Betaine alleviated hepatic triglyceride accumulation and improved antioxidant capacity by decreasing PPAR alpha promoter methylation and upregulating PPAR alpha and its target genes mRNA expression

BACKGROUND: Betaine is a methyl donor and has been considered a lipotropic effect substance. But its mechanism remains unclear. Hepatic steatosis is associated with abnormal expression of genes involved in hepatic lipid metabolism. DNA methylation contributes to the disregulation of gene expression. Here we hypothesized that betaine supplement and subsequent DNA methylation modifications alter the expression of genes that are involved in hepatic lipid metabolism and hence alleviate hepatic triglyceride accumulation. METHODS: Male wild-type (WT) C57BL/6 mice (n = 6) were fed with the AIN-93 G diet. ApoE-/- mice (n = 12), weight-matched with the WT mice, were divided into two groups (n = 6 per group), and fed with the AIN-93 G diet and AIN-93 G supplemented with 2% betaine/100 g diet. Seven weeks after the intervention, mice were sacrificed. Liver betaine, choline, homocysteine concentration were measured by HPLC. Liver oxidants activity and triglyceride level were assessed by ultraviolet spectrophotometry. Finally, hepatic PPAR alpha gene and its target gene expression levels and the methylation status of the PPAR alpha gene were determined.
RESULTS: ApoE-/- mice had higher hepatic triglyceride and lower GSH-Px activity when compared with the WT mice. Betaine intervention reversed triglyceride deposit, enhanced SOD and GSH-Px activity in the liver. Interestingly, mice fed on betaine-supplemented diet showed a dramatic increase of hepatic choline concentration and a decrease of betaine and homocysteine concentration relative to the WT mice and the ApoE-/- mice absent with betaine intervention. Expression of PPAR alpha and CPT1 were decreased and expression of FAS was markedly increased in ApoE-/- mice. In parallel, PPAR alpha promoter methylation level were slightly decreased in ApoE-/- mice though without significance. Betaine supplement upregulated expression of PPAR alpha and its target genes (CPT1, CYP2E1) and reversed hypermethylation of PPAR alpha promoter of ApoE-/- mice. Furthermore, PPAR alpha methylation was positively correlated with hepatic betaine concentration.
CONCLUSIONS: Our findings indicate that betaine supplement could alleviate hepatic triglyceride accumulation and improve antioxidant capacity by decreasing PPAR alpha promoter methylation and upregulating PPAR alpha and its target genes mRNA expression.

Wang, L., et al., Betaine supplement alleviates hepatic triglyceride accumulation of apolipoprotein E deficient mice via reducing methylation of peroxisomal proliferator-activated receptor alpha promoter. Lipids Health Dis, 2013. 12(1): p. 34

Increased risk of low birth weight with increased umbilical choline and betaine in venous UC blood

Background: Low birth weight (LBW) is associated with increased morbidity and mortality for the newborn and risk of chronic disease in adulthood. Choline plays an essential role in the integrity of cell membranes, methylation reactions and memory development. We examined whether choline, betaine and dimethylglycine (DMG) concentrations were associated with LBW in Dutch women.
Methods: Blood was sampled from umbilical cords (UC) at delivery in singleton pregnancies (n=1126). Maternal blood was sampled at 30-34 weeks of gestational age (n=366). We calculated birth weights standardized for gestational age (SBW) and defined LBW as SBW </= 2500 grams.
Results: Maternal concentrations were lower compared to UC concentrations and not associated with birth weight. UC choline and betaine were inversely associated with birth weight (beta= -60 (-89, -31) and beta= -65 (-94, -36)), whereas UC DMG was positively associated with birth weight (beta= 35 (6.1, 63)). Odds ratios for LBW were 4.12 (1.15, 14.78) , 5.68 (1.24, 25.91), and 0.48 (0.09, 2.65) for the highest UC choline, betaine and DMG quartile respectively compared to the lowest quartiles.
Conclusions: We observed an increased risk of low birth weight with increased umbilical choline and betaine in venous UC blood. These results might reflect a change in choline consumption or metabolism or a disturbed placental function.

Hogeveen, M., et al., Umbilical choline and related methylamines betaine and dimethylglycine in relation to birth weight. Pediatr Res, 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