Nonalcoholic Steatohepatitis is associated with a state of betaine-insufficiency

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) develops from a complex process, which includes changes in the liver methylome. Betaine plays a pivotal role in the regulation of methylogenesis. We performed a two-stage case-control study, which included patients with biopsy-proven NAFLD in order to explore circulating levels of betaine and its association with the histological spectrum. We also explored the association between a missense p.Ser646Pro variant in DMGDH (dimethylglycine dehydrogenase mitochondrial) and NAFLD severity (n =390).
RESULTS: In the discovery phase (n = 48), betaine levels were associated with the disease severity (p = 0.0030), including liver inflammation (Spearman R: - 0.51, p = 0.001), ballooning degeneration (R: - 0.50, p = 0.01), and fibrosis (R: - 0.54, p = 0.0008). Betaine levels were significantly decreased in nonalcoholic steatohepatitis (NASH) in comparison with nonalcoholic fatty liver (NAFL). Further replication (n = 51) showed that betaine levels were associated with advanced NAFLD (p = 0.0085), and patients with NASH had a 1.26-fold decrease in betaine levels compared with those with NAFL. The rs1805074 was significantly associated with the disease severity (p = 0.011).
CONCLUSION: NAFLD severity is associated with a state of betaine-insufficiency.

Sookoian, S., et al., Nonalcoholic Steatohepatitis is associated with a state of betaine-insufficiency. Liver Int, 2016.

Maternal betaine supplementation during gestation enhances gene expression in skeletal muscle of newborn piglets

Betaine has been widely used in animal and human nutrition to promote muscle growth and performance, yet it remains unknown whether maternal betaine supplementation during gestation affects the metabolic characteristics of neonatal skeletal muscles. In the present study, feeding sows with betaine-supplemented diets throughout gestation significantly up-regulated the expression of mtDNA-encoded OXPHOS genes including COX1, COX2 and ND5, in the muscle of newborn piglets, which was associated with enhanced mitochondrial COX enzyme activity. Concurrently, maternal betaine supplementation increased plasma betaine concentration and muscle expression of methyl transfer enzymes, BHMT and GNMT in offspring piglets. Nevertheless, Dnmt3a was down-regulated at the level of both mRNA and protein, which was associated with hypomethylated mtDNA D-loop region. These results suggest that maternal betaine supplementation during gestation enhances expression of mtDNA-encoded genes through D-loop DNA hypomethylation in skeletal muscle of newborn piglets.

Jia, Y., et al., Maternal betaine supplementation during gestation enhances expression of mtDNA-encoded genes through D-loop DNA hypomethylation in skeletal muscle of newborn piglets. J Agric Food Chem, 2015.

The beneficial effects of betaine on dysfunctional adipose tissue and N6-methyladenosine mRNA methylation requires the AMP-activated protein kinase alpha1 subunit

The current study was conducted to determine whether betaine could improve fatty acid oxidation, mitochondrial function and N6-methyladenosine (m6A) mRNA methylation in adipose tissue in high-fat-induced mice and how AMP-activated protein kinase alpha1 subunit (AMPKalpha1) was involved. AMPKalpha1 knockout mice and wild-type mice were fed either a low-fat diet, high-fat diet or high-fat diet supplemented with betaine in the drinking water for 8weeks. Our results showed that mitochondrial genes (PGC1alpha) and beta-oxidation-related genes (CPT1a) at protein level were increased in wild-type mice supplemented with betaine when compared with those in mice with high-fat diet. Betaine also decreased FTO expression and improved m6A methylation in adipose tissue of wild-type mice with high-fat diet. However, betaine failed to exert the abovementioned effects in AMPKalpha1 knockout mice. In adipocytes isolated from mice with high-fat diet, betaine treatment increased lipolysis and lipid oxidation. Moreover, betaine decreased FTO expression and increased m6A methylation. However, while AMPKalpha1 was knockdown, no remarkable changes in adipocytes were observed under betaine treatment. Our results indicated that betaine supplementation rectified mRNA hypomethylation and high FTO expression induced by high-fat diet, which may contribute to its beneficial effects on impaired adipose tissue function. Our results suggested that the AMPKalpha1 subunit is required for the beneficial effects of betaine on dysfunctional adipose tissue and m6A methylation. These results may provide the foundation for a mechanism that links m6A methylation status in RNA, AMPKalpha1 phosphorylation and dysfunctional adipose tissue induced by high-fat diet.

