De novo phosphatidylcholine synthesis in the small intestinal epithelium is required for normal dietary lipid handling and maintenance of the mucosal barrier.

Cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTα) is the rate limiting enzyme in the major pathway for de novo phosphatidylcholine (PC) synthesis. When CTα is deleted specifically in intestinal epithelial cells of adult mice (CTαIKO mice) fed a high-fat diet they present with weight loss, lipid malabsorption, and high postprandial GLP-1 levels. The current study aimed to characterize the changes that occur in the small intestines of CTαIKO mice using transcriptomics and to determine whether intestinal function could be rescued in CTαIKO mice. We found that impaired de novo PC synthesis in the gut is linked to lower abundance of transcripts related to lipid metabolism and higher abundance of transcripts related to ER stress and cell death, together with loss of goblet cells from the small intestinal epithelium. Furthermore, impaired movement of fatty acids from the intestinal lumen into enterocytes was observed in isolated intestinal sacs derived from CTαIKO mice, a model that excludes factors such as bile, gastric emptying, the nervous system, and circulating hormones. Antibiotic treatment prevented acute weight loss and normalized jejunum TG concentrations after refeeding but did not prevent ER stress or loss of goblet cells in CTαIKO mice. Dietary PC supplementation partially prevented loss of goblet cells but was unable to normalize jejunal TG concentrations after refeeding in CTαIKO mice. High postprandial plasma GLP-1 levels were present in CTαIKO mice regardless of antibiotic treatment, dietary PC content, or dietary fat content. Together, these data show that there is a specific requirement from de novo PC synthesis in maintaining small intestinal homeostasis, including dietary lipid uptake, normal hormone secretion, and barrier function.

Dietary phosphatidylcholine supplementation reduces atherosclerosis in Ldlr-/- male mice2.

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.

Dietary Choline or Trimethylamine N-oxide Supplementation Does Not Influence Atherosclerosis Development in Ldlr-/- and Apoe-/- Male Mice.

BACKGROUND: Choline, an essential nutrient, is required for cell membranes, lipoprotein secretion, and methyl-group metabolism. Recently, it has been proposed that excess dietary choline consumption is metabolized to trimethylamine (TMA) by the gut microbiota; TMA is then oxidized to trimethylamine N-oxide (TMAO) in the liver. Epidemiological studies have clearly shown a positive correlation between plasma TMAO concentrations and cardiovascular events. Furthermore, some studies have shown an association between excess dietary choline, plasma TMAO concentrations, and atherosclerotic lesion size in apoE knockout (Apoe-/-) mice. OBJECTIVE: The aim of this study was to further investigate the relation between dietary choline and atherosclerosis in 2 atherogenic mouse models, the LDL receptor knockout (Ldlr-/-) and Apoe-/- mice. METHODS: Six feeding trials were performed in Ldlr-/- (40% high-fat diet) and Apoe-/- (unpurified diet) male mice, aged 8-10 wk. Mice randomly received control diet (0.1% choline), or choline- (1% choline), betaine- (0.1% choline and 0.9% betaine), or TMAO- (0.1% choline and 0.12% or 0.2% TMAO) supplemented diet for ≤28 wk. After the dietary intervention, the animals were killed and tissues and blood collected. Aortic atherosclerotic plaque area, plasma lipids, and choline metabolites were quantified. RESULTS: In Ldlr-/- mice, dietary supplementation for 8 wk with choline or TMAO increased plasma TMAO concentrations by 1.6- and 4-fold, respectively. After 16 wk, there was a 2-fold increase in plasma TMAO after dietary TMAO supplementation. In Apoe-/- mice, dietary supplementation with choline, betaine, or TMAO for 12 wk did not increase plasma TMAO concentrations. However, choline and TMAO supplementation for 28 wk significantly increased plasma TMAO concentrations by 1.8- and 1.5-fold, respectively. Contrary to predictions, atherosclerotic lesion size was not altered by any of the dietary interventions, irrespective of mouse model. CONCLUSIONS: In our study, high intakes of dietary choline or TMAO supplementation did not influence atherosclerosis development in Ldlr-/- or Apoe-/- male mice.

Hepatic Expression of PEMT, but Not Dietary Choline Supplementation, Reverses the Protection against Atherosclerosis in Pemt-/-/Ldlr-/- Mice.

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.

Dietary creatine supplementation lowers hepatic triacylglycerol by increasing lipoprotein secretion in rats fed high-fat diet.

Recent studies have shown that dietary creatine supplementation can prevent lipid accumulation in the liver. Creatine is a small molecule that plays a large role in energy metabolism, but since the enzyme creatine kinase is not present in the liver, the classical role in energy metabolism does not hold in this tissue. Fat accumulation in the liver can lead to the development of nonalcoholic fatty liver disease (NAFLD), a progressive disease that is prevalent in humans. We have previously reported that creatine can directly influence lipid metabolism in cell culture to promote lipid secretion and oxidation. Our goal in the current study was to determine whether similar mechanisms that occur in cell culture were present in vivo. We also sought to determine whether dietary creatine supplementation could be effective in reversing steatosis. Sprague-Dawley rats were fed a high-fat diet or a high-fat diet supplemented with creatine for 5 weeks. We found that rats supplemented with creatine had significantly improved rates of lipoprotein secretion and alterations in mitochondrial function that were consistent with greater oxidative capacity. We also find that introducing creatine into a high-fat diet halted hepatic lipid accumulation in rats with fatty liver. Our results support our previous report that liver cells in culture with creatine secrete and oxidize more oleic acid, demonstrating that dietary creatine can effectively change hepatic lipid metabolism by increasing lipoprotein secretion and oxidation in vivo. Our data suggest that creatine might be an effective therapy for NAFLD.

Choline deficiency impairs intestinal lipid metabolism in the lactating rat.

Choline is a precursor to phosphatidylcholine (PC), a structural molecule in cellular membranes that is crucial for cell growth and function. PC is also required for the secretion of lipoprotein particles from liver and intestine. Choline requirements are increased during lactation when maternal choline is supplied to the offspring through breast milk. To investigate the effect of dietary choline on intestinal lipid metabolism during lactation, choline-supplemented (CS), phosphatidylcholine-supplemented (PCS) or choline-deficient (CD) diets were fed to dams during the suckling period. CD dams had lower plasma triacylglycerol, cholesterol and apoB in the fasted state and following a fat-challenge (P < .05). There was a higher content of neutral lipids and lower content of PC in the intestine of CD dams, compared with CS and PCS fed animals (P < .05). In addition, there was lower (P < .05) villus height in CD dams, which indicated a reduced absorptive surface area in the intestine. Choline is critical for the absorption of fat in lactating rats and choline deficiency alters intestinal morphology and impairs chylomicron secretion by limiting the supply of PC.