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Dennis E. Vance - One of the best experts on this subject based on the ideXlab platform.
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Implication of Phosphatidylethanolamine N-Methyltransferase in adipocyte differentiation.
Biochimica et biophysica acta. Molecular basis of disease, 2020Co-Authors: Natalia Presa, Dennis E. Vance, Jelske N. Van Der Veen, Asier Dominguez-herrera, Antonio Gómez-muñozAbstract:Abstract Phosphatidylethanolamine N-Methyltransferase (PEMT) is a small integral membrane protein that converts Phosphatidylethanolamine (PE) into phosphatidylcholine (PC). It has been previously reported that, unexpectedly, PEMT deficiency protected from high-fat diet (HFD)-induced obesity and insulin resistance, pointing to a possible role of this enzyme in the regulation of adipose cell metabolism. Using mouse 3T3-L1 preadipocytes as a biological system, we demonstrate that PEMT expression is strongly increased during the differentiation of preadipocytes into mature adipose cells. Knockdown of PEMT reduced the expression of early and late adipogenic markers, inhibited lipid droplet formation, reduced triacylglycerol content and decreased the levels of leptin release from the adipocytes, suggesting that PEMT is a novel and relevant regulator of adipogenesis. Investigation into the mechanisms whereby PEMT regulates adipocyte differentiation revealed that extracellularly regulated kinases (ERK1/2) and AKT are essential factors in this process. Specifically, the activities of ERK1/2 and AKT, which are decreased during adipocyte differentiation, were elevated upon Pemt knockdown. Moreover, treatment of cells with exogenous ceramide 1-phosphate (C1P), which we reported to be a negative regulator of adipogenesis, decreased PEMT expression, suggesting that PEMT is also a relevant factor in the anti-adipogenic action of C1P. Altogether, the data presented here identify PEMT as a novel regulator of adipogenesis and a mediator of the anti-adipogenic action of C1P.
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A role for phosphatidylcholine and Phosphatidylethanolamine in hepatic insulin signaling.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2019Co-Authors: Jelske N. Van Der Veen, Susanne Lingrell, Nicholas Mccloskey, Nicholas D. Leblond, Danny Galleguillos, Yuan Y. Zhao, Jonathan M. Curtis, Simonetta Sipione, Morgan D. Fullerton, Dennis E. VanceAbstract: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-bo...
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Vitamin E alleviates non-alcoholic fatty liver disease in Phosphatidylethanolamine N-Methyltransferase deficient mice.
Biochimica et biophysica acta. Molecular basis of disease, 2018Co-Authors: Natalia Presa, Dennis E. Vance, Susanne Lingrell, Zamaneh Kassiri, Antonio Gómez-muñoz, Robin D. Clugston, Samuel E. Kelly, Alfred H. Merrill, Sayantan Jana, Rene L JacobsAbstract:Abstract 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.
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Fenofibrate, but not ezetimibe, prevents fatty liver disease in mice lacking Phosphatidylethanolamine N-Methyltransferase
Journal of lipid research, 2017Co-Authors: Jelske N. Van Der Veen, Dennis E. Vance, Susanne Lingrell, Xia Gao, Abhijit Takawale, Zamaneh Kassiri, Rene L JacobsAbstract: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.
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Pioglitazone attenuates hepatic inflammation and fibrosis in Phosphatidylethanolamine N-Methyltransferase-deficient mice
American journal of physiology. Gastrointestinal and liver physiology, 2016Co-Authors: Jelske N. Van Der Veen, Susanne Lingrell, Xia Gao, Ariel D. Quiroga, Abhijit Takawale, Edward A. Armstrong, Jerome Y. Yager, Zamaneh Kassiri, Richard Lehner, Dennis E. VanceAbstract:Phosphatidylethanolamine N-Methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt−/− mice are protected against high-fat diet (HFD)-induced obesity a...
Rene L Jacobs - One of the best experts on this subject based on the ideXlab platform.
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Vitamin E alleviates non-alcoholic fatty liver disease in Phosphatidylethanolamine N-Methyltransferase deficient mice.
