Gene response elements, genetic polymorphisms and epigenetics influence the human dietary requirement for choline

Recent progress in the understanding of the human dietary requirement for choline highlights the importance of genetic variation and epigenetics in human nutrient requirements. Choline is a major dietary source of methyl-groups (one of choline's metabolites, betaine, participates in the methylation of homocysteine to form methionine); also choline is needed for the biosynthesis of cell membranes, bioactive phospholipids and the neurotransmitter acetylcholine. A recommended dietary intake for choline in humans was set in 1998, and a portion of the choline requirement can be met via endogenous de novo synthesis of phosphatidylcholine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) in the liver. Though many foods contain choline, many humans do not get enough in their diets. When deprived of dietary choline, most adult men and postmenopausal women developed signs of organ dysfunction (fatty liver, liver or muscle cell damage, and reduces the capacity to handle a methionine load, resulting in elevated homocysteine). However, only a portion of premenopausal women developed such problems. The difference in requirement occurs because estrogen induces expression of the PEMT gene and allows premenopausal women to make more of their needed choline endogenously. In addition, there is significant variation in the dietary requirement for choline that can be explained by common polymorphisms in genes of choline and folate metabolism. Choline is critical during fetal development, when it alters DNA methylation and thereby influences neural precursor cell proliferation and apoptosis. This results in long term alterations in brain structure and function, specifically memory function.     

Phosphatidylcholine homeostasis and liver failure

In mammals, the only endogenous pathway for choline biosynthesis is the methylation of phosphatidylethanolamine to phosphatidylcholine (PC) by phosphatidylethanolamine N-methyltransferase (PEMT) coupled to PC degradation. Complete choline deprivation in mice by feeding Pemt(-/-) mice a choline-deficient (CD) diet decreases hepatic PC by 50% and is lethal within 5 days. PC secretion into bile is mediated by a PC-specific flippase, multiple drug-resistant protein 2 (MDR2). Here, we report that mice that lack both PEMT and MDR2 and are fed a CD diet survive for >90 days. Unexpectedly, the amount of PC also decreases by 50% in the livers of Mdr2(-/-)/Pemt(-/-) mice. The Mdr2(-/-)/Pemt(-/-) mice adapt to the severe choline deprivation via choline recycling by induction of phospholipase A(2), choline kinase, and CTP:phosphocholine cytidylyltransferase activities and by a strikingly decreased expression of choline oxidase. The ability of Mdr2(-/-)/Pemt(-/-) mice to survive complete choline deprivation suggests that acute lethality in CD-Pemt(-/-) mice results from rapid depletion of hepatic PC via biliary secretion.     

Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-D(9)-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.     

Sexually Dimorphic Activation of Liver and Brain Phosphatidylethanolamine N-Methyltransferase by Dietary Choline Deficiency

Phosphatidylethanolamine N-methyltransferase (PEMT) activity was measured by a radioenzymatic assay in homogenates of brain and liver obtained from Sprague Dawley rats fed a choline-free or control (0.3 g/kg of choline chloride) diet for seven days. Choline deficiency increased PEMT activity in the liver of male rats by 34% but had no effect on hepatic PEMT in females. In contrast, brain PEMT activity was increased in brain of choline deficient females (by 49%) but was unaltered in males. Activation of the PE methylation pathway in female brain may constitute a compensatory mechanism to sustain PC synthesis during choline deficiency.     

Cell density is a critical determinant of aromatase expression in adipose stromal cells

Obesity is associated with an increased risk of breast cancer among postmenopausal women. This is at least partly due to excessive estrogen production in adipose tissue of obese women. Aromatase, the key enzyme in estrogen biosynthesis, is an important target in endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer. In this study we show that high confluency of human adipose stromal cells (ASCs) cultured in vitro can significantly stimulate aromatase gene expression and reduce the expression of breast tumor suppressor BRCA1 and members of the NR4A orphan nuclear family. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Nurr1, a member of the NR4A family, substantially increased aromatase expression. Lastly, we found that the cell density-triggered inducibility of aromatase expression varies in ASCs isolated from different disease-free individuals. Our finding highlights the impact of increased cell number on estrogen biosynthesis as in the case of excessive adiposity.     

