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D E Vance

Publications and source records attributed to D E Vance.

At least 37 records · Page 2Linked to original sources
PubMed1999

Subcellullar localization, developmental expression and characterization of a liver triacylglycerol hydrolase.

The mechanism and enzymic activities responsible for the lipolysis of stored cytosolic triacylglycerol in liver and its re-esterification remain obscure. A candidate enzyme for lipolysis, a microsomal triacylglycerol hydrolase (TGH), was recently purified to homogeneity from pig liver and its kinetic properties were determined [Lehner and Verger (1997) Biochemistry 36, 1861-1868]. We have characterized the enzyme with regard to its species distribution, subcellular localization, developmental expression and reaction with lipase inhibitors. The hydrolase co-sediments with endoplasmic reticulum elements and is associated with isolated liver fat droplets. Immunocytochemical studies localize TGH exclusively to liver cells surrounding capillaries. Both TGH mRNA and protein are expressed in rats during weaning. The enzyme covalently binds tetrahydrolipstatin, an inhibitor of lipases and of triacylglycerol hydrolysis. The enzyme is absent from liver-derived cell lines (HepG2 and McArdle RH7777) known to be impaired in very-low-density lipoprotein (VLDL) assembly and secretion. The localization and developmental expression of TGH are consistent with a proposed role in triacylglycerol hydrolysis and with the proposal that some of the resynthesized triacylglycerol is utilized for VLDL secretion.

Animals↗
PubMed1999

Identification of three novel cDNAs for human phosphatidylethanolamine N-methyltransferase and localization of the human gene on chromosome 17p11.2.

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.

Abnormalities, Multiple↗
PubMed1999

Diminished expression of phosphatidylethanolamine N-methyltransferase 2 during hepatocarcinogenesis.

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.

Animals↗
PubMed1999

The unique acyl chain specificity of biliary phosphatidylcholines in mice is independent of their biosynthetic origin in the liver.

The liver synthesizes phosphatidylcholine (PC) de novo from choline via the CDP-choline pathway, and from phosphatidylethanolamine (PE) via the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. Significant amounts of PC, which are highly specific in their acyl chain composition, are secreted into bile by the liver. To determine whether either of the 2 PC biosynthetic routes is sufficient to provide physiological PC concentrations in bile, or is responsible for the unique acyl chain composition of bile PC, we analyzed gallbladder bile composition in mice that synthesized PC either via the PEMT pathway (induced by feeding a choline-deficient diet) or the CDP-choline pathway (based on genetic PEMT-deficiency). The PC concentration in gallbladder bile of mice that synthesize PC mainly via the CDP-choline pathway was comparable with control mice that synthesize PC via both pathways, whereas it was reduced by approximately 40% in mice that synthesize PC via the PEMT pathway. The acyl chain composition of bile PC was similar irrespective of the active PC biosynthetic pathway in the liver. These data demonstrate that the CDP-choline pathway alone, but not the PEMT pathway alone, can account for physiological concentrations of PC in gallbladder bile. Moreover, the specificity of biliary PC fatty acyl composition is determined independently from the synthetic origin of PC.

Animals↗
PubMed1999

Expression of phosphatidylethanolamine N-methyltransferase in Yoshida ascites hepatoma cells and the livers of host rats.

Previous studies have implicated phosphatidylethanolamine N-methyltransferase-2 (PEMT2) in the regulation of non-neoplastic liver growth [Tessitore,L., Cui,Z. and Vance,E. (1997) Biochem. J., 322, 151-154]. We have now investigated whether or not PEMT2 is also involved in the control of proliferation of hepatoma cells growing in an animal and cell death by apoptosis in the liver of tumor-bearing rats. PEMT activity was barely detectable and PEMT2 protein was absent in hepatoma cells growing exponentially in vivo whereas CTP:phosphocholine cytidylyltransferase (CT) activity and expression were high. The lack of PEMT2 corresponded with the absence of its mRNA. Both PEMT2 protein and mRNA appeared when cells entered the stationary phase of tumor growth and, in parallel, CT expression decreased. The host liver first became hyperplastic and exhibited a slight increase in CT activity and decrease in PEMT2 expression. During the stationary phase of hepatoma growth the host liver regressed and eventually became hypoplastic following induction of apoptosis. The appearance of apoptosis in the host liver was associated with a marked reduction in both CT activity and expression as well as an enhancement of PEMT activity and PEMT2 expression. McArdle RH7777 hepatoma cells underwent apoptosis when transfected with cDNA for PEMT2. The evidence supports the proposal that PEMT2 may have a role in the regulation of 'in vivo' hepatoma and hepatocyte cell division as well as hepatocyte cell death by apoptosis.

Animals↗
PubMed1998

Biochemical and evolutionary significance of phospholipid methylation.

