Two zebrafish cacna1s loss-of-function variants provide models of mild and severe CACNA1S-related myopathy.

CACNA1S-related myopathy, due to pathogenic variants in the CACNA1S gene, is a recently described congenital muscle disease. Disease associated variants result in loss of gene expression and/or reduction of Cav1.1 protein stability. There is an incomplete understanding of the underlying disease pathomechanisms and no effective therapies are currently available. A barrier to the study of this myopathy is the lack of a suitable animal model that phenocopies key aspects of the disease. To address this barrier, we generated knockouts of the two zebrafish CACNA1S paralogs, cacna1sa and cacna1sb. Double knockout fish exhibit severe weakness and early death, and are characterized by the absence of Cav1.1 α1 subunit expression, abnormal triad structure, and impaired excitation-contraction coupling, thus mirroring the severe form of human CACNA1S-related myopathy. A double mutant (cacna1sa homozygous, cacna1sb heterozygote) exhibits normal development, but displays reduced body size, abnormal facial structure, and cores on muscle pathologic examination, thus phenocopying the mild form of human CACNA1S-related myopathy. In summary, we generated and characterized the first cacna1s zebrafish loss-of-function mutants, and show them to be faithful models of severe and mild forms of human CACNA1S-related myopathy suitable for future mechanistic studies and therapy development.

Methyl donor micronutrients, hypothalamic development and programming for metabolic disease.

Nutriture in utero is essential for fetal brain development through the regulation of neural stem cell proliferation, differentiation, and apoptosis, and has a long-lasting impact on risk of disease in offspring. This review examines the role of maternal methyl donor micronutrients in neuronal development and programming of physiological functions of the hypothalamus, with a focus on later-life metabolic outcomes. Although evidence is mainly derived from preclinical studies, recent research shows that methyl donor micronutrients (e.g., folic acid and choline) are critical for neuronal development of energy homeostatic pathways and the programming of characteristics of the metabolic syndrome in mothers and their children. Both folic acid and choline are active in one-carbon metabolism with their impact on epigenetic modification of gene expression. We conclude that an imbalance of folic acid and choline intake during gestation disrupts DNA methylation patterns affecting mechanisms of hypothalamic development, and thus elevates metabolic disease risk. Further investigation, including studies to determine translatability to humans, is required.

Monoallelic loss-of-function BMP2 variants result in BMP2-related skeletal dysplasia spectrum.

PURPOSE: Bone morphogenic proteins (BMPs) regulate gene expression that is related to many critical developmental processes, including osteogenesis for which they are named. In addition, BMP2 is widely expressed in cells of mesenchymal origin, including bone, cartilage, skeletal and cardiac muscle, and adipose tissue. It also participates in neurodevelopment by inducing differentiation of neural stem cells. In humans, BMP2 variants result in a multiple congenital anomaly syndrome through a haploinsufficiency mechanism. We sought to expand the phenotypic spectrum and highlight phenotypes of patients harboring monoallelic missense variants in BMP2. METHODS: We used retrospective chart review to examine phenotypes from an international cohort of 18 individuals and compared these with published cases. Patient-derived missense variants were modeled in zebrafish to examine their effect on the ability of bmp2b to promote embryonic ventralization. RESULTS: The presented cases recapitulated existing descriptions of BMP2-related disorders, including craniofacial, cardiac, and skeletal anomalies and exhibit a wide phenotypic spectrum. We also identified patients with neural tube defects, structural brain anomalies, and endocrinopathies. Missense variants modeled in zebrafish resulted in loss of protein function. CONCLUSION: We use this expansion of reported phenotypes to suggest multidisciplinary medical monitoring and management of patients with BMP2-related skeletal dysplasia spectrum.

Methylenetetrahydrofolate reductase deficiency and high-dose FA supplementation disrupt embryonic development of energy balance and metabolic homeostasis in zebrafish.

