RESUMO
Obesity is a major risk factor for type 2 diabetes, coronary heart disease, and strok. These diseases are associated with profound alterations in gene expression in metabolic tissues. Epigenetic-mediated regulation of gene expression is one mechanism through which environmental factors, such as diet, modify gene expression and disease predisposition. However, epigenetic control of gene expression in obesity and insulin resistance is not fully characterized. We discovered that liver-specific stearoyl-CoA desaturase-1 (Scd1) knockout mice (LKO) fed a high-carbohydrate low-fat diet exhibit dramatic changes in hepatic gene expression and metabolites of the folate cycle and one-carbon metabolism respectively for the synthesis of S-adenosylmethionine (SAM). LKO mice show an increased ratio of S-adenosylmethionine to S-adenosylhomocysteine, a marker for increased cellular methylation capacity. Furthermore, expression of DNA and histone methyltransferase genes is up-regulated while the mRNA and protein levels of the non-DNA methyltransferases including phosphatidylethanolamine methyltransferase (PEMT), Betaine homocysteine methyltransferase (Bhmt), and the SAM-utilizing enzymes such as glycine-N-methyltransferase (Gnmt) and guanidinoacetate methyltransferase (Gamt) are generally down-regulated. Feeding LKO mice a high carbohydrate diet supplemented with triolein, but not tristearin, and increased endogenous hepatic synthesis of oleate but not palmitoleate in Scd1 global knockout mice normalized one carbon gene expression and metabolite levels. Additionally, changes in one carbon gene expression are independent of the PGC-1α-mediated ER stress response previously reported in the LKO mice. Together, these results highlight the important role of oleate in maintaining one-carbon cycle homeostasis and point to observed changes in one-carbon metabolism as a novel mediator of the Scd1 deficiency-induced liver phenotype.
Assuntos
Diabetes Mellitus Tipo 2 , Ácido Oleico , Camundongos , Animais , Ácido Oleico/metabolismo , S-Adenosilmetionina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Fígado/metabolismo , Carboidratos , Camundongos Knockout , Obesidade/metabolismo , Carbono/metabolismo , Fosfatidiletanolamina N-Metiltransferase/metabolismoRESUMO
The major facilitator superfamily domain 2a protein was identified recently as a lysophosphatidylcholine (LPC) symporter with high affinity for LPC species enriched with DHA (LPC-DHA). To test the hypothesis that reproductive state and choline intake influence plasma LPC-DHA, we performed a post hoc analysis of samples available through 10 weeks of a previously conducted feeding study, which provided two doses of choline (480 and 930 mg/d) to non-pregnant (n 21), third-trimester pregnant (n 26), and lactating (n 24) women; all participants consumed 200 mg of supplemental DHA and 22 % of their daily choline intake as 2H-labelled choline. The effects of reproductive state and choline intake on total LPC-DHA (expressed as a percentage of LPC) and plasma enrichments of labelled LPC and LPC-DHA were assessed using mixed and generalised linear models. Reproductive state interacted with time (P = 0·001) to influence total LPC-DHA, which significantly increased by week 10 in non-pregnant women, but not in pregnant or lactating women. Contrary to total LPC-DHA, patterns of labelled LPC-DHA enrichments were discordant between pregnant and lactating women (P < 0·05), suggestive of unique, reproductive state-specific mechanisms that result in reduced production and/or enhanced clearance of LPC-DHA during pregnancy and lactation. Regardless of the reproductive state, women consuming 930 v. 480 mg choline per d exhibited no change in total LPC-DHA but higher d3-LPC-DHA (P = 0·02), indicating that higher choline intakes favour the production of LPC-DHA from the phosphatidylethanolamine N-methyltransferase pathway of phosphatidylcholine biosynthesis. Our results warrant further investigation into the effect of reproductive state and dietary choline on LPC-DHA dynamics and its contribution to DHA status.
Assuntos
Colina/administração & dosagem , Ácidos Docosa-Hexaenoicos/sangue , Fosfatidilcolinas/sangue , Reprodução/fisiologia , Adulto , Deutério , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Feminino , Genótipo , Humanos , Lactação/sangue , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Gravidez , Terceiro Trimestre da GravidezRESUMO
Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt-/- mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt-/- mice. Treatment with vitamin E (0.5â¯g/kg) for 3â¯weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt-/- mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.