All nucleated mammalian cells synthesize phosphatidylcholine from choline via the CDP-choline pathway. Hepatocytes have a second pathway for the synthesis of phosphatidylcholine, a stepwise methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase and encoded by the Pempt gene. We report that when Pempt-deficient mice were fed a choline-deficient diet for 3 days, severe liver pathology occurred apparently due to a lack of phosphatidylcholine biosynthesis. The hepatic concentration of phosphatidylcholine decreased by 50% compared with wild type mice on the diet. The levels of plasma triacylglycerols and cholesterol were decreased by greater than 90% in the Pempt-deficient mice. We suggest that the Pempt gene has been maintained during evolution to provide phosphatidylcholine when dietary choline is insufficient, as might occur during starvation or pregnancy.

Animals↗
PubMed1998

Roles for the methylation of phosphatidylethanolamine.

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.

Animals↗
PubMed1997

Role of lipoproteins in the delivery of lipids to axons during axonal regeneration.

Nerve fiber elongation involves the input of lipids to the growing axons. Since cell bodies are often a great distance from the regenerating tips, alternative sources of lipids have been proposed. We previously demonstrated that axonal synthesis of phosphatidylcholine is required for axonal growth (Posse de Chaves, E., Vance, D. E., Campenot, R. B. and Vance, J. E. (1995) J. Cell Biol. 128, 913-918; Posse de Chaves, E., Vance, D. E., Campenot, R. B. and Vance, J. E. (1995) Biochem. J. 312, 411-417). In contrast, cholesterol is not made in axons. We now show that when compartmented cultures of rat sympathetic neurons are incubated with pravastatin, in the absence of exogenously supplied lipids, cholesterol synthesis is inhibited and axonal growth is impaired. The addition of cholesterol to the axons or cell bodies of neurons treated with this inhibitor restores normal axonal elongation. Similarly, a supply of cholesterol via lipoproteins restores normal axonal growth. In contrast, lipoproteins do not provide axons with sufficient phosphatidylcholine for normal elongation when axonal phosphatidylcholine synthesis is inhibited. Thus, our studies support the idea that during axonal regeneration lipoproteins can be taken up by axons from the microenvironment and supply sufficient cholesterol, but not phosphatidylcholine, for growth. We also show that neither apoE nor apoA-I within the lipoproteins is essential for axonal growth.

Animals↗
PubMed1997

Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase.

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.

Animals↗
PubMed1997

Phosphatidylethanolamine N-methyltransferase from liver.

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.

Animals↗
PubMed1997

Induction of hepatocyte proliferation after partial hepatectomy is accompanied by a markedly reduced expression of phosphatidylethanolamine N-methyltransferase-2.

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.

Animals↗
PubMed1997

Inverse correlation between expression of phosphatidylethanolamine N-methyltransferase-2 and growth rate of perinatal rat livers.

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.

Animals↗
PubMed1997

Transient inactivation of phosphatidylethanolamine N-methyltransferase-2 and activation of cytidine triphosphate: phosphocholine cytidylyltransferase during non-neoplastic liver growth.

Phosphatidylethanolamine N-methyltransferase-2 (PEMT2) may contribute to the control of hepatocyte cell division, since its inactivation is associated with several types of liver proliferation including tumorigenesis [Cui, Houweling and Vance (1994) J. Biol. Chem. 269, 24531-24533]. To determine if the inactivation of PEMT2 was involved in non-neoplastic proliferation of hepatocytes, we studied the expression of this enzyme in a model of lead nitrate-induced liver proliferation in vivo in rats. A maximal decrease in PEMT activity (60%) and loss of PEMT2 protein (95%) coincided with maximal DNA synthesis and maximal cytidine triphosphate:phosphocholine cytidylyltransferase activity 36 h and 48 h after lead nitrate stimulation in male and female livers respectively. The decrease in expression of PEMT2 corresponded to a decrease in its mRNA. Compared with males, female rats exhibited a 12 h delay in the peak of DNA synthesis, in cytidylyltransferase activity and in the minimum of PEMT2 expression. Supplementation of the rats with dietary choline shifted the female pattern of PEMT2 inactivation, DNA synthesis and activation of cytidylyltransferase to 12 h earlier so that it was similar to the time frame of the expression of these activities in males. These results are consistent with the proposal that the inactivation of PEMT2 may have a role in the regulation of non-neoplastic growth of liver.

Animals↗
PubMed1997

Elevation of ceramide within distal neurites inhibits neurite growth in cultured rat sympathetic neurons.

Sphingolipids are abundant constituents of neuronal membranes and have been implicated in intracellular signaling. We show that two analogs of glycosphingolipid biosynthetic intermediates, fumonisin B1 (which inhibits dihydroceramide synthesis) and DL-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) (which inhibits glucosylceramide synthesis) decrease glycosphingolipid synthesis in rat sympathetic neurons. Although both fumonisin and PPMP inhibit glycosphingolipid synthesis, these inhibitors have differential effects on ceramide metabolism in axons. threo-PPMP, but not erythro-PPMP or fumonisin, induces an accumulation of [3H]palmitate-labeled ceramide and impairs axonal growth. Moreover, exogenously added, cell-permeable C6-ceramide, but not C6-dihydroceramide, mimicks the effect of PPMP. Our studies suggest that the lipid second messenger ceramide acts in distal axons, but not cell bodies, as a negative regulator of neurite growth.