Biochimica et biophysica acta. Molecular basis of disease, 2018Co-Authors: Natalia Presa, Dennis E. Vance, Susanne Lingrell, Zamaneh Kassiri, Antonio Gómez-muñoz, Robin D. Clugston, Samuel E. Kelly, Alfred H. Merrill, Sayantan Jana, Rene L JacobsAbstract:Abstract 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.
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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
Molecular Genetics and Metabolism, 2017Co-Authors: Rene L Jacobs, Hua Jiang, John P Kennelly, David J Orlicky, Robert H Allen, Sally P Stabler, Kenneth N MacleanAbstract:Classical homocystinuria (HCU) due to inactivating mutation of cystathionine β-synthase (CBS) is a poorly understood life-threatening inborn error of sulfur metabolism. A previously described cbs-/- mouse model exhibits a semi-lethal phenotype due to neonatal liver failure. The transgenic HO mouse model of HCU exhibits only mild liver injury and recapitulates multiple aspects of the disease as it occurs in humans. Disruption of the methionine cycle in HCU has the potential to impact multiple aspect of phospholipid (PL) metabolism by disruption of both the Kennedy pathway and Phosphatidylethanolamine N-Methyltransferase (PEMT) mediated synthesis of phosphatidylcholine (PC). Comparative metabolomic analysis of HO mouse liver revealed decreased levels of choline, and choline phosphate indicating disruption of the Kennedy pathway. Alterations in the relative levels of multiple species of PL included significant increases in PL degradation products consistent with enhanced membrane PL turnover. A significant decrease in PC containing 20:4n6 which primarily formed by the methylation of Phosphatidylethanolamine to PC was consistent with decreased flux through PEMT. Hepatic expression of PEMT in both the cbs-/- and HO models is post-translationally repressed with decreased levels of PEMT protein and activity that inversely-correlates with the scale of liver injury. Failure to induce further repression of PEMT in HO mice by increased homocysteine, methionine and S-adenosylhomocysteine or depletion of glutathione combined with examination of multiple homocysteine-independent models of liver injury indicated that repression of PEMT in HCU is a consequence rather than a cause of liver injury. Collectively, our data show significant alteration of a broad range of hepatic PL and choline metabolism in HCU with the potential to contribute to multiple aspects of pathogenesis in this disease.
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Fenofibrate, but not ezetimibe, prevents fatty liver disease in mice lacking Phosphatidylethanolamine N-Methyltransferase
Journal of lipid research, 2017Co-Authors: Jelske N. Van Der Veen, Dennis E. Vance, Susanne Lingrell, Xia Gao, Abhijit Takawale, Zamaneh Kassiri, Rene L JacobsAbstract: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.
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Lack of Phosphatidylethanolamine N-Methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle.
Biochimica et biophysica acta, 2015Co-Authors: Guergana Tasseva, Dennis E. Vance, Rene L Jacobs, Jelske N. Van Der Veen, Susanne Lingrell, Jean E. VanceAbstract:Phosphatidylethanolamine N-Methyltransferase (PEMT) converts Phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt(-/-) mice than in Pemt(+/+) mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt(-/-) mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source.
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Lack of Phosphatidylethanolamine N-Methyltransferase alters hepatic phospholipid composition and induces endoplasmic reticulum stress.