Assuntos
Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Vitamina E/metabolismo , Vitamina E/farmacologia , Ceramidase Ácida , Animais , Antioxidantes/farmacologia , Colesterol/metabolismo , Dieta Hiperlipídica , Suplementos Nutricionais , Modelos Animais de Doenças , Progressão da Doença , Fígado Gorduroso/metabolismo , Fibrose/tratamento farmacológico , Inflamação/tratamento farmacológico , Resistência à Insulina , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfotransferases (Aceptor do Grupo Álcool) , RNA Mensageiro , Vitamina E/administração & dosagemRESUMO
Background: Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine to phosphatidylcholine. Pemt-/-/low density lipoprotein receptor (Ldlr)-/- mice have significantly reduced plasma lipids and are protected against atherosclerosis. Recent studies have shown that choline can be metabolized by the gut flora into trimethylamine-N-oxide (TMAO), which is an emerging risk factor for atherosclerosis. Objective: The objective of this study was to determine whether ectopic hepatic PEMT expression or choline supplementation would promote atherosclerosis in Pemt-/-/Ldlr-/- mice. Methods: Male 8- to 10-wk-old Pemt+/+/Ldlr-/- (SKO) and Pemt-/-/Ldlr-/- (DKO) mice were injected with an adeno-associated virus (AAV) expressing green fluorescent protein (GFP) or human PEMT and fed a Western diet (40% of calories from fat, 0.5% cholesterol) for 8 wk. In a separate experiment, 8- to 10-wk-old SKO and half of the DKO male mice were fed a Western diet with normal (3 g/kg) choline for 12 wk. The remaining DKO mice [choline-supplemented (CS) DKO] were fed a CS Western diet (10 g choline/kg). Plasma lipid concentrations, choline metabolites, and aortic atherosclerosis were measured. Results: Plasma cholesterol, plasma TMAO, and aortic atherosclerosis were reduced by 60%, 40%, and 80%, respectively, in DKO mice compared with SKO mice. AAV-PEMT administration increased plasma cholesterol and TMAO by 30% and 40%, respectively, in DKO mice compared with AAV-GFP-treated DKO mice. Furthermore, AAV-PEMT-injected DKO mice developed atherosclerotic lesions similar to SKO mice. In the second study, there was no difference in atherosclerosis or plasma cholesterol between DKO and CS-DKO mice. However, plasma TMAO concentrations were increased 2.5-fold in CS-DKO mice compared with DKO mice. Conclusions: Reintroducing hepatic PEMT reversed the atheroprotective phenotype of DKO mice. Choline supplementation did not increase atherosclerosis or plasma cholesterol in DKO mice. Our data suggest that plasma TMAO does not induce atherosclerosis when plasma cholesterol is low. Furthermore, this is the first report to our knowledge that suggests that de novo choline synthesis alters TMAO status.
Assuntos
Aterosclerose/metabolismo , Colesterol/sangue , Colina/farmacologia , Fígado/metabolismo , Metilaminas/sangue , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Receptores de LDL/metabolismo , Animais , Aorta , Aterosclerose/etiologia , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Colesterol na Dieta/administração & dosagem , Colina/metabolismo , Dieta Ocidental , Suplementos Nutricionais , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidiletanolamina N-Metiltransferase/farmacologia , Fosfatidiletanolaminas/metabolismoRESUMO
Demand for the vital nutrient choline is high during lactation; however, few studies have examined choline metabolism and requirements in this reproductive state. The present study sought to discern the effects of lactation and varied choline intake on maternal biomarkers of choline metabolism and breast milk choline content. Lactating (n=28) and control (n=21) women were randomized to 480 or 930 mg choline/day for 10-12 weeks as part of a controlled feeding study. During the last 4-6 weeks, 20% of the total choline intake was provided as an isotopically labeled choline tracer (methyl-d9-choline). Blood, urine and breast milk samples were collected for choline metabolite quantification, enrichment measurements, and gene expression analysis of choline metabolic genes. Lactating (vs. control) women exhibited higher (P < .001) plasma choline concentrations but lower (P ≤ .002) urinary excretion of choline metabolites, decreased use of choline as a methyl donor (e.g., lower enrichment of d6-dimethylglycine, P ≤ .08) and lower (P ≤ .02) leukocyte expression of most choline-metabolizing genes. A higher choline intake during lactation differentially influenced breast milk d9- vs. d3-choline metabolite enrichment. Increases (P ≤ .03) were detected among the d3-metabolites, which are generated endogenously via the hepatic phosphatidylethanolamine N-methyltransferase (PEMT), but not among the d9-metabolites generated from intact exogenous choline. These data suggest that lactation induces metabolic adaptations that increase the supply of intact choline to the mammary epithelium, and that extra maternal choline enhances breast milk choline content by increasing supply of PEMT-derived choline metabolites. This trial was registered at clinicaltrials.gov as NCT01127022.