Cyclin D1 expression is dependent on estrogen receptor function in tamoxifen-resistant breast cancer cells

The development of resistance to tamoxifen, the most common antiestrogen used in the treatment of breast cancer, is a frequent and severe clinical problem. Tamoxifen-resistant tumors are still capable of responding to other hormonal therapies such as those that downregulate estrogen receptor expression. Mechanisms leading to acquisition of tamoxifen-resistant but hormone-sensitive growth are not completely understood. In tamoxifen-sensitive breast cancer cells, tamoxifen inhibits, whereas estrogen induces, expression of cyclin D1, a key cell cycle regulatory protein. Ectopic expression of cyclin D1 can lead to antiestrogen resistance. Thus, to determine whether cyclin D1 is involved in the growth of tamoxifen-resistant cells, we developed several tamoxifen-resistant variants from MCF-7 cells. These variants grow in the absence of estrogen or in the presence of tamoxifen, but their growth is inhibited by estrogen receptor downregulators. We show here that cyclin D1 expression is maintained at comparable levels in all tamoxifen-resistant variants, whereas pS2, another estrogen-regulated protein, is not. The addition of physiological levels of estrogen further stimulates cyclin D1 expression and proliferation. In contrast, treatment with estrogen receptor downregulators decreases cyclin D1 expression and proliferation. Thus, changes in cyclin D1 expression upon second-line hormonal therapy may predict hormonal sensitivity of tamoxifen-resistant tumors. These studies suggest that estrogen receptor mediates cyclin D1 expression and growth of tamoxifen-resistant tumors.     

Estrogen deficiency worsens steatohepatitis in mice fed high-fat and high-cholesterol diet

Recent studies indicate an accelerated progression of nonalcoholic steatohepatitis (NASH) in postmenopausal women. Hypercholesterolemia, an important risk factor for NASH progression, is often observed after menopause. This study examined the effects of estrogen on NASH in ovariectomized (OVX) mice fed a high-fat and high-cholesterol (HFHC) diet. To investigate the effects of estrogen deficiency, OVX mice and sham-operated (SO) mice were fed normal chow or HFHC diet for 6 wk. Next, to investigate the effects of exogenous estrogen replenishment, OVX mice fed with HFHC diet were treated with implanted hormone release pellets (containing 17β-estradiol or placebo vehicle) for 6 wk. OVX mice on the HFHC diet showed enhanced liver injury with increased liver macrophage infiltration and elevated serum cholesterol levels compared with SO-HFHC mice. Hepatocyte monocyte chemoattractant protein-1 (MCP1) protein expression in OVX-HFHC mice was also enhanced compared with SO-HFHC mice. In addition, hepatic inflammatory gene expressions, including monocytes chemokine (C-C motif) receptor 2 (CCR2), were significantly elevated in OVX-HFHC mice. Estrogen treatment improved serum cholesterol levels, liver injury, macrophage infiltration, and inflammatory gene expressions in OVX-HFHC mice. Moreover, the elevated expression of liver CCR2 and MCP1 were decreased by estrogen treatment in OVX-HFHC mice, whereas low-density lipoprotein dose dependently enhanced CCR2 expression in THP1 monocytes. Our study demonstrated that estrogen deficiency accelerated NASH progression in OVX mice fed HFHC diet and that this effect was improved by estrogen therapy. Hypercholesterolemia in postmenopausal women would be a potential risk factor for NASH progression.     

Localization-independent regulation of homocysteine secretion by phosphatidylethanolamine N-methyltransferase

Genetic ablation of phosphatidylethanolamine N-methyltransferase (PEMT) in mice causes a 50% reduction in plasma homocysteine (Hcy) levels. Because hyperhomocysteinemia is an independent risk factor for cardiovascular disease, resolution of the molecular basis for this reduction is of significant clinical interest. The PEMT pathway is a metabolically channeled process localized to the endoplasmic reticulum (ER). To assess the importance of PEMT localization for Hcy homeostasis, we identified and ablated the minimal ER targeting motif. Mutagenesis of a conserved, C-terminal lysine residue (197) relocalized the enzyme to the Golgi, demonstrating that Lys-197 is essential for targeting PEMT to the ER. To evaluate the functional significance of PEMT localization, hepatoma cell lines were generated that stably expressed either ER- or Golgi-localized PEMT only. Intriguingly, stable expression of PEMT in either the ER or the Golgi caused increased Hcy secretion. Moreover, PEMT-mediated Hcy secretion correlated with the methyltransferase activity of the enzyme, independently of subcellular localization. Thus, our data suggest that Hcy homeostasis is regulated concomitantly with PEMT activity but independently of PEMT localization.     

