Genetic Variation in Choline-Metabolizing Enzymes Alters Choline Metabolism in Young Women Consuming Choline Intakes Meeting Current Recommendations.

Single nucleotide polymorphisms (SNPs) in choline metabolizing genes are associated with disease risk and greater susceptibility to organ dysfunction under conditions of dietary choline restriction. However, the underlying metabolic signatures of these variants are not well characterized and it is unknown whether genotypic differences persist at recommended choline intakes. Thus, we sought to determine if common genetic risk factors alter choline dynamics in pregnant, lactating, and non-pregnant women consuming choline intakes meeting and exceeding current recommendations. Women (n = 75) consumed 480 or 930 mg choline/day (22% as a metabolic tracer, choline-d9) for 10-12 weeks in a controlled feeding study. Genotyping was performed for eight variant SNPs and genetic differences in metabolic flux and partitioning of plasma choline metabolites were evaluated using stable isotope methodology. CHKA rs10791957, CHDH rs9001, CHDH rs12676, PEMT rs4646343, PEMT rs7946, FMO3 rs2266782, SLC44A1 rs7873937, and SLC44A1 rs3199966 altered the use of choline as a methyl donor; CHDH rs9001 and BHMT rs3733890 altered the partitioning of dietary choline between betaine and phosphatidylcholine synthesis via the cytidine diphosphate (CDP)-choline pathway; and CHKA rs10791957, CHDH rs12676, PEMT rs4646343, PEMT rs7946 and SLC44A1 rs7873937 altered the distribution of dietary choline between the CDP-choline and phosphatidylethanolamine N-methyltransferase (PEMT) denovo pathway. Such metabolic differences may contribute to disease pathogenesis and prognosis over the long-term.

Genetic impairments in folate enzymes increase dependence on dietary choline for phosphatidylcholine production at the expense of betaine synthesis.

Although single nucleotide polymorphisms (SNPs) in folate-mediated pathways predict susceptibility to choline deficiency during severe choline deprivation, it is unknown if effects persist at recommended intakes. Thus, we used stable isotope liquid chromatography-mass spectrometry (LC-MS) methodology to examine the impact of candidate SNPs on choline metabolism in a long-term, randomized, controlled feeding trial among pregnant, lactating, and nonpregnant (NP) women consuming 480 or 930 mg/d choline (22% as choline-d9, with d9 indicating a deuterated trimethyl amine group) and meeting folate-intake recommendations. Variants impairing folate metabolism, methylenetetrahydrofolate reductase (MTHFR) rs1801133, methionine synthase (MTR) rs1805087 [wild-type (WT)], MTR reductase (MTRR) rs1801394, and methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) rs2236225, influenced choline dynamics, frequently through interactions with reproductive state and choline intake, with fewer genotypic alterations observed among pregnant women. Women with these variants partitioned more dietary choline toward phosphatidylcholine (PC) biosynthesis via the cytidine diphosphate (CDP)-choline pathway at the expense of betaine synthesis even when use of betaine as a methyl donor was increased. Choline intakes of 930 mg/d restored partitioning of dietary choline between betaine and CDP-PC among NP (MTHFR rs1801133 and MTR rs1805087 WT) and lactating (MTHFD1 rs2236225) women with risk genotypes. Overall, our findings indicate that loss-of-function variants in folate-metabolizing enzymes strain cellular PC production, possibly via impaired folate-dependent phosphatidylethanolamine-N-methyltransferase (PEMT)-PC synthesis, and suggest that women with these risk genotypes may benefit from choline intakes exceeding current recommendations.-Ganz, A. B., Shields, K., Fomin, V. G., Lopez, Y. S., Mohan, S., Lovesky, J., Chuang, J. C., Ganti, A., Carrier, B., Yan, J., Taeswuan, S., Cohen, V. V., Swersky, C. C., Stover, J. A., Vitiello, G. A., Malysheva, O. V., Mudrak, E., Caudill, M. A. Genetic impairments in folate enzymes increase dependence on dietary choline for phosphatidylcholine production at the expense of betaine synthesis.

Complementary Cholesterol-Lowering Response of a Phytosterol/α-Lipoic Acid Combination in Obese Zucker Rats.