Folic acid (synthetic folate, FA) is consumed in excess in North America and may interact with common pathogenic variants in methylenetetrahydrofolate reductase (MTHFR); the most prevalent inborn error of folate metabolism with wide-ranging obesity-related comorbidities. While preclinical murine models have been valuable to inform on diet-gene interactions, a recent Folate Expert panel has encouraged validation of new animal models. In this study, we characterized a novel zebrafish model of mthfr deficiency and evaluated the effects of genetic loss of mthfr function and FA supplementation during embryonic development on energy homeostasis and metabolism. mthfr-deficient zebrafish were generated using CRISPR mutagenesis and supplemented with no FA (control, 0FA) or 100 µm FA (100FA) throughout embryonic development (0-5 days postfertilization). We show that the genetic loss of mthfr function in zebrafish recapitulates key biochemical hallmarks reported in MTHFR deficiency in humans and leads to greater lipid accumulation and aberrant cholesterol metabolism as reported in the Mthfr murine model. In mthfr-deficient zebrafish, energy homeostasis was also impaired as indicated by altered food intake, reduced metabolic rate and lower expression of central energy-regulatory genes. Microglia abundance, involved in healthy neuronal development, was also reduced. FA supplementation to control zebrafish mimicked many of the adverse effects of mthfr deficiency, some of which were also exacerbated in mthfr-deficient zebrafish. Together, these findings support the translatability of the mthfr-deficient zebrafish as a preclinical model in folate research.

X-linked myotubular myopathy is associated with epigenetic alterations and is ameliorated by HDAC inhibition.

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention.

Natural history of a mouse model of X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a severe monogenetic disorder of the skeletal muscle. It is caused by loss-of-expression/function mutations in the myotubularin (MTM1) gene. Much of what is known about the disease, as well as the treatment strategies, has been uncovered through experimentation in pre-clinical models, particularly the Mtm1 gene knockout mouse line (Mtm1 KO). Despite this understanding, and the identification of potential therapies, much remains to be understood about XLMTM disease pathomechanisms, and about the normal functions of MTM1 in muscle development. To lay the groundwork for addressing these knowledge gaps, we performed a natural history study of Mtm1 KO mice. This included longitudinal comparative analyses of motor phenotype, transcriptome and proteome profiles, muscle structure and targeted molecular pathways. We identified age-associated changes in gene expression, mitochondrial function, myofiber size and key molecular markers, including DNM2. Importantly, some molecular and histopathologic changes preceded overt phenotypic changes, while others, such as triad structural alternations, occurred coincidentally with the presence of severe weakness. In total, this study provides a comprehensive longitudinal evaluation of the murine XLMTM disease process, and thus provides a critical framework for future investigations.

Folate dose and form during pregnancy may program maternal and fetal health and disease risk.

The role of folate, in its synthetic and bioactive form, as an in utero modifier of metabolic outcomes in mothers and offspring is examined in this review. During pregnancy, a continuum of adaptive changes occurs to support maternal and fetal requirements. However, an unfavorable in utero environment may lead to permanent changes in cellular and physiological functions, adversely affecting the development of the child and postpartum health of the mother. In North American countries, synthetic folic acid (FA) is overconsumed by pregnant women, and uncertainty exists about its potential unintended health effects. Because the metabolism of FA is different than that of other folate forms, it may modulate disease risk differently. The bioactive form of folate, 5-methyltetrahydrofolic acid, has emerged as a popular alternative to FA, but clinical studies comparing their effects during pregnancy are limited. Current evidence points to the need for caution when maternal intake of either folate form exceed recommended amounts. Research directed toward defining an optimal folate dose and form for healthy pregnancy and long-term metabolic outcomes in mothers and children is urgently needed.

High Intakes of [6S]-5-Methyltetrahydrofolic Acid Compared with Folic Acid during Pregnancy Programs Central and Peripheral Mechanisms Favouring Increased Food Intake and Body Weight of Mature Female Offspring.

