Neurobiology of eating behavior, nutrition, and health.

Eating behavior and food-related decision making are among the most complex of the motivated behaviors, and understanding the neurobiology of eating behavior, and its developmental dynamics, is critical to advancing the nutritional sciences and public health. Recent advances from both human and animal studies are revealing that individual capacity to make health-promoting food decisions varies based on biological and physiological variation in the signaling pathways that regulate the homeostatic, hedonic, and executive functions; past developmental exposures and current life-stage; the food environment; and complications of chronic disease that reinforce the obese state. Eating rate drives increased calorie intake and represents an important opportunity to lower rates of food consumption and energy intake through product reformulation. Understanding human eating behaviors and nutrition in the context of neuroscience can strengthen the evidence base from which dietary guidelines are derived and can inform policies, practices, and educational programs in a way that increases the likelihood they are adopted and effective for reducing rates of obesity and other diet-related chronic disease.

Sucrose Intake Elevates Erythritol in Plasma and Urine in Male Mice.

BACKGROUND: Elevated serum erythritol concentration is a predictive biomarker of diabetes and cardiovascular incidence and complications. Erythritol is synthesized endogenously from glucose, but little is known regarding the origin of elevated circulating erythritol in vivo. OBJECTIVES: In vitro evidence indicates that intracellular erythritol is elevated by high-glucose cell culture conditions and that final step of erythritol synthesis is catalyzed by the enzymes sorbitol dehydrogenase (SORD) and alcohol dehydrogenase (ADH) 1. The purpose of this study was to determine whether dietary intake and/or diet-induced obesity affect erythritol synthesis in mice and whether this relationship is modified by the loss of the enzymes SORD or ADH1. METHODS: First, 8-wk-old male Sord+/+, Sord-/-, Adh1+/+, and Adh1-/- mice were fed either low-fat diet (LFD) with 10% fat-derived calories or diet-induced obesity high-fat diet (HFD) with 60% fat-derived calories for 8 wk. Plasma and tissue erythritol concentrations were measured using gas chromatography-mass spectrometry. Second, male wild-type 8-wk-old C57BL/6J mice were fed LFD or HFD with plain drinking water or 30% sucrose water for 8 wk. Blood glucose and plasma and urinary erythritol concentrations were measured in nonfasted and fasted samples. Tissue erythritol was measured after killing. Finally, male Sord+/+ and Sord-/- mice were fed LFD with 30% sucrose water for 2 wk; then, nonfasted plasma, urine, and tissue erythritol concentrations were quantified. RESULTS: Plasma and tissue erythritol concentrations were not affected by loss of Sord or Adh1 in mice fed LFD or HFD. In wild-type mice, consumption of 30% sucrose water significantly elevated plasma and urinary erythritol concentrations on both LFD-fed and HFD-fed mice compared with that of plain water. Sord genotype did not affect plasma or urinary erythritol concentration in response to sucrose feeding, but Sord-/- mice had reduced kidney erythritol content compared with wild-type littermates in response to sucrose. CONCLUSIONS: Sucrose intake, not HFD, elevates erythritol synthesis and excretion in mice. Loss of ADH1 or SORD does not significantly affect erythritol concentration in mice.

Chronic Dietary Erythritol Exposure Elevates Plasma Erythritol Concentration in Mice but Does Not Cause Weight Gain or Modify Glucose Homeostasis.

BACKGROUND: Erythritol is both a common nonnutritive sweetener and an endogenous product of glucose metabolism. Recent reports suggest that elevated plasma erythritol is a predictive biomarker of cardiometabolic disease onset and complications. OBJECTIVES: Although short-term erythritol consumption has been evaluated, the effect of chronically elevated circulating erythritol on adiposity and glucose metabolism has not. This study investigated the effect of longer-term erythritol consumption on weight gain and glucose tolerance in young/adolescent mice. METHODS: Four erythritol supplementation experiments were completed and analyzed separately in male C57BL/6J mice. In experiments 1 and 2, mice aged 8 wk or 20 wk, respectively, were randomly allocated to consume 16% fat diet (LFD) or LFD with 40 g/kg erythritol. In experiments 3 and 4, mice aged 8 wk or 20 wk were fed 45% fat diet (HFD) or HFD with 40 g/kg erythritol (HFD + ERY). In each experiment, we compared the effect of erythritol consumption on plasma erythritol, body weight and composition, glucose tolerance, and brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression. We also investigated relative endogenous tissue erythritol concentrations in a subset of control (LFD or HFD) mice in experiments 1 and 3. RESULTS: There was no effect of erythritol supplementation on body weight or glucose tolerance in experiments 1-3. In experiment 4, in the 20-wk-old mice fed HFD or HFD + ERY, there was a significant interaction of time and erythritol on body weight (P < 0.0001), but the main effect of diet was not significant. Plasma erythritol was elevated 40-fold in mice consuming erythritol-supplemented diets relative to mice consuming LFD or HFD controls. We found no effect of chronic erythritol consumption on BAT UCP1 protein concentrations. Liver and kidney tissue contained significantly higher endogenous erythritol than quadriceps and visceral adipose (P < 0.001) in young mice fed LFD and HFD. CONCLUSIONS: In young/adolescent mice, prolonged erythritol consumption did not significantly affect body weight, composition, or glucose tolerance.

