AMPK/mTOR downregulated autophagy enhances aberrant endometrial decidualization in folate-deficient pregnant mice.

Existing evidence suggests that adverse pregnancy outcomes are closely related to dietary factors. Folate plays an important role in neural tube formation and fetal growth, folate deficiency is a major risk factor of birth defects. Our early studies showed that folate deficiency could impair enddecidualization, however, the mechanism is still unclear. Dysfunctional autophagy is associated with many diseases. Here, we aimed to evaluate the adverse effect of folate deficiency on endometrial decidualization, with a particular focus on endometrial cell autophagy. Mice were fed with no folate diet in vivo and the mouse endometrial stromal cell was cultured in a folate-free medium in vitro. The decrease of the number of endometrial autophagosomes and the protein expressions of autophagy in the folate-deficient group indicated that autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation were inhibited. Autophagic flux examination using mCherry-GFP-LC3 transfection showed that the fusion of autophagosomes with lysosomes was inhibited by folate deficiency. Autophagy inducer rapamycin could reverse the impairment of folate deficiency on endometrial decidualization. Moreover, folate deficiency could reduce autophagy by disrupting AMPK/mTOR signaling, resulting in aberrant endometrial decidualization and adverse pregnancy outcomes. Further co-immunoprecipitation examination showed that decidual marker protein Hoxa10 could interact with autophagic marker protein Cathepsin L, and the interaction was notably reduced by folate deficiency. In conclusion, AMPK/mTOR downregulated autophagy was essential for aberrant endometrial decidualization in early pregnant mice, which could result in adverse pregnancy outcomes. This provided some new clues for understanding the causal mechanisms of birth defects induced by folate deficiency.

A proton-coupled folate transporter mutation causing hereditary folate malabsorption locks the protein in an inward-open conformation.

The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.

The deficit in folate and vitamin B12 triggers liver macrovesicular steatosis and inflammation in rats with dextran sodium sulfate-induced colitis.

The risks of nonalcoholic steatohepatitis (NASH) and deficiency in vitamin B12 and folate (methyl donor deficiency, MDD) are increased in inflammatory bowel disease (IBD). We investigated the influence of MDD on NASH in rats with DSS-induced colitis. Two-month-old male Wistar rats were subjected to MDD diet and/or ingestion of DSS and compared to control animals. We studied steatosis, inflammation, fibrosis, plasma levels of metabolic markers, cytokines and lipopolysaccharide, and inflammatory pathways in liver. MDD triggered a severe macrovesicular steatosis with inflammation in DSS animals that was not observed in animals subjected to DSS or MDD only. The macrovesicular steatosis was closely correlated to folate, vitamin B12, homocysteine plasma level and liver S-adenosyl methionine/S-adenosyl homocysteine (SAM/SAH) ratio. Liver inflammation was evidenced by activation of nuclear factor kappa B (NFκB) pathway and nuclear translocation of NFκB phospho-p65. MDD worsened the increase of interleukin 1-beta (IL-1ß) and abolished the increase of IL10 produced by DSS colitis. It increased monocyte chemoattractant protein 1 (MCP-1). MDD triggers liver macrovesicular steatosis and inflammation through imbalanced expression of IL-1ß vs. IL10 and increase of MCP-1 in DSS colitis. Our results suggest evaluating whether IBD patients with MDD and increase of MCP-1 are at higher risk of NASH.

Folate Repletion after Deficiency Induces Irreversible Genomic and Transcriptional Changes in Human Papillomavirus Type 16 (HPV16)-Immortalized Human Keratinocytes.

Supplementation of micronutrients like folate is a double-edged sword in terms of their ambivalent role in cell metabolism. Although several epidemiological studies support a protective role of folate in carcinogenesis, there are also data arguing for an opposite effect. To address this issue in the context of human papillomavirus (HPV)-induced transformation, the molecular events of different folate availability on human keratinocytes immortalized by HPV16 E6 and E7 oncoproteins were examined. Several sublines were established: Control (4.5 µM folate), folate deficient (0.002 µM folate), and repleted cells (4.5 µM folate). Cells were analyzed in terms of oncogene expression, DNA damage and repair, karyotype changes, whole-genome sequencing, and transcriptomics. Here we show that folate depletion irreversibly induces DNA damage, impairment of DNA repair fidelity, and unique chromosomal alterations. Repleted cells additionally underwent growth advantage and enhanced clonogenicity, while the above mentioned impaired molecular properties became even more pronounced. Overall, it appears that a period of folate deficiency followed by repletion can shape immortalized cells toward an anomalous phenotype, thereby potentially contributing to carcinogenesis. These observations should elicit questions and inquiries for broader additional studies regarding folate fortification programs, especially in developing countries with micronutrient deficiencies and high HPV prevalence.