Biochimica et biophysica acta, 2015Co-Authors: Xia Gao, Dennis E. Vance, Jelske N. Van Der Veen, Jean E. Vance, Aducio Thiesen, Rene L JacobsAbstract:Abstract Background & aims Endoplasmic reticulum (ER) stress is associated with development of steatohepatitis. Phosphatidylethanolamine N-Methyltransferase (PEMT) is a hepatic enzyme located on the ER and mitochondria-associated membranes and catalyzes phosphatidylcholine (PC) synthesis via methylation of Phosphatidylethanolamine (PE). We hypothesized that PEMT deficiency in mice alters ER phospholipid content, thereby inducing ER stress and sensitizing the mice to diet-induced steatohepatitis. Methods PC and PE mass were measured in hepatic ER fractions from chow-fed and high fat-fed Pemt−/− and Pemt+/+ mice. Proteins implicated in ER stress and the unfolded protein response (UPR) were assessed in mouse livers and in McArdle-RH7777 hepatoma cells that expressed or lacked PEMT. The chemical chaperone 4-phenyl butyric acid was administered to cells and HF-fed Pemt−/− mice to alleviate ER stress. Results In chow-fed Pemt−/− mice, the hepatic PC/PE ratio in the ER was lower than in Pemt+/+ mice, and levels of ER stress markers, CHOP and BIP, were higher without activation of the UPR. In livers of HF-fed Pemt−/− mice the ER had a lower PC/PE ratio, and exhibited more ER stress and UPR activation. Similarly, the UPR was repressed in McArdle cells expressing PEMT compared with those lacking PEMT, with concomitantly lower levels of CHOP and BIP. 4-Phenyl butyric acid attenuated activation of the UPR and ER stress in McArdle cells lacking PEMT, but not the hepatic ER stress in HF-fed Pemt−/− mice. Conclusion PEMT deficiency reduces the PC/PE ratio in the ER and induces ER stress, which sensitizes the mice to HF-induced steatohepatitis.
Zheng Cui - One of the best experts on this subject based on the ideXlab platform.
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Diminished expression of Phosphatidylethanolamine N-Methyltransferase 2 during hepatocarcinogenesis
Biochemical Journal, 1998Co-Authors: Luciana Tessitore, Zheng Cui, Irma Dianzani, Dennis E. VanceAbstract:Phosphatidylethanolamine N-Methyltransferase (PEMT) is a liver-specific enzyme that converts Phosphatidylethanolamine into phosphatidylcholine. At least two forms of PEMT are present in hepatocytes. However, PEMT activity is negligible in two hepatoma cell lines. Previous studies have indicated an inverse relationship between the expression of one form, PEMT2, and the rate of liver growth, suggesting that this enzyme might be involved in inhibition of hepatocyte proliferation. We have now investigated the expression of PEMT2 at various stages of hepatocarcinogenesis induced by chemical carcinogens. Expression of PEMT2 protein was decreased in liver samples that contained the first detectable proliferative lesions. At later stages of carcinogenesis, PEMT2 expression was obliterated. PEMT activity decreased, the levels of PEMT2 mRNA decreased and there was an increase in the activity of CTP:phosphocholine cytidylyltransferase, a key regulatory enzyme in the CDP-choline pathway of phosphatidylcholine biosynthesis. Southern blot analyses of restriction fragments of DNA showed no changes in the PEMT gene in hepatocarcinoma compared with normal liver. A role for PEMT2 in the control of hepatocyte proliferation remains an intriguing possibility.
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Phosphatidylethanolamine N-Methyltransferase from liver.
Biochimica et Biophysica Acta, 1997Co-Authors: Dennis E. Vance, Christopher J. Walkey, Zheng CuiAbstract:Phosphatidylethanolamine N-Methyltransferase (PEMT) converts Phosphatidylethanolamine to phosphatidylcholine. Most PEMT activity (PEMT1) is associated with endoplasmic reticulum. A second form of the enzyme (PEMT2) has been localized to the mitochondria-associated membrane. PEMT2 is a 22.5-kDa protein that has been purified from rat liver. The rat liver PEMT2 cDNA and the murine PEMT gene have been cloned and characterized. The PEMT gene encodes both forms of the enzyme. Deletion of the PEMT gene eliminates all activity in liver that converts Phosphatidylethanolamine to phosphatidylcholine. The activity of PEMT is regulated by supply of the substrates, Phosphatidylethanolamine and S-adenosylmethionine, and by the product S-adenosylhomocysteine. The expression of the gene is regulated during development and by the supply of choline in the diet. There is reciprocal regulation of the Kennedy pathway for phosphatidylcholine biosynthesis (via CDP-choline) and Phosphatidylethanolamine N-Methyltransferase. Several experimental approaches suggest that this enzyme might play a role in regulation of hepatocyte growth and cell division.
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Inverse correlation between expression of Phosphatidylethanolamine N-Methyltransferase-2 and growth rate of perinatal rat livers.
