Phosphatidylcholine homeostasis and liver failure

In mammals, the only endogenous pathway for choline biosynthesis is the methylation of phosphatidylethanolamine to phosphatidylcholine (PC) by phosphatidylethanolamine N-methyltransferase (PEMT) coupled to PC degradation. Complete choline deprivation in mice by feeding Pemt(-/-) mice a choline-deficient (CD) diet decreases hepatic PC by 50% and is lethal within 5 days. PC secretion into bile is mediated by a PC-specific flippase, multiple drug-resistant protein 2 (MDR2). Here, we report that mice that lack both PEMT and MDR2 and are fed a CD diet survive for >90 days. Unexpectedly, the amount of PC also decreases by 50% in the livers of Mdr2(-/-)/Pemt(-/-) mice. The Mdr2(-/-)/Pemt(-/-) mice adapt to the severe choline deprivation via choline recycling by induction of phospholipase A(2), choline kinase, and CTP:phosphocholine cytidylyltransferase activities and by a strikingly decreased expression of choline oxidase. The ability of Mdr2(-/-)/Pemt(-/-) mice to survive complete choline deprivation suggests that acute lethality in CD-Pemt(-/-) mice results from rapid depletion of hepatic PC via biliary secretion.     

A role for high density lipoproteins in hepatic phosphatidylcholine homeostasis

Choline is (95%) found largely in the biosphere as a component of phosphatidylcholine (PC) which is made from choline via the CDP-choline pathway. Animals obtain choline from both the diet and via endogenous biosynthesis that involves the conversion of phosphatidylethanolamine into PC by phosphatidylethanolamine N-methyltransferase (PEMT), followed by PC catabolism. We have uncovered a striking gender-specific conservation of choline in female mice that does not occur in male mice. Female Pemt(-/-) mice maintained hepatic PC/total choline levels during the first day of choline deprivation and escaped liver damage whereas male Pemt(-/-) mice did not. Plasma PC levels in high-density lipoproteins (HDLs) were higher in male Pemt(-/-) mice than those in females before choline deprivation. Interestingly, after choline deprivation for 1 day, female, but not male, Pemt(-/-) mice increased HDL-PC levels. Glybenclamide, an inhibitor of PC efflux mediated by ABC transporters, eliminated this response to choline deprivation in females. These data suggest that (i) increased PC efflux from extra-hepatic tissues to HDLs in the circulation provided sufficient choline for the liver and compensated for loss of hepatic PC during the initial stages of choline deprivation in female, but not male, Pemt(-/-) mice, and (ii) plasma HDL in female mice has an important function in maintenance of hepatic PC as an acute response to severe choline deprivation.     

The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are major phospholipids in mammalian membranes. In liver, PC is synthesized via the choline pathway or by methylation of PE via phosphatidylethanolamine N-methyltransferase (PEMT). Pemt(-/-) mice fed a choline-deficient (CD) diet develop rapid steatohepatitis leading to liver failure. Steatosis is observed in CD mice that lack both PEMT and multiple drug-resistant protein 2 (MDR2), required for PC secretion into bile. We demonstrate that liver failure in CD-Pemt(-/-) mice is due to loss of membrane integrity caused by a decreased PC/PE ratio. The CD-Mdr2(-/-)/Pemt(-/-) mice escape liver failure by maintaining a normal PC/PE ratio. Manipulation of PC/PE levels suggests that this ratio is a key regulator of cell membrane integrity and plays a role in the progression of steatosis into steatohepatitis. The results have clinical implications as patients with nonalcoholic steatohepatitis have a decreased ratio of PC to PE compared to control livers.     

