The metabolic burden of methyl donor deficiency with focus on the betaine homocysteine methyltransferase pathway

Methyl groups are important for numerous cellular functions such as DNA methylation, phosphatidylcholine synthesis, and protein synthesis. The methyl group can directly be delivered by dietary methyl donors, including methionine, folate, betaine, and choline. The liver and the muscles appear to be the major organs for methyl group metabolism. Choline can be synthesized from phosphatidylcholine via the cytidine-diphosphate (CDP) pathway. Low dietary choline loweres methionine formation and causes a marked increase in S-adenosylmethionine utilization in the liver. The link between choline, betaine, and energy metabolism in humans indicates novel functions for these nutrients. This function appears to goes beyond the role of the nutrients in gene methylation and epigenetic control. Studies that simulated methyl-deficient diets reported disturbances in energy metabolism and protein synthesis in the liver, fatty liver, or muscle disorders. Changes in plasma concentrations of total homocysteine (tHcy) reflect one aspect of the metabolic consequences of methyl group deficiency or nutrient supplementations. Folic acid supplementation spares betaine as a methyl donor. Betaine is a significant determinant of plasma tHcy, particularly in case of folate deficiency, methionine load, or alcohol consumption. Betaine supplementation has a lowering effect on post-methionine load tHcy. Hypomethylation and tHcy elevation can be attenuated when choline or betaine is available.

Obeid, R., The metabolic burden of methyl donor deficiency with focus on the betaine homocysteine methyltransferase pathway. Nutrients, 2013. 5(9): p. 3481-95

Alleviation of alcoholic liver injury by betaine involves an enhancement of antioxidant defense via regulation of sulfur amino acid metabolism

Previous studies suggested that the hepatoprotective activity of betaine is associated with its effects on sulfur amino acid metabolism. We examined the mechanism by which betaine prevents the progression of alcoholic liver injury and its therapeutic potential. Rats received a liquid ethanol diet for 6 wk. Ethanol consumption elevated serum triglyceride and TNFalpha levels, alanine aminotransferase and aspartate aminotransferase activities, and lipid accumulation in liver. The oxyradical scavenging capacity of liver was reduced, and expression of CD14, TNFalpha, COX-2, and iNOS mRNAs was induced markedly. These ethanol-induced changes were all inhibited effectively by betaine supplementation. Hepatic S-adenosylmethionine, cysteine, and glutathione levels, reduced in the ethanol-fed rats, were increased by betaine supplementation. Methionine adenosyltransferase and cystathionine gamma-lyase were induced, but cysteine dioxygenase was down-regulated, which appeared to account for the increment in cysteine availability for glutathione synthesis in the rats supplemented with betaine. Betaine supplementation for the final 2 wk of ethanol intake resulted in a similar degree of hepatoprotection, revealing its potential therapeutic value in alcoholic liver. It is concluded that the protective effects of betaine against alcoholic liver injury may be attributed to the fortification of antioxidant defense via improvement of impaired sulfur amino acid metabolism.

Jung, Y.S., et al., Alleviation of alcoholic liver injury by betaine involves an enhancement of antioxidant defense via regulation of sulfur amino acid metabolism. Food Chem Toxicol, 2013

Betaine supplementation decreases plasma homocysteine in healthy adult participants: a meta-analysis

OBJECTIVE: Betaine supplementation has been shown to be an effective agent for decreasing plasma homocysteine in healthy adults. Studies in healthy volunteers show that 6 g/d of betaine lowers plasma homocysteine concentrations by 5% to 20%. The purpose of this study was to perform a meta-analysis of randomized placebo-controlled trials that used daily betaine supplementation to identify the range in betaine's effects on lowering homocysteine. METHODS: Five randomized controlled trials published between 2002 and 2010 were identified using MEDLINE and a manual search. All 5 studies used health adult participants who were supplemented with at least 4 g/d of betaine for between 6 and 24 weeks. A meta-analysis was carried out using a random-effects model, and the overall effect size was calculated for changes in plasma homocysteine. RESULTS: The pooled estimate of effect for betaine supplementation on plasma homocysteine was a reduction of 1.23 mumol/L, which was statistically significant (95% confidence interval, - 1.61 to - 0.85; P = .01). CONCLUSION: Supplementation with at least 4g/d of betaine for a minimum of 6 weeks can lower plasma homocysteine.

McRae, M.P., Betaine supplementation decreases plasma homocysteine in healthy adult participants: a meta-analysis. J Chiropr Med, 2013. 12(1): p. 20-5

Betaine raises brain serotonin levels and produces antidepressant-like effects in rats

The purpose of the present study was to examine the effect of Lycii Radicis Cortex (LRC) and betaine (BT) on immobility and neurochemical change in the forced swimming test (FST) in the rat. LRC, BT or fluoxentine was administered intraperitoneally to Sprague-Dawley rats three times (1, 5 and 23.5 h) before the FST. To investigate antidepressant-like effect, serotonin (5-HT) and norepinephrine (NE) were examined in the hippocampus and hypothalamus of rats. LRC (100 mg/kg) and BT (30, 100 mg/kg) significantly decreased the immobility time in the FST. LRC (100 mg/kg) significantly increased both 5-HT and NE levels in the hypothalamus of rats exposed to FST. BT (100 mg/kg) significantly increased 5-HT levels in the hypothalamus and hippocampus of rats. Taken together, these results demonstrated that improvement in the behavioral changes after LRC and BT administration may be mediated by elevation of 5-HT level in the hypothalamus and hippocampus, indicating a possible antidepressant-like activity. The present results suggest that the efficacy of LRC and BT in an animal model of depression may provide anti-depressant effects in human, which remains to be determined.

