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Dietary recommendations for cheese usually promote low (LOW)- over regular (REG)-fat versions due to the saturated fat. Conversely, epidemiological evidence shows that cheese consumption associates with reduced risk of type 2 diabetes. To investigate how cheese influences diabetes-related outcomes, a feeding trial comparing LOW and REG cheese was conducted in high-fat, lard-based diet (HFD)-fed insulin-resistant rats followed by evaluation of potential mechanisms. After 4 weeks of HFD, LOW or REG was added at 7 and 10 g/100 g diet, respectively, for another 8 weeks. Following either an oral glucose or insulin tolerance test to assess glucoregulation, rats were euthanized and serum was collected for metabolomic and lipid analyses. Hepatic tissue was used to measure glucoregulatory enzymes and lipid content. Both LOW and REG improved insulin sensitivity without effect on oral glucose tolerance, insulin secretion or body weight. Serum metabolomics identified 33 metabolites of interest, with 21 being phosphatidylcholines (PCs) or lysophosphatidylcholines (LPCs). HFD rats had significantly reduced LPC C16:1, C17:0, C18:1, C20:3 and C24:0, and these effects were normalized by LOW or REG cheese. Fourteen PC species were lowest in the HFD group and normalized by cheese feeding. Serum choline was elevated sevenfold in HFD- but not cheese-fed rats compared with rats fed low-fat diet. Liver triglyceride was elevated by LOW feeding. In conclusion, inclusion of both LOW and REG cheeses in the diet of insulin-resistant rats improves in vivo glucoregulation. This is associated with altered phospholipid metabolism, including cheese-mediated normalization of species that are decreased by high-fat feeding., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Classical homocystinuria (HCU) due to inactivating mutation of cystathionine β-synthase (CBS) is a poorly understood life-threatening inborn error of sulfur metabolism. A previously described cbs-/- mouse model exhibits a semi-lethal phenotype due to neonatal liver failure. The transgenic HO mouse model of HCU exhibits only mild liver injury and recapitulates multiple aspects of the disease as it occurs in humans. Disruption of the methionine cycle in HCU has the potential to impact multiple aspect of phospholipid (PL) metabolism by disruption of both the Kennedy pathway and phosphatidylethanolamine N-methyltransferase (PEMT) mediated synthesis of phosphatidylcholine (PC). Comparative metabolomic analysis of HO mouse liver revealed decreased levels of choline, and choline phosphate indicating disruption of the Kennedy pathway. Alterations in the relative levels of multiple species of PL included significant increases in PL degradation products consistent with enhanced membrane PL turnover. A significant decrease in PC containing 20:4n6 which primarily formed by the methylation of phosphatidylethanolamine to PC was consistent with decreased flux through PEMT. Hepatic expression of PEMT in both the cbs-/- and HO models is post-translationally repressed with decreased levels of PEMT protein and activity that inversely-correlates with the scale of liver injury. Failure to induce further repression of PEMT in HO mice by increased homocysteine, methionine and S-adenosylhomocysteine or depletion of glutathione combined with examination of multiple homocysteine-independent models of liver injury indicated that repression of PEMT in HCU is a consequence rather than a cause of liver injury. Collectively, our data show significant alteration of a broad range of hepatic PL and choline metabolism in HCU with the potential to contribute to multiple aspects of pathogenesis in this disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) method was developed and validated to simultaneously quantify six aqueous choline-related compounds and eight major phospholipids classes in a single run. HILIC chromatography was coupled to positive ion electrospray mass spectrometry. A combination of multiple scan modes including precursor ion scan, neutral loss scan and multiple reaction monitoring was optimized for the determination of each compound or class in a single LC/MS run. This work developed a simplified extraction scheme in which both free choline and related compounds along with phospholipids were extracted into a homogenized phase using chloroform/methanol/water (1:2:0.8) and diluted into methanol for the analysis of target compounds in a variety of sample matrices. The analyte recoveries were evaluated by spiking tissues and food samples with two isotope-labeled internal standards, PC-d(3) and Cho-d(3). Recoveries of between 90% and 115% were obtained by spiking a range of sample matrices with authentic standards containing all 14 of the target analytes. The precision of the analysis ranged from 1.6% to 13%. Accuracy and precision was comparable to that obtained by quantification of selected phospholipid classes using (31)P NMR. A variety of sample matrices including egg yolks, human diets and animal tissues were analyzed using the validated method. The measurements of total choline in selected foods were found to be in good agreement with values obtained from the USDA choline database., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Biological methylation reactions and homocysteine (Hcy) metabolism are intimately linked. In previous work, we have shown that phosphatidylethanolamine N-methyltransferase, an enzyme that methylates phosphatidylethanolamine to form phosphatidylcholine, plays a significant role in the regulation of plasma Hcy levels through an effect on methylation demand (Noga, A. A., Stead, L. M., Zhao, Y., Brosnan, M. E., Brosnan, J. T., and Vance, D. E. (2003) J. Biol. Chem. 278, 5952-5955). We have further investigated methylation demand and Hcy metabolism in liver-specific CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) knockout mice, since flux through the phosphatidylethanolamine N-methyltransferase pathway is increased 2-fold to meet hepatic demand for phosphatidylcholine. Our data show that plasma Hcy is elevated by 20-40% in mice lacking hepatic CTalpha. CTalpha-deficient hepatocytes secrete 40% more Hcy into the medium than do control hepatocytes. Liver activity of betaine:homocysteine methyltransferase and methionine adenosyltransferase are elevated in the knockout mice as a mechanism for maintaining normal hepatic S-adenosylmethionine and S-adenosylhomocysteine levels. These data suggest that phospholipid methylation in the liver is a major consumer of AdoMet and a significant source of plasma Hcy.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Purpose: To determine the effect of feeding buttermilk-derived choline metabolites on the immune system development in Sprague–Dawley rat pups. Methods: Sprague–Dawley dams were randomized to one of the three diets containing 1.7 g/kg choline: 1-Control (100% free choline (FC)), 2-Buttermilk (BM, 37% phosphatidylcholine (PC), 34% sphingomyelin (SM), 17% glycerophosphocholine (GPC), 7% FC, 5% phosphocholine), and 3-Placebo (PB, 50% PC, 25% FC, 25% GPC) until the end of the lactation period. At weaning, pups continued on the same diet as their mom. Cell phenotypes and cytokine production by mitogen-stimulated splenocytes isolated from 3- and 10-week-old pups were measured. Results: At 3 weeks, BM-pups had a higher proportion of cytotoxic T cells (CTL; CD3 + CD8 +) while both BM- and PB-pups had an increased proportion of cells expressing CD28 + , CD86 + and CD27 + (all p > 0.05). Following ConA stimulation, splenocytes from BM- and PB-pups produced more TNF-α and IFN-γ and after LPS stimulation produced more IL-10 and TNF-α (all p > 0.05). Starting at week 6 of age, BM-pups had a higher body weight. At 10 weeks, both the BM- and PB-pups had a higher proportion of CTL expressing CD27 +. After ConA stimulation, splenocytes from BM- and PB-pups produced more IL-2, IFN-γ and IL-6 and more IL-10 after LPS stimulation (all p > 0.05). Conclusion: The proportion of lipid soluble forms of choline in the diet during lactation and weaning periods influence the immune system development in rat offspring. [ABSTRACT FROM AUTHOR]