Assuntos
Colina/administração & dosagem , Suplementos Nutricionais , Lactação/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Leite Humano/química , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Colina/análise , Colina/sangue , Colina/metabolismo , Estudos de Coortes , Deutério , Indução Enzimática , Feminino , Humanos , Lactação/sangue , Lactação/urina , Leucócitos/enzimologia , Leucócitos/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Glândulas Mamárias Humanas/enzimologia , Glândulas Mamárias Humanas/metabolismo , Leite Humano/metabolismo , New York , Fosfatidiletanolamina N-Metiltransferase/química , Fosfatidiletanolamina N-Metiltransferase/genética , RNA Mensageiro/metabolismo , Recomendações Nutricionais , Adulto JovemRESUMO
Maternal choline supplementation (MCS) induces lifelong cognitive benefits in the Ts65Dn mouse, a trisomic mouse model of Down syndrome and Alzheimer's disease. To gain insight into the mechanisms underlying these beneficial effects, we conducted a study to test the hypothesis that MCS alters choline metabolism in adult Ts65Dn offspring. Deuterium-labeled methyl-d9-choline was administered to adult Ts65Dn and disomic (2N) female littermates born to choline-unsupplemented or choline-supplemented Ts65Dn dams. Enrichment of d9-choline metabolites (derived from intact choline) and d3 + d6-choline metabolites [produced when choline-derived methyl groups are used by phosphatidylethanolamine N-methyltransferase (PEMT)] was measured in harvested tissues. Adult offspring (both Ts65Dn and 2N) of choline-supplemented (vs. choline-unsupplemented) dams exhibited 60% greater (P≤0.007) activity of hepatic PEMT, which functions in de novo choline synthesis and produces phosphatidylcholine (PC) enriched in docosahexaenoic acid. Higher (P<0.001) enrichment of PEMT-derived d3 and d6 metabolites was detected in liver, plasma, and brain in both genotypes but to a greater extent in the Ts65Dn adult offspring. MCS also yielded higher (P<0.05) d9 metabolite enrichments in liver, plasma, and brain. These data demonstrate that MCS exerts lasting effects on offspring choline metabolism, including up-regulation of the hepatic PEMT pathway and enhanced provision of choline and PEMT-PC to the brain.
Assuntos
Colina/farmacocinética , Síndrome de Down/metabolismo , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Animais , Colina/administração & dosagem , Colina/farmacologia , Colina/uso terapêutico , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/metabolismo , Síndrome de Down/tratamento farmacológico , Feminino , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilcolinas/metabolismo , Gravidez , Distribuição TecidualRESUMO
BACKGROUND: Although biomarkers of choline metabolism are altered by pregnancy, little is known about the influence of human pregnancy on the dynamics of choline-related metabolic processes. OBJECTIVE: This study used stable isotope methodology to examine the effects of pregnancy on choline partitioning and the metabolic activity of choline-related pathways. DESIGN: Healthy third-trimester pregnant (n = 26; initially week 27 of gestation) and nonpregnant (n = 21) women consumed 22% of their total choline intake (480 or 930 mg/d) as methyl-d9-choline for the final 6 wk of a 12-wk feeding study. RESULTS: Plasma d9-betaine:d9-phosphatidylcholine (PC) was lower (P ≤ 0.04) in pregnant than in nonpregnant women, suggesting greater partitioning of choline into the cytidine diphosphate-choline (CDP-choline) PC biosynthetic pathway relative to betaine synthesis during pregnancy. Pregnant women also used more choline-derived methyl groups for PC synthesis via phosphatidylethanolamine N-methyltransferase (PEMT) as indicated by comparable increases in PEMT-PC enrichment in pregnant and nonpregnant women despite unequal (pregnant > nonpregnant; P < 0.001) PC pool sizes. Pregnancy enhanced the hydrolysis of PEMT-PC to free choline as shown by greater (P < 0.001) plasma d3-choline:d3-PC. Notably, d3-PC enrichment increased (P ≤ 0.011) incrementally from maternal to placental to fetal compartments, signifying the selective transfer of PEMT-PC to the fetus. CONCLUSIONS: The enhanced use of choline for PC production via both the CDP-choline and PEMT pathways shows the substantial demand for choline during late pregnancy. Selective partitioning of PEMT-PC to the fetal compartment may imply a unique requirement of PEMT-PC by the developing fetus.