Supplementation with [6S]-5-methyltetrahydrofolic acid (MTHF) is recommended as an alternative to folic acid (FA) in prenatal supplements. This study compared equimolar gestational FA and MTHF diets on energy regulation of female offspring. Wistar rats were fed an AIN-93G diet with recommended (2 mg/kg diet) or 5-fold (5X) intakes of MTHF or FA. At weaning, female offspring were fed a 45% fat diet until 19 weeks. The 5X-MTHF offspring had higher body weight (>15%), food intake (8%), light-cycle energy expenditure, and lower activity compared to 5X-FA offspring (p < 0.05). Both the 5X offspring had higher plasma levels of the anorectic hormone leptin at birth (60%) and at 19 weeks (40%), and lower liver weight and total liver lipids compared to the 1X offspring (p < 0.05). Hypothalamic mRNA expression of leptin receptor (ObRb) was lower, and of suppressor of cytokine signaling-3 (Socs3) was higher in the 5X-MTHF offspring (p < 0.05), suggesting central leptin dysregulation. In contrast, the 5X-FA offspring had higher expression of genes encoding for dopamine and GABA- neurotransmitter receptors (p < 0.01), consistent with their phenotype and reduced food intake. When fed folate diets at the requirement level, no differences were found due to form in the offspring. We conclude that MTHF compared to FA consumed at high levels in the gestational diets program central and peripheral mechanisms to favour increased weight gain in the offspring. These pre-clinical findings caution against high gestational intakes of folates of either form and encourage clinical trials examining their long-term health effects when consumed during pregnancy.

High Choline Intake during Pregnancy Reduces Characteristics of the Metabolic Syndrome in Male Wistar Rat Offspring Fed a High Fat But Not a Normal Fat Post-Weaning Diet.

Maternal choline intakes are below recommendations, potentially impairing the child's later-life metabolic health. This study aims to elucidate the interaction between the choline content of the gestational diet (GD) and fat content of the post-weaning diet (PWD) on metabolic phenotype of male Wistar rats. Pregnant Wistar rats were fed a standard rodent diet (AIN-93G) with either recommended choline (RC, 1 g/kg diet choline bitartrate) or high choline (HC, 2.5-fold). Male pups were weaned to either a normal (16%) fat (NF) or a high (45%) fat (HF) diet for 17 weeks. Body weight, visceral adiposity, food intake, energy expenditure, plasma hormones, triglycerides, and hepatic fatty acids were measured. HC-HF offspring had 7% lower body weight but not food intake, and lower adiposity, plasma triglycerides, and insulin resistance compared to RC-HF. They also had increased hepatic n-3 fatty acids and a reduced n-6/n-3 and C 18:1 n-9/C18:0 ratios. In contrast, HC-NF offspring had 6-8% higher cumulative food intake and body weight, as well as increased leptin and elevated hepatic C16:1 n-7/C16:0 ratio compared to RC-NF. Therefore, gestational choline supplementation associated with improved long-term regulation of several biomarkers of the metabolic syndrome in male Wistar rat offspring fed a HF, but not a NF, PWD.

Choline and Folic Acid in Diets Consumed during Pregnancy Interact to Program Food Intake and Metabolic Regulation of Male Wistar Rat Offspring.

