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Nonalcoholic fatty liver (NAFL) is a common liver disease, associated with insulin resistance. Betaine has been tested as a treatment for NAFL in animal models and in small clinical trials, with mixed results. The present study aims to determine whether betaine treatment would prevent or treat NAFL in mice and to understand how betaine reverses hepatic insulin resistance. Male mice were fed a moderate high-fat diet (mHF) containing 20% of calories from fat for 7 (mHF) or 8 (mHF8) mo without betaine, with betaine (mHFB), or with betaine for the last 6 wk (mHF8B). Control mice were fed standard chow containing 9% of calories from fat for 7 mo (SF) or 8 mo (SF8). HepG2 cells were made insulin resistant and then studied with or without betaine, mHF mice had higher body weight, fasting glucose, insulin, and triglycerides and greater hepatic fat than SF mice. Betaine reduced fasting glucose, insulin, triglycerides, and hepatic fat. In the mHF8B group, betaine treatment significantly improved insulin resistance and hepatic steatosis. Hepatic betaine content significantly decreased in mHF and increased significantly in mHFB. Betaine treatment reversed the inhibition of hepatic insulin signaling in mHF and in insulin-resistant HepG2 cells, including normalization of insulin receptor substrate 1 (IRS1) phosphorylation and of downstream signaling pathways for gluconeogenesis and glycogen synthesis. Betaine treatment prevents and treats fatty liver in a moderate high-dietary-fat model of NAFL in mice. Betaine also reverses hepatic insulin resistance in part by increasing the activation of IRS1, with resultant improvement in downstream signaling pathways. high fat; hepatic steatosis; insulin receptor substrate; triglyceride doi: 10.1152/ajpgi.00249.2010.

An emerging goal of medical nutrition therapy is to tailor dietary advice to an individual's genetic profile. In the United States and elsewhere, 'nutrigenetic' services are available over the Internet without the direct involvement of a health care professional. Among the genetic variants most commonly assessed by these companies are those found in genes that influence cardiovascular disease risk. However, the interpretation of DNA-based data is complex. The goal of this paper is to carefully examine nutritional genomics as a potential tool for targeted medical nutrition therapy. The approach is to use heart health susceptibility genes and their common genetic variants as the model. Key words: heart health susceptiblity genes, medical nutrition therapy, nutritional genomics

Glycine N-methyltransferase (GNMT) is a key regulatory protein in folate metabolism, methionine availability, and transmethylation reactions. Perturbations in GNMT may lead to aberrations in homocysteine metabolism, a marker of numerous pathologies. The primary objective of this study was to examine the influence of the GNMT 1289 C [right arrow] T alone, and in combination with the methylenetetrahydrofolate reductase (MTHFR) 677 C [right arrow] T variant, on plasma total homocysteine concentrations in healthy young women (n = 114). Plasma total homocysteine was measured at baseline (wk 0) and after 2 wk of controlled folate restriction (135/[micro]g/d as dietary folate equivalents). Plasma homocysteine concentrations did not differ among the GNMT C1289T genotypes at baseline. However, after folate restriction, women with the GNMT 1289 TT genotype (n = 16) had higher (P = 0.019) homocysteine concentrations than women with the CT (n = 51) or CC (n = 47) genotype. The influence of the GNMT 1289 C [right arrow] T variant on homocysteine was dependent on the MTHFR C677T genotype. In subjects with the MTHFR 677 CC genotype, homocysteine was greater (P [less than or equal to] 0.05) for GNMT 1289 TT subjects relative to 1289 CT or CC subjects. However, in subjects with the MTHFR 677 TT genotype, plasma homocysteine concentrations did not differ among the GNMT C1289T genotypes. Overall, these data suggest that the GNMT 1289 C [right arrow] T polymorphism influences plasma homocysteine and is responsive to folate intake. KEY WORDS: * glycine N-methyltransferase * methylenetetrahydrofolate reductase * methioninesynthase reductase * homocysteine * folate * women