Biochimica et biophysica acta, 1997Co-Authors: Zheng Cui, You-jun Shen, Dennis E. VanceAbstract:Abstract Our previous studies have implicated the liver-specific Phosphatidylethanolamine N -methyltransferase-2 (PEMT2) in suppression of hepatocarcinoma proliferation (Cui et al. (1994) J. Biol. Chem. 269, 24531–24533). It was not known if this phenomenon in cell culture had relevance to liver growth and PEMT2 expression in an intact animal. Hence, we investigated the relationship between normal proliferation of liver and the expression of PEMT2 during the perinatal period of developing rats. PEMT2 protein was completely absent, and PEMT activity was very low, in prenatal livers in which liver growth is rapid. At birth, a decrease of liver growth coincided with the rapid appearance in liver of a high level of PEMT2 protein that was sustained throughout adult life. Northern blots revealed that the postnatal expression of PEMT2 correlated with the level of its mRNA. Immunohistochemical staining of liver sections showed a distinctive pattern of PEMT2 expression at birth. A high level of PEMT2 was expressed in defined extranuclear regions of hepatocytes from newborn rats whereas the protein was dispersed in the extranuclear areas in adult hepatocytes. The inverse correlation between the rate of liver growth and PEMT2 expression together with other results suggest that this enzyme, or its product, is involved in control of normal liver proliferation. © 1997 Elsevier Science B.V. All rights reserved.
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Induction of hepatocyte proliferation after partial hepatectomy is accompanied by a markedly reduced expression of Phosphatidylethanolamine N-Methyltransferase-2
Biochimica et biophysica acta, 1997Co-Authors: Martin Houweling, Luciana Tessitore, Zheng Cui, Dennis E. VanceAbstract:Abstract Expression of Phosphatidylethanolamine N-Methyltransferase (PEMT)-2 in rat hepatoma cells caused an increase in the time for cell division from 18 to 50 h [Cui et al. (1994) J. Biol. Chem. 269, 24531–24533]. We investigated whether or not a similar inverse relationship might exist for liver proliferation in vivo. Thus, partial hepatectomized rats were used to investigate the expression of PEMT2 during liver regeneration. Enhanced biosynthesis of phosphatidylcholine after partial hepatectomy was due to increased activity and amount of CTP:phosphocholine cytidylyltransferase. On the other hand the total activity of PEMT was markedly decreased during the first days of rat liver regeneration. Maximal decrease of total PEMT activity (45%) and loss of PEMT2 protein (90%) coincided with maximal DNA synthesis and CTP:phosphocholine cytidylyltransferase activity 24 h after partial hepatectomy in both male and female rats. Supplementing dietary choline in the diets of female rats shifted this pattern from 24 h to 36 h after partial hepatectomy, whereas the pattern in male rats was not affected. Northern blot studies showed that the amount of PEMT2 mRNA was decreased accordingly, suggesting regulation of the amount and activity of PEMT2 at a pre-translational level. Thus, our data show a reciprocal regulation of CTP:phosphocholine cytidylyltransferase and PEMT2 at the level of gene expression in regenerating rat liver. These results implicate PEMT2 in the regulation of hepatocyte cell growth in a physiologically relevant model. © 1997 Elsevier Science B.V. All rights reserved.
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Expression of Phosphatidylethanolamine N-Methyltransferase-2 is markedly enhanced in long term choline-deficient rats.
The Journal of biological chemistry, 1996Co-Authors: Zheng Cui, Dennis E. VanceAbstract:Abstract When rats are fed a choline-deficient (CD) diet, acute fatty liver develops along with other biochemical changes. However, when choline deficiency is prolonged, the growth rate of CD rats is similar to that of control rats fed a choline-supplemented diet. Furthermore, CD rats maintain their levels of choline-containing lipids, such as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. The mechanism for this compensation in CD rats was investigated. We screened the major tissues for the activities of two important enzymes involved in the biosynthesis of phosphatidylcholine, CTP:phosphocholine cytidylyltransferase (CT) and Phosphatidylethanolamine N-Methyltransferase (PEMT). Only the livers of CD rats had higher specific enzyme activities of PEMT and CT than control animals. The amount of PEMT2, one of two PEMTs in liver, increased 5-fold in CD rats after 6 weeks on the CD diet. A similar increase in the level of PEMT2 mRNA suggested that this activation was due to enhanced expression of the PEMT2 gene in CD livers. The labeling of phosphatidycholine in isolated hepatocytes from CD rats was consistent with the conversion of PE to PC being increased as a result of a higher expression of liver PEMT. We conclude that activation of PE methylation at the level of gene expression may be the mechanism by which CD rats compensate for the lack of dietary choline.
