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.     

Head group specificity in the requirement of phosphatidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes

We have demonstrated that hepatic very low density lipoprotein (VLDL) secretion requires active phosphatidylcholine (PC) synthesis via either the CDP-choline pathway or phosphatidylethanolamine (PE) methylation pathway (Yao, Z., and Vance, D.E. (1988) J. Biol. Chem. 263, 2998-3004). In the present work, the head group specificity of phospholipid synthesis required for lipoprotein secretion was investigated in cultured hepatocytes isolated from choline-deficient rats. When N-monomethylethanolamine (0.1 mM) or N,N-dimethylethanolamine (0.1 mM) was added to the culture medium, the cells synthesized correspondingly phosphatidylmonomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). However, the synthesis of PDME could correct the impaired VLDL secretion only to a limited extent, whereas the synthesis of PMME inhibited VLDL secretion. Although dimethylethanolamine did not promote VLDL secretion as well as choline, dimethylethanolamine altered the increased triacylglycerol synthesis in the choline-deficient cells as effectively as choline. Supplementation of the culture medium with ethanolamine (0.1 mM) had little effect on cellular PE or PC levels, nor was normal VLDL secretion resumed. However, the amounts of cellular PC and PE were both decreased when the medium was supplemented with N-monomethylethanolamine or N,N-dimethylethanolamine. These results suggest that the choline head group moiety of PC is specifically required for normal VLDL secretion and cannot be replaced with ethanolamine, monomethylethanolamine, or dimethylethanolamine. In addition, the impaired VLDL secretion from the choline-deficient hepatocytes could also be corrected by supplementation of betaine (0.2 mM) and homocysteine (0.2 mM), indicating the utilization of a methyl group from betaine for PC formation via methylation of PE.     

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.     

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.     

Sexually Dimorphic Activation of Liver and Brain Phosphatidylethanolamine N-Methyltransferase by Dietary Choline Deficiency

Phosphatidylethanolamine N-methyltransferase (PEMT) activity was measured by a radioenzymatic assay in homogenates of brain and liver obtained from Sprague Dawley rats fed a choline-free or control (0.3 g/kg of choline chloride) diet for seven days. Choline deficiency increased PEMT activity in the liver of male rats by 34% but had no effect on hepatic PEMT in females. In contrast, brain PEMT activity was increased in brain of choline deficient females (by 49%) but was unaltered in males. Activation of the PE methylation pathway in female brain may constitute a compensatory mechanism to sustain PC synthesis during choline deficiency.     

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.     

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.     

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.     

An unexpected requirement for phosphatidylethanolamine N-methyltransferase in the secretion of very low density lipoproteins

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine (PC). We investigated whether there was diminished secretion of lipoproteins from hepatocytes derived from mice that lacked PEMT (Pemt(-/-)) compared with Pemt(+/+) mice. Hepatocytes were incubated with 0.75 mm oleate, the media were harvested, and triacylglycerol (TG), PC, apolipoprotein (apo) B100, and apoB48 were isolated and quantified. Compared with hepatocytes from Pemt(+/+) mice, hepatocytes from Pemt(-/-) mice secreted 50% less TG, whereas secretion of PC was unaffected. Fractionation of the secreted lipoproteins on density gradients demonstrated that the decrease in TG was in the very low density lipoprotein (VLDL)/low density lipoprotein fractions. The secretion of apoB100 was decreased by approximately 70% in VLDLs/low density lipoproteins, whereas there was no significant decrease in apoB48 secretion in any fraction. Transfection of McArdle hepatoma cells (that lack PEMT) with PEMT cDNA enhanced secretion of TG in the VLDLs. Because the levels of PC in the hepatocytes and hepatoma cells were unaffected by the lack of PEMT expression, there appears to be an unexpected requirement for PEMT in the secretion of apoB100-containing VLDLs.     

Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-D(9)-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.     

