofolate (5-MeTHF), and dimethylglycine (DMG) were measured. Differential expression analysis and weighted gene correlation network analysis (WGCNA) were used to compare gene expression between children later diagnosed as typical development (TD), Non-TD and ASD, and to one-carbon metabolites. Using differential gene expression analysis, six transcripts (TGR-AS1, SQSTM1, HLA-C, and RFESD) were associated with child outcomes (ASD, Non-TD, and TD) with genome-wide significance. Genes nominally differentially expressed between ASD and TD significantly overlapped with seven high confidence ASD genes. WGCNA identified co-expressed gene modules significantly correlated with 5-MeTHF, folic acid, DMG, and betaine. A module enriched in DNA methylation functions showed a suggestive protective association with folic acid/5-MeTHF concentrations and ASD risk. Maternal plasma betaine and DMG concentrations were associated with a block of co-expressed genes enriched for adaptive immune, histone modification, and RNA processing functions. These results suggest that the prenatal maternal blood transcriptome is a sensitive indicator of gestational one-carbon metabolite status and changes relevant to children's later neurodevelopmental outcomes. LAY SUMMARY: Pregnancy is a time when maternal nutrition could interact with genetic risk for autism spectrum disorder. Blood samples collected during pregnancy from mothers who had a prior child with autism were examined for gene expression and nutrient metabolites, then compared to the diagnosis of the child at age three. Expression differences in gene pathways related to the immune system and gene regulation were observed for pregnancies of children with autism and non-typical neurodevelopment and were associated with maternal nutrients.

Knowledge gaps in understanding the metabolic and clinical effects of excess folates/folic acid: a summary, and perspectives, from an NIH workshop.

Folate, an essential nutrient found naturally in foods in a reduced form, is present in dietary supplements and fortified foods in an oxidized synthetic form (folic acid). There is widespread agreement that maintaining adequate folate status is critical to prevent diseases due to folate inadequacy (e.g., anemia, birth defects, and cancer). However, there are concerns of potential adverse effects of excess folic acid intake and/or elevated folate status, with the original concern focused on exacerbation of clinical effects of vitamin B-12 deficiency and its role in neurocognitive health. More recently, animal and observational studies have suggested potential adverse effects on cancer risk, birth outcomes, and other diseases. Observations indicating adverse effects from excess folic acid intake, elevated folate status, and unmetabolized folic acid (UMFA) remain inconclusive; the data do not provide the evidence needed to affect public health recommendations. Moreover, strong biological and mechanistic premises connecting elevated folic acid intake, UMFA, and/or high folate status to adverse health outcomes are lacking. However, the body of evidence on potential adverse health outcomes indicates the need for comprehensive research to clarify these issues and bridge knowledge gaps. Three key research questions encompass the additional research needed to establish whether high folic acid or total folate intake contributes to disease risk. 1) Does UMFA affect biological pathways leading to adverse health effects? 2) Does elevated folate status resulting from any form of folate intake affect vitamin B-12 function and its roles in sustaining health? 3) Does elevated folate intake, regardless of form, affect biological pathways leading to adverse health effects other than those linked to vitamin B-12 function? This article summarizes the proceedings of an August 2019 NIH expert workshop focused on addressing these research areas.

Associations between Plasma Choline Metabolites and Genetic Polymorphisms in One-Carbon Metabolism in Postmenopausal Women: The Women's Health Initiative Observational Study.