Assuntos
Colina/metabolismo , Dieta , Suplementos Nutricionais , Fenômenos Fisiológicos da Nutrição Materna , Troca Materno-Fetal , Gravidez/metabolismo , Adulto , Betaína/sangue , Colina/administração & dosagem , Colina/análogos & derivados , Colina/sangue , Deutério , Feminino , Sangue Fetal , Humanos , Hidrólise , Metilação , Fosfatidilcolinas/sangue , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Placenta/metabolismo , Gravidez/sangue , Terceiro Trimestre da Gravidez , Adulto JovemRESUMO
BACKGROUND: Phosphatidylcholine (PC) produced via the S-adenosylmethionine-dependent phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is enriched with docosahexaenoic acid (DHA). DHA plays a critical role in fetal development and is linked to health endpoints in adulthood. It is unknown whether choline, which can serve as a source of S-adenosylmethionine methyl groups, influences PC-DHA or the PC:PE ratio in pregnant and nonpregnant women. OBJECTIVE: This study tested whether choline intake affects indicators of choline-related lipid metabolism, including erythrocyte and plasma PC-DHA and PC:PE ratios, in pregnant women in the third trimester and nonpregnant women. DESIGN: Pregnant (n = 26) and nonpregnant (n = 21) women consumed 480 or 930 mg choline/d and a daily DHA supplement for 12 wk. Blood was collected at baseline and at the midpoint and end of the study. PC-DHA was analyzed as the proportion of total PC fatty acids. RESULTS: Pregnant women had greater (P = 0.002) PC-DHA concentrations than did nonpregnant women at baseline. The proportion of erythrocyte and plasma PC-DHA increased (P ≤ 0.002) in pregnant and nonpregnant women regardless of choline intake. However, in nonpregnant women, consumption of 930 mg choline/d led to greater (P < 0.001) erythrocyte PC-DHA and a more rapid increase (P < 0.001) in plasma PC-DHA. Lower (P = 0.001-0.024) erythrocyte and plasma PC:PE in pregnant women was not modified by choline intake. CONCLUSIONS: A higher choline intake may increase PEMT activity, resulting in greater PC-DHA enrichment of the PC molecule in nonpregnant women. Increased production of PC-DHA during pregnancy indicates elevated PEMT activity and a higher demand for methyl donors. This trial was registered at clinicaltrials.gov as NCT01127022.
Assuntos
Colina/farmacologia , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/sangue , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipotrópicos/farmacologia , Fosfatidilcolinas/sangue , Gravidez/sangue , Eritrócitos/metabolismo , Feminino , Humanos , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Fosfatidiletanolaminas/sangue , Gravidez/metabolismo , Terceiro Trimestre da GravidezRESUMO
Biosynthesis of hepatic choline via phosphatidylethanolamine N-methyltransferase (PEMT) plays an important role in the development of type 2 diabetes and obesity. We investigated the mechanism(s) by which choline modulates insulin sensitivity. PEMT wild-type (Pemt(+/+)) and knock-out (Pemt(-/-)) mice received either a high fat diet (HF; 60% kcal of fat) or a high fat, high choline diet (HFHC; 4 g of choline/kg of HF diet) for 1 week. Hepatic insulin signaling and glucose and lipid homeostasis were investigated. Glucose and insulin intolerance occurred in Pemt(-/-) mice fed the HFHC diet, but not in their Pemt(-/-) littermates fed the HF diet. Plasma glucagon was elevated in Pemt(-/-) mice fed the HFHC diet compared with Pemt(-/-) mice fed the HF diet, concomitant with increased hepatic expression of glucagon receptor, phosphorylated AMP-activated protein kinase (AMPK), and phosphorylated insulin receptor substrate 1 at serine 307 (IRS1-s307). Gluconeogenesis and mitochondrial oxidative stress were markedly enhanced, whereas glucose oxidation and triacylglycerol biosynthesis were diminished in Pemt(-/-) mice fed the HFHC diet. A glucagon receptor antagonist (2-aminobenzimidazole) attenuated choline-induced hyperglycemia and insulin intolerance and blunted up-regulation of phosphorylated AMPK and IRS1-s307. Choline induces glucose and insulin intolerance in Pemt(-/-) mice through modulating plasma glucagon and its action in liver.