Chronic ethanol consumption induces histopathological changes and increases nitric oxide generation in the rat liver

In the present work we evaluated the effect of ethanol consumption in histopathological liver changes and several biochemical biomarkers employed in the detection of hepatic dysfunction. Male Wistar rats were treated with ethanol 20% (vol/vol) for 6 weeks. Histopathological investigation of livers from ethanol-treated animals revealed steatosis. Indices of hepatic function (transaminases) and mitochondrial respiration were not altered in ethanol-treated rats. Chronic ethanol consumption did not alter malondialdehyde (MDA) levels in the liver. Ethanol consumption induced a significant increase on hepatic nitrite and nitrate levels. Treatment with ethanol increased both mRNA expression and immunostaining of iNOS, but not eNOS. Finally, ethanol consumption did not alter hepatic levels of metalloproteinase (MMP)-2 and MMP-9. We conclude that alterations on biochemical biomarkers (nitrite and nitrate levels) and histopathology occurred in ethanol-treated rats, supporting the practice of including both types of evaluation in toxicity studies to detect potential ethanol-related hepatic effects. In our model of ethanol consumption, histopathological liver changes were accompanied by elevation in nitrite and nitrate levels indicating increased nitric oxide (NO) generation. Since iNOS-derived NO contributes to hepatic injury, the increased levels of NO described in our study might contribute to a progressive hepatic damage. Therefore, increases in NO generation may be an early indicator of ethanol-induced liver damage.     

Effects of recombinant human keratinocyte growth factor on surfactant, plasma, and liver phospholipid homeostasis in hyperoxic neonatal rats

Respiratory distress and bronchopulmonary dysplasia (BPD) are major problems in preterm infants that are often addressed by glucocorticoid treatment and increased oxygen supply, causing catabolic and injurious side effects. Recombinant human keratinocyte growth factor (rhKGF) is noncatabolic and antiapoptotic and increases surfactant pools in immature lungs. Despite its usefulness in injured neonatal lungs, the mechanisms of improved surfactant homeostasis in vivo and systemic effects on lipid homeostasis are unknown. We therefore exposed newborn rats to 85% vs. 21% oxygen and treated them systemically with rhKGF for 48 h before death at 7 days. We determined type II pneumocyte (PN-II) proliferation, surfactant protein (SP) mRNA expression, and the pulmonary metabolism of individual phosphatidylcholine (PC) species using [D(9)-methyl]choline and tandem mass spectrometry. In addition, we assessed liver and plasma lipid metabolism, addressing PC synthesis de novo, the liver-specific phosphatidylethanolamine methyl transferase (PEMT) pathway, and triglyceride concentrations. rhKGF was found to maintain PN-II proliferation and increased SP-B/C expression and surfactant PC in both normoxic and hyperoxic lungs. We found increased total PC together with decreased [D(9)-methyl]choline enrichment, suggesting decreased turnover rather than increased secretion and synthesis as the underlying mechanism. In the liver, rhKGF increased PC synthesis, both de novo and via PEMT, underlining the organotypic differences of rhKGF actions on lipid metabolism. rhKGF increased the hepatic secretion of newly synthesized polyunsaturated PC, indicating improved systemic supply with choline and essential fatty acids. We suggest that rhKGF has potential as a therapeutic agent in neonates by improving pulmonary and systemic PC homeostasis.     

Elevated autophagic sequestration of mitochondria and lipid droplets in steatotic hepatocytes of chronic ethanol-treated rats: an immunohistochemical and electron microscopic study

