A Physiologically Relevant Dose of 50% Egg-Phosphatidylcholine Is Sufficient in Improving Gut Permeability while Attenuating Immune Cell Dysfunction Induced by a High-Fat Diet in Male Wistar Rats.

BACKGROUND: Obesity is associated with increased intestinal permeability and a diminished immune response. Phosphatidylcholine (PC), a form of choline found in eggs, has been shown to beneficially modulate T-cell response in the context of obesity when provided as the sole form of choline in the diet. OBJECTIVE: This study aimed to determine the impact of varying doses of PC as part of a high-fat diet (HFD) on immune cell function and intestinal permeability. METHODS: Male Wistar rats 4 wk of age were randomly assigned to consume 1 of 6 diets for 12 wk containing the same amount of total choline but differing in the forms of choline: 1-control low-fat (CLF, 20% fat, 100% free choline [FC]); 2-control high-fat (CHF, 50% fat, 100% FC); 3-100% PC (100PC, 50% fat, 100% egg-PC); 4-75% PC (75PC, 50% fat, 75% egg-PC+25% FC); 5-50% PC (50PC, 50% fat, 50% egg-PC+50% FC); and 6-25% PC (25PC; 50% fat, 25% egg-PC+75% FC). Intestinal permeability was measured by fluorescein isothiocyanate-dextran. Immune function was assessed by ex vivo cytokine production of splenocytes and cells isolated from the mesenteric lymph node (MLN) after stimulation with different mitogens. RESULTS: Feeding the CHF diet increased intestinal permeability compared with the CLF diet, and doses of PC 50% or greater returned permeability to levels similar to that of the CLF diet. Feeding the CHF diet lowered splenocyte production of interleukin (IL)-1ß, IL-2, IL-10, and tumor necrosis factor-alpha, and MLN production of IL-2 compared with the CLF group. The 50PC diet most consistently significantly improved cytokine levels (IL-2, IL-10, tumor necrosis factor-alpha) compared with the CHF diet. CONCLUSIONS: Our results show that a dose of 50% of total choline derived from egg-PC can ameliorate HFD-induced intestinal permeability and immune cell dysfunction.

Hepatocyte-derived DPP4 regulates portal GLP-1 bioactivity, modulates glucose production, and when absent influences NAFLD progression.

Elevated circulating dipeptidyl peptidase-4 (DPP4) is a biomarker for liver disease, but its involvement in gluconeogenesis and metabolic associated fatty liver disease progression remains unclear. Here, we identified that DPP4 in hepatocytes but not TEK receptor tyrosine kinase-positive endothelial cells regulates the local bioactivity of incretin hormones and gluconeogenesis. However, the complete absence of DPP4 (Dpp4-/-) in aged mice with metabolic syndrome accelerates liver fibrosis without altering dyslipidemia and steatosis. Analysis of transcripts from the livers of Dpp4-/- mice displayed enrichment for inflammasome, p53, and senescence programs compared with littermate controls. High-fat, high-cholesterol feeding decreased Dpp4 expression in F4/80+ cells, with only minor changes in immune signaling. Moreover, in a lean mouse model of severe nonalcoholic fatty liver disease, phosphatidylethanolamine N-methyltransferase mice, we observed a 4-fold increase in circulating DPP4, in contrast with previous findings connecting DPP4 release and obesity. Last, we evaluated DPP4 levels in patients with hepatitis C infection with dysglycemia (Homeostatic Model Assessment of Insulin Resistance > 2) who underwent direct antiviral treatment (with/without ribavirin). DPP4 protein levels decreased with viral clearance; DPP4 activity levels were reduced at long-term follow-up in ribavirin-treated patients; but metabolic factors did not improve. These data suggest elevations in DPP4 during hepatitis C infection are not primarily regulated by metabolic disturbances.

The Lipid-Soluble Forms of Choline Enhance Ex Vivo Responses from the Gut-Associated Immune System in Young Female Rat Offspring.