BACKGROUND: North American women consume high folic acid (FA), but most are not meeting the adequate intakes for choline. High-FA gestational diets induce an obesogenic phenotype in rat offspring. It is unclear if imbalances between FA and other methyl-nutrients (i.e., choline) account for these effects. OBJECTIVE: This study investigated the interaction of choline and FA in gestational diets on food intake, body weight, one-carbon metabolism, and hypothalamic gene expression in male Wistar rat offspring. METHODS: Pregnant Wistar rats were fed an AIN-93G diet with recommended choline and FA [RCRF; 1-fold, control] or high (5-fold) FA with choline at 0.5-fold [low choline and high folic acid (LCHF)], 1-fold [recommended choline and high folic acid (RCHF)], or 2.5-fold [high choline and high folic acid (HCHF)]. Male offspring were weaned to an RCRF diet for 20 wk. Food intake, weight gain, plasma energy-regulatory hormones, brain and plasma one-carbon metabolites, and RNA sequencing (RNA-seq) in pup hypothalamuses were assessed. RESULTS: Adult offspring from LCHF and RCHF, but not HCHF, gestational diets had 10% higher food intake and weight gain than controls (P < 0.01). HCHF newborn pups had lower plasma insulin and leptin compared with LCHF and RCHF pups (P < 0.05), respectively. Pup brain choline (P < 0.05) and betaine (P < 0.01) were 22-33% higher in HCHF pups compared with LCHF pups; methionine was ∼23% lower after all high FA diets compared with RCRF (P < 0.01). LCHF adult offspring had lower brain choline (P < 0.05) than all groups and lower plasma 5-methyltetrahydrofolate (P < 0.05) than RCRF and RCHF groups. HCHF adult offspring had lower plasma cystathionine (P < 0.05) than LCHF adult offspring and lower homocysteine (P < 0.01) than RCHF and RCRF adult offspring. RNA-seq identified 144 differentially expressed genes in the hypothalamus of HCHF newborns compared with controls. CONCLUSIONS: Increased choline in gestational diets modified the programming effects of high FA on long-term food intake regulation, plasma energy-regulatory hormones, one-carbon metabolism, and hypothalamic gene expression in male Wistar rat offspring, emphasizing a need for more attention to the choline and FA balance in maternal diets.

[6S]-5-Methyltetrahydrofolic Acid and Folic Acid Pregnancy Diets Differentially Program Metabolic Phenotype and Hypothalamic Gene Expression of Wistar Rat Dams Post-Birth.

[6S]-5-methyltetrahydrofolic acid (MTHF) is a proposed replacement for folic acid (FA) in diets and prenatal supplements. This study compared the effects of these two forms on maternal metabolism and hypothalamic gene expression. Pregnant Wistar rats received an AIN-93G diet with recommended FA (1X, 2 mg/kg, control), 5X-FA or equimolar levels of MTHF. During lactation they received the control diet and then a high fat diet for 19-weeks post-weaning. Body weight, adiposity, food intake, energy expenditure, plasma hormones, folate, and 1-carbon metabolites were measured. RNA-sequencing of the hypothalamus was conducted at parturition. Weight-loss from weaning to 1-week post-weaning was less in dams fed either form of the 5X vs. 1X folate diets, but final weight-gain was higher in 5X-MTHF vs. 5X-FA dams. Both doses of the MTHF diets led to 8% higher food intake and associated with lower plasma leptin at parturition, but higher leptin at 19-weeks and insulin resistance at 1-week post-weaning. RNA-sequencing revealed 279 differentially expressed genes in the hypothalamus in 5X-MTHF vs. 5X-FA dams. These findings indicate that MTHF and FA differ in their programing effects on maternal phenotype, and a potential adverse role of either form when given at the higher doses.

Folic acid content of diet during pregnancy determines post-birth re-set of metabolism in Wistar rat dams.

Maternal metabolism begins to return to homeostasis (re-set) following birth and is accelerated by lactation. Delay in metabolic re-set may contribute to postpartum weight retention and later-life metabolic consequences. Folic acid (FA) is essential during pregnancy but inadequate intakes may alter 1-carbon metabolism, consequently affecting energy homeostatic systems. Our objectives were to examine the effects of FA content 1)below and 2)above requirements during pregnancy on the re-set of body weight, markers of hepatic 1-carbon metabolism and central and peripheral energy metabolic pathways in Wistar rat mothers early post-weaning (PW) compared to pregnant controls. Pregnant Wistar rats were fed an AIN-93G diet with FA at 0X, 1X (control, 2 mg FA/kg) or a range above requirements at 2.5X, 5X or 10X recommended levels then the control diet during lactation up to 1 week PW. Dams fed below (0X) or above (5X and 10X) FA requirements had delayed weight-loss from weaning up to 1 week PW, higher plasma insulin and HOMA-IR and changes in glucose and lipid metabolism-regulating genes in muscle, but not liver or adipose tissue compared to controls. Expression of folate-related genes in liver were lower in high FA fed dams. Central food intake neurons were not affected by FA diets. In conclusion, intakes of FA below (0X) or above (5X, 10X) requirements during pregnancy delayed weight-loss, dysregulated 1-carbon pathways in the liver and peripheral energy metabolic pathways in the Wistar rat mother up to 4 weeks after dietary exposure; potentially programming long-term negative metabolic effects and that of her future offspring.