To investigate the cholesterol-lowering effectiveness of a phytosterol/α-lipoic acid (PS/αLA) therapy, thirty-two male Zucker rats were randomly assigned to 1 of 4 diets for 30 days: (i) high fat diet (HF, 40% energy from fat); (ii) HF diet supplemented with 3% phytosterols; (iii) HF diet supplemented with 0.25% αLA; or (iv) HF diet supplemented with PS (3%) and αLA (0.25%, PS/αLA). Compared with the HF diet, combination PS/αLA proved more effective in reducing non-HDL cholesterol (-55%) than either the PS (-24%) or the αLA (-25%) therapies alone. PS supplementation did not affect LDL particle number, however, αLA supplementation reduced LDL particle number when supplemented alone (-47%) or in combination with PS (-54%). Compared with the HF-fed animals, evidence of increased HDL-particle number was evident in all treatment groups to a similar extent (21-22%). PS-mediated interruption of intestinal cholesterol absorption was evident by increased fecal cholesterol loss (+52%) and compensatory increase in HMG-CoA reductase mRNA (1.6 fold of HF), however, αLA supplementation did not affect fecal cholesterol loss. Hepatic mRNA and protein expression patterns suggested that αLA modulated multiple aspects of cholesterol homeostasis including reduced synthesis (HMG-CoA reductase mRNA, 0.7 fold of HF), reduced bile acid synthesis (CYP7a1 expression, 0.17 of HF), and increased cholesterol clearance (reduced PCSK9 mRNA, 0.5 fold of HF; increased LDLr protein, 2 fold of HF). Taken together, this data suggests that PS and αLA work through unique and complementary mechanisms to provide a superior and more comprehensive cholesterol lowering response than either therapy alone.

Disease-Associated Single-Nucleotide Polymorphisms From Noncoding Regions in Juvenile Idiopathic Arthritis Are Located Within or Adjacent to Functional Genomic Elements of Human Neutrophils and CD4+ T Cells.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is considered a complex disease in which the environment interacts with inherited genes to produce a phenotype that shows broad interindividual variability. Twenty-four regions of genetic risk for JIA were identified in a recent genome-wide association study (GWAS); however, as is typical of the results of GWAS, most of the regions of genetic risk (22 of 24) were in noncoding regions of the genome. This study was undertaken to identify functional elements (other than genes) that might be located within the regions of genetic risk. METHODS: We used paired-end RNA sequencing to identify noncoding RNAs (ncRNAs) located within 5 kb of disease-associated single-nucleotide polymorphisms (SNPs). In addition, we used chromatin immunoprecipitation (ChIP) followed by sequencing to identify epigenetic marks associated with enhancer function (H3K4me1 and H3K27ac) in human neutrophils to determine whether enhancer-associated histone marks were enriched in the linkage disequilibrium blocks that encompassed the 22 SNPs identified in the GWAS. RESULTS: In human neutrophils, we identified H3K4me1 and/or H3K27ac marks in 15 of the 22 regions previously identified as risk loci for JIA. In CD4+ T cells, 18 regions had H3K4me1 and/or H3K27ac marks. In addition, we identified ncRNA transcripts at the rs4705862 and rs6894249 loci in human neutrophils. CONCLUSION: Much of the genetic risk for JIA lies within or adjacent to regions of neutrophil and CD4+ T cell genomes that carry epigenetic marks associated with enhancer function and/or ncRNA transcripts. These findings are consistent with the hypothesis that JIA is fundamentally a disorder of gene regulation that includes both the innate and the adaptive immune system. Elucidating the specific roles of these noncoding elements within leukocyte genomes will be critical to our understanding of JIA pathogenesis.

Alpha-lipoic acid supplementation reduces mTORC1 signaling in skeletal muscle from high fat fed, obese Zucker rats.