Norma M. Giusto - One of the best experts on this subject based on the ideXlab platform.
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Light Activation of Phosphatidylethanolamine N-Methyltransferase in Rod Outer Segments and its Modulation by Association States of Transducin
Experimental eye research, 1999Co-Authors: Marta Elena Roque, Gabriela Alejandra Salvador, Norma M. GiustoAbstract:Phosphatidylethanolamine N-Methyltransferase (PE N-MTase) is the enzyme responsible for the synthesis of phosphatidylcholine from Phosphatidylethanolamine by successive transfer of methyl groups. This enzyme is present in bovine rod outer segments (ROS) and it is the only pathway for the synthesis of phosphatidylcholine in the outer segment of rod photoreceptor cells. In dark-adapted ROS membranes PE N-MTase activity is stimulated by 100% when ROS membranes are incubated under light condition. To determine whether the retinal G protein, transducin (Gt), intervenes in the regulation of PE N-MTase in these membranes, the effects of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and guanosine 5'-O-(2-thiodiphosphate (GDPbetaS) on the enzyme activity were examined. In dark, GTPgammaS which induces dissociation of Gt, stimulates the enzyme activity mimicking the stimulation by light. On the contrary, GDPbetaS stabilizes the inactive state of Gt, inhibiting the stimulation by light of PE N-MTase without affecting basal activities. In addition, adenosine 5'-diphosphate (ADP)-ribosylation by cholera and pertussis toxin was studied. ADP-ribosylation of ROS membrane with pertussis toxin, which stabilizes transducin in its inactive state, prevents the light-induced increase in PE N-MTase activity. On the contrary ADP-ribosylation with cholera toxin stimulates the enzyme activity. Our findings therefore suggest that light-stimulated effect of PE N-MTase activity is transducin-mediated.
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PHOSPHORYLATION OF ROD OUTER SEGMENT PROTEINS MODULATES Phosphatidylethanolamine N-Methyltransferase AND PHOSPHOLIPASE A2 ACTIVITIES IN PHOTORECEPTOR MEMBRANES
Comparative biochemistry and physiology. Part B Biochemistry & molecular biology, 1998Co-Authors: P.i. Castagnet, Marta Elena Roque, Susana J. Pasquaré, Norma M. GiustoAbstract:Abstract The activities of enzymes involved in lipid metabolism—phospholipase A 2 (PLA 2 ) and Phosphatidylethanolamine N-Methyltransferase (PE N-MTase)—were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA 2 activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA 2 activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA 2 activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA 2 activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA 2 activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.
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Phosphatidylethanolamine N-Methyltransferase activity in isolated rod outer segments from bovine retina.