Maternal Choline Intake Programs Hypothalamic Energy Regulation and Later-Life Phenotype of Male Wistar Rat Offspring.

SCOPE: High-folic-acid diets during pregnancy result in obesity in the offspring, associated with altered DNA-methylation of hypothalamic food intake neurons. Like folic acid, the methyl-donor choline modulates foetal brain development, but its long-term programing effects on energy regulation remain undefined. This study aims to describe the effect of choline intake during pregnancy on offspring phenotype and hypothalamic energy-regulatory mechanisms. METHODS AND RESULTS: Wistar rat dams are fed an AIN-93G diet with recommended choline (RC, 1 g kg-1 diet), low choline (LC, 0.5-fold), or high choline (HC, 2.5-fold) during pregnancy. Male pups are terminated at birth and 17 weeks post-weaning. Brain 1-carbon metabolites, body weight, food intake, energy expenditure, plasma hormones, and protein expression of hypothalamic neuropeptides are measured. HC pups have higher expression of the orexigenic neuropeptide-Y neurons at birth, consistent with higher cumulative food intake and body weight gain post-weaning compared to RC and LC offspring. LC pups have lower leptin receptor expression at birth and lower energy expenditure and activity during adulthood. CONCLUSION: Choline content of diets that are consumed by rats during pregnancy affects the later-life phenotype of offspring, associated with altered in utero programing of hypothalamic food intake regulation.

Gestational folic acid content alters the development and function of hypothalamic food intake regulating neurons in Wistar rat offspring post-weaning.

Background: Folic acid plays an important role in early brain development of offspring, including proliferation and differentiation of neural stem cells known to impact the function of food intake regulatory pathways. Excess (10-fold) intakes of folic acid in the gestational diet have been linked to increased food intake and obesity in male rat offspring post-weaning.Objective: The present study examined the effects of folic acid content in gestational diets on the development and function of two hypothalamic neuronal populations, neuropeptide Y (NPY) and pro-opiomelanocortin (POMC), within food intake regulatory pathways of male Wistar rat offspring at birth and post-weaning.Results: Folic acid fed at 5.0-fold above recommended levels (5RF) to Wistar dams during pregnancy increased the number of mature NPY-positive neurons in the hypothalamus of male offspring, compared to control (RF), 0RF, 2.5RF, and 10RF at birth. Folic acid content had no effect on expression and maturation of POMC-positive neurons. Body weight and food intake were higher in all treatment groups (2.5-, 5.0-, and 10.0-fold folic acid) from birth to 9 weeks post-weaning compared to control. Increased body weight and food intake at 9-weeks post-weaning were accompanied by a reduced activation of POMC neurons in the arcuate nucleus (ARC).Conclusion: Gestational folic acid content modulates expression of mature hypothalamic NPY-positive neurons at birth and activation of POMC-positive neurons at 9-weeks post-weaning in the ARC of male Wistar rat offspring which may contribute to higher body weight and food intake later in life.

Maternal fat-soluble vitamins, brain development, and regulation of feeding behavior: an overview of research.