High concentration of sugars is genotoxic to folate-deficient cells.

Patients with type 2 diabetes mellitus (T2DM) are associated with an elevated, but poorly understood baseline of genomic instability (GIN). Expert panels are still debating on whether hyperglycemia is the key element in conferring this high GIN. Since high blood glucose and low blood folate are prevalent in T2DM, we hypothesized that high glucose may work with low folate to induce GIN. Using NCM460, CCD841 and L02 cell lines as in vitro cell models, we investigated the genotoxic effects of high sugars (HS; 1-2% glucose, fructose, galactose or sucrose) alone or in combination with folate deficiency (23 nM, FD) over a course of 7 days by the cytokinesis block micronucleus assay. We found that HS is nongenotoxic to NCM460, CCD841 and L02 cells. However, the combination of HS and FD induced significantly higher levels of micronuclei, nucleoplasmic bridges and nuclear buds. Our in vitro work demonstrates that HS is non-genotoxic under folate repletive condition, but is genotoxic under FD condition. These results provide preclinal proof of concept that concomitant hyperglycemia and low folate may explain, at least in part, the high baseline of GIN in T2DM patients, suggesting that folate levels should be kept under control in order to limit the risk of GIN and carcinogenesis in T2DM.

A case of subacute combined degeneration of the spinal cord due to folic acid and copper deficiency.

Subacute combined degeneration of the spinal cord (SACD) is a rare neurologic disorder manifesting progressive symptoms of paresthesia and spastic paralysis. Herein we present an autopsy case of SACD caused by folic acid and copper deficiency. A 16-year-old male presented with gradually worsening unsteady gait, and bladder and rectal dysfunction. He had a medical history of T-cell acute lymphoblastic leukemia (T-ALL), diagnosed 1.5 years previously. The patient had undergone chemotherapy, including methotrexate, as well as allogeneic bone mallow transplantation. Laboratory tests revealed normal vitamin B12 and methylmalonic acid concentration, but reduced serum copper, ceruloplasmin and folic acid concentrations. Magnetic resonance imaging revealed symmetrical T2 signal hyperintensities in the posterior and lateral spinal cord. The patient was treated with oral copper, oral folate, and intravenous vitamin B12. A month after this treatment, the patient's symptoms were unchanged, and 2 months later he died of acute adrenal insufficiency. The pathological findings of the spinal cord were compatible with SACD. Because SACD is usually reversible with early treatment, it should be suspected in high-risk patients undergoing chemotherapy or those who are malnourished with characteristic symptoms of SACD, even in young patients.

Widening the phenotypic spectrum - Non epileptic presentation of folate transporter deficiency.

INTRODUCTION: Folate is essential for production of DNA, neurotransmitters and myelin and regulation of genetic activity. A specific transporter protein is required to transport folate from blood to CSF. Various inherited brain-specific folate transport defects have been recognized due to mutation in Folate Receptor alpha (FOLR1). FOLR1 mutation is one of the vitamin responsive encephalopathies and is inherited as an autosomal recessive condition. It has a wide spectrum of phenotype, commonly presenting as epileptic encephalopathy. Less frequently the condition may manifest with subtle hypotonia, movement disorder as tremors, ataxia or intellectual disability and autistic spectrum disorder. We present a case of folate transporter deficiency with non-epileptic manifestations, presenting with tremors, speech delay and stable white matter changes in MRI brain. OBJECTIVE: We present a case of Folate transporter defect with Non-epileptic presentation. CONCLUSION: Folate transporter deficiency has a wide range of presenting symptoms. Presentation with slowly progressive atypical symptoms, stable white matter changes in brain MRI that does not fit a specific diagnosis, should raise a high suspicion of FOLR1 mutation, even in absence of seizures. Since folate transporter deficiency is a treatable neurodegenerative disorder, early diagnosis and supplementation with folinic acid is vital.

[Neurological pathology associated with vitamin B group deficiency: thiamine, folate and cobalamin]. / Patología neurológica por déficit de vitaminas del grupo B: tiamina, folato y cobalamina.