The phosphatidylethanolamine N-methyltransferase pathway is quantitatively not essential for biliary phosphatidylcholine secretion

The phosphatidylethanolamine N-methyltransferase (PEMT) pathway of phosphatidylcholine (PC) biosynthesis is not essential for the highly specific acyl chain composition of biliary PC. We evaluated whether the PEMT pathway is quantitatively important for biliary PC secretion in mice under various experimental conditions. Biliary bile salt and PC secretion were determined in mice in which the gene encoding PEMT was inactivated (Pemt(-/-)) and in wild-type mice under basal conditions, during acute metabolic stress (intravenous infusion of the bile salt tauroursodeoxycholate), and during chronic metabolic stress (feeding a taurocholate-containing diet for 1 week). The activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of PC biosynthesis via the CDP-choline pathway, and the abundance of multi-drug-resistant protein 2 (Mdr2; encoded by the Abcb4 gene), the canalicular membrane flippase essential for biliary PC secretion, were determined. Under basal conditions, Pemt(-/-) and wild-type mice exhibited similar biliary secretion rates of bile salt and PC ( approximately 145 and approximately 28 nmol/min/100 g body weight, respectively). During acute or chronic bile salt administration, the biliary PC secretion rates increased similarly in Pemt(-/-) and control mice. Mdr2 mRNA and protein abundance did not differ between Pemt(-/-) and wild-type mice. The cytidylyltransferase activity in hepatic lysates was increased by 20% in Pemt(-/-) mice fed the basal (bile salt-free) diet (P < 0.05). We conclude that the biosynthesis of PC via the PEMT pathway is not quantitatively essential for biliary PC secretion under acute or chronic bile salt administration.     

Choline cannot be replaced by propanolamine in mice

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.     

A gender-specific role for phosphatidylethanolamine N-methyltransferase-derived phosphatidylcholine in the regulation of plasma high density and very low density lipoproteins in mice

Phosphatidylethanolamine N-methyltransferase (PEMT)is involved in a secondary pathway for production of phosphatidylcholine (PC) in liver. We fed Pemt-/-mice a high fat/high cholesterol diet for 3 weeks to determine whether or not PC derived from PEMT is required for very low density lipoprotein secretion. Lipid analyses of plasma and liver indicated that male Pemt-/- mice accumulated triacylglycerols in their livers and were unable to secrete the same amount of triacylglycerols from the liver as did Pemt+/+ mice. Plasma levels of triacylglycerol and both apolipoproteins B100 and B48 were significantly decreased only in male Pemt-/- mice. Experiments in which mice were injected with Triton WR1339 showed that, whereas hepatic apoB100 secretion was decreased in male Pemt-/- mice, the decrease in plasma apoB48 in male Pemt-/- mice was not due to reduced secretion. Moreover, female and, to a lesser extent, male Pemt-/- mice showed a striking 40% decrease in plasma PC and cholesterol in high density lipoproteins. These results suggest that, even though the content of hepatic PC was normal in PEMT-deficient mice, plasma lipoprotein levels were profoundly altered in a gender-specific manner.     

EFFECTS OF N-METHYLAMINOETHANOL, AND N,N-DIMETHYL-AMINOETHANOL IN THE DIET OF PREGNANT RATS ON NEONATAL RAT BRAIN CHOLINERGIC AND PHOSPHOLIPID PROFILE

Abstract— Pregnant rats were fed for 15 days predelivery until 15 days postpartum a choline (Ch)-deficient diet (CD diet) or a CD diet supplemented with 0.8% Ch-CI (CS), 1% N -methylaminoethanol (MME) or 1% N,N -dimethylaminoethanol (DME). Gestation and parturition of the pregnant rats proceeded normally. However, all the pups born of dams fed the MME diet, and most of those born of dams fed the DME diet, died within 36 h of birth. No histological or cytological alterations were detected in the brain of the pups. Levels of Ch and acetylcholine (ACh) were elevated in the brain of pups born of dams fed the MME and DME diets, but not the CS diet. The content of total phospholipids in the brain of the pups was not altered by the diet fed to the dams. However, the phosphatidyl-Ch and phosphatidylaminoethanol (PAE) contents in the brain of the MME- and DME-exposed pups were markedly reduced. At the same time, significant amounts of DME, phosphatidyl-N-monomethylaminoethanol (PMME) and of phosphatidyl- N,N -dimethylaminoethanol (PDME) were present in the same brain areas. These results are evaluated and discussed in terms of providing a cause for the death of the MME- and DME-exposed neonatal rats.     