Pyruvate Kinase M2 Supports Muscle Progenitor Cell Proliferation but Is Dispensable for Skeletal Muscle Regeneration after Injury.

BACKGROUND: Skeletal muscle progenitor cells (MPCs) repair damaged muscle postinjury. Pyruvate kinase M2 (PKM2) is a glycolytic enzyme (canonical activity) that can also interact with other proteins (noncanonical activity) to modify diverse cellular processes. Recent evidence links PKM2 to MPC proliferation. OBJECTIVES: This study aimed to understand cellular roles for PKM2 in MPCs and the necessity of PKM2 in MPCs for muscle regeneration postinjury. METHODS: Cultured, proliferating MPCs (C2C12 cells) were treated with a short hairpin RNA targeting PKM2 or small molecules that selectively affect canonical and noncanonical PKM2 activity (shikonin and TEPP-46). Cell number was measured, and RNA-sequencing and metabolic assays were used in follow-up experiments. Immunoprecipitation coupled to proteomics was used to identify binding partners of PKM2. Lastly, an MPC-specific PKM2 knockout mouse was generated and challenged with a muscle injury to determine the impact of PKM2 on regeneration. RESULTS: When the noncanonical activity of PKM2 was blocked or impaired, there was an increase in reactive oxygen species concentrations (1.6-2.0-fold, P < 0.01). Blocking noncanonical PKM2 activity also increased lactate excretion (1.2-1.6-fold, P < 0.05) and suppressed mitochondrial oxygen consumption (1.3-1.6-fold, P < 0.01). Glutamate dehydrogenase 1 (GLUD1) was identified as a PKM2 binding partner and blocking noncanonical PKM2 activity increased GLUD activity (1.5-1.6-fold, P < 0.05). Mice with an MPC-specific PKM2 deletion did not demonstrate impaired muscle regeneration. CONCLUSIONS: The results suggest that the noncanonical activity of PKM2 is important for MPC proliferation in vitro and demonstrate GLUD1 as a PKM2 binding partner. Because no impairments in muscle regeneration were detected in a mouse model, the endogenous environment may compensate for loss of PKM2.

Reduced Shmt2 Expression Impairs Mitochondrial Folate Accumulation and Respiration, and Leads to Uracil Accumulation in Mouse Mitochondrial DNA.

BACKGROUND: Adequate cellular thymidylate (dTMP) pools are essential for preservation of nuclear and mitochondrial genome stability. Previous studies have indicated that disruption in nuclear dTMP synthesis leads to increased uracil misincorporation into DNA, affecting genome stability. To date, the effects of impaired mitochondrial dTMP synthesis in nontransformed tissues have been understudied. OBJECTIVES: This study aimed to determine the effects of decreased serine hydroxymethyltransferase 2 (Shmt2) expression and dietary folate deficiency on mitochondrial DNA (mtDNA) integrity and mitochondrial function in mouse tissues. METHODS: Liver mtDNA content, and uracil content in liver mtDNA, were measured in Shmt2+/- and Shmt2+/+ mice weaned onto either a folate-sufficient control diet (2 mg/kg folic acid; C) or a modified diet lacking folic acid (0 mg/kg folic acid) for 7 wk. Shmt2+/- and Shmt2+/+ mouse embryonic fibroblast (MEF) cells were cultured in defined culture medium containing either 0 or 25 nM folate (6S-5-formyl-tetrahydrofolate, folinate) to assess proliferative capacity and mitochondrial function. Chi-square tests, linear mixed models, and 2-factor ANOVA with Tukey post hoc analyses were used to analyze data. RESULTS: Shmt2 +/- mice exhibited a 48%-67% reduction in SHMT2 protein concentrations in tissues. Interestingly, Shmt2+/- mice consuming the folate-sufficient C diet exhibited a 25% reduction in total folate in liver mitochondria. There was also a >20-fold increase in uracil in liver mtDNA in Shmt2+/- mice consuming the C diet, and dietary folate deficiency also increased uracil content in mouse liver mtDNA from both Shmt2+/+ and Shmt2+/- mice. Furthermore, decreased Shmt2 expression in MEF cells reduced cell proliferation, mitochondrial membrane potential, and oxygen consumption rate. CONCLUSIONS: This study demonstrates that Shmt2 heterozygosity and dietary folate deficiency impair mitochondrial dTMP synthesis in mice, as evidenced by the increased uracil in mtDNA. In addition, Shmt2 heterozygosity impairs mitochondrial function in MEF cells. These findings suggest that elevated uracil in mtDNA may impair mitochondrial function.