BACKGROUND: In humans, the development of gut-associated lymphoid tissue (GALT) occurs in the first years of life and can be influenced by diet. OBJECTIVES: The objective of this study was to determine the effect of dietary choline on the development of gut-associated lymphoid tissue (GALT). METHODS: Three feeding trials were conducted in female Sprague-Dawley rats. Beginning 3 d before parturition (studies 1 and 3) or at day 10 of gestation (study 2), control dams consumed a 100% free choline (FC) diet until the end of the lactation period. In studies 1 and 3, test dams consumed a high-glycerophosphocholine (HGPC) diet [75% glycerophosphocholine (GPC), 12.5% phosphatidylcholine (PC), 12.5% FC] and a 100% PC diet, respectively (both 1 g of choline/kg diet). In study 2, test dams consumed a high-sphingomyelin (SM) and PC (SMPC) diet (34% SM, 37% PC, 17% GPC, 7% FC, 5% phosphocholine) or a 50% PC diet (50% PC, 25% FC, 25% GPC), both 1.7 g of choline/kg diet. Immune cell phenotypes and ex vivo cytokine production by mitogen-stimulated immune cells were measured. RESULTS: Feeding of the HGPC diet lowered T-cell IL-2 (44%), IFN-γ (34%), and TNF-α (55%) production in mesenteric lymph nodes (MLNs) compared with control. Feeding both SMPC and 50% PC diets during the lactation and weaning periods increased IL-2 (54%) and TNF-α (46%) production after T-cell stimulation compared with control. There was a lower production of IL-2 (46%), IL-6 (66%), and TNF-α (45%), and a higher production of IL-10 (44%) in both SMPC and 50% PC groups following ovalbumin stimulation compared with control in MLNs. Feeding a diet containing 100% PC increased the production of IFN-γ by 52% after T-cell stimulation compared with control. CONCLUSION: Feeding a diet containing a mixture of choline forms with a high content of lipid-soluble forms during both the lactation and weaning periods enhances ex vivo immune responses from the GALT in female Sprague-Dawley offspring.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

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.

Two-Week Isocaloric Time-Restricted Feeding Decreases Liver Inflammation without Significant Weight Loss in Obese Mice with Non-Alcoholic Fatty Liver Disease.

Prolonged, isocaloric, time-restricted feeding (TRF) protocols can promote weight loss, improve metabolic dysregulation, and mitigate non-alcoholic fatty liver disease (NAFLD). In addition, 3-day, severe caloric restriction can improve liver metabolism and glucose homeostasis prior to significant weight loss. Thus, we hypothesized that short-term, isocaloric TRF would improve NAFLD and characteristics of metabolic syndrome in diet-induced obese male mice. After 26 weeks of ad libitum access to western diet, mice either continued feeding ad libitum or were provided with access to the same quantity of western diet for 8 h daily, over the course of two weeks. Remarkably, this short-term TRF protocol modestly decreased liver tissue inflammation in the absence of changes in body weight or epidydimal fat mass. There were no changes in hepatic lipid accumulation or other characteristics of NAFLD. We observed no changes in liver lipid metabolism-related gene expression, despite increased plasma free fatty acids and decreased plasma triglycerides in the TRF group. However, liver Grp78 and Txnip expression were decreased with TRF suggesting hepatic endoplasmic reticulum (ER) stress and activation of inflammatory pathways may have been diminished. We conclude that two-week, isocaloric TRF can potentially decrease liver inflammation, without significant weight loss or reductions in hepatic steatosis, in obese mice with NAFLD.

The marine compound and elongation factor 1A1 inhibitor, didemnin B, provides benefit in western diet-induced non-alcoholic fatty liver disease.