The mammalian target of rapamycin (mTOR) signaling pathway is hyperactive in liver, adipose and skeletal muscle tissues of obese rodents. Alpha-lipoic acid (αLA) has been well accepted as a weight-loss treatment, though there are limited studies on its effect on mTOR signaling in high-fat fed, obese rodents. Therefore, the goal of this study was to determine mTOR signaling and oxidative protein alterations in skeletal muscle of high-fat fed, obese rats after αLA supplementation. Phosphorylation of the mTOR substrate, eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) and eIF4B were significantly reduced (p < 0.05) in muscle from αLA supplemented rats. Activation of AMP-activated protein kinase (AMPK), an mTOR inhibitory kinase, was higher (p < 0.05) in the αLA group. Protein expression of markers of oxidative metabolism, acetyl CoA carboxylase (ACC), cytochrome c oxidase IV (COX IV), peroxisome proliferator-activated receptor (PPAR), and PPAR gamma coactivator 1-alpha (PGC-1α) were significantly higher (p < 0.05) after αLA supplementation compared to non-supplemented group. Our findings show that αLA supplementation limits the negative ramifications of consuming a high fat diet on skeletal muscle markers of oxidative metabolism and mTORC1 signaling.

Alpha-lipoic acid reduces LDL-particle number and PCSK9 concentrations in high-fat fed obese Zucker rats.

We characterized the hypolipidemic effects of alpha-lipoic acid (LA, R-form) and examined the associated molecular mechanisms in a high fat fed Zucker rat model. Rats (n = 8) were assigned to a high fat (HF) diet or the HF diet with 0.25% LA (HF-LA) for 30 days and pair fed to remove confounding effects associated with the anorectic properties of LA. Compared with the HF controls, the HF-LA group was protected against diet-induced obesity (102.5±3.1 vs. 121.5±3.6,% change BW) and hypercholesterolemia with a reduction in total-C (-21%), non-HDL-C (-25%), LDL-C (-16%), and total LDL particle number (-46%) and an increase in total HDL particles (∼22%). This cholesterol-lowering response was associated with a reduction in plasma PCSK9 concentration (-70%) and an increase in hepatic LDLr receptor protein abundance (2 fold of HF). Compared with the HF-fed animals, livers of LA-supplemented animals were protected against TG accumulation (-46%), likely through multiple mechanisms including: a suppressed lipogenic response (down-regulation of hepatic acetyl-CoA carboxylase and fatty acid synthase expression); enhanced hepatic fat oxidation (increased carnitine palmitoyltransferase Iα expression); and enhanced VLDL export (increased hepatic diacylglycerol acyltransferase and microsomal triglyceride transfer protein expression and elevated plasma VLDL particle number). Study results also support an enhanced fatty acid uptake (2.8 fold increase in total lipase activity) and oxidation (increased CPT1ß protein abundance) in muscle tissue in LA-supplemented animals compared with the HF group. In summary, in the absence of a change in caloric intake, LA was effective in protecting against hypercholesterolemia and hepatic fat accumulation under conditions of strong genetic and dietary predisposition toward obesity and dyslipidemia.

Folate intake, MTHFR genotype, and sex modulate choline metabolism in mice.

Choline and folate are interrelated in 1-carbon metabolism, mostly because of their shared function as methyl donors for homocysteine remethylation. Folate deficiency and mutations of methylenetetrahydrofolate reductase (MTHFR) reduce the availability of a major methyl donor, 5-methyltetrahydrofolate, which in turn may lead to compensatory changes in choline metabolism. This study investigated the hypothesis that reductions in methyl group supply, either due to dietary folate deficiency or Mthfr gene deletion, would modify tissue choline metabolism in a sex-specific manner. Mthfr wild type (+/+) or heterozygous (+/-) knockout mice were randomized to a folate-deficient or control diet for 8 wk during which time deuterium-labeled choline (d9-choline) was consumed in the drinking water (~10 µmol/d). Mthfr heterozygosity did not alter brain choline metabolite concentrations, but it did enhance their labeling in males (P < 0.05) and tended to do so in females (P < 0.10), a finding consistent with greater turnover of dietary choline in brains of +/- mice. Dietary folate deficiency in females yielded 52% higher (P = 0.027) hepatic glycerophosphocholine, which suggests that phosphatidylcholine (PtdCho) degradation was enhanced. Labeling of the hepatic PtdCho in d3 form was also reduced (P < 0.001) in females, which implies that fewer of the dietary choline-derived methyl groups were used for de novo PtdCho biosynthesis under conditions of folate insufficiency. Males responded to folate restriction with a doubling (P < 0.001) of hepatic choline dehydrogenase transcripts, a finding consistent with enhanced conversion of choline to the methyl donor, betaine. Collectively, these data show that several adaptations in choline metabolism transpire as a result of mild perturbations in folate metabolism, presumably to preserve methyl group homeostasis.