Choline redistribution during adaptation to choline deprivation

Choline is an important nutrient for mammals. Choline can also be generated by the catabolism of phosphatidylcholine synthesized in the liver by the methylation of phosphatidylethanolamine by phosphatidylethanolamine N-methyltransferase (PEMT). Complete choline deprivation is achieved by feeding Pemt(-)(/)(-) mice a choline-deficient diet and is lethal due to liver failure. Mice that lack both PEMT and MDR2 (multiple drug-resistant protein 2) successfully adapt to choline deprivation via hepatic choline recycling. We now report another mechanism involved in this adaptation, choline redistribution. Normal levels of choline-containing metabolites were maintained in the brains of choline-deficient Mdr2(-)(/)(-)/Pemt(-)(/)(-) mice for 90 days despite continued choline consumption via oxidation. Choline oxidase activity had not been previously detected in the brain. Plasma levels of choline were also maintained for 90 days, whereas plasma phosphatidylcholine levels decreased by >60%. The injection of [(3)H]choline into Mdr2(-)(/)(-)/Pemt(-)(/)(-) mice revealed a redistribution of choline among tissues. Although CD-Pemt(-)(/)(-) mice failed to adapt to choline deprivation, choline redistribution was also initiated in these mice. The data suggest that adaptation to choline deprivation is not restricted to liver via choline recycling but also occurs in the whole animal via choline redistribution.     

A role for hepatic scavenger receptor class B, type I in decreasing high density lipoprotein levels in mice that lack phosphatidylethanolamine N-methyltransferase

Phosphatidylethanolamine N-methyltransferase (PEMT) is a liver-specific enzyme that converts phosphatidylethanolamine to phosphatidylcholine (PC). Mice that lack PEMT have reduced plasma levels of PC and cholesterol in high density lipoproteins (HDL). We have investigated the mechanism responsible for this reduction with experiments designed to distinguish between a decreased formation of HDL particles by hepatocytes or an increased hepatic uptake of HDL lipids. Therefore, we analyzed lipid efflux to apoA-I and HDL lipid uptake using primary cultured hepatocytes isolated from Pemt(+/+) and Pemt(-/-) mice. Hepatic levels of the ATP-binding cassette transporter A1 are not significantly different between Pemt genotypes. Moreover, hepatocytes isolated from Pemt(-/-) mice released cholesterol and PC into the medium as efficiently as did hepatocytes from Pemt(+/+) mice. Immunoblotting of liver homogenates showed a 1.5-fold increase in the amount of the scavenger receptor, class B, type 1 (SR-BI) in Pemt(-/-) compared with Pemt(+/+) livers. In addition, there was a 1.5-fold increase in the SR-BI-interacting protein PDZK1. Lipid uptake experiments using radiolabeled HDL particles revealed a greater uptake of [(3)H]cholesteryl ethers and [(3)H]PC by hepatocytes derived from Pemt(-/-) compared with Pemt(+/+) mice. Furthermore, we observed an increased association of [(3)H]cholesteryl ethers in livers of Pemt(-/-) compared with Pemt(+/+) mice after tail vein injection of [(3)H]HDL. These results strongly suggest that PEMT is involved in the regulation of plasma HDL levels in mice, mainly via HDL lipid uptake by SR-BI.     

Insights into the requirement of phosphatidylcholine synthesis for liver function in mice