Phosphatidylethanolamine N‐methyltransferase (PEMT) is a hepatic integral membrane protein localized to the endoplasmic reticulum (ER). PEMT catalyzes approximately 30% of hepatic phosphatidylcholine (PC) biosynthesis. Pemt–/– mice fed a high‐fat diet (HFD) develop steatohepatitis. Interestingly, portions of the ER located close to the canaliculus are enriched in PEMT. Phospholipid balance and asymmetrical distribution by adenosine triphosphatase phospholipid transporting 8B1 (ATP8B1) on the canalicular membrane is required for membrane integrity and biliary processes. We hypothesized that PEMT is an important supplier of PC to the canaliculus and that PEMT activity is critical for the maintenance of canalicular membrane integrity and bile formation following HFD feeding when there is an increase in overall hepatic PC demand. Pemt+/+ and Pemt–/– mice were fed a chow diet, an HFD, or a choline‐supplemented HFD. Plasma and hepatic indices of liver function and parameters of bile formation were determined. Pemt–/– mice developed cholestasis, i.e, elevated plasma bile acid (BA) concentrations and decreased biliary secretion rates of BAs and PC, during HFD feeding. The maximal BA secretory rate was reduced more than 70% in HFD‐fed Pemt–/– mice. Hepatic ABCB11/bile salt export protein, responsible for BA secretion, was decreased in Pemt–/– mice and appeared to be retained intracellularly. Canalicular membranes of HFD‐fed Pemt–/– mice contained fewer invaginations and displayed a smaller surface area than Pemt+/+ mice. Choline supplementation (CS) prevented and reversed the development of HFD‐induced cholestasis. Conclusion: We propose that hepatic PC availability is critical for bile formation. Dietary CS might be a potential noninvasive therapy for a specific subset of patients with cholestasis. Mice with impaired phosphatidylcholine synthesis develop NAFLD and cholestasis when fed a high fat diet. We propose that hepatic PC availability is critical for bile formation. As such, dietary choline supplementation might be a potential non‐invasive therapy for specific cholestasis patients. [ABSTRACT FROM AUTHOR]

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]