Wheat flour fortification with iron and other micronutrients for reducing anaemia and improving iron status in populations.

BACKGROUND: Anaemia is a condition where the number of red blood cells (and consequently their oxygen-carrying capacity) is insufficient to meet the body's physiological needs. Fortification of wheat flour is deemed a useful strategy to reduce anaemia in populations. OBJECTIVES: To determine the benefits and harms of wheat flour fortification with iron alone or with other vitamins and minerals on anaemia, iron status and health-related outcomes in populations over two years of age. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, CINAHL, 21 other databases and two trials registers up to 21 July 2020, together with contacting key organisations to identify additional studies. SELECTION CRITERIA: We included cluster- or individually-randomised controlled trials (RCTs) carried out among the general population from any country, aged two years and above. The interventions were fortification of wheat flour with iron alone or in combination with other micronutrients. We included trials comparing any type of food item prepared from flour fortified with iron of any variety of wheat DATA COLLECTION AND ANALYSIS: Two review authors independently screened the search results and assessed the eligibility of studies for inclusion, extracted data from included studies and assessed risks of bias. We followed Cochrane methods in this review. MAIN RESULTS: Our search identified 3538 records, after removing duplicates. We included 10 trials, involving 3319 participants, carried out in Bangladesh, Brazil, India, Kuwait, Philippines, South Africa and Sri Lanka. We identified two ongoing studies and one study is awaiting classification. The duration of interventions varied from 3 to 24 months. One study was carried out among adult women and one trial among both children and nonpregnant women. Most of the included trials were assessed as low or unclear risk of bias for key elements of selection, performance or reporting bias. Three trials used 41 mg to 60 mg iron/kg flour, three trials used less than 40 mg iron/kg and three trials used more than 60 mg iron/kg flour. One trial used various iron levels based on type of iron used: 80 mg/kg for electrolytic and reduced iron and 40 mg/kg for ferrous fumarate. All included studies contributed data for the meta-analyses. Iron-fortified wheat flour with or without other micronutrients added versus wheat flour (no added iron) with the same other micronutrients added Iron-fortified wheat flour with or without other micronutrients added versus wheat flour (no added iron) with the same other micronutrients added may reduce by 27% the risk of anaemia in populations (risk ratio (RR) 0.73, 95% confidence interval (CI) 0.55 to 0.97; 5 studies, 2315 participants; low-certainty evidence). It is uncertain whether iron-fortified wheat flour with or without other micronutrients reduces iron deficiency (RR 0.46, 95% CI 0.20 to 1.04; 3 studies, 748 participants; very low-certainty evidence) or increases haemoglobin concentrations (in g/L) (mean difference MD 2.75, 95% CI 0.71 to 4.80; 8 studies, 2831 participants; very low-certainty evidence). No trials reported data on adverse effects in children (including constipation, nausea, vomiting, heartburn or diarrhoea), except for risk of infection or inflammation at the individual level. The intervention probably makes little or no difference to the risk of Infection or inflammation at individual level as measured by C-reactive protein (CRP) (mean difference (MD) 0.04, 95% CI -0.02 to 0.11; 2 studies, 558 participants; moderate-certainty evidence). Iron-fortified wheat flour with other micronutrients added versus unfortified wheat flour (nil micronutrients added) It is unclear whether wheat flour fortified with iron, in combination with other micronutrients decreases anaemia (RR 0.77, 95% CI 0.41 to 1.46; 2 studies, 317 participants; very low-certainty evidence). The intervention probably reduces the risk of iron deficiency (RR 0.73, 95% CI 0.54 to 0.99; 3 studies, 382 participants; moderate-certainty evidence) and it is unclear whether it increases average haemoglobin concentrations (MD 2.53, 95% CI -0.39 to 5.45; 4 studies, 532 participants; very low-certainty evidence). No trials reported data on adverse effects in children. Nine out of 10 trials reported sources of funding, with most having multiple sources. Funding source does not appear to have distorted the results in any of the assessed trials. AUTHORS' CONCLUSIONS: Fortification of wheat flour with iron (in comparison to unfortified flour, or where both groups received the same other micronutrients) may reduce anaemia in the general population above two years of age, but its effects on other outcomes are uncertain. Iron-fortified wheat flour in combination with other micronutrients, in comparison with unfortified flour, probably reduces iron deficiency, but its effects on other outcomes are uncertain. None of the included trials reported data on adverse side effects except for risk of infection or inflammation at the individual level. The effects of this intervention on other health outcomes are unclear. Future studies at low risk of bias should aim to measure all important outcomes, and to further investigate which variants of fortification, including the role of other micronutrients as well as types of iron fortification, are more effective, and for whom.