Phosphatidylcholine (PC) is made in the liver by the CDP-choline pathway and via phosphatidylethanolamine N-methyltransferase (PEMT), which catalyzes the conversion of phosphatidylethanolamine to PC. Unexpectedly, hepatic apolipoprotein B-100 secretion is inhibited in male, but not female, Pemt-/- mice (Noga, A. A., Y. Zhao, and D. E. Vance. 2002. J. Biol. Chem. 277: 42358-42365; Noga, A. A., and D. E. Vance. 2003. J. Biol. Chem. 278: 21851-21859). To gain further insight into this process, we compared PC metabolism in male and female mice fed chow or a high-fat/high-cholesterol (HF/HC) diet. Immunoblot analyses demonstrated that twice as much PEMT2 was present in livers from female compared with male mice. In contrast, assays of CTP:phosphocholine cytidylyltransferase from livers of Pemt+/+ mice demonstrated more active cytidylyltransferase in male than in female mice. Secretion of PEMT-derived PC into lipoproteins was examined in vivo by injection of mice with [methyl-3H]methionine in the presence of Triton WR1339. The PEMT-derived PC shifts to smaller-sized particles in response to a HF/HC diet, but only in male mice. Secretion of PEMT-derived PC into bile was enhanced in mice fed a HF/HC diet. These results demonstrate that the synthesis and targeting of PC produced by the PEMT pathway in the livers of mice differs in a gender- and diet-specific manner.     

Reduced early alcohol-induced liver injury in CD14-deficient mice

Activation of Kupffer cells by gut-derived endotoxin is associated with alcohol-induced liver injury. Recently, it was shown that CD14-deficient mice are more resistant to endotoxin-induced shock than wild-type controls. Therefore, this study was designed to investigate the role of CD14 receptors in early alcohol-induced liver injury using CD14 knockout and wild-type BALB/c mice in a model of enteral ethanol delivery. Animals were given a high-fat liquid diet continuously with ethanol or isocaloric maltose-dextrin as control for 4 wk. The liver to body weight ratio in wild-type mice (5.8 +/- 0.3%) was increased significantly by ethanol (7.3 +/- 0.2%) but was not altered by ethanol in CD14-deficient mice. Ethanol elevated serum alanine aminotransferase levels nearly 3-fold in wild-type mice, but not in CD14-deficient mice. Wild-type and knockout mice given the control high-fat diet had normal liver histology, whereas ethanol caused severe liver injury (steatosis, inflammation, and necrosis; pathology score = 3.8 +/- 0.4). In contrast, CD14-deficient mice given ethanol showed minimal hepatic changes (score = 1.6 +/- 0.3, p < 0.05). Additionally, NF-kappa B, TGF-beta, and TNF-alpha were increased significantly in wild-type mice fed ethanol but not in the CD14 knockout. Thus, chronic ethanol feeding caused more severe liver injury in wild-type than CD14 knockouts, supporting the hypothesis that endotoxin acting via CD14 plays a major role in the development of early alcohol-induced liver injury.     

Alpha-tocopherol modulates Cyp3a expression, increases gamma-CEHC production, and limits tissue gamma-tocopherol accumulation in mice fed high gamma-tocopherol diets

Although all forms of vitamin E are absorbed, the liver preferentially secretes alpha-, but not gamma-tocopherol, into plasma. Liver alpha-tocopherol secretion is under the control of the alpha-tocopherol transfer protein (TTP). Therefore, to assess gamma-tocopherol bioactivities Ttpa-/-, +/- and +/+ mice were fed for 5 weeks diets containing gamma-tocopherol 550 (gamma-T550), gamma-tocopherol 60 (gamma-T60) mg/kg that also contained trace amounts of alpha-tocopherol, a vitamin E-deficient diet, or a control diet. Plasma and tissues from mice fed gamma-T550 diets were found to contain similar gamma- and alpha-tocopherol concentrations despite the high dietary gamma-tocopherol content; nervous tissues contained almost no gamma-tocopherol. Liver vitamin E metabolites (carboxyethyl hydroxychromans, CEHCs) were also measured. In mice with widely ranging liver alpha- (from 0.7 to 16 nmol/g) and gamma-tocopherol concentrations (0 to 13 nmol/g), hepatic alpha-CEHC was undetectable, but gamma-CEHC concentrations (0.1 to 0.8 nmol/g) were correlated with both alpha- and gamma-tocopherol concentrations (P < 0.004). Hepatic cytochrome P450s (CYPs) involved in vitamin E metabolism, Cyp4f and Cyp3a, were also measured. There were no variations in Cyp4f protein expression as related to diet or mouse genotype. However, Cyp3a was correlated (P < 0.0001) with liver alpha-, but not gamma-tocopherol concentrations. These data support the hypothesis that alpha-tocopherol modulates xenobiotic metabolism by increasing Cyp3a expression, gamma-CEHC formation, and the excretion of both gamma-tocopherol and gamma-CEHC.     