Assuntos
Colina/administração & dosagem , Glucagon/fisiologia , Resistência à Insulina , Fígado/efeitos dos fármacos , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Animais , Sequência de Bases , Colina/farmacologia , Primers do DNA , Gluconeogênese/efeitos dos fármacos , Teste de Tolerância a Glucose , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidiletanolamina N-Metiltransferase/genéticaRESUMO
In 1850, Theodore Gobley, working in Paris, described a substance, 'lecithine', which he named after the Greek 'lekithos' for egg yolk. Adolph Strecker noted in 1862 that when lecithin from bile was heated, it generated a new nitrogenous chemical that he named 'choline'. Three years later, Oscar Liebreich identified a new substance, 'neurine', in the brain. After a period of confusion, neurine and choline were found to be the same molecule, and the name choline was adapted. Lecithin was eventually characterized chemically as being phosphatidylcholine. In 1954, Eugene Kennedy described the cytidine 5-dihphosphocholine pathway by which choline is incorporated into phosphatidylcholine. A second route, the phosphatidylethanolamine-N-methyltransferase pathway, was identified by Jon Bremer and David Greenberg in 1960. The role of choline as part of the neurotransmitter acetylcholine was established by Otto Loewi and Henry Dale. Working in the 1930s at the University of Toronto, Charles Best showed that choline prevented fatty liver in dogs and rats. The importance of choline as an essential nutrient for human health was determined in the 1990s through controlled feeding studies in humans. Recently, an understanding of the role of genetic variation in setting the dietary requirement for choline in people is being unraveled.
Assuntos
Colina/química , Colina/história , Colina/farmacologia , Acetilcolina/metabolismo , Animais , Bile/metabolismo , Cães , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/prevenção & controle , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Lecitinas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neurotransmissores/metabolismo , Valor Nutritivo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferase/metabolismo , RatosRESUMO
The reactions of the methionine/homocysteine pathway are mediated by several enzymes, including phosphatidylethanolamine N-methyltransferase, cystathionine ß-synthase, and betaine-homocysteine methyltransferase. Homocysteine homeostasis is regulated by these enzymes. We hypothesized here that the protein and folic acid content in the maternal diet affects methionine/homocysteine metabolism in the progeny. To test this hypothesis, pregnant rats were fed a diet with normal protein and normal folic acid levels (a modified casein-based AIN-93G diet), a protein-restricted and normal folic acid diet, a protein-restricted and folic acid-supplemented diet, or a normal protein and folic acid-supplemented diet. The progeny were fed either the modified AIN-93G diet or a high-fat lard-based diet. Progeny were analyzed for expression of the phosphatidylethanolamine N-methyltransferase, cystathionine ß-synthase, and betaine-homocysteine methyltransferase genes in the liver and for serum homocysteine concentration. Interactions between prenatal and postnatal nutrition were also determined. The progeny of the dams fed the diets supplemented with folic acid showed decreased expression of all 3 genes (P < .001). An interaction effect between the protein and folic acid content in the maternal diet contributed to this down-regulation (P < .001), and the postweaning diet modified these effects. Serum homocysteine concentrations were approximately 15% higher in the male rats (P < .01), but neither prenatal nutrition nor the postweaning diet affected it significantly. We conclude that maternal diet during gestation has an important effect on the transcription level of these 3 genes, but changes in gene expression were not associated with significant changes in progeny homocysteine concentrations.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Cistationina beta-Sintase/metabolismo , Dieta , Ácido Fólico/farmacologia , Expressão Gênica/efeitos dos fármacos , Metiltransferases/metabolismo , Gravidez , Animais , Betaína-Homocisteína S-Metiltransferase/genética , Betaína-Homocisteína S-Metiltransferase/metabolismo , Cistationina beta-Sintase/genética , Dieta Hiperlipídica , Dieta com Restrição de Proteínas , Gorduras na Dieta/farmacologia , Proteínas Alimentares/farmacologia , Suplementos Nutricionais , Regulação para Baixo , Feminino , Homeostase , Homocisteína/sangue , Fígado/metabolismo , Masculino , Metionina/metabolismo , Metiltransferases/genética , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Fenômenos Fisiológicos da Nutrição Pré-Natal , Ratos Wistar , Complexo Vitamínico B/farmacologia , DesmameRESUMO
Phosphatidylcholine (PC) is synthesized from choline via the CDP-choline pathway. Liver cells can also synthesize PC via the sequential methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). The current study investigates whether or not hepatic PC biosynthesis is linked to diet-induced obesity. Pemt(+/+) mice fed a high fat diet for 10 weeks increased in body mass by 60% and displayed insulin resistance, whereas Pemt(-/-) mice did not. Compared with Pemt(+/+) mice, Pemt(-/-) mice had increased energy expenditure and maintained normal peripheral insulin sensitivity; however, they developed hepatomegaly and steatosis. In contrast, mice with impaired biosynthesis of PC via the CDP-choline pathway in liver became obese when fed a high fat diet. We, therefore, hypothesized that insufficient choline, rather than decreased hepatic phosphatidylcholine, was responsible for the lack of weight gain in Pemt(-/-) mice despite the presence of 1.3 g of choline/kg high fat diet. Supplementation with an additional 2.7 g of choline (but not betaine)/kg of diet normalized energy metabolism, weight gain, and insulin resistance in high fat diet-fed Pemt(-/-) mice. Furthermore, Pemt(+/+) mice that were fed a choline-deficient diet had increased oxygen consumption, had improved glucose tolerance, and gained less weight. Thus, de novo synthesis of choline via PEMT has a previously unappreciated role in regulating whole body energy metabolism.