DNA methylation is a heritable epigenetic feature that is associated with transcriptional silencing, X-chromosome inactivation, genetic imprinting, and genomic stability. The addition of the methyl group is catalyzed by a family of DNA methyltransferases whose co-substrates are DNA and S-adenosylmethionine, the latter being derived from the methionine cycle. Aberrant DNA methylation is linked to numerous pathologies, including cancer. The purpose of this review is to describe DNA methylation and its functions, to examine the relationship between dietary methyl insufficiency and DNA methylation, and to evaluate the associations between DNA methylation and cancer. Key words: DNA methylation, folate, cancer, gene expression

The presence of folic acid in enriched cereal grain products and the higher bioavailability of folic acid than food folate led to the expression of the 1998 folate RDA, 400 [micro]g/d, as dietary folate equivalents (DFE). DFE are defined as: [mu]g natural food folate + 1.7 [micro]g synthetic folic acid. The 1.7 multiplier was based on assumptions that added folic acid was 85% available and food folate was 50% available. The 85/50 ratio also inferred that the bioavailability of food folate was ~60% relative to added folic acid. The objective of this long-term controlled feeding study was to assess the dietary folate equivalency of folic acid. After a 2-wk period of folate restriction, women (n = 42, 18-45 y old) consumed either 400 or 800 [micro]g DFE/d derived from various combinations of food folate and folic acid for 12 wk. Folic acid was converted to DFE using the 1.7 multiplier from the DFE calculation and was consumed with a meal throughout the treatment period. Folate status response to the various treatments was assessed during wk 12-14. Serum folate, RBC folate, and plasma total homocysteine did not differ among the 400 [micro]g DFE/d groups or among the 800 [micro]g DFE/d groups. In contrast, consumption of 800 [micro]g DFE/d led to higher (P [less than or equal to] 0.05) serum and RBC folate than consumption of 400 [micro]g DFE/d. These data support the validity of the 1.7 multiplier in the DFE equation and suggest that food folate bioavailability is ~60% that of added folic acid when consumed as part of a mixed diet. KEY WORDS: * folate * bioavailability * folic acid * dietary folate equivalent * DFE

Population-based studies report differences in folate status indicators among Mexican American (MA), African American (AA) and Caucasian (CA) women. It is unclear, however, whether these differences are due to variations in dietary folate intake. The present study was designed to investigate the influence of ethnicity/race on folate status parameters in MA, AA, and CA women (18-45 y; n = 14 in each group) under conditions of strictly controlled folate intake. In addition, the adequacy of the 1998 folate U.S. recommended dietary allowance (RDA), 400/[micro]g/d as dietary folate equivalents (DFE), for non-Caucasian women was assessed. Subjects (n = 42) with the methylenetetrahydrofolate reductase 677 CC genotype consumed a low-folate diet (135 [micro]g DFE/d) for 7 wk followed by repletion with 400 (7 MA, 7 AA, 7 CA) or 800/Lg DFE/d (7 MA, 7 AA, 7 CA) for 7 wk. AA women had lower (P [less than or equal to] 0.05) blood folate concentrations and excreted less (P [less than or equal to] 0.05) urinary folate throughout folate depletion and repletion with 400 and/or 800 [micro] DFE/d compared with MA and/or CA women. MA women had lower (P [less than or equal to] 0.05) plasma total homocysteine (tHcy) throughout folate depletion and during repletion with 400 [micro]g DFE/d relative to the other ethnic/racial groups. Repletion with the 1998 folate U.S. RDA led to normal blood folate and plasma tHcy for all 3 ethnic/racial groups. Collectively, these data demonstrate that ethnicity/race is an important determinant of folate status under conditions of strictly controlled dietary folate intake and support the adequacy of the 1998 folate U.S. RDA for the 3 largest ethnic/racial groups in the United States. KEY WORDS: * ethnicity * race * homocysteine * folate * humans * RDA