The "fragility" of the nervous system, especially concerning to its nutrition and metabolism, explains why vitamin deficits are an important cause of neurological pathology. Some deficiency diseases, which can be very severe and irreversible, are still present in our environment; diagnosis, which must be early so as not to delay treatment, can be difficult if we do not have them in mind. In this review we address the most relevant neurological diseases associated with thiamine, folate and cobalamin deficiency, and we focus especially combined subacute degeneration and Wernicke-Korsakoff syndrome.

La "fragilidad" del sistema nervioso, en especial en lo referente a su nutrición y metabolismo, explica que los déficits vitamínicos sean una causa importante de patología neurológica. Algunas enfermedades carenciales, que pueden ser muy graves e irreversibles, aún se presentan en nuestro entorno. Su diagnóstico, que debe ser precoz para no retrasar el tratamiento, puede ser complicado si no las tenemos en mente. En esta revisión abordamos las enfermedades neurológicas más relevantes asociadas al déficit de tiamina, folatos y cobalamina, deteniéndonos especialmenteen la degeneración combinada subaguda y el síndrome de Wernicke-Korsakoff.

Folate deficiency increases chromosomal damage and mutations in hematopoietic cells in the transgenic mutamouse model.

Folate deficiency causes megaloblastic anemia and neural tube defects, and is also associated with some cancers. In vitro, folate deficiency increases mutation frequency and genome instability, as well as exacerbates the mutagenic potential of known environmental mutagens. Conversely, it remains unclear whether or not elevated folic acid (FA) intakes are beneficial or detrimental to the induction of DNA mutations and by proxy human health. We used the MutaMouse transgenic model to examine the in vivo effects of FA deficient, control, and supplemented diets on somatic DNA mutant frequency (MF) and genome instability in hematopoietic cells. We also examined the interaction between FA intake and exposure to the known mutagen N-ethyl-N-nitrosourea (ENU) on MF. Male mice were fed the experimental diets for 20 weeks from weaning. Half of the mice from each diet group were gavaged with 50 mg/kg body weight ENU after 10 weeks on diet and remained on their respective diet for an additional 10 weeks. Mice fed a FA-deficient diet had a 1.3-fold increase in normochromatic erythrocyte micronucleus (MN) frequency (P = 0.034), and a doubling of bone marrow lacZ MF (P = 0.035), compared to control-fed mice. Mice exposed to ENU showed significantly higher bone marrow lacZ and Pig-a MF, but there was no effect of FA intake on ENU-induced MF. These data indicate that FA deficiency increases mutations and MN formation in highly proliferative somatic cells, but that FA intake does not mitigate ENU-induced mutations. Also, FA intake above adequacy had no beneficial or detrimental effect on mutations or MN formation. Environ. Mol. Mutagen. 59:366-374, 2018. © 2018 Her Majesty the Queen in Right of Canada 2018.

Craniofacial structure alterations of foetuses from folic acid deficient pregnant mice.

INTRODUCTION: Craniofacial development in mammals is a complex process that involves a coordinated series of molecular and morphogenetic events. Folic acid (FA) deficiency has historically been associated with congenital spinal cord malformations, but the effect that a maternal diet deficient in FA has on the development of other structures has been poorly explored. In the present study, the objective was to describe and quantify the alterations of craniofacial structures presented in mouse foetuses from dams fed a FA deficient (FAD) diet compared with controls that were given a regular maternal diet. MATERIAL AND METHODS: E17 mouse foetuses were removed from dams that were fed with a control diet or with a FAD diet for several weeks. Foetuses with maternal FAD diets were selected for the study when they showed an altered tongue or mandible. Histological sections were used to quantify the dimensions of the head, tongue, mandibular bone and masseter muscle areas using ImageJ software. The muscles of the tongue, suprahyoid muscles, lingual septum, submandibular ducts, and lingual arteries were also analysed. RESULTS: The heads of malformed foetuses were smaller than the heads of the controls, and they showed different types of malformations: microglossia with micrognathia (some of which were combined with cleft palate) and aglossia with either micrognathia or agnathia. Lingual and suprahyoid muscles were affected in different forms and degrees. We also found alterations in the lingual arteries and in the ducts of the submandibular glands. Summarised we can state that pharyngeal arches-derived structures were affected, and the main malformations observed corroborate the vulnerability of cranial neural crest cells to FA deficiency. CONCLUSION: The present study reveals alterations in the development of craniofacial structures in FAD foetuses. This study provides a new focus for the role of FA during embryological development.

Folate deficiency inhibits the PCP pathway and alters genomic methylation levels during embryonic development.