Zhou, X., et al., The beneficial effects of betaine on dysfunctional adipose tissue and N6-methyladenosine mRNA methylation requires the AMP-activated protein kinase alpha1 subunit. J Nutr Biochem, 2015

FTO-dependent function of N6-methyladenosine is involved in the hepatoprotective effects of betaine on adolescent mice

Nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease among children and adolescents in the developed world. Betaine, as a methyl donor, recently has been demonstrated to exert its hepatoprotective effects through rectifying the genomic DNA hypomethylation state. However, whether betaine supplementation affects N6-methyladenosine (m6A) mRNA methylation in NAFLD is still unknown. We conducted the current study to investigate the effects of betaine supplementation during adolescence on high-fat diet-induced pathological changes in liver of mice, and we further identified the effects of betaine supplementation on expression of the fat mass and obesity-associated gene (FTO) and hepatic m6A mRNA methylation. Our results showed that betaine supplementation across adolescence significantly alleviated high-fat-induced impairment of liver function and morphology as well as ectopic fat accumulation. Surprisingly, no significant effects on serum TG and NEFA level, as well as fat mass, were observed in mice supplemented with betaine. We also found that high-fat diet upregulated ACC1 and FAS gene expression and downregulated HSL and ATGL gene expression. However, these alterations were rectified by betaine supplementation. Moreover, an m6A hypomethylation state and increased FTO expression were detected in mice fed with high-fat diet, while betaine supplementation prevented these changes. Our results suggested that betaine supplementation during adolescence could protect mice from high-fat-induced NAFLD by decreasing de novo lipogenesis and increasing lipolysis. Furthermore, a novel FTO-dependent function of m6A may involve in the hepatoprotective effects of betaine.

Chen, J., et al., FTO-dependent function of N6-methyladenosine is involved in the hepatoprotective effects of betaine on adolescent mice. J Physiol Biochem, 2015.

Maternal betaine supplementation enhances betaine/methionine metabolism and DNA methyltransferase expression in the hippocampus of neonatal piglets

PURPOSE: The adequate supply of methyl donors is critical for the normal development of brain. The purpose of the present study was to investigate the effects of maternal betaine supplementation on hippocampal gene expression in neonatal piglets and to explore the possible mechanisms.
METHODS: Gestational sows were fed control or betaine-supplemented (3 g/kg) diets throughout the pregnancy. Immediately after birth, male piglets were killed, and the hippocampus was dissected for analyses. The mRNA abundance was determined by reverse transcription real-time polymerase chain reaction. Protein content was measured by Western blot, and DNA methylation was detected by methylated DNA immunoprecipitation assay.
RESULTS: Prenatal betaine supplementation did not alter the body weight or the hippocampus weight, but increased the hippocampal DNA content as well as the mRNA expression of proliferation-related genes. Prenatal betaine supplementation increased serum level of methionine (P < 0.05) and up-regulated (P < 0.05) the mRNA and protein expression of betaine-homocysteine methyltransferase, glycine N-methyltransferase and DNA methyltransferase 1 in the neonatal hippocampus. Hippocampal expression of insulin growth factor II (IGF2) and its receptors IGF1R and IGF2R were all significantly up-regulated (P < 0.05) in betaine-treated group, together with a significant activation (P < 0.01) of the downstream extracellular signal-regulated kinase 1/2. Moreover, the differentially methylated region (DMR) 1 and 2 on IGF2 locus was found to be hypermethylated (P < 0.05) in the hippocampus of betaine-treated piglets.
CONCLUSIONS: These results indicate that maternal betaine supplementation enhances betaine/methionine metabolism and DNA methyltransferase expression, causes hypermethylation of DMR on IGF2 gene, which was associated with augmented expression of IGF2 and cell proliferation/anti-apoptotic markers in the hippocampus of neonatal piglets.

Li, X., et al., Dietary betaine supplementation to gestational sows enhances hippocampal IGF2 expression in newborn piglets with modified DNA methylation of the differentially methylated regions. Eur J Nutr, 2014.

Maternal dietary betaine supplementation modifies hepatic expression of cholesterol metabolic genes via epigenetic mechanisms in newborn piglets

To elucidate the effects of maternal dietary betaine supplementation on hepatic expression of cholesterol metabolic genes in newborn piglets and the involved epigenetic mechanisms, we fed gestational sows with control or betaine-supplemented diets (3 g/kg) throughout pregnancy. Neonatal piglets born to betaine-supplemented sows had higher serum methionine concentration and hepatic content of betaine, which was associated with significantly up-regulated hepatic expression of glycine N-methyltransferase. Prenatal betaine exposure increased hepatic cholesterol content and modified the hepatic expression of cholesterol metabolic genes in neonatal piglets. Sterol regulatory element-binding protein 2 was down-regulated at both mRNA and protein levels, while 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) was down-regulated at the mRNA level, but up-regulated at the protein level, in betaine-exposed piglets. The transcriptional repression of HMGCR was associated with CpG island hypermethylation and higher repressive histone mark H3K27me3 (histone H3 lysine 27 trimethylation) on the promoter, whereas increased HMGCR protein content was associated with significantly decreased expression of miR-497. Furthermore, LDL receptor was significantly down-regulated at both mRNA and protein levels in the liver of betaine-exposed piglets, which was associated with promoter CpG hypermethylation. In addition, the expression of cholesterol-27alpha-hydroxylase (CYP27alpha1) was up-regulated at both mRNA and protein levels, while the expression of cholesterol-7alpha-hydroxylase (CYP7alpha1) was increased at the mRNA level, but unchanged at the protein level associated with increased expression of miR-181. These results indicate that maternal betaine supplementation increases hepatic cholesterol content in neonatal piglets through epigenetic regulations of cholesterol metabolic genes, which involve alterations in DNA and histone methylation and in the expression of microRNA targeting these genes.