The interaction of a dietary excess of vitamin A (retinoid) and deficiency of methyl-donor compounds was examined in murine early-organogenesis embryonic development. Female mice were fed one of six diets from the time of vaginal plug detection until gestational d 8.0, when embryos were removed and grown in whole embryo culture for 46 h, using serum from rats fed the same diet for 36 d as the culture medium. The six diets were either methyl-donor deficient (designated -FCM: devoid of folic acid, choline and supplemental L-methionine, but having methionine as a component of the protein portion of the diet) or methyl-donor sufficient (designated +FCM: containing folic acid, choline and L-methionine supplementation), in combination with one of three concentrations of retinyl palmitate (0.016, 0.416 or 4.016 g/kg diet). The high dose of retinyl palmitate induced a failure of anterior neuropore closure and hypoplasia of the visceral arches, both of which were significantly ameliorated by simultaneous administration of the methyl-donor-deficient diet. The primary acidic retinoid detected in the rat serum was 9,13-di-cis-retinoic acid, although we hypothesize that teratogenic retinoids were formed by embryonic biotransformation of the retinyl esters to toxic metabolites. Biochemical measurements of metabolites in relevant pathways were performed. We propose that the amelioration of these malformations may be used to determine biochemical pathways critical for retinoid teratogenesis. KEY WORDS: * vitamin A * retinoid * methylation * neural tube * teratogenesis

A common genetic variant in the methylenetetrahydrofolate reductase (MTHFR) gene involving a cytosine to thymidine (C [right arrow] T) transition at nucleotide 677 is associated with reduced enzyme activity, altered folate status and potentially higher folate requirements. The objectives of this study were to investigate the effect of the MTHFR 677 T allele on folate status variables in Mexican women (n = 43; 18-45 y) and to assess the adequacy of the 1998 folate U.S. Recommended Dietary Allowance (RDA), 400 [micro]g/d as dietary folate equivalents (DFE). Subjects (14 CC, 12 CT, 17 TT genotypes) consumed a low folate diet (135 [micro]g/d DFE) for 7 wk followed by repletion with 400 [micro]g/d DFE (7 CC, 6 CT, 9 TT) or 800 [micro]g/d DFE (7 CC, 6 CT, 8 TT) for 7 wk. Throughout repletion with 400 [micro]g/d DFE, the TT genotype had lower (P [less than or equal to] 0.05) serum folate and higher (P [less than or equal to] 0.05) plasma total homocysteine (tHcy) concentrations than the CC genotype. CT heterozygotes did not differ (P > 0.05) in their response relative to the CC genotype. Throughout repletion with 800 [micro]g/d DFE, the CT genotype had lower (P [less than or equal to] 0.05) serum folate concentrations and excreted less (P [less than or equal to] 0.05) urinary folate than the CC genotype. However, there were no differences (P > 0.05) in the measured variables between the TT and CC genotypes. Repletion with 400 [micro]g/d DFE led to normal blood folate and desirable plasma tHcy concentrations, regardless of MTHFR C677T genotype. Collectively, these data demonstrate that the MTHFR C [right arrow] T variant modulates folate status response to controlled folate intakes and support the adequacy of the 1998 folate U.S. RDA for all three MTHFR C677T genotypes. KEY WORDS: * folate * homocysteine * human * MTHFR genotype * RDA