Experimental eye research, 1995Co-Authors: Marta Elena Roque, Norma M. GiustoAbstract:Phosphatidylcholine (PC) can be synthesized in isolated rod outer segments from bovine retina by successive transfer of methyl groups from S-adenosyl-L-methionine (SAM) to Phosphatidylethanolamine (PE), with the intermediate formation of phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME). This reaction is time-protein-and SAM concentration-dependent. Phosphatidylethanolamine N-Methyltransferase (PE N-MTase) has two pH optima, 8.5 and 10, at low (10 microM) and high (200 microM) SAM concentrations and requires magnesium ions for full activity. When ROS membranes were incubated at 5 to 200 microM SAM concentrations at pH 8.5 or pH 10, the major methylated product was PMME, followed by PC and PDME. The apparent Kms for SAM at pH 8.5 and at pH 10 were similar (37 and 38 microM, respectively). The Vmax was 13 pmol h-1 (mg protein)-1 at pH 8.5 and 12.50 pmol h-1 (mg protein)-1 at pH 10. Pulse-chase experiments demonstrated a precursor-product relationship with [3H]PC as the end product. The level of PE N-Mtase activity in the purified ROS preparation obtained from crude ROS fractions by discontinuous sucrose gradient centrifugation, was as high as 65% of the level found in the microsomal fraction obtained from the remainder of the retinas. The presence of microsomal and mitochondrial marker enzymes, however, was minimal in the ROS preparation. The radioactivity incorporated into ROS PC was measured in an upper and lower band of PC obtained by two-dimensional TLC. We found that the amount of [methyl-3H] groups incorporated into the upper PC band was 2.5-fold greater than that incorporated into the lower one. The fatty acid composition of the upper band was very different from that of the lower band, the former being enriched in very long-chain polyunsaturated fatty acids and the latter in saturated fatty acids. Phosphatidyl-ethanolamine N-Methyltransferase activity increased in the presence of exogenous phospholipid substrates. PDME being augmented ten-fold and PC eight-fold when the incubations were carried out in the presence of PMME and PDME, respectively. At a 2 mM concentration, S-adenosyl-L-homocysteine (SAH) inhibited the methyl groups' incorporation into the endogenous phospholipids by 40%. When ROS membranes were selectively depleted of soluble or peripheral and soluble proteins, the PE N-MTase activity remained mainly associated to the membrane, suggesting that this enzyme (s) is an intrinsic membrane protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Christopher J. Walkey - One of the best experts on this subject based on the ideXlab platform.
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Identification of three novel cDNAs for human Phosphatidylethanolamine N-Methyltransferase and localization of the human gene on chromosome 17p11.2
Biochimica et biophysica acta, 1999Co-Authors: Christopher J. Walkey, David J. Shields, Dennis E. VanceAbstract:Abstract Phosphatidylethanolamine is converted to phosphatidylcholine in mammalian liver by the enzyme Phosphatidylethanolamine N-Methyltransferase (PEMT). A form of the enzyme (PEMT2) has been isolated from rat liver, the cDNA cloned and expressed and the murine gene has been characterized and disrupted. Several lines of evidence suggested that PEMT2 might have a role in hepatocyte proliferation and liver cancer. Hence, we decided to investigate the human form of the enzyme. Unexpectedly, we cloned and expressed three novel human cDNAs encoding PEMT2. These forms differ from each other in the 5′-region with the point of divergence being 15 nucleotides upstream of the putative translation initiation codon. The remainder of the three cDNAs was identical. Expression of the coding region of the cDNAs in McArdle rat hepatoma cells resulted in three stable cell lines that showed a 27- to 115-fold elevation of PEMT activity compared to vector-transfected control cell lines. Screening of somatic cell hybrid panels, radiation hybrid panel mapping and fluorescent in situ hybridization mapping localized the human gene for PEMT2 to chromosome 17p11.2. The identification of three different human cDNAs for PEMT2 suggests that understanding the function of PEMT2 will be more complicated than anticipated.
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Roles for the methylation of Phosphatidylethanolamine.
Current opinion in lipidology, 1998Co-Authors: Dennis E. Vance, Christopher J. WalkeyAbstract:The methylation of Phosphatidylethanolamine is an auxiliary pathway for phosphatidylcholine biosynthesis in liver. Two forms of the enzyme, Phosphatidylethanolamine N-Methyltransferase, which catalyses this reaction, are located on the endoplasmic reticulum and mitochondria-associated membranes. Both forms are encoded by a single murine gene, Pempt, located on chromosome 11. The expression of the gene begins at birth. An inverse relationship exists between the rate of liver growth and the expression of Phosphatidylethanolamine N-Methyltransferase. However, disruption of the Pempt gene does not alter liver growth in mice or cause any other obvious phenotype.