Recent research shows a link between vitamin intake during pregnancy and offspring health. Inadequate intakes of water-soluble vitamins during pregnancy lead to obesity and characteristics of the metabolic syndrome, concurrent with altered developments in food intake regulatory pathways. Few studies, however, have reported on the effects of fat-soluble vitamins (A, D, E, and K) on the development of food intake regulatory pathways. The majority of studies to date have focused on associations between inadequate and high intakes of folic acid and vitamin D and neurocognitive development of the offspring. Hence, the objective of this review is to present an evaluation of the role of maternal vitamins A, D, E, and K in brain development and function of neural pathways that regulate feeding behaviors. PubMed and Google Scholar were searched from 1975 through September, 2016. Most studies supporting a role for fat-soluble vitamins in regulating brain development and associated behaviors have been conducted in animal and cell models, leaving uncertain their relevance to neurocognitive development and function in humans. Nevertheless, although current research on defining the role of maternal fat-soluble vitamins in offspring's brain development is limited, it is sufficient to warrant further investigations on their impact when intake amounts during pregnancy are not only inadequate but also exceed requirements.

Role of maternal vitamins in programming health and chronic disease.

Vitamin consumption prior to and during pregnancy has increased as a result of proactive recommendations by health professionals, wide availability of vitamin supplements, and liberal food-fortification policies. Folic acid, alone or in combination with other B vitamins, is the most recommended vitamin consumed during pregnancy because deficiency of this vitamin leads to birth defects in the infant. Folic acid and other B vitamins are also integral components of biochemical processes that are essential to the development of regulatory systems that control the ability of the offspring to adapt to the external environment. Although few human studies have investigated the lasting effects of high vitamin intakes during pregnancy, animal models have shown that excess vitamin supplementation during gestation is associated with negative metabolic effects in both the mothers and their offspring. This research from animal models, combined with the recognition that epigenetic regulation of gene expression is plastic, provides evidence for further examination of these relationships in the later life of pregnant women and their children.

High vitamin A intake during pregnancy modifies dopaminergic reward system and decreases preference for sucrose in Wistar rat offspring.

High multivitamin (HV) content in gestational diets has long-term metabolic effects in rat offspring. These changes are associated with in utero modifications of gene expression in hypothalamic food intake regulation. However, the role of fat-soluble vitamins in mediating these effects has not been explored. Vitamin A is a plausible candidate due to its role in gene methylation. Vitamin A intake above requirements during pregnancy affects the development of neurocircuitries involved in food intake and reward regulation. Pregnant Wistar rats were fed AIN-93G diets with the following content: recommended multivitamins (1-fold multivitamins: RV), high vitamin A (10-fold vitamin A: HA) or HV with only recommended vitamin A (10-fold multivitamins, 1-fold vitamin A: HVRA). Body weight, food intake and preference, mRNA expression and DNA methylation of hippocampal dopamine-related genes were assessed in male offspring brains at different developmental windows: birth, weaning and 14weeks postweaning. HA offspring had changes in dopamine-related gene expression at all developmental windows and DNA hypermethylation in the dopamine receptor 2 promoter region compared to RV offspring. Furthermore, HA diet lowered sucrose preference but had no effect on body weight and expression of hypothalamic genes. In contrast, HVRA offspring showed only at adulthood changes in expression of hippocampal genes and a modest effect on hypothalamic genes. High vitamin A intake alone in gestational diets has long-lasting programming effects on the dopaminergic system that are further translated into decreased sucrose preference but not food intake.

A gestational diet high in fat-soluble vitamins alters expression of genes in brain pathways and reduces sucrose preference, but not food intake, in Wistar male rat offspring.