Phosphatidylcholine is an essential phospholipid that is synthesized by 2 different pathways, the CDP-choline pathway and the methylation of phosphatidylethanolamine by phosphatidylethanolamine N-methyltransferase (PEMT). Recent studies have suggested that PEMT is an important consumer of methyl groups from S-adenosylmethionine (SAM) and is a major determinant of homocysteine pools. Diabetes and all-trans-retinoic acid (ATRA) have been shown to alter the activities of several enzymes involved in methyl group metabolism. Thus, we investigated how diabetes and ATRA, individually and together, affect SAM-dependent phospholipid methylation. Rats received a single injection of streptozotocin (60 mg/kg body wt) or vehicle followed by administration of ATRA (30 mumol/kg body wt) or vehicle for 5 d. The hepatic activity of PEMT increased 50% in both diabetic rat groups, whereas administration of ATRA was without effect. In diabetic rats, plasma total homocysteine decreased 30-35% in all treatment groups as compared with the control group. Thus, alterations in the activity of PEMT were not directly correlated to changes in homocysteine concentrations. Moreover, treatment of diabetic rats with insulin prevented the increase in PEMT activity and abundance. Because these observations support an increased need for SAM-dependent phosphatidylcholine synthesis, this may also indicate an increased choline requirement in diabetes. [ABSTRACT FROM AUTHOR]

Folate is an essential B vitamin required for the maintenance of AdoMet-dependent methylation. The liver is responsible for many methylation reactions that are used for post-translational modification of proteins, methylation of DNA, and the synthesis of hormones, creatine, carnitine, and phosphatidylcholine. Conditions where methylation capacity is compromised, including folate deficiency, are associated with impaired phosphatidylcholine synthesis resulting in non-alcoholic fatty liver disease and steatohepatitis. In addition, folate intake and folate status have been associated with changes in the expression of genes involved in lipid metabolism, obesity, and metabolic syndrome. In this review, we provide insight on the relationship between folate and lipid metabolism, and an outlook for the future of lipid-related folate research. © 2013 BioFactors, 40(3):277-283, 2014 [ABSTRACT FROM AUTHOR]

5-Aminoimidazole-4-carboxamide- 1-β-D-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK. [ABSTRACT FROM AUTHOR]

Based on a growing need for additional dietary sources of choline, this research developed a choline rich cereal based functional food. Egg yolk lipids were extracted using ethanol to produce an egg yolk extract (EYE) of high choline content. Corn meal, wheat flour, sugar, salt and EYE were mixed and extrudates made using a twin-screw extruder with an exit temperature of 120 °C. Products were packaged and stored at −20 °C and room temperature for 12 weeks. Initial processing decreased the total fat and total choline content by up to 30% and 19% respectively. Similarly, tocopherols, tocotrienols and cholesterol decreased after processing. However, the total fat and choline content and tocopherols and tocotrienols did not significantly change during the storage stability study. A shelf-stable choline rich (115 mg/30 g serving) functional food product was developed that could provide a way for consumers to meet their choline requirement. [ABSTRACT FROM AUTHOR]

Background & aims Endoplasmic reticulum (ER) stress is associated with development of steatohepatitis. Phosphatidylethanolamine N-methyltransferase (PEMT) is a hepatic enzyme located on the ER and mitochondria-associated membranes and catalyzes phosphatidylcholine (PC) synthesis via methylation of phosphatidylethanolamine (PE). We hypothesized that PEMT deficiency in mice alters ER phospholipid content, thereby inducing ER stress and sensitizing the mice to diet-induced steatohepatitis. Methods PC and PE mass were measured in hepatic ER fractions from chow-fed and high fat-fed Pemt −/− and Pemt +/+ mice. Proteins implicated in ER stress and the unfolded protein response (UPR) were assessed in mouse livers and in McArdle-RH7777 hepatoma cells that expressed or lacked PEMT. The chemical chaperone 4-phenyl butyric acid was administered to cells and HF-fed Pemt −/− mice to alleviate ER stress. Results In chow-fed Pemt −/− mice, the hepatic PC/PE ratio in the ER was lower than in Pemt +/+ mice, and levels of ER stress markers, CHOP and BIP, were higher without activation of the UPR. In livers of HF-fed Pemt −/− mice the ER had a lower PC/PE ratio, and exhibited more ER stress and UPR activation. Similarly, the UPR was repressed in McArdle cells expressing PEMT compared with those lacking PEMT, with concomitantly lower levels of CHOP and BIP. 4-Phenyl butyric acid attenuated activation of the UPR and ER stress in McArdle cells lacking PEMT, but not the hepatic ER stress in HF-fed Pemt −/− mice. Conclusion PEMT deficiency reduces the PC/PE ratio in the ER and induces ER stress, which sensitizes the mice to HF-induced steatohepatitis . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]