Ethanol-induced hepatic steatosis may induce the progression of alcoholic liver disease. The involvement of autophagic clearance of damaged mitochondria (mitophagy) and lipid droplets (LDs) (lipophagy) in chronic ethanol-induced hepatic steatosis is not clearly understood. Adult Wistar rats were fed either 5 % ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 10 weeks. Light microscopy showed marked steatosis in hepatocytes of ethanol-treated rats (ETRs), which was further revealed by transmission electron microscopy (TEM), where significant numbers of large LDs and damaged mitochondria were detected in steatotic hepatocytes. Moreover, TEM demonstrated that hepatocyte steatosis was associated with greatly enhanced autophagic vacuole (AV) formation compared to control hepatocytes. Mitochondria and LDs were the predominant contents of AVs in steatotic hepatocytes. Immunohistochemistry of LC3, a specific marker of early AVs (autophagosomes), demonstrated an extensive punctate pattern in hepatocytes of ETRs, while LC3 puncta were much less frequent in control hepatocytes. This was confirmed by immunoelectron microscopy (IEM), which showed localization of LC3 to autophagosomes sequestering damaged mitochondria and LDs. In addition, IEM revealed that PINK1 (a sensor of mitochondrial damage and marker of mitophagy) was overexpressed in mitochondria of ETRs. Enhanced autophagic lysosomal activity was evidenced by increased immunolabeling of LAMP-2, a marker of late AVs (autolysosomes) in hepatocytes of ETRs and colocalization of LC3 and lysosomal cathepsins using double immunofluorescence labeling. Increased AVs in hepatocytes of ETRs reflect ethanol toxicity and could represent a possible protective mechanism via stimulation of mitophagy and lipophagy.     

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.     

Evaluating the effects of refined carbohydrate and fat diets with acute ethanol consumption using a mouse model of alcoholic liver injury

Alcoholism is a multifactorial and complex disorder responsible for 5.9% of deaths worldwide. Excessive consumption of ethanol (Et-OH) induces alcoholic liver disease (ALD), a condition comprising a spectrum of clinical signs and morphological changes, ranging from fatty liver (steatosis) to more severe forms of chronic liver injury. Secondary cofactors, such as nutritional and hepatotoxic comorbid conditions, can also contribute to liver disease development. Here we investigated the effects in the progression of ALD following short-term exposure to diet high in refined carbohydrates (HC), a high-sugar and -butter (HSB) hypercaloric diet and acute Et-OH consumption. HSB diet increased the body weight (BW) and adiposity independently of acute Et-OH consumption. HC diet did not affect BW but increased the adiposity, while acute Et-OH alone did not affect BW and adiposity. All groups of mice developed steatosis except the control group. Exposure to acute Et-OH and HSB diet increased the number of neutrophils and macrophages, and apoptosis in the liver. This combination also increased the number of circulating neutrophils and reduced mononuclear cells in the blood. Thus, short-term exposure to HSB diet and acute Et-OH intake is linked to increased liver injury. These findings offer important clues to understand the hepatic injuries associated with short exposure to hypercaloric diets and acute Et-OH.     

Differences in hepatic and metabolic changes after acute and chronic alcohol consumption.

Hepatic metabolism of ethanol to acetaldehyde by the alcohol dehydrogenase pathway is associated with the generation of reducing equivalents as NADH. Conversely, reducing equivalents are consumed when ethanol oxidation is catalyzed by the NADPH dependent microsomal ethanol oxidizing system. Since the major fraction of ethanol metabolism proceeds via alcohol dehydrogenase and since the oxidation of acetaldehyde also generates NADH, an excess of reducing equivalents is produced. This explains a variety of effects following acute ethanol administration, including hyperlactacidemia, hyperuricemia, enhanced lipogenesis and depressed lipid oxidation. To the extent that ethanol is oxidized by the alternate microsomal ethanol oxidizing system pathway, it slows the metabolism of other microsomal substrates. Following chronic ethanol consumption, adaptive microsomal changes prevail, which include enhanced ethanol and drug metabolism, and increased lipoprotein production. Severe hepatic lesions (alcoholic hepatitis and cirrhosis) develop after prolonged ethanol consumption in baboons. These injurious alterations are not prevented by nutritionally adequate diets and can therefore be ascribed to ethanol rather than to dietary inadequacy.     

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.