The incorporation of monomethylethanolamine and dimethylethanolamine in fetal brain aggregating cell culture

Fetal rat brain aggregating cell cultures were exposed to varying concentrations of [³H]monomethylethanolamine (MME) and [³H] dimethylethanolamine (DME). The rate of labeling of water-soluble compounds was more rapid and the amount of radioactivity present was greater than in the lipids. After a 72 hour incubation in the presence of millimolar concentrations of these nitrogenous bases, the major water-soluble products were the phosphorylated form of the bases. Little label was associated with the free bases or their cytidyl derivate. In the phospholipids, 97% of the radioactivity was recovered in phosphatidylmonomethylethanolamine (PMME) and 3% in phosphatidyldimethylethanolamine (PDME) or 95% in PDME and 5% in phosphatidylcholine (PC) after growth in presence of [³H]MME and [³H]DME respectively. The rate of formation of the radioactive products increased as function of the concentration of the nitrogenous base added up to 4 mM, the highest concentration employed. There was no significant difference in the pattern of labeling with cells grown in media devoid of methionine or choline. The turnover of the water-soluble metabolites was more rapid than in the phospholipids where an apparent half-life of 24 hours was calculated.Abbreviations PMT phospholipid-N-methyltransferase - AdoMet S-adenosyl-L-methionine - EA ethanolamine - MME N-monomethylethanolamine - DME N,N-dimethylethanolamine - CH choline - PE phosphatidylethanolamine - PMME phosphatidylmonomethylethanolamine - PDME phosphatidyldimethylethanolamine - PC phosphatidylcholine - PS phosphatidylserine - CAPS cyclohexylaminopropane sulfonic acid     

Effect of folic acid deficiency upon lymphocyte subsets from lymphoid organs in mice.

1. Three groups of weanling C57BL/6 female mice were fed one of two folate-deficient diets (0 and 0.1 mg folic acid/kg diet) or a normal folate-containing diet (2 mg folic acid/kg diet) for 8 weeks. A control pair-fed group was introduced with the most severe folate-deficient diet. Seven mice were fed the 0 mg folic acid/kg diet for 8 weeks, then rehabilitated (R) on the 2 mg folic acid/kg diet for 10 days. 2. Mice fed 0 mg folic acid/kg diet were severely folate-deficient (SFD), whereas mice fed 0.1 mg folic acid/kg diet were moderately folate-deficient (MFD), as shown by their folate status parameters. 3. Thymus weight, thymocyte content and positive immature CD4+8+ cells were decreased in SFD mice compared to controls. These values were normalized after 10 days of rehabilitation. 4. Mesenteric lymph node cells were apparently not affected by folate deficiency. 5. The proportion of Thy-1+ splenocytes was mildly lower in SFD mice than in controls. In R mice, mean spleen weight and spleen cellularity were increased compared to the other groups, but the proportions of Thy-1+, CD4+8- and CD4-8+ cells were markedly lower than control values.     

Intraduodenal infusion of lysophosphatidylcholine restores the intestinal absorption of vitamins A and E in rats fed a low-zinc diet