Cai, D., et al., Maternal dietary betaine supplementation modifies hepatic expression of cholesterol metabolic genes via epigenetic mechanisms in newborn piglets. Br J Nutr, 2014: p. 1-10

Betaine supplementation in maternal diet modulates the epigenetic regulation of hepatic gluconeogenic genes in neonatal piglets.

In this study, gestational sows were fed control or betaine-supplemented diets (3 g/kg) throughout the pregnancy, and the newborn piglets were used to elucidate whether maternal dietary betaine affected offspring hepatic gluconeogenic genes through epigenetic mechanisms. Neonatal piglets born to betaine-supplemented sows had significantly higher serum and hepatic betaine contents, together with significantly greater expression of methionine metabolic enzymes in the liver. Interestingly, significantly higher serum concentrations of lactic acid and glucogenic amino acids, including serine, glutamate, methionine and histidine, were detected in the piglets born to betaine-supplemented sows, which were coincident with higher hepatic glycogen content and PEPCK1 enzyme activity, as well as greater protein expression of gluconeogenic enzymes, pyruvate carboxylase (PC), cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK1), mitochondrional phosphoenolpyruvate carboxykinase (PEPCK2) and fructose-1, 6-bisphosphatase (FBP1). Moreover, maternal betaine significantly changed the methylation status of both CpGs and histones on the promoter of gluconeogenic genes. The lower PEPCK1 mRNA was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3, while the up-regulated PEPCK2 and FBP1 mRNA was associated with DNA hypomethylation and more enriched activation histone mark H3K4me3. Furthermore, the expression of two miRNAs predicted to target PC and 6 miRNAs predicted to target PEPCK1 was dramatically suppressed in the liver of piglets born to betaine-supplemented sows. Our results provide the first evidence that maternal betaine supplementation affects hepatic gluconeogenic genes expression in newborn piglets through enhanced hepatic methionine metabolism and epigenetic regulations, which involve DNA and histone methylations, and possibly miRNAs-mediated post-transcriptional mechanism.

Cai, D., et al., Betaine supplementation in maternal diet modulates the epigenetic regulation of hepatic gluconeogenic genes in neonatal piglets. PLoS One, 2014. 9(8): p. e105504

The relationship between betaine, homocysteine, and BHMT expression in hibernating and active mammalian brain

Elevated homocysteine is an important risk factor that increases cerebrovascular and neurodegenerative disease morbidity. In mammals, B vitamin supplementation can reduce homocysteine levels. Whether, and how, hibernating mammals, that essentially stop ingesting B vitamins, maintain homocysteine metabolism and avoid cerebrovascular impacts and neurodegeneration remain unclear. Here, we compare homocysteine levels in the brains of torpid bats, active bats and rats to identify the molecules involved in homocysteine homeostasis. We found that homocysteine does not elevate in torpid brains, despite declining vitamin B levels. At low levels of vitamin B6 and B12, we found no change in total expression level of the two main enzymes involved in homocysteine metabolism (methionine synthase and cystathionine beta-synthase), but a 1.85-fold increase in the expression of the coenzyme-independent betaine-homocysteine S-methyltransferase (BHMT). BHMT expression was observed in the amygdala of basal ganglia and the cerebral cortex where BHMT levels were clearly elevated during torpor. This is the first report of BHMT protein expression in the brain and suggests that BHMT modulates homocysteine in the brains of hibernating bats. BHMT may have a neuroprotective role in the brains of hibernating mammals and further research on this system could expand our biomedical understanding of certain cerebrovascular and neurodegenerative disease processes.