Assuntos
Colina/biossíntese , Dieta , Obesidade/enzimologia , Obesidade/prevenção & controle , Fosfatidiletanolamina N-Metiltransferase/deficiência , Animais , Betaína/administração & dosagem , Betaína/farmacologia , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Suplementos Nutricionais , Metabolismo Energético/efeitos dos fármacos , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/complicações , Fígado Gorduroso/enzimologia , Fígado Gorduroso/patologia , Comportamento Alimentar/efeitos dos fármacos , Resistência à Insulina , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Obesidade/induzido quimicamente , Obesidade/complicações , Fenótipo , Fosfatidilcolinas/biossíntese , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Aumento de Peso/efeitos dos fármacosRESUMO
Phosphatidylcholine is made in all nucleated mammalian cells via the CDP-choline pathway. Another major pathway for phosphatidylcholine biosynthesis in liver is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We have now identified 3T3-L1 adipocytes as a cell culture model that expresses PEMT endogenously. We have found that PEMT mRNA and protein levels increased dramatically in 3T3-L1 cells upon differentiation to adipocytes. 5'-Deletion analysis of the PEMT promoter-luciferase constructs stably expressed in 3T3-L1 adipocytes identified a regulatory region between -471 and -371 bp (relative to the transcriptional start site). Competitive and supershift assays demonstrated binding sites for transcription factors Sp1, Sp3 (-408 to -413), and YY1 (-417 to -420). During differentiation of 3T3-L1 cells to adipocytes, the amount of Sp1 protein decreased by approximately 50% just prior to activation of PEMT. Transduction of 3T3-L1 adipocytes with retrovirus containing Sp1 cDNA demonstrated that Sp1 inhibited PEMT transcriptional activity. Similarly, short hairpin RNA directed against Sp1 in 3T3-L1 adipocytes enhanced PEMT transcriptional activation. Chromatin immunoprecipitation assays confirmed that Sp1 binds to the PEMT promoter, and this interaction decreases upon differentiation to adipocytes. These experiments directly link increased PEMT expression in adipocytes to decreased transcriptional expression of Sp1. In addition, our data established that Sp1 binding was required for tamoxifen-mediated inhibition of Pemt promoter activity.
Assuntos
Adipócitos/metabolismo , Fígado/metabolismo , Fosfatidiletanolamina N-Metiltransferase/genética , Fator de Transcrição Sp1/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Sequência de Bases , Sítios de Ligação/genética , Diferenciação Celular , Primers do DNA/genética , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/metabolismo , Tamoxifeno/farmacologia , Transcrição Gênica , Fator de Transcrição YY1/metabolismoRESUMO
Homocysteine is a metabolic intermediate in methyl group metabolism that is dependent on a number of nutritional B-vitamin cofactors. An emerging aspect of homocysteine metabolism is its relation to health and disease. Perturbations of homocysteine metabolism, particularly intracellular and subsequently circulating accumulation of homocysteine (i.e., hyperhomocysteinemia), are associated with vascular disease risk, as well as other pathologies. However, intervention with B-vitamin supplementation has been shown to successfully restore normal homocysteine concentrations, but without concomitant reductions in disease risk. Thus, the mechanistic relation between homocysteine balance and disease states, as well as the value of homocysteine management, remains an area of intense investigation.