Inhibition of eukaryotic elongation factor 1A1 (EEF1A1) with the marine compound didemnin B decreases lipotoxic HepG2 cell death in vitro and improves early stage non-alcoholic fatty liver disease (NAFLD) in young genetically obese mice. However, the effects of didemnin B on NAFLD in a model of long-term diet-induced obesity are not known. We investigated the effects of didemnin B on NAFLD severity and metabolic parameters in western diet-induced obese mice, and on the cell types that contribute to liver inflammation and fibrosis in vitro. Male 129S6 mice were fed either standard chow or western diet for 26 weeks, followed by intervention with didemnin B (50 µg/kg) or vehicle by intraperitoneal (i.p.) injection once every 3 days for 14 days. Didemnin B decreased liver and plasma triglycerides, improved oral glucose tolerance, and decreased NAFLD severity. Moreover, didemnin B moderately increased hepatic expression of genes involved in ER stress response (Perk, Chop), and fatty acid oxidation (Fgf21, Cpt1a). In vitro, didemnin B decreased THP-1 monocyte proliferation, disrupted THP-1 monocyte-macrophage differentiation, decreased THP-1 macrophage IL-1ß secretion, and decreased hepatic stellate cell (HSteC) proliferation and collagen secretion under both basal and lipotoxic (high fatty acid) conditions. Thus, didemnin B improves hepatic steatosis, glucose tolerance, and blood lipids in obesity, in association with moderate, possibly hormetic, upregulation of pathways involved in cell stress response and energy balance in the liver. Furthermore, it decreases the activity of the cell types implicated in liver inflammation and fibrosis in vitro. These findings highlight the therapeutic potential of partial protein synthesis inhibition in the treatment of NAFLD.

Hepatic PEMT activity mediates liver health, weight gain, and insulin resistance.

Phosphatidylethanolamine (PE) N-methyltransferase (PEMT) accounts for ∼30% of hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected against diet-induced obesity (DIO) and insulin resistance (IR) but develop nonalcoholic fatty liver disease (NAFLD) associated with a decreased PC:PE ratio. We investigated whether the lack of hepatic PEMT or the lack of PEMT in other tissues (where it is expressed at low levels) is responsible for or contributes to the protection against DIO and IR in Pemt-/- mice. Furthermore, we investigated whether decreasing PEMT expression with antisense oligonucleotides (ASOs) would result in metabolic benefits in both lean and obese mice without negatively impacting liver health. We both restored hepatic PEMT in Pemt-/- mice via adeno-associated virus delivery and decreased hepatic PEMT with ASOs in wild-type and ob/ob mice. Weight gain, insulin sensitivity, and indices of liver function were determined. We report that the protection against DIO and IR and the development of NAFLD is dependent on hepatic PEMT activity. NAFLD, associated with a significant decrease in the hepatic PC:PE ratio, was exacerbated by PEMT deficiency in obese mice, suggesting that phospholipid insufficiency promotes NAFLD progression during obesity or overnutrition. Hepatic PEMT is critical for maintaining phospholipid balance, which is crucial for a healthy liver.-Wan, S., van der Veen, J. N., Bakala N'Goma, J.-C., Nelson, R. C., Vance, D. E., Jacobs, R. L. Hepatic PEMT activity mediates liver health, weight gain, and insulin resistance.

Impaired Hepatic Phosphatidylcholine Synthesis Leads to Cholestasis in Mice Challenged With a High-Fat Diet.

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.

A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected from obesity and whole-body insulin resistance. However, Pemt-/- mice develop severe nonalcoholic steatohepatitis (NASH). Because NASH is often associated with hepatic insulin resistance, we investigated whether the increased insulin sensitivity in Pemt-/- mice was restricted to nonhepatic tissues or whether the liver was also insulin sensitive. Strikingly, the livers of Pemt-/- mice compared with those of Pemt+/+ mice were not insulin resistant, despite elevated levels of hepatic triacylglycerols and diacylglycerols, as well as increased hepatic inflammation and fibrosis. Endogenous glucose production was lower in Pemt-/- mice under both basal and hyperinsulinemic conditions. Experiments in primary hepatocytes and hepatoma cells revealed improved insulin signaling in the absence of PEMT, which was not due to changes in diacylglycerols, ceramides, or gangliosides. On the other hand, the phospholipid composition in hepatocytes seems critically important for insulin signaling such that lowering the PC:phosphatidylethanolamine (PE) ratio improves insulin signaling. Thus, treatments to reduce the PC:PE ratio in liver may protect against the development of hepatic insulin resistance.-Van der Veen, J. N., Lingrell, S., McCloskey, N., LeBlond, N. D., Galleguillos, D., Zhao, Y. Y., Curtis, J. M., Sipione, S., Fullerton, M. D., Vance, D. E., Jacobs, R. L. A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice.