Zhang, Y., et al., Homocysteine homeostasis and betaine-homocysteine s-methyltransferase expression in the brain of hibernating bats. PLoS One, 2013. 8(12): p. e85632

Betaine attenuates hepatic steatosis by reducing methylation of the MTTP promoter and elevating genomic methylation in mice fed a high fat diet

Aberrant DNA methylation contributes to the abnormality of hepatic gene expression, one of the main factors in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Betaine is a methyl donor and has been considered to be a lipotropic agent. However, whether betaine supplementation improves NAFLD via its effect on the DNA methylation of specific genes and the genome has not been explored. Male C57BL/6 mice were fed either a control diet (CD) or high fat diet (HFD) supplemented with 0%, 1% and 2% betaine in water (wt/vol) for 12 weeks. Betaine supplementation ameliorated HFD-induced hepatic steatosis in a dose-dependent manner. HFD upregulated FAS and ACOX mRNA expression and downregulated PPARα, ApoB and MTTP mRNA expression; however, these alterations were reversed by betaine supplementation except ApoB. MTTP mRNA expression was negatively correlated with the DNA methylation of its CpG sites at -184, -156, -63 and -60. Methylation of these CpG sites was lower in both the 1% and 2% betaine-supplemented groups than in the HFD group (averages; 25.55% and 14.33% vs. 30.13%). In addition, both 1% and 2% betaine supplementation significantly restored the methylation capacity (SAM concentration and SAM/SAH ratios) and genomic methylation level which had been decreased by HFD (0.37% and 0.47% vs. 0.25%). These results suggest that the regulation of aberrant DNA methylation by betaine might be a possible mechanism of the improvements in NAFLD upon betaine supplementation.

Wang, L.-j., et al., Betaine attenuates hepatic steatosis by reducing methylation of the MTTP promoter and elevating genomic methylation in mice fed a high fat diet. The Journal of Nutritional Biochemistry, 2013

Survival and psychomotor development with early betaine treatment in patients with severe methylenetetrahydrofolate reductase deficiency

IMPORTANCE The impact of betaine treatment on outcome in patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency is presently unclear.
OBJECTIVE To investigate the effect of betaine treatment on development and survival in patients with severe MTHFR deficiency. DATA SOURCES MEDLINE, EMBASE, and Cochrane databases between January 1960 and December 2012. STUDY SELECTION Studies that described patients with severe MTHFR deficiency who received betaine treatment.
DATA EXTRACTION AND SYNTHESIS We identified 15 case reports and case series, totaling 36 patients. Data included the following: (1) families with 2 or more patients with severe MTHFR deficiency, of whom at least 1 received betaine, or (2) single patients with severe MTHFR deficiency treated with betaine. To define severe MTHFR deficiency, methionine, homocysteine, MTHFR enzyme activity in fibroblasts, or mutations (in the MTHFR gene) had to be described as well as the effect of treatment (survival and/or psychomotor development). We compared the outcome in treated vs untreated patients and early- vs late-treated patients. Sensitivity analysis was performed to address definition of early treatment. To further assess the impact of treatment on mortality, we performed a subanalysis in families with at least 1 untreated deceased patient.
MAIN OUTCOMES AND MEASURES Survival and psychomotor development.
RESULTS Eleven of 36 patients (31%) died. All deaths occurred in patients who did not receive treatment or in patients in whom treatment was delayed. In contrast, all 5 early-treated patients survived. Subgroup analysis of patients with deceased siblings-their genotypically identical controls-revealed that betaine treatment prevented mortality (P = .002). In addition, psychomotor development in surviving patients treated with betaine was normal in all 5 early-treated patients but in none of the 19 surviving patients with delayed treatment (P < .001).
CONCLUSIONS AND RELEVANCE Early betaine treatment prevents mortality and allows normal psychomotor development in patients with severe MTHFR deficiency, highlighting the importance of timely recognition through newborn screening.

Diekman, E.F., et al., Survival and Psychomotor Development With Early Betaine Treatment in Patients With Severe Methylenetetrahydrofolate Reductase Deficiency. JAMA Neurol, 2013

High fat-sucrose diet-induced liver fat accumulation was prevented by methyl donor supplementation

Non-alcoholic fatty liver disease is a primary hepatic manifestation of obesity and an important adverse metabolic syndrome trait. Animal models of diet-induced obesity promote liver fat accumulation putatively associated with alterations in epigenetic profile. Dietary methyl donor-supplementation may protect against this disturbance during early developmental stages affecting the molecular basis of gene regulation. The aim of this study was to investigate the transcriptomic and epigenetic mechanisms implicated in liver fat accumulation as a result of an obesogenic diet and the putative preventive role of dietary methyl donors. Forty-eight male Wistar rats were assigned into four dietary groups for 8weeks; control, control methyl-donor-supplemented with a dietary cocktail containing betaine, choline, vitamin B12 and folic acid, high-fat-sucrose and high-fat-sucrose methyl-donor-supplemented. Liver fat accumulation induced by a HFS diet was prevented by methyl donor supplementation in HFS-fed animals. A liver mRNA microarray, subsequently validated by real time-qPCR, showed modifications in some biologically relevant genes involved in obesity development and lipid metabolism (Lepr, Srebf2, Agpat3 and Esr1). Liver global DNA methylation was decreased by methyl donor supplementation in control-fed animals. Methylation levels of specific CpG sites from Srebf2, Agpat3 and Esr1 promoter regions showed changes due to the obesogenic diet and the supplementation with methyl donors. Interestingly, Srebf2 CpG23_24 methylation levels (-167bp and -156bp with respect to the transcriptional start site) correlated with HDLc plasma levels, whereas Esr1 CpG14 (-2623bp) methylation levels were associated with body and liver weights and fat content. Furthermore HFS diet-induced liver fat accumulation was prevented by methyl donor supplementation. In conclusion, both obesogenic diet and methyl donor supplementation modified the mRNA hepatic profile as well as the methylation of specific gene promoters and total DNA.