Unexpected roles for ADH1 and SORD in catalyzing the final step of erythritol biosynthesis.

The low-calorie sweetener erythritol is endogenously produced from glucose through the pentose phosphate pathway in humans. Erythritol is of medical interest because elevated plasma levels of this polyol are predictive for visceral adiposity gain and development of type 2 diabetes. However, the mechanisms behind these associations remain unknown because the erythritol biosynthesis pathway, particularly the enzyme catalyzing the final step of erythritol synthesis (reduction of erythrose to erythritol), is not characterized. In this study, we purified two enzymes from rabbit liver capable of catalyzing the conversion of erythrose to erythritol: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Both recombinant human ADH1 and SORD reduce erythrose to erythritol, using NADPH as a co-factor, and cell culture studies indicate that this activity is primarily NADPH-dependent. We found that ADH1 variants vary markedly in both their affinity for erythrose and their catalytic capacity (turnover number). Interestingly, the recombinant protein produced from the ADH1B2 variant, common in Asian populations, is not active when NADPH is used as a co-factor in vitro We also confirmed SORD contributes to intracellular erythritol production in human A549 lung cancer cells, where ADH1 is minimally expressed. In summary, human ADH1 and SORD catalyze the conversion of erythrose to erythritol, pointing to novel roles for two dehydrogenase proteins in human glucose metabolism that may contribute to individual responses to diet. Proteomics data are available via ProteomeXchange with identifier PXD015178.

The Role of Brain Barriers in Maintaining Brain Vitamin Levels.

It is increasingly recognized that tissue-specific nutrient deficiencies can exist in the absence of whole-body deficiency and that these deficiencies may result from disease or disease-related physiological processes. Brain and central nervous system tissues require adequate nutrient levels to function. Many nutrients are concentrated in the cerebrospinal fluid relative to the serum in healthy individuals, and other nutrients resist depletion in the presence of whole-body nutrient depletion. The endothelial, epithelial, and arachnoid brain barriers work in concert to selectively transport, concentrate, and maintain levels of the specific nutrients required by the brain while also blocking the passage of blood-borne toxins and pathogens to brain and central nervous system tissues. These barriers preserve nutrient levels within the brain and actively concentrate nutrients within the cerebrospinal fluid and brain. The roles of physical and energetic barriers, including the blood-brain and blood-nerve barriers, in maintaining brain nutrient levels in health and disease are discussed.

Mammalian metabolism of erythritol: a predictive biomarker of metabolic dysfunction.

PURPOSE OF REVIEW: To summarize recent advances in our understanding of mammalian erythritol metabolism and its use as a predictive biomarker of cardiometabolic disease risk. RECENT FINDINGS: Elevated serum erythritol predicts future central adiposity gain and type 2 diabetes mellitus in healthy adults. Erythritol is a newly recognized human metabolic product of glucose, synthesized through the pentose phosphate pathway. The final conversion of this metabolic pathway is catalyzed by the enzymes sorbitol dehydrogenase and alcohol dehydrogenase 1. Erythritol is also a well characterized nonnutritive sweetener. Recent studies show that dietary erythritol can be metabolized to erythrose or erythronate in humans before excretion. SUMMARY: Elevated serum erythritol predicts risk for cardiometabolic disease, but more research is required to maximize its utility as a biomarker, including characterizing the determinants of endogenous erythritol synthesis from glucose. New insights into dietary erythritol metabolism also highlight the need to evaluate the effects of long-term erythritol consumption.

Emerging Concepts in Nutrient Needs.

Dietary reference intakes (DRIs) are quantitative, nutrient intake-based standards used for assessing the diets and specific nutrient intakes of healthy individuals and populations and for informing national nutrition policy and nutrition programs. Because nutrition needs vary by age, sex, and physiological state, DRIs are often specified for healthy subgroups within a population. Diet is known to be the leading modifiable risk factor for chronic disease, and the prevalence of chronic disease is growing in all populations globally and across all subgroups, but especially in older adults. It is known that nutrient needs can change in some chronic disease and other clinical states. Disease states and/or disease treatment can cause whole-body or tissue-specific nutrient depletion or excess, resulting in the need for altered nutrient intakes. In other cases, disease-related biochemical dysfunction can result in a requirement for a nonessential nutrient, rendering it as conditionally essential, or result in toxicity for a food component at levels usually tolerated by healthy people, as seen in inborn errors of metabolism. Here we summarize examples from a growing body of literature of disease-altering nutrient requirements, supporting the need to give more consideration to special nutrient requirements in disease states.

p53 Disruption Increases Uracil Accumulation in DNA of Murine Embryonic Fibroblasts and Leads to Folic Acid-Nonresponsive Neural Tube Defects in Mice.