Expression of sex steroid receptors and IGF-1 mRNA in breast tissue--effects of hormonal treatment

The mechanisms behind increased breast tissue proliferation and a possibly increased breast cancer risk in women using hormonal contraception (HC) and hormonal replacement therapy (HRT) are incompletely understood. We analyzed breast tissue from 20 premenopausal and seven postmenopausal women undergoing reduction mammoplasties for estrogen receptor (ER) and progesterone receptor (PR) content as well as mRNA levels for ER, PR and insulin-like growth factor-1 (IGF-1). The receptor values were correlated to IGF-1 mRNA concentrations and levels of steroid and peptide hormones and SHBG. In women using HC, we found significantly lower ER values (p=0.02) but non-significantly lower ER mRNA levels compared to those in naturally cycling women. PR and PR mRNA were no different. Women on HC displayed a higher breast tissue proliferation (p=0.05) expressed as Ki-67, MIB-1 positivity, which was correlated with IGF-1 mRNA (r_s=0.82, p=0.04). Since the concentration of sex steroid receptors in breast tissue is comparatively low and steroid receptors are down-regulated during hormonal treatment, mechanisms other than direct sex steroid receptor action are likely to be present. Our results suggest a role for IGF-1 in the proliferative response of breast tissue during exogenous hormonal treatment.     

New insights into the molecular mechanisms underlying effects of estrogen on cholesterol gallstone formation

Epidemiological and clinical studies have found that at all ages women are twice as likely as men to form cholesterol gallstones, and this gender difference begins since puberty and continues through the childbearing years, which highlight the importance of female sex hormones. Estrogen is a crucial hormone in human physiology and regulates a multitude of biological processes. The actions of estrogen have traditionally been ascribed to two closely related classical nuclear hormone receptors, estrogen receptor 1 (ESR1) and ESR2. Recent studies have revealed that the increased risk for cholesterol gallstones in women vs. men is related to differences in how the liver metabolizes cholesterol in response to estrogen. A large number of human and animal studies have proposed that estrogen increases the risk of developing cholesterol gallstones by increasing the hepatic secretion of biliary cholesterol, which, in turn, leads to an increase in cholesterol saturation of bile. Furthermore, it has been identified that hepatic ESR1, but not ESR2, plays a major role in cholesterol gallstone formation in mice in response to high doses of 17β-estradiol. The mechanisms mediating estrogen's action have become more complicated with the recent identification of a novel estrogen receptor, G protein-coupled receptor 30 (GPR30), a member of the seven-transmembrane G protein-coupled receptor superfamily. In this review, we provide an overview of the evidence for the lithogenic actions of estrogen through ESR1 and discuss the cellular and physiological actions of GPR30 in estrogen-dependent processes and the relationship between GPR30 and classical ESR1 on gallstone formation.     

Effects of recombinant human keratinocyte growth factor on surfactant, plasma, and liver phospholipid homeostasis in hyperoxic neonatal rats

Respiratory distress and bronchopulmonary dysplasia (BPD) are major problems in preterm infants that are often addressed by glucocorticoid treatment and increased oxygen supply, causing catabolic and injurious side effects. Recombinant human keratinocyte growth factor (rhKGF) is noncatabolic and antiapoptotic and increases surfactant pools in immature lungs. Despite its usefulness in injured neonatal lungs, the mechanisms of improved surfactant homeostasis in vivo and systemic effects on lipid homeostasis are unknown. We therefore exposed newborn rats to 85% vs. 21% oxygen and treated them systemically with rhKGF for 48 h before death at 7 days. We determined type II pneumocyte (PN-II) proliferation, surfactant protein (SP) mRNA expression, and the pulmonary metabolism of individual phosphatidylcholine (PC) species using [D(9)-methyl]choline and tandem mass spectrometry. In addition, we assessed liver and plasma lipid metabolism, addressing PC synthesis de novo, the liver-specific phosphatidylethanolamine methyl transferase (PEMT) pathway, and triglyceride concentrations. rhKGF was found to maintain PN-II proliferation and increased SP-B/C expression and surfactant PC in both normoxic and hyperoxic lungs. We found increased total PC together with decreased [D(9)-methyl]choline enrichment, suggesting decreased turnover rather than increased secretion and synthesis as the underlying mechanism. In the liver, rhKGF increased PC synthesis, both de novo and via PEMT, underlining the organotypic differences of rhKGF actions on lipid metabolism. rhKGF increased the hepatic secretion of newly synthesized polyunsaturated PC, indicating improved systemic supply with choline and essential fatty acids. We suggest that rhKGF has potential as a therapeutic agent in neonates by improving pulmonary and systemic PC homeostasis.     

Metabotropic Glutamate Receptor 1 Expression and Its Polymorphic Variants Associate with Breast Cancer Phenotypes

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.