Cordero, P., et al., Transcriptomic and epigenetic changes in early liver steatosis associated to obesity: Effect of dietary methyl donor supplementation. Mol Genet Metab, 2013

Maternal methyl metabolism, offspring DNA methylation, and epigenetic consequences on later disease susceptibility

Evidence is growing for the long-term effects of environmental factors during early-life on later disease susceptibility. It is believed that epigenetic mechanisms (changes in gene function not mediated by DNA sequence alteration), particularly DNA methylation, play a role in these processes. This paper reviews the current state of knowledge of the involvement of C1 metabolism and methyl donors and cofactors in maternal diet-induced DNA methylation changes in utero as an epigenetic mechanism. Methyl groups for DNA methylation are mostly derived from the diet and supplied through C1 metabolism by way of choline, betaine, methionine or folate, with involvement of riboflavin and vitamins B6 and B12 as cofactors. Mouse models have shown that epigenetic features, for example DNA methylation, can be altered by periconceptional nutritional interventions such as folate supplementation, thereby changing offspring phenotype. Evidence of early nutrient-induced epigenetic change in human subjects is scant, but it is known that during pregnancy C1 metabolism has to cope with high fetal demands for folate and choline needed for neural tube closure and normal development. Retrospective studies investigating the effect of famine or season during pregnancy indicate that variation in early environmental exposure in utero leads to differences in DNA methylation of offspring. This may affect gene expression in the offspring. Further research is needed to examine the real impact of maternal nutrient availability on DNA methylation in the developing fetus.

Dominguez-Salas, P., et al., Maternal nutritional status, C(1) metabolism and offspring DNA methylation: a review of current evidence in human subjects. Proc Nutr Soc, 2012. 71(1): p. 154-65

Lower level of serum betaine is associated with an increased risk of microangiopathy in diabetics

BACKGROUND: Diabetes represents one of the greatest medical and socioeconomic emergencies worldwide and pathogenesis involved is complicated. The effect of methyl donors and genetic polymorphisms in metabolic enzymes on the risk of microangiopathy in patients with diabetes is not well understood. This study aims to investigate the association of homocysteine, choline, and betaine levels and phosphatidylethanolamine N-methyltransferase (PEMT) G774C (rs12325817) genotypes with the risk of diabetes and its related microangiopathic complications.
METHODS: Between January 2009 and June 2010, 184 diabetic patients and 188 non-diabetic control subjects were enrolled in the hospital-based case-control study. Serum concentrations of betaine and choline were determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). Serum concentration of homocysteine was assayed using HPLC. PEMT gene mutations were detected by polymerase chain reaction and restriction fragment length polymorphism.
RESULTS: After adjustment for the potential confounders, serum total homocysteine had a significant dose-dependent positive association, and serum choline had an inverse association with the risks of diabetes and its microangiopathic complications (both p < 0.001). Although serum betaine was not associated with the risk of diabetes, it had a significant inverse association with diabetic microangiopathy. Compared with GG genotype, the CC genotype of PEMT G774C was associated with a decreased risk of diabetes (OR 0.559, 95%CI 0.338, 0.926) and its microangiopathy (OR 0.452, 95%CI 0.218, 0.937).
CONCLUSION: The GG genotype of the PEMT G774C polymorphism, higher level of serum homocysteine, and lower level of serum betaine are associated with an increased risk of microangiopathy in patients with diabetes.