BACKGROUND: Neural tube defects (NTDs) occur in nervous tissue during embryogenesis when the neural tube fails to close. Approximately 70% of all human NTDs can be prevented by folic acid (FA). Altered expression and/or function of the tumor suppressor protein p53 can lead to NTDs in mouse models. OBJECTIVES: The aim of this study was to determine if dietary FA could rescue p53-/--induced NTDs in mice, and to determine the effect loss of p53 has on pathways in folate 1-carbon metabolism. METHODS: p53+/- female mice were randomly allocated and weaned onto either an FA-sufficient diet (2 mg/kg folic acid; +FA), or an FA-deficient diet (-FA). After 8 wk, the females were time-mated to p53-/- males. Embryos were examined at E12.5 for NTDs. Folate enzyme concentrations, nucleotide synthesis, uracil accumulation in DNA, and proliferation were measured in primary murine embryonic fibroblasts (MEFs). The "n - 1" chi-square test was used to compare NTD percentages, whereas all other data were analyzed by Student t test, except where noted a multilevel-fit model was used. RESULTS: NTD rates of litters from dams consuming the +FA diet (20/46; 43%) did not differ from those of litters from dams consuming the -FA diet (14/35; 40%) (P > 0.05). p53-/- MEFs had 55% higher rates of folate-dependent de novo dTMP synthesis, a ∼2-fold higher accumulation of uracil in DNA, and a ∼30% higher rate of proliferation (P ≤ 0.05) than p53+/- MEFs independent of folate. CONCLUSIONS: p53-related NTDs are not FA responsive. Increased dTMP synthesis in p53-/- MEFs might not have been sufficient to meet the demands for thymidine triphosphate (dTTP) synthesis as evidenced by the elevated amounts of uracil in DNA. This study provides additional evidence that elevated uracil in DNA is a risk factor for NTDs.

Wheat flour fortification with iron for reducing anaemia and improving iron status in populations.