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1053/jada.2003.50030 Byline: Setareh Torabian Esfahani, Edward A Cogger, Marie A Caudill Abstract: Objective To determine the prevalence of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms in women of different ethnic groups and to relate these common mutations to plasma homocysteine, red cell folate, and serum folate. Design A one-time fasting blood sample was obtained for MTHFR genotype (C677T and A1298C) determinations (n=433). Serum folate, red cell folate, and homocysteine analyses were performed in nonfolic acid supplement users (n=215). Subjects/setting This study involved 433 women from four ethnic groups, including 193 Hispanic women of Mexican descent, 139 white women, 53 Asian women of mixed descent, and 48 African American women. Statistical Analyses Performed I.sup.2, t Test, and analysis of variance were used. Results Mexican women (18.1%) had a higher frequency of the 677 TT genotype compared with white (7.2%), Asian (3.8%), and African American (0%) women. White women (7.9%) had a higher frequency of the 1298 CC genotype than the other ethnic groups (range=1.9% to 2.6%). The frequency of compound heterozygosity (677 CT + 1298 AC) was higher in Mexican (17.6%) and white (15.1%) women than Asian and African American ([approximately equal to]4% to 6%) women. In the era of folic acid fortification, neither genotype, independently or together, was associated with homocysteine or blood folate concentrations when ethnic groups were combined. In Mexican women, however, a linear trend (P[less than or equal to].05) was detected for the C677T variants with the lowest red cell folate in the TT genotype. Applications/conclusions These data demonstrate ethnic differences in genetic polymorphisms that are diet responsive and may be useful when investigating ethnic variations in chronic disease, developmental anomalies, and folate requirements. J Am Diet Assoc. 2003;103:200-207. Author Affiliation: S. T. Esfahani, at the time of this research, was a graduate student of Agricultural Sciences in the Human Nutrition and Food Science Department, Cal Poly Pomona University, Pomona, CA, and is now a doctoral candidate of Public Health at Loma Linda University, Loma Linda, CA. E. A. Cogger is a professor in the Animal and Veterinary Sciences Department at Cal Poly Pomona University, and M. A. Caudill is an associate professor in the Human Nutrition and Food Science Department at Cal Poly Pomona University, Pomona, CA Article Note: (footnote) [star] Supported in part by the National Institutes of Health (S06GM53933) and the California Agricultural Research Initiative.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1053/jada.2003.50030 Byline: Setareh Torabian Esfahani, Edward A Cogger, Marie A Caudill Abstract: Objective To determine the prevalence of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms in women of different ethnic groups and to relate these common mutations to plasma homocysteine, red cell folate, and serum folate. Design A one-time fasting blood sample was obtained for MTHFR genotype (C677T and A1298C) determinations (n=433). Serum folate, red cell folate, and homocysteine analyses were performed in nonfolic acid supplement users (n=215). Subjects/setting This study involved 433 women from four ethnic groups, including 193 Hispanic women of Mexican descent, 139 white women, 53 Asian women of mixed descent, and 48 African American women. Statistical Analyses Performed I.sup.2, t Test, and analysis of variance were used. Results Mexican women (18.1%) had a higher frequency of the 677 TT genotype compared with white (7.2%), Asian (3.8%), and African American (0%) women. White women (7.9%) had a higher frequency of the 1298 CC genotype than the other ethnic groups (range=1.9% to 2.6%). The frequency of compound heterozygosity (677 CT + 1298 AC) was higher in Mexican (17.6%) and white (15.1%) women than Asian and African American ([approximately equal to]4% to 6%) women. In the era of folic acid fortification, neither genotype, independently or together, was associated with homocysteine or blood folate concentrations when ethnic groups were combined. In Mexican women, however, a linear trend (P[less than or equal to].05) was detected for the C677T variants with the lowest red cell folate in the TT genotype. Applications/conclusions These data demonstrate ethnic differences in genetic polymorphisms that are diet responsive and may be useful when investigating ethnic variations in chronic disease, developmental anomalies, and folate requirements. J Am Diet Assoc. 2003;103:200-207. Author Affiliation: S. T. Esfahani, at the time of this research, was a graduate student of Agricultural Sciences in the Human Nutrition and Food Science Department, Cal Poly Pomona University, Pomona, CA, and is now a doctoral candidate of Public Health at Loma Linda University, Loma Linda, CA. E. A. Cogger is a professor in the Animal and Veterinary Sciences Department at Cal Poly Pomona University, and M. A. Caudill is an associate professor in the Human Nutrition and Food Science Department at Cal Poly Pomona University, Pomona, CA Article Note: (footnote) [star] Supported in part by the National Institutes of Health (S06GM53933) and the California Agricultural Research Initiative.