Our previous work has shown that the lymphatic absorptions of lipids and lipid-soluble vitamins, retinol and alpha-tocopherol (alphaTP), are lowered markedly in rats fed a low-zinc (LZ) diet in parallel with lower lymphatic phospholipid outputs. Phosphatidylcholine (PC), when infused enterally, restored the absorptions of fat and retinol, but further lowered the absorption of alphaTP in rats fed the LZ diet. This study was conducted to determine whether a luminal infusion of lysophosphatidylcholine, a product of PC hydrolysis by pancreatic phospholipase A2 (PLA2), would simultaneously restore the absorptions of retinol and alphaTP in LZ rats. Rats were trained to consume two meals per day and were divided into two groups. One group was fed an AIN-93G diet containing a LZ (3.0 mg Zn/kg), and the other was fed the same diet, but containing adequate zinc (AZ; 30.0 mg Zn/kg) for 6 weeks. Rats with lymph cannula were infused at 3.0 ml/hr for 8 hr with a lipid emulsion containing retinol, alphaTP, and 14C-labeled triolein (14C-oleic acid) with or without 1-oleoyl-2-hydroxy phosphatidylcholine (lysoPC) in 24 ml of PBS (pH 6.5). When the lipid emulsion without lysoPC was infused, the absorptions of retinol and alphaTP were significantly lower in LZ rats (retinol, 13.2+/-1.5 nmol; alphaTP, 430.6+/-66.8 nmol) than in AZ rats (retinol, 18.2+/-1.0 nmol; alphaTP, 543.8+/-58.9 nmol). The lower absorptions of the vitamins in LZ rats occurred in parallel with a significant decrease in 14C-oleic acid absorption. When the emulsion containing lysoPC was infused, however, absorptions of the vitamins (retinol, 18.4+/-3.0 nmol; alphaTP, 777.2+/-92.1 nmol) in LZ rats were restored completely to the control levels (retinol, 20.4+/-2.8 nmol; alphaTP, 756.3+/-136.1 nmol). The results suggest that the luminal hydrolysis of PC to lysoPC by PLA2 may be impaired in LZ rats, resulting in impaired absorption of fat and the fat-soluble vitamins.     

Dietary α-tocopherol and atorvastatin reduce high-fat-induced lipid accumulation and down-regulate CD36 protein in the liver of guinea pigs

The increased uptake and storage of lipids in the liver are important features of steatotic liver diseases. The fatty acid translocase/scavenger receptor cluster of differentiation (CD)36 facilitates the hepatic uptake of lipids. We investigated if RRR-α-tocopherol (αT) alone or in combination with atorvastatin (ATV) is capable of preventing hepatic lipid accumulation via down-regulation of CD36. To this end, Dunkin Hartley guinea pigs were fed a control diet (5% fat); or a high-fat control diet (21% fat, 0.15% cholesterol); or a high-fat control diet fortified with αT (250 mg/kg diet), ATV (300 mg/kg diet) or both ATV+αT for 6 weeks. Hepatic triacylglycerols, hepatic protein and mRNA expression of CD36 as well as the mRNA expression of the controlling nuclear receptors LXRα, PXR and PPARγ were determined. Animals fed the high-fat control diet accumulated significantly more triacylglycerols in the liver than control animals. This was significantly reduced by ATV and numerically by αT and ATV+αT. Hepatic CD36 protein concentrations were significantly higher in the high-fat than in the control group, and both αT and ATV reduced CD36 expression to the level observed in the control group. However, no synergistic effect of the combined treatment was observed. Neither CD36 mRNA nor that of the nuclear receptors (LXRα, PXR and PPARγ) differed between groups, suggesting a posttranslational regulatory mechanism. Our results indicate that orally administered ATV and αT individually, but not synergistically, prevent diet-induced lipid accumulation in the liver of guinea pigs by down-regulation of hepatic CD36 protein.     

A diet high in cholesterol and deficient in vitamin E induces lipid peroxidation but does not enhance antioxidant enzyme expression in rat liver

Expression of antioxidant enzymes (AOE), an important mechanism in the protection against oxidative stress, could be modified by the redox status of the cells. The aim of this project was to evaluate the role of vitamin E deficiency in association with a high-cholesterol diet in the hepatic lipid peroxidation and the expression of AOE. Two groups of 6 male rats were fed with a high-cholesterol or a high-cholesterol vitamin E-deficient diet. All animals were sacrificed at 72 days of treatment. Liver lipid peroxidation index (Malondialdehyde; MDA) and hepatic AOE were evaluated. Total liver RNA was extracted, and the steady state messenger RNA (mRNA) levels of glutathion peroxydase, manganese superoxide dismutase, Cu/Zn superoxide dismutase and catalase were examined by northern blot. After 72 days on the diet, a significant increase in the lipid peroxidation index was observed in the vitamin E deficient group (MDA : 4.45 +/- 0.29 nmol/mg protein versus 3.65 +/- 0.1 nmol/mg protein in vitamin E normal group). Despite this oxidative stress, the activities and mRNA levels of liver AOE were not significantly different in the 2 groups. These preliminary results show that chronic vitamin E deficiency associated with high cholesterol diet is able to increase lipid peroxidation without modulation of AOE expression and activity in the liver. This suggests that beneficial effects of dietary vitamin E are due to a plasma antioxidant effect or a cell mediated action, rather than to a specific modulation of cellular enzymes.     