BACKGROUND: Anaemia is a condition where the number of red blood cells (and consequently their oxygen-carrying capacity) is insufficient to meet the body's physiologic needs. Fortification of wheat flour is deemed a useful strategy to reduce anaemia in populations. OBJECTIVES: To determine the benefits and harms of wheat flour fortification with iron alone or with other vitamins and minerals on anaemia, iron status and health-related outcomes in populations over two years of age. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, CINAHL, and other databases up to 4 September 2019. SELECTION CRITERIA: We included cluster- or individually randomised controlled trials (RCT) carried out among the general population from any country aged two years and above. The interventions were fortification of wheat flour with iron alone or in combination with other micronutrients. Trials comparing any type of food item prepared from flour fortified with iron of any variety of wheat were included. DATA COLLECTION AND ANALYSIS: Two review authors independently screened the search results and assessed the eligibility of studies for inclusion, extracted data from included studies and assessed risk of bias. We followed Cochrane methods in this review. MAIN RESULTS: Our search identified 3048 records, after removing duplicates. We included nine trials, involving 3166 participants, carried out in Bangladesh, Brazil, India, Kuwait, Phillipines, Sri Lanka and South Africa. The duration of interventions varied from 3 to 24 months. One study was carried out among adult women and one trial among both children and nonpregnant women. Most of the included trials were assessed as low or unclear risk of bias for key elements of selection, performance or reporting bias. Three trials used 41 mg to 60 mg iron/kg flour, two trials used less than 40 mg iron/kg and three trials used more than 60 mg iron/kg flour. One trial employed various iron levels based on type of iron used: 80 mg/kg for electrolytic and reduced iron and 40 mg/kg for ferrous fumarate. All included studies contributed data for the meta-analyses. Seven studies compared wheat flour fortified with iron alone versus unfortified wheat flour, three studies compared wheat flour fortified with iron in combination with other micronutrients versus unfortified wheat flour and two studies compared wheat flour fortified with iron in combination with other micronutrients versus fortified wheat flour with the same micronutrients (but not iron). No studies included a 'no intervention' comparison arm. None of the included trials reported any other adverse side effects (including constipation, nausea, vomiting, heartburn or diarrhoea). Wheat flour fortified with iron alone versus unfortified wheat flour (no micronutrients added) Wheat flour fortification with iron alone may have little or no effect on anaemia (risk ratio (RR) 0.81, 95% confidence interval (CI) 0.61 to 1.07; 5 studies; 2200 participants; low-certainty evidence). It probably makes little or no difference on iron deficiency (RR 0.43, 95% CI 0.17 to 1.07; 3 studies; 633 participants; moderate-certainty evidence) and we are uncertain about whether wheat flour fortified with iron increases haemoglobin concentrations by an average 3.30 (g/L) (95% CI 0.86 to 5.74; 7 studies; 2355 participants; very low-certainty evidence). No trials reported data on adverse effects in children, except for risk of infection or inflammation at the individual level. The intervention probably makes little or no difference to risk of Infection or inflammation at individual level as measured by C-reactive protein (CRP) (moderate-certainty evidence). Wheat flour fortified with iron in combination with other micronutrients versus unfortified wheat flour (no micronutrients added) Wheat flour fortified with iron, in combination with other micronutrients, may or may not decrease anaemia (RR 0.95, 95% CI 0.69 to 1.31; 2 studies; 322 participants; low-certainty evidence). It makes little or no difference to average risk of iron deficiency (RR 0.74, 95% CI 0.54 to 1.00; 3 studies; 387 participants; moderate-certainty evidence) and may or may not increase average haemoglobin concentrations (mean difference (MD) 3.29, 95% CI -0.78 to 7.36; 3 studies; 384 participants; low-certainty evidence). No trials reported data on adverse effects in children. Wheat flour fortified with iron in combination with other micronutrients versus fortified wheat flour with same micronutrients (but not iron) Given the very low certainty of the evidence, the review authors are uncertain about the effects of wheat flour fortified with iron in combination with other micronutrients versus fortified wheat flour with same micronutrients (but not iron) in reducing anaemia (RR 0.24, 95% CI 0.08 to 0.71; 1 study; 127 participants; very low-certainty evidence) and in reducing iron deficiency (RR 0.42, 95% CI 0.18 to 0.97; 1 study; 127 participants; very low-certainty evidence). The intervention may make little or no difference to the average haemoglobin concentration (MD 0.81, 95% CI -1.28 to 2.89; 2 studies; 488 participants; low-certainty evidence). No trials reported data on the adverse effects in children. Eight out of nine trials reported source of funding with most having multiple sources. Funding source does not appear to have distorted the results in any of the assessed trials. AUTHORS' CONCLUSIONS: Eating food items containing wheat flour fortified with iron alone may have little or no effect on anaemia and probably makes little or no difference in iron deficiency. We are uncertain on whether the intervention with wheat flour fortified with iron increases haemoglobin concentrations improve blood haemoglobin concentrations. Consuming food items prepared from wheat flour fortified with iron, in combination with other micronutrients, has little or no effect on anaemia, makes little or no difference to iron deficiency and may or may not improve haemoglobin concentrations. In comparison to fortified flour with micronutrients but no iron, wheat flour fortified with iron with other micronutrients, the effects on anaemia and iron deficiency are uncertain as certainty of the evidence has been assessed as very low. The intervention may make little or no difference to the average haemoglobin concentrations in the population. None of the included trials reported any other adverse side effects. The effects of this intervention on other health outcomes are unclear.

Folate rescues vitamin B12 depletion-induced inhibition of nuclear thymidylate biosynthesis and genome instability.

Clinical vitamin B12 deficiency can result in megaloblastic anemia, which results from the inhibition of DNA synthesis by trapping folate cofactors in the form of 5-methyltetrahydrofolate (5-methylTHF) and subsequent inhibition of de novo thymidylate (dTMP) biosynthesis. In the cytosol, vitamin B12 functions in the remethylation of homocysteine to methionine, which regenerates THF from 5-methylTHF. In the nucleus, THF is required for de novo dTMP biosynthesis, but it is not understood how 5-methylTHF accumulation in the cytosol impairs nuclear dTMP biosynthesis. The impact of vitamin B12 depletion on nuclear de novo dTMP biosynthesis was investigated in methionine synthase-null human fibroblast and nitrous oxide-treated HeLa cell models. The nucleus was the most sensitive cellular compartment to 5-methylTHF accumulation, with levels increasing greater than fourfold. Vitamin B12 depletion decreased de novo dTMP biosynthesis capacity by 5-35%, whereas de novo purine synthesis, which occurs in the cytosol, was not affected. Phosphorylated histone H2AX (γH2AX), a marker of DNA double-strand breaks, was increased in vitamin B12 depletion, and this effect was exacerbated by folate depletion. These studies also revealed that 5-formylTHF, a slow, tight-binding inhibitor of serine hydroxymethyltransferase (SHMT), was enriched in nuclei, accounting for 35% of folate cofactors, explaining previous observations that nuclear SHMT is not a robust source of one-carbons for de novo dTMP biosynthesis. These findings indicate that a nuclear 5-methylTHF trap occurs in vitamin B12 depletion, which suppresses de novo dTMP biosynthesis and causes DNA damage, accounting for the pathophysiology of megaloblastic anemia observed in vitamin B12 and folate deficiency.