Folate deficiency results in abnormal embryonic development, but the underlying mechanisms remain to be comprehensively investigated. Mutation of Vangl genes belonging to the planar cell polarity (PCP) pathway is associated with abnormal embryonic development, but the effect of folate deficiency on the PCP pathway is unclear. In this study, we found that folate deficiency inhibited Vangl gene expression and Vangl protein binding to the ligand Dvl. As a methyl donor, folate can chemically alter the DNA methylation levels of genomic sequences. Here, reduced representation bisulfite sequencing (RRBS) was employed to detect the methylation profiles of mouse embryos. The results confirmed that folate deficiency affected the genomic methylation levels of mouse embryos, which resulted in down-regulation of key genes involved in embryonic development. Gene ontology (GO) analysis suggested that the genes located in the differentially methylated regions (DMRs) are primarily involved in biological regulation, cellular processes, development, metabolism, and signaling pathways. The data revealed that folate deficiency inhibits the PCP pathway and alters genomic methylation profiles, which may be the underlying mechanisms through which folate deficiency impairs embryonic development.

Folic acid is necessary for proliferation and differentiation of C2C12 myoblasts.

Folic acid, a water soluble B vitamin, plays an important role in cellular metabolic activities, such as functioning as a cofactor in one-carbon metabolism for DNA and RNA synthesis as well as nucleotide and amino acid biosynthesis in the body. A lack of dietary folic acid can lead to folic acid deficiency and result in several health problems, including macrocytic anemia, elevated plasma homocysteine, cardiovascular disease, birth defects, carcinogenesis, muscle weakness, and walking difficulty. However, the effect of folic acid deficiency on skeletal muscle development and its molecular mechanisms are unknown. We, therefore, investigated the effect of folic acid deficiency on myogenesis in skeletal muscle cells and found that folic acid deficiency induced proliferation inhibition and cell cycle breaking as well as cellular senescence in C2C12 myoblasts, implying that folic acid deficiency influences skeletal muscle development. Folic acid deficiency also inhibited differentiation of C2C12 myoblasts and induced deregulation of the cell cycle exit and many cell cycle regulatory genes. It inhibited expression of muscle-specific marker MyHC as well as myogenic regulatory factor (myogenin). Moreover, immunocytochemistry and Western blot analyses revealed that DNA damage was more increased in folic acid-deficient medium-treated differentiating C2C12 cells. Furthermore, we found that folic acid resupplementation reverses the effect on the cell cycle and senescence in folic acid-deficient C2C12 myoblasts but does not reverse the differentiation of C2C12 cells. Altogether, the study results suggest that folic acid is necessary for normal development of skeletal muscle cells.

Folate deficiency induces mitotic aberrations and chromosomal instability by compromising the spindle assembly checkpoint in cultured human colon cells.

Folates comprise the essential B9 vitamin that act as cofactors and cosubstrates in one-carbon metabolism for both biosynthesis and methylation of DNA and RNA. Folate deficiency (FD) has been shown to induce chromosomal instability (CIN), yet the underlying mechanisms are poorly understood. Here, we used human NCM460 colon mucosal cells as a model to investigate the effect of FD on spindle assembly checkpoint (SAC), a cell-cycle regulatory pathway preventing CIN during mitosis. Cells were maintained in medium containing 1.36 (FD) and 2260 nM (control, FC) folate for 21 days. CIN was measured by cytokinesis-block micronucleus assay; mitotic infidelity was determined by aberrant mitosis analysis; SAC activity was assessed by nocodazole-challenge assay, and the expression of core SAC genes was examined by real-time quantitative PCR (RT-qPCR). We found that, relative to FC, FD significantly induced CIN in a time-dependent way (P < 0.01). Mitotic cells cultured in FD medium had significant higher frequencies of misalignment, misegregation and spindle multipolarity than those cultured in FC medium (P < 0.01). FD-induced SAC impairment and overriding, resulting premature mitotic exit and cell multinucleation (P < 0.05). Moreover, FD deregulated the expression of several key SAC genes (P < 0.01). Overall, these data are the first to demonstrate that FD substantially compromises SAC network which predisposes cells to mitotic aberrations and CIN. These results establish a new link between folate metabolism and SAC signalling, two pathways that are highly relevant for tumorigenesis.

Finding the genetic mechanisms of folate deficiency and neural tube defects-Leaving no stone unturned.