Assuntos
Homocisteína/metabolismo , Hiper-Homocisteinemia/metabolismo , Guanidinoacetato N-Metiltransferase/metabolismo , Humanos , Hiper-Homocisteinemia/tratamento farmacológico , Fosfatidiletanolamina N-Metiltransferase/metabolismo , S-Adenosilmetionina/metabolismo , Complexo Vitamínico B/uso terapêuticoRESUMO
Phospholipid N-methyltransferase (PLMT) enzymes catalyze the S-adenosylmethionine-dependent methylation of ethanolamine-containing phospholipids to produce the abundant membrane lipid phosphatidylcholine (PtdCho). In mammals and yeast, PLMT activities are required for the de novo synthesis of the choline headgroup found in PtdCho. PLMT enzyme activities have also been reported in plants, yet their roles in PtdCho biosynthesis are less clear because most plants can produce the choline headgroup entirely via soluble substrates, initiated by the methylation of free ethanolamine-phosphate. To gain further insights into the function of PLMT enzymes in plants, we isolated PLMT cDNAs from Arabidopsis and soybean (Glycine max) based upon primary amino acid sequence homology to the rat PLMT, phosphatidylethanolamine N-methyltransferase. Using a heterologous yeast expression system, it was shown that plant PLMTs methylate phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine but cannot utilize phosphatidylethanolamine as a substrate. Identification of an Arabidopsis line containing a knock-out dissociator transposon insertion within the single copy AtPLMT gene allowed us to investigate the consequences of loss of PLMT function. Although the accumulation of the PLMT substrates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine was considerably elevated in the atplmt knock-out line, PtdCho levels remained normal, and no obvious differences were observed in plant morphology or development under standard growth conditions. However, because the metabolic routes through which PtdCho is synthesized in plants vary greatly among differing species, it is predicted that the degree with which PtdCho synthesis is dependent upon PLMT activities will also vary widely throughout the plant kingdom.
Assuntos
Arabidopsis/enzimologia , Glycine max/enzimologia , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , DNA de Plantas/genética , Cinética , Mutagênese Insercional , Fosfatidilcolinas/biossíntese , Fosfatidiletanolamina N-Metiltransferase/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Glycine max/genéticaRESUMO
BACKGROUND: Chronic ethanol consumption coupled with folate deficiency leads to rapid liver fat accumulation and progression to alcoholic steatohepatitis (ASH). However, the specific effects of alcohol on key liver lipid metabolic pathways involved in fat accumulation are unknown. It is unclear whether lipid synthesis, lipid export, or a combination of both is contributing to hepatic steatosis in ASH. METHODS: In this study we estimated the flux of fatty acids (FA) through the stearoyl-CoA desaturase (SCD), phosphatidylethanolamine-N-methyltransferase (PEMT), and FA elongation pathways in relation to liver triacylglycerol (TG) content in Yucatan micropigs fed a 40% ethanol folate-deficient diet with or without supplementation with S-adenosyl methionine (SAM) compared with controls. Flux through the SCD and PEMT pathways was used to assess the contribution of lipid synthesis and lipid export respectively on the accumulation of fat in the liver. Liver FA composition within TG, cholesterol ester (CE), phosphatidylethanolamine, and phosphatidylcholine classes was quantified by gas chromatography. RESULTS: Alcoholic pigs had increased liver TG content relative to controls, accompanied by increased flux through the SCD pathway as indicated by increases in the ratios of 16:1n7 to 16:0 and 18:1n9 to 18:0. Conversely, flux through the elongation and PEMT pathways was suppressed by alcohol, as indicated by multiple metabolite ratios. SAM supplementation attenuated the TG accumulation associated with alcohol. CONCLUSIONS: These data provide an in vivo examination of liver lipid metabolic pathways confirming that both increased de novo lipogenesis (e.g., lipid synthesis) and altered phospholipid metabolism (e.g., lipid export) contribute to the excessive accumulation of lipids in liver affected by ASH.
