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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. [ABSTRACT FROM AUTHOR]

The increased prevalence of obesity and diabetes in human populations can induce the deposition of fat (triacylglycerol) in the liver (steatosis). The current view is that most hepatic triacylglycerols are derived from fatty acids released from adipose tissue. In this study, we show that phosphatidylcholine (PC), an important structural component of cell membranes and plasma lipoproteins, can be a precursor of ∼65% of the triacylglycerols in liver. Mice were injected with [3H]PC-labeled high density lipoproteins (HDLs). Hepatic uptake of HDL-PC was ∼10 μmol/day, similar to the rate of hepatic de novo PC synthesis. Consistent with this finding, measurement of the specific radioactivity of PC in plasma and liver indicated that 50% of hepatic PCis derived from the circulation. Moreover, one-third of HDLderived PC was converted into triacylglycerols. Importantly, ∼65% of the total hepatic pool of triacylglycerol appears to be derived from hepatic PC, half of which is derived from HDL. Thus, lipoprotein-associated PC should be considered a quantitatively significant source of triacylglycerol for the etiology of hepatic steatosis. [ABSTRACT FROM AUTHOR]

Recent studies have indicated that direct intestinal secretion of plasma cholesterol significantly contributes to fecal neutral sterol loss in mice. The physiological relevance of this novel route, which represents a part of the reverse cholesterol transport pathway, has not been directly established in vivo as yet. We have developed a method to quantify the fractional and absolute contributions of several cholesterol fluxes to total fecal neutral sterol loss in vivo in mice, by assessing the kinetics of orally and intravenously administered stable isotopically labeled cholesterol combined with an isotopic approach to assess the fate of de novo synthesized cholesterol. Our results show that trans-intestinal cholesterol excretion significantly contributes to removal of blood-derived free cholesterol in C57B16/J mice (33% of 231 μmol/kg/day) and that pharmacological activation of LXR with T0901317 strongly stimulates this pathway (63% of 706 μmol/ kg/day). Trans-intestinal cholesterol excretion is impaired in mice lacking Abcg5 (-4%), suggesting that the cholesterol transporting Abcg5/Abcg8 heterodimer is involved in this pathway. Our data demonstrate that intestinal excretion represents a quantitatively important route for fecal removal of neutral sterols independent of biliary secretion in mice. This pathway is sensitive to pharmacological activation of the LXR system. These data support the concept that the intestine substantially contributes to reverse cholesterol transport. [ABSTRACT FROM AUTHOR]

Background & Aims: Conversion into bile acids represents an important route to remove excess cholesterol from the body. Rev-erbα is a nuclear receptor that participates as one of the clock genes in the control of circadian rhythmicity and plays a regulatory role in lipid metabolism and adipogenesis. Here, we investigate a potential role for Rev-erbα in the control of bile acid metabolism via the regulation of the neutral bile acid synthesis pathway. Methods: Bile acid synthesis and CYP7A1 gene expression were studied in vitro and in vivo in mice deficient for or over expressing Rev-erbα. Results: Rev-erbα-deficient mice display a lower synthesis rate and an impaired excretion of bile acids into the bile and feces. Expression of CYP7A1, the rate-limiting enzyme of the neutral pathway, is decreased in livers of Rev-erbα-deficient mice, whereas adenovirus-mediated hepatic Rev-erbα overexpression induces its expression. Moreover, bile acid feeding resulted in a more pronounced suppression of hepatic CYP7A1 expression in Rev-erbα-deficient mice. Hepatic expression of E4BP4 and the orphan nuclear receptor small heterodimer partner (SHP), both negative regulators of CYP7A1 expression, is increased in Rev-erbα-deficient mice. Promoter analysis and chromatin immunoprecipitation experiments demonstrated that SHP and E4BP4 are direct Rev-erbα target genes. Finally, the circadian rhythms of liver CYP7A1, SHP, and E4BP4 messenger RNA levels were perturbed in Rev-erbα-deficient mice. Conclusions: These data identify a role for Rev-erbα in the regulatory loop of bile acid synthesis, likely acting by regulating both hepatic SHP and E4BP4 expression. [Copyright &y& Elsevier]

Choline is an essential nutrient required for various biological processes. Eggs, dairy, and meat are rich in phosphatidylcholine (PC), whereas cereal and legumes are rich in free choline. Excess dietary choline leads to increase plasma trimethylamine N-oxide (TMAO). Epidemiological studies suggest that plasma TMAO is a biomarker for atherosclerosis and it has been suggested that a lower intake of eggs and meat would reduce choline consumption and thus reduce atherosclerosis development. To investigate whether the form of dietary choline influences atherosclerosis development in Ldlr-/-, we randomly fed Ldlr-/-male mice (aged 8 - 10 wk) one of the three 40% (calories) high fat diets (with 0.5% w/w of cholesterol): Control (0.1% w/w free-choline, CON), choline-supplemented (0.4% free-choline, CS), or PC-supplemented (0.1% free-choline and 0.3% choline from PC, PCS). After 12-wk of dietary intervention, the animals were euthanized and tissues and blood collected. Aortic atherosclerotic plaque area, plasma choline, lipid metabolites, and spleen and peripheral blood cell phenotypes were quantified. Surprisingly, the PCS group had significantly lower atherosclerotic lesions while having 2-fold higher plasma TMAO levels compared with both CON and CS groups (P<0.05). In the fasting state, we found that PCS decreased plasma very low-density lipoprotein-cholesterol (VLDL-C) and apolipoprotein B48 (APOB48), and increased plasma high-density lipoprotein-cholesterol (HDL-C). However, very low-density lipoprotein (VLDL) secretion was not affected by dietary treatment. We observed lower levels of circulating pro-atherogenic chemokines in the PCS group. Our study suggests that increased dietary PC intake does not induce a pro-atherogenic phenotype. [ABSTRACT FROM AUTHOR]

Coenzyme Q (CoQ) is an essential redox-active lipid, and its deficiency is implicated in numerous diseases. However, it is largely unknown what regulates CoQ content. To investigate cellular CoQ regulation, we performed a genome-wide screen (~5,000 S. cerevisiae single gene mutants) to isolate genes critical for CoQ homeostasis, and measured CoQ content in each mutant using HPLC-EC detection. Some 30 knockout mutants were identified with significantly higher CoQ content compared to WT including the cho2 mutant. CHO2 encodes a phosphatidylethanolamine (PE) methyltransferase that catalyzes the conversion of PE to phosphatidylcholine (PC). Homologs of CHO2 are found in mammals (PEMT) indicating there may be role for this gene in CoQ regulation in other species. The cho2 mutant contained concentrations of CoQ five times greater than WT, and a significantly increased rate of CoQ synthesis. To investigate if Pemt–/– mice also display elevated CoQ content akin to yeast cho2 mutants, we analyzed the CoQ content in liver and adipose tissue from Pemt+/+ and Pemt–/– mice. In these tissues, CoQ content was approximately double in Pemt–/– compared to Pemt+/+. Taken together, our data suggests a novel role for phosphatidylethanolamine methyltransferases in CoQ content regulation. [ABSTRACT FROM AUTHOR]