Neural tube defects (NTDs) occur secondary to failed closure of the neural tube between the third and fourth weeks of gestation. The worldwide incidence ranges from 0.3 to 200 per 10,000 births with the United States of American NTD incidence at around 3-6.3 per 10,000 dependent on race and socioeconomic background. Human NTD incidence has fallen by 35-50% in North America due to mandatory folic acid fortification of enriched cereal grain products since 1998. The US Food and Drug Administration has approved the folic acid fortification of corn masa flour with the goal to further reduce the incidence of NTDs, especially among individuals who are Hispanic. However, the genetic mechanisms determining who will benefit most from folate enrichment of the diet remains unclear despite volumes of literature published on studies of association of genes with functions related to folate metabolism and risk of human NTDs. The advances in omics technologies provides hypothesis-free tools to interrogate every single gene within the genome of NTD affected individuals to discover pathogenic variants and methylation targets throughout the affected genome. By identifying genes with expression regulated by presence of folate through transcriptome profiling studies, the genetic mechanisms leading to human NTDs due to folate deficiency may begin to be more efficiently revealed.

Folate deficiency disturbs hsa-let-7 g level through methylation regulation in neural tube defects.

Folic acid deficiency during pregnancy is believed to be a high-risk factor for neural tube defects (NTDs). Disturbed epigenetic modifications, including miRNA regulation, have been linked to the pathogenesis of NTDs in those with folate deficiency. However, the mechanism by which folic acid-regulated miRNA influences this pathogenesis remains unclear. It is believed that DNA methylation is associated with dysregulated miRNA expression. To clarify this issue, here we measured the methylation changes of 22 miRNAs in 57 human NTD cases to explore whether such changes are involved in miRNA regulation in NTD cases through folate metabolism. In total, eight of the 22 miRNAs tested reduced their methylation modifications in NTD cases, which provide direct evidence of the roles of interactions between DNA methylation and miRNA level in these defects. Among the findings, there was a significant association between folic acid concentration and hsa-let-7 g methylation level in NTD cases. Hypomethylation of hsa-let-7 g increased its own expression level in both NTD cases and cell models, which indicated that hsa-let-7 g methylation directly regulates its own expression. Overexpression of hsa-let-7 g, along with its target genes, disturbed the migration and proliferation of SK-N-SH cells, implying that hsa-let-7 g plays important roles in the prevention of NTDs by folic acid. In summary, our data suggest a relationship between aberrant methylation of hsa-let-7 g and disturbed folate metabolism in NTDs, implying that improvements in nutrition during early pregnancy may prevent such defects, possibly via the donation of methyl groups for miRNAs.

Nutritional role of folate.

Folate functions as a coenzyme to transfer one-carbon units that are necessary for deoxythymidylate synthesis, purine synthesis, and various methylation reactions. Ingested folate becomes a functional molecule through intestinal absorption, circulation, transport to cells, and various modifications to its structure. Associations between nutritional folate status and chronic diseases such as cardiovascular disease, cancer, and cognitive dysfunction have been reported. It has also been reported that maternal folate nutritional status is related to the risk of neural tube defects (NTDs) in the offspring. It has also been recommended that folate be consumed in the diet to promote the maintenance of good health. To reduce the risk of NTDs, supplementation with folic acid (a synthetic form of folate) during the periconceptional period has also been recommended. This paper describes the basic features and nutritional role of folate.

Role of folate in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of chronic liver conditions that are characterized by steatosis, inflammation, fibrosis, and liver injury. The global prevalence of NAFLD is rapidly increasing in proportion to the rising incidence of obesity and type 2 diabetes. Because NAFLD is a multifaceted disorder with many underlying metabolic abnormalities, currently, there is no pharmacological agent that is therapeutically approved for the treatment of this disease. Folate is a water-soluble B vitamin that plays an essential role in one-carbon transfer reactions involved in nucleic acid biosynthesis, methylation reactions, and sulfur-containing amino acid metabolism. The liver is the primary organ responsible for storage and metabolism of folates. Low serum folate levels have been observed in patients with obesity and diabetes. It has been reported that a low level of endogenous folates in rodents perturbs folate-dependent one-carbon metabolism, and may be associated with development of metabolic diseases such as NAFLD. This review highlights the biological role of folate in the progression of NAFLD and its associated metabolic complications including obesity and type 2 diabetes. Understanding the role of folate in metabolic disease may position this vitamin as a potential therapeutic for NAFLD.

Five lipoxygenase hypomethylation mediates the homocysteine effect on Alzheimer's phenotype.