Chen, L., et al., Higher homocysteine and lower betaine increases the risk of microangiopathy in patients with diabetes mellitus carrying the GG genotype of PEMT G774C. Diabetes Metab Res Rev, 2013

You are what your mother ate? A commentary on Dominguez-Salas, P., et al., Am J Clin Nutr, 2013. 97(6): p. 1217-27

First brought to scientific attention several decades ago by the report of a dose-response, inverse relation between birth weight and adult risk of cardiometabolic diseases (1), evidence that early nutrition, stress, and other similar environmental exposures can have lingering impacts on later health outcomes has now been widely documented in human populations and in experimental animal model work (2). A central problem in this field is identifying the mechanisms that underlie the biological “memory” of early nutrition and other exposures. Early proposals focused on growth alterations or changes in the function of organs such as the kidneys or liver, which it was speculated would be short-changed under conditions of fetal nutritional stress. Although this likely helps to explain some of the human findings (3), more recent work has consolidated around identifying epigenetic changes induced by prenatal or maternal experiences, which provide particularly attractive candidates (4, 5). Epigenetic modifications typically involve chemical changes to the chromatin that influence which genes can be expressed, silencing or amplifying gene production in a targeted fashion. DNA methylation refers to the addition of a methyl group to cytosine nucleotides in the vicinity of a gene promoter, which impedes gene transcription. It is among the best studied of epigenetic processes and one that is chemically stable enough to potentially account for late-life effects of early nutrition.

A long-running study in Keneba, Gambia, provides an unusual opportunity to probe the effect of maternal diet as an inducer of epigenetic change in offspring in a quasi-experimental fashion. Longitudinal studies of nutrition, growth, and health have been conducted in this region for more than 60 y (6). An important feature of the local ecology is a strong seasonal change in nutritional stress that is secondary to the annual harvest cycle and compounded by seasonal changes in workload in the fields and infectious disease. During the rainy season, workloads are high and crop stores are generally running thin, and, as a result, adults typically lose weight during this season (7). Not surprisingly, rainy-season birth weights also tend to be smaller.

A recent study of children in Keneba by Waterland et al (8) showed that methylation at putative “metastable epialleles”—regions where gene promoter methylation is stochastically rather than genetically based and environmentally sensitive during early development—was contingent on season of birth (8). The goal of the study was to evaluate whether a climatically driven seasonal change in nutritional stress experienced around the time of conception would predict differences in methylation in adulthood. This would help to establish that methylation at those loci was not simply genetically based while also demonstrating the stability of the induced changes. The authors did find differences in epigenetic marking by season of birth but were somewhat surprised to find that methylation was increased during the nutritionally stressful rainy season, when food supplies dwindle. This finding ran counter to their expectations because it suggested that methylation was in fact enhanced at times when one might expect the diet to be most deficient in methyl donors and related cofactors that are required of methylation. The authors speculated that seasonal changes in dietary availability of essential nutrients, rather than gross changes in calories or macronutrients, might account for the apparent increased methyl transfer capacity during times of dietary dearth.

A new study in this issue of the Journal by the same group (9) takes an important step toward evaluating this idea by measuring seasonal changes in dietary intake of methyl donors and related cofactors and at the same time by evaluating their circulating concentrations. The authors weighed household food intake and analyzed the concentrations of choline, betaine, folate, methionine, riboflavin, and vitamins B-6 and B-12 in 98 locally common foods to estimate intake of these nutrients. To evaluate whether dietary intake predicted circulating concentrations, the authors also collected monthly blood samples and measured blood levels of the same nutrients, along with several metabolites involved in methyl transfer. Their findings were a bit mixed, but they did find seasonal changes in intake of these essential nutrients and also changes in circulating biomarkers. In some instances, but not all, these changes were correlated. The authors also found evidence for seasonal shifts in the body’s reliance on different methyl donor pathways. Of importance, the changes in circulating biomarkers were generally in agreement with the finding of an increased capacity for methylation during the difficult rainy season.

Where does this new study leave us? To the extent that seasonal changes in dietary intake have remained relatively constant across a generation or so, the authors’ findings bolster the case that dietary methyl donors, rather than energy or macronutrient intake, help to explain season-of-birth–related variation in epigenetic state in the adult population. This is important and suggests that this population provides a natural experiment for probing the dietary determinants of long-term epigenetic programming in humans with greater specificity, and with an unusually strong basis for causal inference owing to the fact that dietary changes are driven primarily by climate and season rather than by more inherently confounded factors like socioeconomic status. Because methyl donor intake is elevated when energy and macronutrient intakes are most compromised, there is also greater assurance that the relations truly reflect an effect specific to these essential nutrients rather than being secondary to greater energy or macronutrient intake generally.

Although these factors are apparently important locally, it is not yet clear how important dietary intake of methyl donors and related cofactors are likely to be as influences on health in populations with diverse diets and ecologies. It seems unlikely, for instance, that the finding that helped launch this field—a dose-response relation between birth weight and cardiovascular disease risk across the entire birth-weight distribution, even in well-nourished, high-income populations—will be explained by gradients of dietary methyl donor intake. Indeed, earlier work in this population failed to find evidence that early-life nutritional stress or growth faltering predicted measures of diabetes and cardiometabolic disease as expected (10), raising questions about how this population might differ from others in which these relations have been found. Perhaps the fact that dietary methyl donor intake peaks during what is otherwise a season of nutritional stress will ultimately help to shed light on this negative finding.