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 (0.5 g/kg) for 3 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.

Liver and plasma lipid changes induced by cyclic fatty acid monomers from heated vegetable oil in the rat.

Cyclic fatty acid monomers (CFAM) generated through domestic or industrial heating of vegetable oils may alter liver enzymes and induce hepatomegaly and steatosis, but the underlying mechanisms are not clearly understood. This study aimed to assess the effects of CFAM on liver and plasma lipids and to determine whether these effects are modulated by dietary lipids. Thirty-six (36) male Wistar rats were fed either of the four isoenergetic diets consisting of canola oil or soybean oil with/without 500 mg/100 g CFAM of total fat for 28 days. Rats fed CFAM had higher liver total lipids (p = 0.03) and triacylglycerols (TAG) (p = 0.02), but less hepatic phosphatidylcholine (p = 0.02) compared to those fed the non-CFAM diets. CFAM did not alter liver phosphatidylethanolamine N-methyltransferase (PEMT) activity and CTP: phosphocholine cytidylyltransferase (CT-α) protein levels. Rats fed CFAM diets had higher levels of plasma total cholesterol (TC), VLDL + LDL cholesterol, higher ratio of TC to HDL cholesterol, and lower levels of HDL cholesterol compared with rats fed non-CFAM diets (p < 0.05). Plasma alanine transaminase (ALT) was decreased with CFAM, but plasma insulin, glucose, and TAG did not vary among the four diet groups (p < 0.05). Rats fed canola oil and CFAM had higher plasma levels of aspartate transaminase (AST) and AST/ALT ratio compared with the other three diet groups. These results indicate that CFAM may provoke an accumulation of TAG in the liver related to a decrease in phosphatidylcholine (PC) levels, but the effect of CFAM on PC concentrations may not occur through impairment of the two main PC biosynthesis pathways.

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.

Feeding a Mixture of Choline Forms to Lactating Dams Improves the Development of the Immune System in Sprague-Dawley Rat Offspring.

Dietary choline is essential during lactation, but few studies have examined the implications of feeding a mixture of choline forms on immune function. This study investigates the impact of feeding lactating dams different mixtures of choline forms, similar to those in human diets, on the development and later immune function of suckled offspring. Sprague-Dawley lactating dams (n = 6/diet) were randomized to consume one of three diets, containing 1 g/kg choline: Control (100% free choline (FC)), Mixed Choline (MC: 50% phosphatidylcholine (PC), 25% FC, 25% glycerophosphocholine (GPC)), or High GPC (HGPC: 75% GPC, 12.5% PC, 12.5% FC). At weaning, female pups (n = 2/dam) were fed the Control diet until 10 weeks. At 3 weeks, MC and HGPC pups were heavier and their splenocytes had a higher proportion of helper T cells expressing CD25 and CD28 and produced less interferon gamma (IFN-γ) and tumor-necrosis factor-α (TNF-α) after Concanavalin A stimulation vs. Control pups (p < 0.05). At 10 weeks, MC and HGPC offspring had a lower proportion of macrophages and dendritic cells and produced less interleukin (IL)-1ß but more IL-10 after lipopolysaccharide stimulation vs. Control pups (p < 0.05). In summary, feeding mixed choline diets during lactation improved T cell phenotype/function at the end of suckling and programmed a less inflammatory response later in life.