BACKGROUND: Choline plays an integral role in one-carbon metabolism in the body, but it is unclear whether genetic polymorphisms are associated with variations in plasma choline and its metabolites. OBJECTIVES: This study aimed to evaluate the association of genetic variants in choline and one-carbon metabolism with plasma choline and its metabolites. METHODS: We analyzed data from 1423 postmenopausal women in a case-control study nested within the Women's Health Initiative Observational Study. Plasma concentrations of choline, betaine, dimethylglycine (DMG), and trimethylamine N-oxide were determined in 12-h fasting blood samples collected at baseline (1993-1998). Candidate and tagging single-nucleotide polymorphisms (SNPs) were genotyped in betaine-homocysteine S-methyltransferase (BHMT), BHMT2, 5,10-methylenetetrahydrofolate reductase (MTHFR), methylenetetrahydrofolate dehydrogenase (NADP+ dependent 1) (MTHFD1), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR). Linear regression was used to derive percentage difference in plasma concentrations per variant allele, adjusting for confounders, including B-vitamin biomarkers. Potential effect modification by plasma vitamin B-12, vitamin B-6, and folate concentrations and folic-acid fortification periods was examined. RESULTS: The candidate SNP BHMT R239Q (rs3733890) was associated with lower concentrations of plasma betaine and DMG concentrations (-4.00% and -6.75% per variant allele, respectively; both nominal P < 0.05). Another candidate SNP, BHMT2 rs626105 A>G, was associated with higher plasma DMG concentration (13.0%; P < 0.0001). Several tagSNPs in these 2 genes were associated with plasma concentrations after correction for multiple comparisons. Vitamin B-12 status was a significant effect modifier of the association between the genetic variant BHMT2 rs626105 A>G and plasma DMG concentration. CONCLUSIONS: Genetic variations in metabolic enzymes were associated with plasma concentrations of choline and its metabolites. Our findings contribute to the knowledge on the variation in blood nutrient concentrations in postmenopausal women.

Exercise during pregnancy mitigates the adverse effects of maternal obesity on adult male offspring vascular function and alters one-carbon metabolism.

Maternal obesity during pregnancy can adversely affect adult offspring vascular endothelial function. This study examined whether maternal exercise during pregnancy and lactation mitigates the adverse effects of maternal obesity on offspring vascular endothelial function. Female (C57BL/6N) mice were fed from weaning a control diet (10% kcal fat) or western diet (45% kcal fat) to induce excess adiposity (maternal obesity). After 13 weeks, the female mice were bred and maintained on the diets, with and without access to a running wheel (exercise), throughout breeding, pregnancy, and lactation. Offspring were weaned onto the control or western diet and fed for 13 weeks; male offspring were studied. Maternal exercise prevented the adverse effects of maternal obesity on offspring vascular endothelial function. However, this was dependent on offspring diet and the positive effect of maternal exercise was only observed in offspring fed the western diet. This was accompanied by alterations in aorta and liver one-carbon metabolism, suggesting a role for these pathways in the improved endothelial function observed in the offspring. Obesity and exercise had no effect on endothelial function in the dams but did affect aorta and liver one-carbon metabolism, suggesting the phenotype observed in the offspring may be due to obesity and exercise-induced changes in one-carbon metabolism in the dams. Our findings demonstrate that maternal exercise prevented vascular dysfunction in male offspring from obese dams and is associated with alterations in one-carbon metabolism.

Low-vitamin-D diet lowers cerebral serotonin concentration in mature female mice.

Low circulating 25-hydroxyvitamin D (25OHD) is commonly found in obese individuals and is often attributed to a volume dilution effect of adipose tissue. However, low vitamin D (LD) intake may contribute to the obesity itself. In this study, we examine whether low vitamin D status contributes to increased food intake and weight gain and can be explained by altered brain serotonin metabolism in 8-month-old female C57BL/6J mice. In a first experiment, mice were fed a 45% high-fat diet (HFD) containing different amounts of vitamin D at low (100 IU/kg), normal (1,000 IU/kg) or high (10,000 IU/kg) intake. After 10 weeks, mice fed LD had greater energy intake, weight gain, total and hepatic fat than the higher vitamin D groups (P < .05). In a second experiment, mice were examined for the central serotonin regulation of food intake after a 10% normal-fat diet (NFD) or 45% HFD containing low (100 IU/kg) or normal (1000 IU/kg) vitamin D. After 10 weeks, both HFD and LD diets attenuated circulating 25OHD concentration. Additionally, LD intake lowered cortical serotonin level, regardless of dietary fat intake (P < .05). In the arcuate and raphe nuclei, gene expression of vitamin D 1α-hydroxylase was lower due to LD during HFD feeding (P < .05). Tryptophan hydroxylase-2 and serotonin reuptake transporter gene expression was not altered due to LD. Overall, these findings suggest that a LD diet alters peripheral 25OHD, reduces central serotonin, and may contribute to weight gain in an obesogenic environment.

High folic acid or folate combined with low vitamin B-12 status: potential but inconsistent association with cognitive function in a nationally representative cross-sectional sample of US older adults participating in the NHANES.