Kim, S.J., et al., Antidepressant-like effects of lycii radicis cortex and betaine in the forced swimming test in rats. Biomol Ther (Seoul), 2013. 21(1): p. 79-83

Betaine supplementation improved body composition, arm size, bench press work capacity, attenuated the rise in urinary HCTL, and tended to improve power but not strength

BACKGROUND: This study investigated the effects of long term betaine supplementation on body composition, performance, and homocysteine thiolactone (HCTL) in experienced strength trained men. METHODS: Twenty-three subjects were matched for training experience (4.8 +/- 2.3 years) and body fat percentage (BF%: 16.9 +/- 8.0%), randomly assigned to either a placebo (PL; n = 12) or betaine group (BET; n = 11; 2.5 g/day), and completed a 6 week periodized training program consisting of 3 two-week micro-cycles. Bench press and back squat training volumes were recorded and changes in training volume were assessed at each micro-cycle. Fasting urine was collected at baseline (BL), weeks 2, 4 and 6, and assayed for HCTL. Subjects were tested prior to and following 6 weeks of treatment. Arm and thigh cross sectional area (CSA) was estimated via girth and skin fold measurements. Body density was estimated via skin fold calipers and used to estimate BF%, fat mass (FM), and lean body mass (LBM). Performance was assessed via vertical jump (VJ), bench press 1 RM (BP), and back squat 1 RM (BS).
RESULTS: Arm CSA increased significantly (p < .05) in BET but not PL. No differences existed between group and time for changes in thigh CSA. Back squat training volume increased significantly (p < .05) for both groups throughout training. Bench press training volume was significantly (p < .05) improved for BET compared to PL at microcycles one and three. Body composition (BF%, FM, LBM) improved significantly (p < .05) in BET but not PL. No differences were found in performance variables (BP, BS, VJ) between groups, except there was a trend (p = .07) for increased VJ power in BET versus PL. A significant interaction (p < .05) existed for HCTL, with increases from BL to week 2 in PL, but not BET. Additionally, HCTL remained elevated at week 4 in PL, but not BET.
CONCLUSION: Six-weeks of betaine supplementation improved body composition, arm size, bench press work capacity, attenuated the rise in urinary HCTL, and tended to improve power (p = .07) but not strength.

Cholewa, J.M., et al., Effects of betaine on body composition, performance, and homocysteine thiolactone. J Int Soc Sports Nutr, 2013. 10(1): p. 39

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

Topical application of betaine limited progression of environmentally induced dry eye

Purpose: To evaluate the efficacy of osmoprotectants on prevention and treatment of dry eye in a murine model. Methods: Dry eye was induced in mice using an intelligently controlled environmental system (ICES). Osmoprotectants betaine, L-carnitine, erythritol, or vehicle (PBS) were topically administered to eyes 4 times daily following two schedules: Schedule 1 (modeling prevention): dosing started at the beginning of housing in ICES and lasted for 21 or 35 days; Schedule 2 (modeling treatment): dosing started after ICES-housed mice developed dry eye (Day 21), continuing till Day 35. Treatment efficacy was evaluated for corneal fluorescein staining; corneal epithelial apoptosis by TUNEL and caspase-3 assays; goblet cell numbers by PAS staining; and expression of inflammatory mediators, TNF-alpha, IL-17, IL-6 or IL-1beta using RT-PCR on Days 0, 14, 21 and/or 35.
Results: Compared to vehicle, prophylactic administration of betaine, L-carnitine or erythritol significantly decreased corneal staining and expression of TNF-alpha and IL-17 on Day 21 (Schedule 1). Treatment of mouse dry eye with osmoprotectants significantly reduced corneal staining on Day 35 compared to Day 21 (Schedule 2). Relative to vehicle, L-carnitine treatment of mouse dry eye for 14 days (Day 21 to 35) resulted in a significant reduction in corneal staining, number of TUNEL-positive cells, and expression of TNF-alpha, IL-17, IL-6, or IL-1beta, as well significantly increased number of goblet cells.
Conclusion: Topical application of betaine, L-carnitine or erythritol systematically limited progression of environmentally induced dry eye. L-carnitine can also reduce the severity of such dry eye conditions.

Chen, W., et al., Efficacy of Osmoprotectants on Prevention and Treatment of Murine Dry Eye. Invest Ophthalmol Vis Sci, 2013

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.

Betaine protects against isoprenaline-induced myocardial infarction

We investigated the antioxidant preventive effect of betaine on isoprenaline-induced myocardial infarction in male albino rats. Isoprenaline induced myocardial infarction was manifested by a moderate elevation in the levels of diagnostic marker enzymes (alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and creatine phosphokinase) and homocysteine in plasma of experimental rats. Significant rise in the level of lipid peroxidation with a concomitant decline in the levels of myocardial non-enzymic (reduced glutathione) and enzymic antioxidants (glutathione peroxidase, glutathione-S-transferase, catalase and superoxide dismutase) was also observed. Oral pretreatment with betaine significantly prevented isoprenaline-induced alterations in the levels of diagnostic marker enzymes and homocysteine in plasma of experimental groups of rats. It counteracted the isoprenaline-induced lipid peroxidation and maintained the myocardial antioxidant defense system at near normal. Histopathological observations also confirmed the protective effect of betaine against isoprenaline-induced myocardial infarction. The results of the present investigation indicate that the protective effect of betaine is probably related to its ability to strengthen the myocardial membrane by its membrane stabilizing action or to a counteraction of free radicals by its antioxidant property.
Ganesan, B., et al., Antioxidant defense of betaine against isoprenaline-induced myocardial infarction in rats. Mol Biol Rep, 2010. 37(3): p. 1319-27