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Disruption of the murine gene encoding Phosphatidylethanolamine N-Methyltransferase
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Christopher J. Walkey, Luis B. Agellon, Leah Rae Donohue, Rod Bronson, Dennis E. VanceAbstract:All nucleated cells make phosphatidylcholine via the CDP-choline pathway. Liver has an alternative pathway in which phosphatidylcholine is made by methylation of Phosphatidylethanolamine catalyzed by Phosphatidylethanolamine N-Methyltransferase (PEMT). We investigated the function of PEMT and its role in animal physiology by targeted disruption of its gene, Pempt2. A targeting vector that interrupts exon 2 was constructed and introduced into mice yielding three genotypes: normal (+/+), heterozygotes (+/−), and homozygotes (−/−) for the disrupted PEMT gene. Only a trace of PE methylation activity remained in Pempt2(−/−) mice. Antibody to one form of the enzyme, PEMT2, indicated complete loss of this protein from Pempt2(−/−) mice and a decrease in Pempt2(+/−) mice, compared with Pempt2(+/+) mice. The levels of hepatic Phosphatidylethanolamine and phosphatidylcholine were minimally affected. The active form of CTP:phosphocholine cytidylyltransferase, the regulated enzyme in the CDP-choline pathway, was increased 60% in the PEMT-deficient mice. Injection of [l-methyl-3H]methionine demonstrated that the in vivo PEMT activity was eliminated in the Pempt2(−/−) mice and markedly decreased in the Pempt2(+/−) mice. This experiment also demonstrated that the choline moiety derived from PEMT in the liver can be distributed via the plasma throughout the mouse where it is found as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. Mice homozygous for the disrupted Pempt2 gene displayed no abnormal phenotype, normal hepatocyte morphology, normal plasma lipid levels and no differences in bile composition. This is the first application of the “knockout mouse” technique to a gene for phospholipid biosynthesis.
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Phosphatidylethanolamine N-Methyltransferase from liver.
Biochimica et Biophysica Acta, 1997Co-Authors: Dennis E. Vance, Christopher J. Walkey, Zheng CuiAbstract:Phosphatidylethanolamine N-Methyltransferase (PEMT) converts Phosphatidylethanolamine to phosphatidylcholine. Most PEMT activity (PEMT1) is associated with endoplasmic reticulum. A second form of the enzyme (PEMT2) has been localized to the mitochondria-associated membrane. PEMT2 is a 22.5-kDa protein that has been purified from rat liver. The rat liver PEMT2 cDNA and the murine PEMT gene have been cloned and characterized. The PEMT gene encodes both forms of the enzyme. Deletion of the PEMT gene eliminates all activity in liver that converts Phosphatidylethanolamine to phosphatidylcholine. The activity of PEMT is regulated by supply of the substrates, Phosphatidylethanolamine and S-adenosylmethionine, and by the product S-adenosylhomocysteine. The expression of the gene is regulated during development and by the supply of choline in the diet. There is reciprocal regulation of the Kennedy pathway for phosphatidylcholine biosynthesis (via CDP-choline) and Phosphatidylethanolamine N-Methyltransferase. Several experimental approaches suggest that this enzyme might play a role in regulation of hepatocyte growth and cell division.
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Characterization of the murine Phosphatidylethanolamine N-Methyltransferase-2 gene.
Journal of lipid research, 1996Co-Authors: Christopher J. Walkey, Zheng Cui, Luis B. Agellon, Dennis E. VanceAbstract:Phosphatidylethanolamine N-Methyltransferase (PEMT) catalyzes the conversion of Phosphatidylethanolamine to phosphatidylcholine in the mammalian liver via three sequential methylations. In the present studies, we cloned and characterized the murine gene for PEMT2, the isoform of the enzyme that localized to the mitochondria-associated membrane. The structure of the gene was determined by analysis of two lambda and three P1 genomic clones, and compared to the known rat PEMT2 cDNA sequence. Southern blotting of mouse genomic DNA indicated that PEMT2 is a single-copy gene. The gene spans at least 35 kb, with seven exons and six introns. Two transcription start sites, 139 and 148 base pairs upstream of the translation start site, were detected by primer extension and reverse transcriptase-polymerase chain reaction. These experiments indicated that the PEMT2 gene is transcribed from a single promoter. Finally, the PEMT2 gene was localized to mouse chromosome 11 by interspecific backcrossing. These experiments represent the first cloning and characterization of a full-length mammalian gene involved in phospholipid biosynthesis.