The critical role of phosphatidylcholine and phosphatidylethanolamine metabolism in health and disease.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in all mammalian cell membranes. In the 1950s, Eugene Kennedy and co-workers performed groundbreaking research that established the general outline of many of the pathways of phospholipid biosynthesis. In recent years, the importance of phospholipid metabolism in regulating lipid, lipoprotein and whole-body energy metabolism has been demonstrated in numerous dietary studies and knockout animal models. The purpose of this review is to highlight the unappreciated impact of phospholipid metabolism on health and disease. Abnormally high, and abnormally low, cellular PC/PE molar ratios in various tissues can influence energy metabolism and have been linked to disease progression. For example, inhibition of hepatic PC synthesis impairs very low density lipoprotein secretion and changes in hepatic phospholipid composition have been linked to fatty liver disease and impaired liver regeneration after surgery. The relative abundance of PC and PE regulates the size and dynamics of lipid droplets. In mitochondria, changes in the PC/PE molar ratio affect energy production. We highlight data showing that changes in the PC and/or PE content of various tissues are implicated in metabolic disorders such as atherosclerosis, insulin resistance and obesity. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Cystathionine beta-synthase deficiency alters hepatic phospholipid and choline metabolism: Post-translational repression of phosphatidylethanolamine N-methyltransferase is a consequence rather than a cause of liver injury in homocystinuria.

Classical homocystinuria (HCU) due to inactivating mutation of cystathionine ß-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.

Fenofibrate, but not ezetimibe, prevents fatty liver disease in mice lacking phosphatidylethanolamine N-methyltransferase.

Mice lacking phosphatidylethanolamine N-methyltransferase (PEMT) are protected from high-fat diet (HFD)-induced obesity and insulin resistance. However, these mice develop severe nonalcoholic fatty liver disease (NAFLD) when fed the HFD, which is mainly due to inadequate secretion of VLDL particles. Our aim was to prevent NAFLD development in mice lacking PEMT. We treated Pemt-/- mice with either ezetimibe or fenofibrate to see if either could ameliorate liver disease in these mice. Ezetimibe treatment did not reduce fat accumulation in Pemt-/- livers, nor did it reduce markers for hepatic inflammation or fibrosis. Fenofibrate, conversely, completely prevented the development of NAFLD in Pemt-/- mice: hepatic lipid levels, as well as markers of endoplasmic reticulum stress, inflammation, and fibrosis, in fenofibrate-treated Pemt-/- mice were similar to those in Pemt+/+ mice. Importantly, Pemt-/- mice were still protected against HFD-induced obesity and insulin resistance. Moreover, fenofibrate partially reversed hepatic steatosis and fibrosis in Pemt-/- mice when treatment was initiated after NAFLD had already been established. Increasing hepatic fatty acid oxidation can compensate for the lower VLDL-triacylglycerol secretion rate and prevent/reverse fatty liver disease in mice lacking PEMT.

Riboflavin Deficiency in Rats Decreases de novo Formate Production but Does Not Affect Plasma Formate Concentration.

Background: The one-carbon metabolism pathway is highly dependent on a number of B vitamins in order to provide one-carbon units for purine and thymidylate biosynthesis as well as homocysteine remethylation. Previous studies have examined folate and vitamin B-12 deficiency and their effects on formate metabolism; as of yet, to our knowledge, no studies on the effects of riboflavin deficiency on formate metabolism have been published.Objective: Our objective was to determine the effects of riboflavin deficiency on formate metabolism.Methods: Weanling male rats were randomly assigned either to control, riboflavin-replete (RR) or to experimental, riboflavin-deficient (RD) versions of the AIN-93G diet for 13 d, at which time a constant infusion of [13C]-formate was carried out to ascertain the effects of deficiency on formate production. Gas chromatography-mass spectrometry was used to measure plasma formate concentration and [13C]-formate enrichment. HPLC, LC-mass spectrometry (MS)/MS, and enzymatic assays were used for the measurement of one-carbon precursors and other metabolites.Results: RD rats had significantly lower rates of formate production (15%) as well as significantly reduced hepatic methylenetetrahydrofolate reductase activity (69%) and protein concentration (54%) compared with RR rats. There was no difference in plasma formate concentrations between the groups. Plasma serine, a potential one-carbon precursor, was significantly higher in RD rats (467 ± 73 µM) than in RR rats (368 ± 52 µM).Conclusions: Although deficiencies in folate and vitamin B-12 lead to major changes in plasma formate concentrations, riboflavin deficiency results in no significant difference; this disagrees with the prediction of a published mathematical model. Our observation of a lower rate of formate production is consistent with a role for flavoproteins in this process.

Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice.

Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease.

The Form of Choline in the Maternal Diet Affects Immune Development in Suckled Rat Offspring.

BACKGROUND: Lipid-soluble phosphatidylcholine (PC) and aqueous free choline are absorbed and metabolized differently, but the metabolic effects of feeding these 2 forms of choline have not been thoroughly investigated. OBJECTIVE: We sought to compare the effects of PC and free choline in the maternal diet on the development of the offspring's immune system. METHODS: During lactation, Sprague-Dawley dams (n= 10) were randomly assigned to 1 of 2 diet groups containing the same concentration of total choline (1 g/kg diet) as free choline (choline bitartrate) or PC (egg lecithin). The splenocytes of pups aged 21 d were isolated and stimulated ex vivo with concanavalin A (ConA) or lipopolysaccharide (LPS), and the choline concentrations of stomach content, plasma, and the spleen were measured. RESULTS: Pups from PC-fed dams had a lower proportion of cells involved in antigen presentation but produced 54% more interleukin (IL)-2, 163% more IL-6, and 107% more IFN-γ after ConA stimulation and 110% more IL-6 and 43% more tumor necrosis factor (TNF)-α after LPS stimulation (allP< 0.05). The PC concentrations were significantly higher in the plasma and spleen of pups from PC-fed dams (P< 0.05). Increasing the supply of PC in the form of lysophosphatidylcholine to splenocytes in vitro increased the rate of proliferation and IL-2 production and the surface expression of CD25, CD28, CD71, and CD152 on CD8+ T cells, suggesting 1 possible mechanism. CONCLUSIONS: The results of this study demonstrate that providing choline to rats in the form of PC (compared to free choline), possibly by increasing the supply of PC to the suckling pups, promotes maturation and improves function of the offspring's immune system.

Pioglitazone attenuates hepatic inflammation and fibrosis in phosphatidylethanolamine N-methyltransferase-deficient mice.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt(-/-) mice are protected against high-fat diet (HFD)-induced obesity and insulin resistance; however, these mice develop nonalcoholic fatty liver disease (NAFLD). We hypothesized that peroxisomal proliferator-activated receptor-γ (PPARγ) activation by pioglitazone might stimulate adipocyte proliferation, thereby directing lipids from the liver toward white adipose tissue. Pioglitazone might also act directly on PPARγ in the liver to improve NAFLD. Pemt(+/+) and Pemt(-/-) mice were fed a HFD with or without pioglitazone (20 mg·kg(-1)·day(-1)) for 10 wk. Pemt(-/-) mice were protected from HFD-induced obesity but developed NAFLD. Treatment with pioglitazone caused an increase in body weight gain in Pemt(-/-) mice that was mainly due to increased adiposity. Moreover, pioglitazone improved NAFLD in Pemt(-/-) mice, as indicated by a 35% reduction in liver weight and a 57% decrease in plasma alanine transaminase levels. Livers from HFD-fed Pemt(-/-) mice were steatotic, inflamed, and fibrotic. Hepatic steatosis was still evident in pioglitazone-treated Pemt(-/-) mice; however, treatment with pioglitazone reduced hepatic fibrosis, as evidenced by reduced Sirius red staining and lowered mRNA levels of collagen type Iα1 (Col1a1), tissue inhibitor of metalloproteinases 1 (Timp1), α-smooth muscle actin (Acta2), and transforming growth factor-ß (Tgf-ß). Similarly, oxidative stress and inflammation were reduced in livers from Pemt(-/-) mice upon treatment with pioglitazone. Together, these data show that activation of PPARγ in HFD-fed Pemt(-/-) mice improved liver function, while these mice were still protected against diet-induced obesity and insulin resistance.