Folate catabolism represents the major route of folate turnover in humans and involves cleavage of the C9-N10 bond producing a pterin and para-aminobenzoylglutamate (pABG). Thus, the quantitation of pABG and its acetylated more predominant counterpart para-acetamidobenzolyglutamate (apABG) may be useful in assessing folate status and requirements. However, until the in vivo fate of dietary pABG is understood, studies using pABG excretion parameters can not be fully interpreted. As part of a larger study, an oral dose (376 nmol or 100 [micro]g) of [[sup.13][C.sub.5]]pABG in 40 mL apple juice was ingested by pregnant women (2nd trimester, n = 2) and nonpregnant controls (n = 2) consuming controlled total folate intakes of 450 or 850 [micro]g/d. Urine collections (24 h) were obtained over the next 4 d and gas chromatography-mass spectrometry was used to measure urinary [[sup.13][C.sub.5]]pABG, [[sup.13][C.sub.5]]apABG and [[sup.13][C.sub.5]]folate. Of the 376 nmol [[sup.13][C.sub.5]]pABG administered, only 17.5 [+ or -] 6.4 nmol; mean [+ or -] SEM) or 4.6 [+ or -] 1.7% of the dose was accounted for in the urine. Most of the excreted [[sup.13][C.sub.5]]pABG, in acetamido form (15.1 [+ or -] 5.3 nmol), was excreted the day after the dose. No urinary [[sup.13][C.sub.5]]folate was detected. Folate intake did not seem to influence the urinary excretion of total pABG derived from oral pABG, whereas pregnancy may lessen total pABG excretion derived from oral pABG. Overall, these results suggest that the contribution of dietary pABG to the urinary excretion of pABG and apABG is small. KEY WORDS: * folate * para-aminobenzoylglutamate * catabolism * stable isotope * humans

Chronic nutritional deficiencies in folate, choline, methionine, vitamin B-6 and/or vitamin B-12 can perturb the complex regulatory network that maintains normal one-carbon metabolism and homocysteine homeostasis. Genetic polymorphisms in these pathways can act synergistically with nutritional deficiencies to accelerate metabolic pathology associated with occlusive heart disease, birth defects and dementia. A major unanswered question is whether homocysteine is causally involved in disease pathogenesis or whether homocysteinemia is simply a passive and indirect indicator of a more complex mechanism. S-Adenosylmethionine and S-adenosylhomocysteine (SAH), as the substrate and product of methyltransferase reactions, are important metabolic indicators of cellular methylation status. Chronic elevation in homocysteine levels results in parallel increases in intracellular SAH and potent product inhibition of DNA methyltransferases. SAH-mediated DNA hypomethylation and associated alterations in gene expression and chromatin structure may provide new hypotheses for pathogenesis of diseases related to homocysteinemia. KEY WORDS: * homocysteine * S-adenosylhomocysteine * S-adenosylmethionine * DNA methylation

Because S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are the substrate and product of essential methyltransferase reactions; the ratio of SAM:SAH is frequently used as an indicator of cellular methylation potential. However, it is not clear from the ratio whether substrate insufficiency, product inhibition or both are required to negatively affect cellular methylation capacity. A combined genetic and dietary approach was used to modulate intracellular concentrations of SAM and SAH. Wild-type (WT) or heterozygous cystathionine [beta]-synthase (CBS +/-) mice consumed a control or methyl-deficient diet for 24 wk. The independent and combined effect of genotype and diet on SAM, SAH and the SAM:SAH ratio were assessed in liver, kidney, brain and testes and were correlated with relative changes in tissue-specific global DNA methylation. The combined results from the different tissues indicated that a decrease in SAM alone was not sufficient to affect DNA methylation in this model, whereas an increase in SAH, either alone or associated with a decrease in SAM, was most consistently associated with DNA hypomethylation. A decrease in SAM:SAH ratio was predictive of reduced methylation capacity only when associated with an increase in SAH; a decrease in the SAM:SAH ratio due to SAM depletion alone was not sufficient to affect DNA methylation in this model. Plasma homocysteine levels were positively correlated with intracellular SAH levels in all tissues except kidney. These results support the possibility that plasma SAH concentrations may provide a sensitive biomarker for cellular methylation status. J. Nutr. 131: 2811-2818, 2001. KEY WORDS: * cystathionine [beta]-synthase * S-adenosylhomocysteine * methylation * homocysteine * mice