Environmental and genetic risk factors are implicated in the pathogenesis of Alzheimer's disease (AD). However, how they interact and influence its pathogenesis remains to be investigated. High level of homocysteine (Hcy) is an AD risk factor and associates with an up-regulation of the ALOX5 gene. In the current paper we investigated whether this activation is responsible for the Hcy effect on the AD phenotype and the mechanisms involved. Triple transgenic mice were randomized to receive regular chow diet, a diet deficient in folate and B vitamins (Diet), which results in high Hcy, or the Diet plus zileuton, a specific ALOX5 inhibitor, for 7 months. Compared with controls, Diet-fed mice had a significant increase in Hcy levels, memory and learning deficits, up-regulation of the ALOX5 pathway, increased Aß levels, tau phosphorylation, and synaptic pathology, which were absent in mice treated with zileuton. In vivo and vitro studies demonstrated that the mechanism responsible was the hypomethylation of the ALOX5 promoter. Our findings demonstrate that the up-regulation of the ALOX5 is responsible for the Hcy-dependent worsening of the AD phenotype in a relevant mouse model of the disease. The discovery of this previously unknown cross-talk between these two pathways could afford novel therapeutic opportunities for treating or halting AD.

Metabolic Reprogramming by Folate Restriction Leads to a Less Aggressive Cancer Phenotype.

Folate coenzymes are involved in biochemical reactions of one-carbon transfer, and deficiency of this vitamin impairs cellular proliferation, migration, and survival in many cell types. Here, the effect of folate restriction on mammary cancer was evaluated using three distinct breast cancer subtypes differing in their aggressiveness and metastatic potential: noninvasive basal-like (E-Wnt), invasive but minimally metastatic claudin-low (M-Wnt), and highly metastatic claudin-low (metM-Wntliver) cell lines, each derived from the same pool of MMTV-Wnt-1 transgenic mouse mammary tumors. NMR-based metabolomics was used to quantitate 41 major metabolites in cells grown in folate-free medium versus standard medium. Each cell line demonstrated metabolic reprogramming when grown in folate-free medium. In E-Wnt, M-Wnt, and metM-Wntliver cells, 12, 29, and 25 metabolites, respectively, were significantly different (P < 0.05 and at least 1.5-fold change). The levels of eight metabolites (aspartate, ATP, creatine, creatine phosphate, formate, serine, taurine and ß-alanine) were changed in each folate-restricted cell line. Increased glucose, decreased lactate, and inhibition of glycolysis, cellular proliferation, migration, and invasion occurred in M-Wnt and metM-Wntliver cells (but not E-Wnt cells) grown in folate-free versus standard medium. These effects were accompanied by altered levels of several folate-metabolizing enzymes, indicating that the observed metabolic reprogramming may result from both decreased folate availability and altered folate metabolism. These findings reveal that folate restriction results in metabolic and bioenergetic changes and a less aggressive cancer cell phenotype. IMPLICATIONS: Metabolic reprogramming driven by folate restriction represents a therapeutic target for reducing the burden of breast cancer. Mol Cancer Res; 15(2); 189-200. ©2016 AACR.

Folate deficiency affects dendritic cell function and subsequent T helper cell differentiation.

Insufficient folate status may be related to the increasing prevalence of immune- or inflammation-related chronic diseases. To investigate the effects of folate on immune regulation, we examined the impact of folate deficiency (FD) on dendritic cell (DC) maturation and function and, thus, T helper (Th) cells differentiation. First, bone marrow-derived DCs (BMDCs) were generated from BALB/c mice bone marrow cells cultured in folate-containing (F-BMDCs) or folate-deficient (FD-BMDCs) medium. FD-BMDC displayed more immature phenotype including reduced levels of major histocompatibility complex class II (MHC II), co-stimulatory molecules and characteristic of higher endocytic activity. FD-BMDC produced less IL-12p70 and proinflammatory cytokines in response to lipopolysaccharide. This aberrant DC maturation due to FD resulted in reduced BMDC-induced Th cell activity and lower IL-2, IFNγ, IL-13 and IL-10 productions. Further in vivo study confirmed significantly lower IFNγ and IL-10 productions by T cells and showed higher splenic naïve Th and lower memory T, effector T and regulatory T cell (Treg) percentages in mice fed with the FD diet for 13 weeks. To investigate the role of DCs on T cell activity, splenic DCs (spDC) from FD mice were cocultured with Th cells. The FD spDC had lower MHC II and CD80 expressions and subsequently impaired DC-induced Th differentiation, shown as decreased cytokine productions. This study demonstrated that folate deficiency impaired DC functions and, thus, Th differentiation and responses, suggesting that folate plays a crucial role in maintaining Th cells homeostasis.