Kuzawa, C.W., You are what your mother ate? Am J Clin Nutr, 2013. 97(6): p. 1157-8.

Maternal BHMT polymorphism 742G>A is associated with decreased risk of having a Down syndrome child

Down syndrome (DS) is the most common form of mental retardation of genetic etiology. Several polymorphisms in genes involved with the folic acid cycle have been associated to the risk of bearing a DS child; however, the results are controversial. Betaine-homocysteine methyltransferase (BHMT) is a key enzyme of folate pathway, and catalyzes the remethylation of homocysteine into methionine. Recent studies suggest that the polymorphism BHMT 742G>A may be associated with a decreased risk of having a DS child. We herein investigate the association of this polymorphism with the occurrence of DS in a Brazilian population. We have genotyped 94 mothers of DS infants (DSM) and 134 control mothers (CM) for this polymorphism through PCR-RFLP, and found significant differences for both BHMT 742G>A genotype (P = 0.04) and allele (P = 0.03) frequencies between DSM and CM. The observed genotypic frequencies were GG = 0.45; GA = 0.45 and AA = 0.10 in CM, and GG = 0.54; GA = 0.38 and AA = 0.02 in DSM. Allelic frequencies were G = 0.68 and A = 0.32 in CM and G = 0.78 and A = 0.22 in DSM. The presence of the mutant BHMT 742 A allele decreases 40 % the risk of bearing a DS child (OR = 0.61; 95 % CI: 0.40-0.93; P = 0.03), and the risk is diminished up to >80 % in association with the homozygous genotype (OR = 0.17; 95 % CI: 0.04-0.80; P = 0.01). Our results indicate that women harboring the single nucleotide polymorphism BHMT 742G>A have a decreased risk of a DS pregnancy, and further studies are necessary to confirm this protective effect.

Amorim, M.R., et al., Betaine-homocysteine methyltransferase 742G>A polymorphism and risk of down syndrome offspring in a Brazilian population. Mol Biol Rep, 2013

Lower seasonal maternal dietary betaine intake and resultant lower blood levels correlates with previously reported reduced DNA methylation in offspring

BACKGROUND: Animal models show that periconceptional supplementation with folic acid, vitamin B-12, choline, and betaine can induce differences in offspring phenotype mediated by epigenetic changes in DNA. In humans, altered DNA methylation patterns have been observed in offspring whose mothers were exposed to famine or who conceived in the Gambian rainy season.

OBJECTIVE:The objective was to understand the seasonality of DNA methylation patterns in rural Gambian women. We studied natural variations in dietary intake of nutrients involved in methyl-donor pathways and their effect on the respective metabolic biomarkers.

DESIGN:In 30 women of reproductive age (18-45 y), we monitored diets monthly for 1 y by using 48-h weighed records to measure intakes of choline, betaine, folate, methionine, riboflavin, and vitamins B-6 and B-12. Blood biomarkers of these nutrients, S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), homocysteine, cysteine, and dimethylglycine were also assessed monthly.

RESULTS:Dietary intakes of riboflavin, folate, choline, and betaine varied significantly by season; the most dramatic variation was seen for betaine. All metabolic biomarkers showed significant seasonality, and vitamin B-6 and folate had the highest fluctuations. Correlations between dietary intakes and blood biomarkers were found for riboflavin, vitamin B-6, active vitamin B-12 (holotranscobalamin), and betaine. We observed a seasonal switch between the betaine and folate pathways and a probable limiting role of riboflavin in these processes and a higher SAM/SAH ratio during the rainy season.

CONCLUSIONS:Naturally occurring seasonal variations in food-consumption patterns have a profound effect on methyl-donor biomarker status. The direction of these changes was consistent with previously reported differences in methylation of metastable epialleles. This trial was registered at www.clinicaltrials.gov as NCT01811641.

Dominguez-Salas, P., et al., DNA methylation potential: dietary intake and blood concentrations of one-carbon metabolites and cofactors in rural African women. Am J Clin Nutr, 2013

High fat diet induces upregulation of BHMT to accommodate dietary fat processing and preserve methionine

Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine beta-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments , using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARalpha) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-alpha-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARalpha-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine.