BACKGROUND: Potential safety concerns relative to impaired cognitive function may exist when high folic acid exposures are combined with low vitamin B-12 status. OBJECTIVES: We aimed to examine the relation of the coexistence of high folate and low vitamin B-12 status with cognitive function, utilizing various definitions of "high" folate status. METHODS: Cross-sectional data from older adults (≥60 y; n = 2420) from the 2011-2014 NHANES were analyzed. High folate status was defined as unmetabolized serum folic acid (UMFA) > 1 nmol/L or serum total folate > 74.1 nmol/L, and low vitamin B-12 status as methylmalonic acid > 271 nmol/L or serum vitamin B-12 < 150 pmol/L. Logistic regression models estimated ORs of scoring low on 1 of 4 cognitive tests: the Digit Symbol Substitution Test (DSST), the Consortium to Establish a Registry for Alzheimer's Disease Delayed Recall (CERAD-DR) and Word Learning tests, and the Animal Fluency test (AF). RESULTS: A significant interaction was observed relative to scoring low on the DSST (<34; UMFA; P-interaction = 0.0071) and AF (serum folate; P-interaction = 0.0078) for low vitamin B-12 and high folate status. Among those with low vitamin B-12, high UMFA or high serum total folate was associated with higher risk of scoring low on the DSST (OR: 2.16; 95% CI: 1.05, 4.47) and the AF (OR: 1.93; 95% CI: 1.08, 3.45). Among those with "normal" vitamin B-12, higher UMFA or serum total folate was protective on the CERAD-DR. In noninteraction models, when high folate and normal vitamin B-12 status was the reference group, low vitamin B-12 combined with high UMFA was associated with greater risk based on the DSST (<34, OR: 2.87; 95% CI: 1.85, 4.45; <40, OR: 2.22; 95% CI: 1.31, 3.75) and AF (OR: 1.97; 95% CI: 1.30, 2.97); but low vitamin B-12 and lower UMFA (OR: 1.69; 95% CI: 1.16, 2.47) was also significantly associated for DSST < 40 risk. CONCLUSIONS: Low vitamin B-12 was associated with cognitive impairment both independently and in an interactive manner with high folate for certain cognitive performance tests among older adults.

Folate Deficiency Inhibits Development of the Mammary Gland and its Associated Lymphatics in FVB Mice.

BACKGROUND: Folate is essential for DNA synthesis, DNA repair, cell proliferation, development, and morphogenesis. Folic acid (FA) is a nutritional supplement used to fortify human diets. OBJECTIVES: We investigated the effects of dietary FA on early mammary gland (MG) development and hyperplasia. METHODS: Study 1: nulliparous female FVB wild-type (WT) mice were fed control (Con; 2 mg FA/kg), deficient (Def; 0 mg FA/kg), excess (Ex; 5 mg FA/kg), or super excess (S-Ex; 20 mg FA/kg) diets for 8 wk before mating to WT or heterozygous FVB/N-Tg[mouse mammary tumor virus long terminal repeat (MMTV)-polyomavirus middle T antigen (PyVT)]634Mul/J (MMTV-PyMT+/-) transgenic males. Dams were fed these diets until they weaned WT or MMTV-PyMT+/- pups, which were fed the dam's diet from postnatal day (PND) 21 to 42. Tissues were collected from female progeny at PNDs 1, 21, and 42. Study 2: Con or Def diets were fed to WT intact females and males from PND 21 to 56, or to ovariectomized females from PND 21 to 77; tissues were collected at PND 56 or 77. Growth of all offspring, development of MGs, MG hyperplasia, supramammary lymph nodes, thymus and spleen, cell proliferation, and expression of MG growth factors were measured. RESULTS: Study 1: Ex or S-Ex did not affect postnatal MG development or hyperplasia. The rate of isometric MG growth (PND 1-21) was reduced by 69% in Def female progeny (P < 0.0001). Similarly, hyperplastic growth in MGs of Def MMTV-PyMT+/- offspring was 18% of Con (P < 0.05). The Def diet reduced supramammary lymph node size by 20% (P < 0.0001) and increased MG insulin-like growth factor 2 mRNA by 200% (P < 0.05) and protein by 130%-150% (P < 0.05). Study 2: the Def diet did not affect MG growth, but it did reduce supramammary lymph node size (P < 0.05), spleen weight (P < 0.001), and thymic medulla area (P < 0.05). CONCLUSIONS: In utero and postnatal folate deficiency reduced the isometric development of the MGs and early MG hyperplasia. Postnatal folate deficiency reduced the development of lymphatic tissues.