Measurement of the urinary folate catabolites, para-aminobenzoylglutamate (pABG) and the more predominant acetylated form, acetamidobenzoylglutamate (apABG), has been used to assess folate requirements in both pregnant and nonpregnant women. Folate catabolite excretion has been reported to be significantly higher in pregnant women (second trimester) compared with nonpregnant controls. The primary goals of this study were to determine if pregnant women in a controlled metabolic study excreted higher quantities of urinary folate catabolites than nonpregnant controls and if catabolite excretion was influenced by folate intake. We evaluated the effect of gestation and folate intake on the urinary excretion of apABG and pABG in pregnant women (n = 12; wk 14-26 gestation) and nonpregnant controls (n = 12) assigned to consume folate levels approximating the current (400[[micro]gram]/d) and previous (800 [[micro]gram]/d) RDA. Subjects were fed a controlled diet containing 120 [[micro]gram] folate/d and either 330 or 730 [[micro]gram] synthetic folic acid/d. In contrast to previously reported data, no differences in mean folate catabolite excretion were detected between pregnant and nonpregnant subjects. Catabolite excretion (pABG + apABG) decreased significantly relative to initial values in pregnant women consuming 450 [[micro]gram] folate/d (-40 [+ or -] 20%; mean [+ or -] SD) and final mean excretion was significantly lower in the pregnant women consuming 450 [[micro]gram] folate/d (86 [+ or -] 32 nmol/d) compared with 850 [[micro]gram] folate/d (148 [+ or -] 20 nmol/d). Data from this study indicate that second trimester pregnant women do not excrete more folate catabolites than nonpregnant controls and that consumption of 450 vs. 850 [[micro]gram] folate/d results in a significant reduction in the quantity of folate catabolites excreted. KEY WORDS: folate requirements catabolites pregnancy humans

A metabolic study (84-d) was conducted to investigate the folate status response of pregnant subjects (n = 12) during their second trimester and nonpregnant controls (n = 12) to folate intakes approximating the current (400 [[micro]gram]/d) and former (800 [[micro]gram]/d) recommended dietary allowance (RDA). The overall goal of the study was to provide metabolic data to assist in the interpretation of the current RDA for folate. Subjects were fed a controlled diet containing 120 [+ or -] 15 [[micro]gram]/d (mean [+ or -] SD) folate and either 330 or 730 [[micro]gram]/d synthetic folic acid. Outcome variables between and within supplementation groups were compared at steady state. Serum folate was higher (P [less than or equal to] 0.05) in pregnant women consuming 850 compared with 450 [[micro]gram]/d (44.6 [+ or -] 13.4, 26.3 [+ or -] 11.3 nmol/L, respectively, mean [+ or -] SD). No differences (P > 0.05) were detected in serum folate between pregnant and nonpregnant women within the same supplementation group. Urinary 5-methyl-tetrahydrofolate excretion was greater (P [less than or equal to] 0.05) in pregnant women consuming 850 compared with 450 [[micro]gram]/d (198.0 [+ or -] 100.4, 9.5 [+ or -] 3.2 nmol/d, respectively). No differences (P > 0.05) in 5-methyl-tetrahydrofolate excretion were detected between pregnant and nonpregnant women within supplementation groups. Differences (P [less than or equal to] 0.05) were not detected in red cell folate between pregnant women consuming either 450 or 850 [[micro]gram]/d (1452.5 [+ or -] 251.8, 1733.5 [+ or -] 208.5 nmol/L, respectively) or between pregnant and nonpregnant women consuming 450 [[micro]gram]/d. Our data suggest that 450 [[micro]gram]/d (dietary folate + synthetic folic acid) is sufficient to maintain folate status in pregnant women. This level of intake equates to [approximately]600 [[micro]gram]/d dietary equivalents, assuming 50 and 75% availability of dietary folate and synthetic folic acid consumed with meals, respectively. KEY WORDS: folate requirements humans pregnant