Arsenic trioxide targets MTHFD1 and SUMO-dependent nuclear de novo thymidylate biosynthesis.

Arsenic exposure increases risk for cancers and is teratogenic in animal models. Here we demonstrate that small ubiquitin-like modifier (SUMO)- and folate-dependent nuclear de novo thymidylate (dTMP) biosynthesis is a sensitive target of arsenic trioxide (As2O3), leading to uracil misincorporation into DNA and genome instability. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and serine hydroxymethyltransferase (SHMT) generate 5,10-methylenetetrahydrofolate for de novo dTMP biosynthesis and translocate to the nucleus during S-phase, where they form a multienzyme complex with thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR), as well as the components of the DNA replication machinery. As2O3 exposure increased MTHFD1 SUMOylation in cultured cells and in in vitro SUMOylation reactions, and increased MTHFD1 ubiquitination and MTHFD1 and SHMT1 degradation. As2O3 inhibited de novo dTMP biosynthesis in a dose-dependent manner, increased uracil levels in nuclear DNA, and increased genome instability. These results demonstrate that MTHFD1 and SHMT1, which are key enzymes providing one-carbon units for dTMP biosynthesis in the form of 5,10-methylenetetrahydrofolate, are direct targets of As2O3-induced proteolytic degradation, providing a mechanism for arsenic in the etiology of cancer and developmental anomalies.

The mitochondrial inner membrane protein MPV17 prevents uracil accumulation in mitochondrial DNA.

Mitochondrial inner membrane protein MPV17 is a protein of unknown function that is associated with mitochondrial DNA (mtDNA)-depletion syndrome (MDS). MPV17 loss-of-function has been reported to result in tissue-specific nucleotide pool imbalances, which can occur in states of perturbed folate-mediated one-carbon metabolism (FOCM), but MPV17 has not been directly linked to FOCM. FOCM is a metabolic network that provides one-carbon units for the de novo synthesis of purine and thymidylate nucleotides (e.g. dTMP) for both nuclear DNA (nuDNA) and mtDNA replication. In this study, we investigated the impact of reduced MPV17 expression on markers of impaired FOCM in HeLa cells. Depressed MPV17 expression reduced mitochondrial folate levels by 43% and increased uracil levels, a marker of impaired dTMP synthesis, in mtDNA by 3-fold. The capacity of mitochondrial de novo and salvage pathway dTMP biosynthesis was unchanged by the reduced MPV17 expression, but the elevated levels of uracil in mtDNA suggested that other sources of mitochondrial dTMP are compromised in MPV17-deficient cells. These results indicate that MPV17 provides a third dTMP source, potentially by serving as a transporter that transfers dTMP from the cytosol to mitochondria to sustain mtDNA synthesis. We propose that MPV17 loss-of-function and related hepatocerebral MDS are linked to impaired FOCM in mitochondria by providing insufficient access to cytosolic dTMP pools and by severely reducing mitochondrial folate pools.

Nuclear Folate Metabolism.

Despite unequivocal evidence that folate deficiency increases risk for human pathologies, and that folic acid intake among women of childbearing age markedly decreases risk for birth defects, definitive evidence for a causal biochemical pathway linking folate to disease and birth defect etiology remains elusive. The de novo and salvage pathways for thymidylate synthesis translocate to the nucleus of mammalian cells during S- and G2/M-phases of the cell cycle and associate with the DNA replication and repair machinery, which limits uracil misincorporation into DNA and genome instability. There is increasing evidence that impairments in nuclear de novo thymidylate synthesis occur in many pathologies resulting from impairments in one-carbon metabolism. Understanding the roles and regulation of nuclear de novo thymidylate synthesis and its relationship to genome stability will increase our understanding of the fundamental mechanisms underlying folate- and vitamin B12-associated pathologies.

Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis.

An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.

Alcohol Dehydrogenase 5 Is a Source of Formate for De Novo Purine Biosynthesis in HepG2 Cells.