Dahlhoff, C., et al., Hepatic Methionine Homeostasis Is Conserved in C57BL/6N Mice on High-Fat Diet Despite Major Changes in Hepatic One-Carbon Metabolism. PLoS One, 2013. 8(3): p. e57387

Betaine prevents vasculature remodeling and inflammatory response during vascular aging

We previously reported that lysophosphatidylcholine (LPC) is a mediator of endothelial dysfunction in expression of adhesion molecules (AMs) during aging. This study aimed at investigating the effects of betaine on LPC-related expression of AMs and the molecular modulation of nuclear factor-kappaB (NF-kappaB) activation in the aorta of aged rats and rat endothelial YPEN-1 cells. The experiment was performed on young (7months) and old (21months) rats; 2 groups of old rats were fed betaine (3 or 6mg*kg-1*day-1 for 10days). Betaine inhibited the expression of LPC-related AMs in the serum and tissue of aged rats, without affecting the elevated levels of serum LPC. Betaine also prevented the generation of reactive species, thereby maintaining the redox status via the enhancement of the thiol status during aging. Furthermore, betaine attenuated NF-kappaB activation via the dephosphorylation of IkappaB kinase (IKK) and mitogen-activated protein kinases (MAPKs) in aged aorta and LPC-treated YPEN-1 cells. Thus, betaine suppressed the LPC-related AM expression associated with NF-kappaB activation via the upregulation of IKK/MAPKs. Our findings provide insights into the prevention of vascular disorders and the development of interventions based on natural compounds, such as betaine.

Lee, E.K., et al., Betaine attenuates lysophosphatidylcholine-mediated adhesion molecules in aged rat aorta: Modulation of the nuclear factor-kappaB pathway. Exp Gerontol, 2013

Betaine homocysteine methyltransferase (BHMT) transcription is decreased in cancer cells due to loss of gene function

Carcinogenesis is a multi-step and multifactorial process. It includes genetic, epigenetic, nutritional and environmental factors, which are closely interconnected. Human hepatocellular carcinoma (HCC) is among the most frequent and lethal cancers. Imbalance in the S-adenosylmethionine (SAM) concentration, the main methyl group donor, strongly influences the development of HCC. Key enzymes of carbon metabolism are greatly reduced in patients with cirrhosis and HCC. These alterations play a role in genetic instability and epigenetic modifications (DNA methylation, and histone modifications), however, the molecular underlying mechanisms are still poorly understood. We aimed to investigate betaine homocysteine methyltransferase (BHMT) expression in HepG2 cells and human hepatocarcinoma tissues. Tumor and surrounding healthy tissue were compared. HepG2 cells and tumor samples showed a strong decrease in BHMT transcripts resulting from the transcription of a splicing variant that contained a frameshift mutation generating a premature termination codon and gene loss of function. This splicing variant, not detected in normal adult and fetal liver, cannot be explained by any mechanism involving the known splicing consensus sequences. BHMT activity was abolished in HepG2 cells and protein expression was detected neither in HepG2 cells nor in five of the six tumor samples investigated. Further investigation is needed to elucidate whether this abnormal BHMT transcription is part of cause or consequence of liver carcinogenesis.

Pellanda, H., Betaine homocysteine methyltransferase (BHMT)-dependent remethylation pathway in human healthy and tumoral liver. Clin Chem Lab Med, 2013. 51(3): p. 617-21

Osmoregulation by betaine involves well-orchestrated gene expression

Betaine critically contributes to the control of hepatocellular hydration and provides protection of the liver from different kinds of stress. To investigate how the hepatocellular hydration state affects gene expression of enzymes involved in the metabolism of betaine and related organic osmolytes we used qRT-PCR gene expression studies in rat hepatoma cells as well as metabolic and gene expression profiling in primary hepatocytes of both wild-type and 5,10-methylenetetrahydrofolate reductase (MTHFR) deficient mice. Anisotonic incubation caused co-ordinated adaptive changes in the expression of various genes involved in betaine metabolism, in particular of betaine homocysteine methyltransferase (BHMT), dimethylglycine dehydrogenase (DMGDH), and sarcosine dehydrogenase (SARDH). The expression of betaine-degrading enzymes was downregulated by cell shrinking and strongly induced by an increase in cell volume under hypotonic conditions. Metabolite concentrations in the culture system changed accordingly. Expression changes were mediated through tyrosine kinases, cyclic nucleotide-dependent protein kinases and JNK-dependent signalling. Assessment of hepatic gene expression using a customised microarray chip showed that hepatic betaine depletion in Mthfr-/- mice was associated with alterations that were comparable to those induced by cell swelling in hepatocytes. In conclusion, the adaptation of hepatocytes to changes in cell volume involves the co-ordinated regulation of betaine synthesis and degradation and concomitant changes in intracellular osmolyte concentrations. The existence of such a well-orchestrated response underlines the importance of cell volume homeostasis for liver function and of methylamine osmolytes such as betaine as hepatic osmolytes.

Hoffmann, L., et al., Osmotic regulation of hepatic betaine metabolism. Am J Physiol Gastrointest Liver Physiol, 2013