Animals↗
PubMed1997

Phosphatidylcholine biosynthesis is required for secretion of truncated apolipoprotein Bs from McArdle RH7777 cells only when a neutral lipid core is formed.

Decreased phosphatidylcholine biosynthesis inhibits the secretion of very low density lipoproteins from hepatocytes (Yao, Z. and D. E. Vance. 1988. J. Biol. Chem. 263: 2998-3004). We have now investigated whether or not phosphatidylcholine biosynthesis is required for secretion of carboxyl-truncated apoBs 15, 18, 23, 28, and 37 from transfected McArdle RH7777 cells. Transfected cells were choline-deprived for 1 day and then choline-supplemented or maintained choline-deficient. Pulse-chase experiments showed that in the presence of 100 microM choline, the secretion of apoBs 28 and 37 was increased by up to 50 and 45%, respectively, whereas the secretion of apoBs 15, 18, and 23 was not affected by the addition of choline. Immunoblot analyses also showed that choline in the medium increased the secretion of apoBs 28 and 37 by 30-50% whereas the secretion of apoBs 15, 18, and 23 was unaffected over 24 h. As only carboxyl-terminal truncations with a size greater than 23% of apoB-100 are able to assemble a neutral lipid core (McLeod, R. S., Y. Zhao, S. L. Selby, J. Westerlund, and Z. Yao. 1994. J. Biol. Chem. 269: 2852-2862), we conclude that only apoB-truncations that assemble a neutral lipid core require phosphatidylcholine synthesis. Other experiments established that the requirement of phosphatidylcholine for apoB secretion is related to the presence of neutral lipid associated with a truncation, rather than the length of apoB.

Animals↗
PubMed1996

Overexpression of phosphatidylethanolamine N-methyltransferase 2 in CHO-K1 cells does not attenuate the activity of the CDP-choline pathway for phosphatidylcholine biosynthesis.

Chinese hamster ovary (CHO) cells express only a trace amount of phosphatidylethanolamine N-methyltransferase (PEMT) activity. CHO cells make their phosphatidylcholine (PC) via the CDP-choline pathway. We investigated whether or not overexpression of PEMT2, an isoform of PEMT, in these cells would down-regulate the activity of the CDP-choline pathway. Transfection of CHO cells with PEMT2 cDNA behind the cytomegalovirus promoter resulted in a series of cell lines that overexpressed PEMT2. Phospholipid metabolism was characterized in cell lines that expressed a medium (281 pmol/min per mg of protein) and a high (1300 pmol/min per mg of protein) level of PEMT activity. The expression of the regulated enzyme (CTP:phosphocholine cytidylyltransferase) in the CDP-choline pathway was increased, not decreased, in these cell lines as judged by immunoblot analysis and enzymic activity. Conversion of phosphatidylethanolamine to PC was enhanced in CHO cells that expressed PEMT2 activity. PC mass was not increased in the transfected compared with the control cells. The rate of PC catabolism made by either the CDP-choline or methylation pathways was unaffected by PEMT2 expression. We conclude that expression of PEMT2 in CHO cells does not down-regulate, but rather enhances, the expression of CTP:phosphocholine cytidylyltransferase.

Animals↗
PubMed1996

A genetic defect in phosphatidylcholine biosynthesis triggers apoptosis in Chinese hamster ovary cells.

We have investigated the cell death of a Chinese hamster ovary mutant (MT-58) with a thermo-sensitive CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of the CDP-choline pathway for phosphatidylcholine biosynthesis (Esko, J. D., Wermuth, M. M., and Raetz, C. R. H. (1981) J. Biol. Chem. 256, 7388-7393). After MT-58 cells were shifted to the restrictive temperature of 40 degrees C, the cytidylyltransferase was inactivated immediately leading to a decrease in phosphatidylcholine biosynthesis and cell death. DNA content and number of cells in the S phase decreased significantly in the dying MT-58 cells according to flow cytometrical analyses. The fragmentation of genomic DNA was detected by DNA ladders in agarose gel and release of the prelabeled genomic DNA into cytosolic fractions 14 h after the temperature shift. The dying cells underwent a dramatic reduction of cellular volume while maintaining the membrane containment of cellular contents. These events indicated that the inactivation of cytidylyltransferase triggered apoptosis in Chinese hamster ovary cells. This is the first report that apoptosis was induced in cultured cells, not by an added agent, but by a mutation in phospholipid biosynthesis.

Animals↗