Assuntos
Alcoolismo/metabolismo , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso/metabolismo , Metabolismo dos Lipídeos/fisiologia , Metabolômica , Animais , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Etanol/metabolismo , Ácidos Graxos/metabolismo , Masculino , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Fosfatidiletanolaminas/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Suínos , Porco Miniatura , Triglicerídeos/metabolismoRESUMO
Choline is an important nutrient for humans and animals. Animals obtain choline from the diet and from the catabolism of phosphatidylcholine made by phosphatidylethanolamine N-methyltransferase (PEMT). The unique model of complete choline deprivation is Pemt(-/-) mice that are fed a choline-deficient diet. This model, therefore, can be used for the examination of choline substitutes in mammalian systems. Recently, propanolamine was found to be a replacement for choline in yeast. Thus, we tested to see whether or not choline can be replaced by propanolamine in mice. Mice were fed a choline-deficient diet and supplemented with either methionine, 2-amino-propanol, 2-amino-isopropanol and 3-amino-propanol. We were unable to detect the formation of any of the possible phosphatidylpropanolamines. Moreover, none of them prevented liver damage, reduction of hepatic phosphatidylcholine levels or fatty liver induced in choline-deficient-Pemt(-/-) mice. These results suggest that choline in mice cannot be replaced by any of the three propanolamine derivatives.
Assuntos
Colina/metabolismo , Propanolaminas/metabolismo , Animais , Colina/administração & dosagem , Deficiência de Colina/induzido quimicamente , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Metionina/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferase/deficiência , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Fosfatidiletanolaminas/metabolismo , Propanolaminas/administração & dosagem , Triglicerídeos/metabolismoRESUMO
BACKGROUND/AIMS: Previous studies in our laboratory implicated ethanol-induced decreases in hepatocellular S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH) ratios in lowering the activity of phosphatidylethanolamine methyltransferase (PEMT), which is associated with the generation of steatosis. Further in vitro studies showed that betaine supplementation could correct these alterations in the ratio as well as attenuate alcoholic steatosis. Therefore, we sought to determine whether the protective effect of betaine is via its effect on PEMT activity. METHODS: Male Wistar rats were fed the Lieber DeCarli control or ethanol diet with or without 1% betaine supplementation for 4 weeks. RESULTS: We observed that ethanol feeding resulted in decreased phosphatidylcholine (PC) production by a PEMT-catalyzed reaction. Betaine supplementation corrected the ethanol-induced decrease in hepatic SAM:SAH ratios and by normalizing PC production via the PEMT-mediated pathway, significantly reduced fatty infiltration associated with ethanol consumption. This restoration of hepatocellular SAM:SAH ratio by betaine supplementation was associated with increases in the activity, enzyme mass and gene expression of the enzyme, betaine homocysteine methyltransferase (BHMT), that remethylates homocysteine. CONCLUSIONS: Betaine, by virtue of promoting an alternate remethylation pathway, restores SAM:SAH ratios that, in turn, correct the defective cellular methylation reaction catalyzed by PEMT resulting in protection against the generation of alcoholic steatosis.
Assuntos
Betaína/administração & dosagem , Fígado Gorduroso Alcoólico/prevenção & controle , Lipotrópicos/administração & dosagem , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Animais , Dieta , Etanol/toxicidade , Fígado Gorduroso Alcoólico/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Fosfatidiletanolamina N-Metiltransferase/análise , Ratos , Ratos Wistar , S-Adenosil-Homocisteína/análise , S-Adenosilmetionina/análise , Triglicerídeos/análiseRESUMO
A diabetic state induces the activity and abundance of glycine N-methyltransferase (GNMT), a key protein in the regulation of folate, methyl group, and homocysteine metabolism. Because the folate-dependent one-carbon pool is a source of methyl groups and 5-methyltetrahydrofolate allosterically inhibits GNMT, the aim of this study was to determine whether folate status has an impact on the interaction between diabetes and methyl group metabolism. Rats were fed a diet containing deficient (0 ppm), adequate (2 ppm), or supplemental (8 ppm) folate for 30 days, after which diabetes was initiated in one-half of the rats by streptozotocin treatment. The activities of GNMT, phosphatidylethanolamine N-methyltransferase (PEMT), and betaine-homocysteine S-methyltransferase (BHMT) were increased about twofold in diabetic rat liver; folate deficiency resulted in the greatest elevation in GNMT activity. The abundance of GNMT protein and mRNA, as well as BHMT mRNA, was also elevated in diabetic rats. The marked hyperhomocysteinemia in folate-deficient rats was attenuated by streptozotocin, likely due in part to increased BHMT expression. These results indicate that a diabetic state profoundly modulates methyl group, choline, and homocysteine metabolism, and folate status may play a role in the extent of these alterations. Moreover, the upregulation of BHMT and PEMT may indicate an increased choline requirement in the diabetic rat.