Stimulation of glutathione synthesis in iron-loaded mice

We have previously reported that the iron-loading of mice, by feeding them carbonyl iron, caused an elevation of hepatic glutathione concentration and an increase in glutathione excretion from the liver (Kawabata, T., Ogino, T. and Awai, M. (1989) Biochim. Biophys. Acta 1004, 89-94). To elucidate the mechanism of glutathione elevation, hepatic cysteine concentration and gamma-glutamylcysteine synthetase (L-glutamate: L-cysteine gamma-ligase (ADP-forming), EC 6.3.2.2) activity were measured and possible changes in cysteine metabolism were also compared between iron-loaded and control mice. Hepatic cysteine concentration was higher in iron-loaded mice (185 +/- 12 nmol/g wet wt.) than in the controls (164 +/- 8 nmol/g wet wt.), and gamma-glutamylcysteine synthetase activity was also elevated in iron-loaded mice (34.3 +/- 3.2 nmol/mg protein per min) compared with the controls (28.6 +/- 3.8 nmol/mg protein per min). A comparison of the metabolic pathways with intravenously injected [35S]cysteine showed that organ distribution of the isotope was not significantly different, and also the rate of [35S]cysteine uptake into the hepatic glutathione fraction exhibited no difference between the two groups of mice. This shows that hepatic cysteine turnover may not be different between the two groups of mice. Since hepatic cysteine concentration was higher in iron-loaded mice, the apparently equal turnover of hepatic cysteine suggests that GSH synthesis may be elevated in iron-loaded mice. The high gamma-glutamylcysteine synthetase activity is suggested to stimulate GSH synthesis in iron-loaded mice.     

Phosphatidylcholine protects against steatosis in mice but not non-alcoholic steatohepatitis

Several studies suggest that low levels of hepatic phosphatidylcholine (PC) play a role in the pathogenesis of non-alcoholic steatohepatitis (NASH). CTP: phosphocholine cytidylyltransferase (CT) is the key regulatory enzyme in the CDP-choline pathway for PC biosynthesis. Liver-specific elimination of CTα (LCTα(-/-)) in mice fed a chow diet decreases very-low-density lipoprotein secretion, reduces lipid efflux from liver, and causes mild steatosis. We fed LCTα(-/-) mice a high fat diet to determine if impaired PC biosynthesis played a role in development of NASH. LCTα(-/-) mice developed NASH within one week of high fat feeding. Hepatic CTα deficiency caused hepatic steatosis, a 2-fold increase in ceramide mass, and a 20% reduction in PC content. In an attempt to prevent NASH, LCTα(-/-) mice were either injected daily with CDP-choline or fed the high fat diet supplemented with betaine. In addition, LCTα(-/-) mice were injected with adenoviruses expressing CTα. CDP-choline injections and adenoviral expression of CTα increased hepatic PC, while dietary betaine supplementation normalized hepatic triacylglycerol but did not alter hepatic PC mass in LCTα(-/-) mice. Interestingly, none of the treatments normalized hepatic ceramide mass or fully prevented the development of NASH in LCTα(-/-) mice. These results show that normalizing the amount of hepatic PC is not sufficient to prevent NASH in LCTα(-/-) mice.     

Impaired de Novo Choline Synthesis Explains Why Phosphatidylethanolamine N-Methyltransferase-deficient Mice Are Protected from Diet-induced Obesity

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.