Background: Formate provides one-carbon units for de novo purine and thymidylate (dTMP) synthesis and is produced via both folate-dependent and folate-independent pathways. Folate-independent pathways are mediated by cytosolic alcohol dehydrogenase 5 (ADH5) and mitochondrial aldehyde dehydrogenase 2 (ALDH2), which generate formate by oxidizing formaldehyde. Formate is a potential biomarker of B-vitamin-dependent one-carbon metabolism.Objective: This study investigated the contributions of ADH5 and ALDH2 to formate production and folate-dependent de novo purine and dTMP synthesis in HepG2 cells.Methods:ADH5 knockout and ALDH2 knockdown HepG2 cells were cultured in folate-deficient [0 nM (6S) 5-formyltetrahydrofolate] or folate-sufficient [25 nM (6S) 5-formyltetrahydrofolate] medium. Purine biosynthesis was quantified as the ratio of [14C]-formate to [3H]-hypoxanthine incorporated into genomic DNA, which indicates the contribution of the de novo purine synthesis pathway relative to salvage synthesis. dTMP synthesis was quantified as the ratio of [14C]-deoxyuridine to [3H]-thymidine incorporation into genomic DNA, which indicates the capacity of de novo dTMP synthesis relative to salvage synthesis.Results: The [14C]-formate-to-[3H]-hypoxanthine ratio was greater in ADH5 knockout than in wild-type HepG2 cells, under conditions of both folate deficiency (+30%; P < 0.001) and folate sufficiency (+22%; P = 0.02). These data indicate that ADH5 deficiency increases the use of exogenous formate for de novo purine biosynthesis. The [14C]-deoxyuridine-to-[3H]-thymidine ratio did not differ between ADH5 knockout and wild-type cells, indicating that ADH5 deficiency does not affect de novo dTMP synthesis capacity relative to salvage synthesis. Under folate deficiency, ALDH2 knockdown cells exhibited a 37% lower ratio of [14C]-formate to [3H]-hypoxanthine (P < 0.001) compared with wild-type HepG2 cells, indicating decreased use of exogenous formate, or increased endogenous formate synthesis, for de novo purine biosynthesis.Conclusions: In HepG2 cells, ADH5 is a source of formate for de novo purine biosynthesis, especially during folate deficiency when folate-dependent formate production is limited. Formate is also shown to be limiting in the growth of HepG2 cells.

Nuclear enrichment of folate cofactors and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protect de novo thymidylate biosynthesis during folate deficiency.

Folate-mediated one-carbon metabolism is a metabolic network of interconnected pathways that is required for the de novo synthesis of three of the four DNA bases and the remethylation of homocysteine to methionine. Previous studies have indicated that the thymidylate synthesis and homocysteine remethylation pathways compete for a limiting pool of methylenetetrahydrofolate cofactors and that thymidylate biosynthesis is preserved in folate deficiency at the expense of homocysteine remethylation, but the mechanisms are unknown. Recently, it was shown that thymidylate synthesis occurs in the nucleus, whereas homocysteine remethylation occurs in the cytosol. In this study we demonstrate that methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), an enzyme that generates methylenetetrahydrofolate from formate, ATP, and NADPH, functions in the nucleus to support de novo thymidylate biosynthesis. MTHFD1 translocates to the nucleus in S-phase MCF-7 and HeLa cells. During folate deficiency mouse liver MTHFD1 levels are enriched in the nucleus >2-fold at the expense of levels in the cytosol. Furthermore, nuclear folate levels are resistant to folate depletion when total cellular folate levels are reduced by >50% in mouse liver. The enrichment of folate cofactors and MTHFD1 protein in the nucleus during folate deficiency in mouse liver and human cell lines accounts for previous metabolic studies that indicated 5,10-methylenetetrahydrofolate is preferentially directed toward de novo thymidylate biosynthesis at the expense of homocysteine remethylation during folate deficiency.

Vitamin B12 status and folic acid supplementation influence mitochondrial heteroplasmy levels in mice.

One-carbon metabolism is a complex network of metabolic reactions that are essential for cellular function including DNA synthesis. Vitamin B12 and folate are micronutrients that are utilized in this pathway and their deficiency can result in the perturbation of one-carbon metabolism and subsequent perturbations in DNA replication and repair. This effect has been well characterized in nuclear DNA but to date, mitochondrial DNA (mtDNA) has not been investigated extensively. Mitochondrial variants have been associated with several inherited and age-related disease states; therefore, the study of factors that impact heteroplasmy are important for advancing our understanding of the mitochondrial genome's impact on human health. Heteroplasmy studies require robust and efficient mitochondrial DNA enrichment to carry out in-depth mtDNA sequencing. Many of the current methods for mtDNA enrichment can introduce biases and false-positive results. Here, we use a method that overcomes these limitations and have applied it to assess mitochondrial heteroplasmy in mouse models of altered one-carbon metabolism. Vitamin B12 deficiency was found to cause increased levels of mitochondrial DNA heteroplasmy across all tissues that were investigated. Folic acid supplementation also contributed to elevated mitochondrial DNA heteroplasmy across all mouse tissues investigated. Heteroplasmy analysis of human data from the Framingham Heart Study suggested a potential sex-specific effect of folate and vitamin B12 status on mitochondrial heteroplasmy. This is a novel relationship that may have broader consequences for our understanding of one-carbon metabolism, mitochondrial-related disease and the influence of nutrients on DNA mutation rates.