High intakes of multivitamins (HV) during pregnancy by Wistar rats increase food intake, body weight, and characteristics of the metabolic syndrome in male offspring. In this study, high-fat soluble vitamins were fed in combination during gestation to test the hypothesis that they partially account for the effects of the HV diet. Pregnant Wistar rats (14-16/group) were fed a recommended multivitamin diet (1-fold all vitamins) or high-fat soluble vitamin diet (HFS; 10-fold vitamins A, D, E, and K) during pregnancy. Offspring body weight, food intake, and preference as well as expression of selected genes in the hypothalamus and hippocampus were evaluated at birth, weaning, and 14 weeks postweaning. Body weight and food intake were not affected but sucrose preference decreased by 4% in those born to dams fed the HFS gestational diet. Gene expressions of the hypothalamic anorexogenic pro-opiomelanocortin (Pomc) and orexogenic neuropeptide Y (Npy) (∼30% p = 0.008, ∼40% p = 0.007) were increased in weaning and adult rats, respectively. Hippocampal dopaminergic genes (35%-50% p < 0.05) were upregulated at birth and 14 weeks postweaning. DNA hypermethylation (2% p = 0.006) was observed in the dopamine receptor 1 (Drd1) promoter region. We conclude that a gestational diet high in vitamins A, D, E, and K does not show the effects of the HV diet on body weight or food intake but may affect the development of higher hedonic regulatory pathways associated with food preference.

A high multivitamin diet fed to Wistar rat dams during pregnancy increases maternal weight gain later in life and alters homeostatic, hedonic and peripheral regulatory systems of energy balance.

High multivitamin (10-fold, HV) and high folic acid (Fol) diets fed to pregnant Wistar rats increase body weight and characteristics of the metabolic syndrome in their offspring. Our objective was to determine the effects of a HV maternal diet on dams and whether methyl vitamins contribute to these effects. Pregnant Wistar rats were fed AIN-93G diets containing either (1) recommended multivitamins (RV, control), (2) HV, (3) HV with recommended Fol (HVRF; 1-fold Fol), or (4) RV with high methyl group vitamins (HMethyl; 10-fold Fol, vitamin B12 and B6). All groups were fed a RV diet during lactation until weaning and a RV high fat (HF; 60% fat) diet for 16 weeks post-weaning. The HV, HVRF and HMethyl diet fed dams gained 45% more weight from 2 to 15 weeks post-weaning and their weight gain (WG) was positively associated with cumulative post-weaning food intake (FI). However, only HV dams had a reduced preference for a sucrose solution, lower mesolimbic dopamine (DA) turnover in the nucleus accumbens (NAc), and higher expression of several genes involved in FI regulation in the arcuate nucleus of the hypothalamus (ARC). Energy conserving peroxisome proliferator-activated receptor (Ppar)-γ in adipose and -α in liver was also greater in these dams consistent with their WG. In conclusion, HV, HVRF and HMethyl maternal diets exacerbate maternal WG when dams are exposed to a HF diet post-weaning. However, the diets differed in their effects on central and peripheral regulatory systems of energy balance.

Methyl vitamins contribute to obesogenic effects of a high multivitamin gestational diet and epigenetic alterations in hypothalamic feeding pathways in Wistar rat offspring.

SCOPE: High multivitamin (HV, tenfold AIN-93G) gestational diets fed to Wistar rats increase food intake, obesity, and characteristics of metabolic syndrome in the offspring. We hypothesized that methyl vitamins, and specifically folate, in the HV gestational diet contribute to the obesogenic phenotypes consistent with their epigenetic effects on hypothalamic food intake regulatory mechanisms. METHODS AND RESULTS: Male offspring of dams fed the AIN-93G diet with high methyl vitamins (HMethyl; tenfold folate, vitamins B12, and B6) (Study 1) and HV with recommended folate (HVRF) (Study 2) were compared with those from HV and recommended vitamin (RV) fed dams. All offspring were weaned to a high fat diet for 8 wks. HMethyl diet, similar to HV, and compared to RV, resulted in higher food intake, body weight, and metabolic disturbances. Removing folate additions to the HV diet in HVRF offspring normalized the obesogenic phenotype. Methyl vitamins, and folate in HV diets, altered hypothalamic gene expression toward increased food intake concurrent with DNA methylation and leptin and insulin receptor signaling dysfunction. CONCLUSION: Methyl vitamins in HV gestational diets contribute to obesogenic phenotypes and epigenetic alterations in the hypothalamic feeding pathways in the offspring. Folate alone accounts for many of these effects.