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.

High homocysteine induces betaine depletion

Betaine is the substrate of the liver- and kidney-specific betaine-homocysteine (Hcy) methyltransferase (BHMT), an alternate pathway for Hcy remethylation. We hypothesized that BHMT is a major pathway for homocysteine removal in cases of hyperhomocysteinaemia (HHcy). Therefore, we measured betaine in plasma and tissues from patients and animal models of HHcy of genetic and acquired cause. Plasma was collected from patients presenting HHcy without any Hcy interfering treatment. Plasma and tissues were collected from rat models of HHcy induced by diet and from a mouse model of cystathionine beta-synthase (CBS) deficiency. S-adenosyl-methionine (AdoMet), S-adenosyl-homocysteine (AdoHcy), methionine, betaine and dimethylglycine (DMG) were quantified by ESI-LC-MS/MS. mRNA expression was quantified using quantitative real-time (QRT)-PCR. For all patients with diverse causes of HHcy, plasma betaine concentrations were below the normal values of our laboratory. In the diet-induced HHcy rat model, betaine was decreased in all tissues analysed (liver, brain, heart). In the mouse CBS deficiency model, betaine was decreased in plasma, liver, heart and brain, but was conserved in kidney. Surprisingly, BHMT expression and activity was decreased in liver. However, in kidney, BHMT and SLC6A12 expression was increased in CBS-deficient mice. Chronic HHcy, irrespective of its cause, induces betaine depletion in plasma and tissues (liver, brain and heart), indicating a global decrease in the body betaine pool. In kidney, betaine concentrations were not affected, possibly due to overexpression of the betaine transporter SLC6A12 where betaine may be conserved because of its crucial role as an osmolyte.

Imbard, A., et al., High homocysteine induces betaine depletion. Biosci Rep, 2015. 35(4).

High homocysteine induces betaine depletion

Betaine is the substrate of the liver- and kidney- specific betaine-homocysteine methyltransferase (BHMT), an alternate pathway for homocysteine remethylation. We hypothesized that BHMT is a major pathway for homocysteine removal in cases of hyperhomocysteinemia (HHcy). Therefore, we measured betaine in plasma and tissues from patients and animal models of HHcy of genetic and acquired cause. Plasma was collected from patients presenting HHcy without any homocysteine interfering treatment. Plasma and tissues were collected from rat models of HHcy induced by diet and from a mouse model of CBS deficiency. S-adenosyl-methionine, S-adenosyl-homocysteine, methionine, betaine, and dimethylglycine were quantified by ESI-LC-MS/MS. mRNA expression was quantified using quantitative real-time PCR. For all patients with diverse causes of HHcy, plasma betaine concentrations were below the normal values of our laboratory. In the diet-induced HHcy rat model, betaine was decreased in all tissues analysed (liver, brain, heart). In the mouse CBS deficiency model, betaine was decreased in plasma, liver, heart and brain, but was conserved in kidney. Surprisingly, BHMT expression and activity was decreased in liver. However, in kidney, BHMT and SLC6A12 expression was increased in CBS-deficient mice. Chronic HHcy, irrespective of its cause, induces betaine depletion in plasma and tissues (liver, brain and heart), indicating a global decrease of the body betaine pool. In kidney, betaine concentrations were not affected, possibly due to overexpression of the betaine transporter SLC6A12 where betaine may be conserved because of its crucial role as an osmolyte.

Imbard, A., et al., High homocysteine induces betaine depletion. Biosci Rep, 2015.

Betaine attenuates alcohol-induced leaky gut through BHMT-mediated catalysis

Alcoholic liver disease (ALD) is a major healthcare challenge worldwide. Emerging evidence reveals that ethanol administration disrupts the intestinal epithelial tight junction (TJ) complex; this defect allows for the paracellular translocation of gut-derived pathogenic molecules to reach the liver to cause inflammation and progressive liver injury. We have previously demonstrated a causative role of impairments in liver transmethylation reactions in the pathogenesis of ALD. We have further shown that treatment with betaine, a methylation agent that normalizes liver methylation potential, can attenuate ethanol-induced liver injury. Herein, we explored whether alterations in methylation reactions play a causative role in disrupting intestinal mucosal barrier function by employing an intestinal epithelial cell line. Monolayers of Caco-2 cells were exposed to ethanol or a-pan methylation reaction inhibitor, tubercidin, in the presence and absence of betaine. The structural and functional integrity of intestinal epithelial barrier was then examined. We observed that exposure to either ethanol or tubercidin disrupted TJ integrity and function by decreasing the localization of TJ protein occludin-1 to the intracellular junctions, reducing transepithelial electrical resistance and increasing dextran influx. All these detrimental effects of ethanol and tubercidin were attenuated by co-treatment with betaine. We further show that the mechanism of betaine protection was through BHMT-mediated catalysis. Collectively, our data suggest a novel mechanism for alcohol-induced gut leakiness and identifies the importance of normal methylation reactions in maintaining TJ integrity. We also propose betaine as a potential therapeutic option for leaky gut in alcohol-consuming patients who are at the risk of developing ALD.

Thomes, P.G., et al., Role of defective methylation reactions in ethanol-induced dysregulation of intestinal barrier integrity. Biochem Pharmacol, 2015.

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

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

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

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 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

Deletion of murine betaine-homocysteine S-methyltransferase in mice perturbs choline and 1-carbon metabolism, resulting in fatty liver and hepatocellular carcinoma

Betaine-homocysteine S-methyltransferase (BHMT) uses betaine to catalyze the degradation of homocysteine (Hcy). There are common genetic polymorphisms in the BHMT gene in humans. To model the phenotype of mice with a loss of BHMT function, we generated the first Bhmt-/- mouse. Deletion of the gene resulted in a 6-fold increase in hepatic and an 8-fold increase in plasma Hcy concentrations, suggesting the importance of BHMT in Hcy removal. Deletion of the gene resulted in a 43% reduction in hepatic S-adenosylmethionine (AdoMet) and a 3-fold increase in hepatic S-adenosylhomocysteine (AdoHcy) concentrations, resulting in a 75% reduction in methylation potential (AdoMet:AdoHcy). Bhmt-/- mice accumulated betaine in most tissues, including a 21-fold increase in the liver concentration compared to wildtype (WT). These mice had lower concentrations of choline, phosphocholine, glycerophosphocholine, phosphatidylcholine and sphingomyelin in several tissues. At 5 weeks of age, Bhmt-/- mice had 36% lower total hepatic phospholipid concentrations and a 6-fold increase in hepatic triacyglycerol concentrations compared to WT, which was due to a decrease in the secretion of very low density lipoproteins. At 1 year of age, 64% of Bhmt-/- mice had visible hepatic tumors. Histopathological analysis revealed that Bhmt-/- mice developed hepatocellular carcinoma (HCC) or carcinoma precursors. These results indicate that BHMT has an important role in Hcy, choline and one-carbon homeostasis. A lack of BHMT also affects susceptibility to fatty liver and HCC. We suggest that functional polymorphisms in BHMT that significantly reduce activity may have similar effects in humans.

Teng, Y.W., et al., Deletion of murine betaine-homocysteine S-methyltransferase in mice perturbs choline and 1-carbon metabolism, resulting in fatty liver and hepatocellular carcinoma. J Biol Chem, 2011. 286: p. 36258-67