Transcriptional regulation of adipogenesis.
Explore the source record for details and available documents.
Adipose Tissue↗
Biomedical subjects
C J Walkey
Publications and source records attributed to C J Walkey.
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↗
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↗
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↗
Why has phosphatidylethanolamine N-methyltransferase survived in evolution?
Explore the source record for details and available documents.
Animals↗
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↗
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↗
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↗
Characterization of the murine phosphatidylethanolamine N-methyltransferase-2 gene.
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.
Animals↗
Overexpression of rat liver CTP:phosphocholine cytidylyltransferase accelerates phosphatidylcholine synthesis and degradation.
Two rat liver cDNAs encoding CTP:phosphocholine cytidylyltransferase (CT-1 and CT-2) were expressed in COS cells. The specific activity of CT in the microsomes increased approximately 20- or 100-fold after transfection with CT-1 or CT-2, respectively, but there was only a 3-5 fold increase in the rate of [3H]choline or [3H]glycerol incorporation into phosphatidylcholine (PC). The phosphocholine pool decreased approximately 40% in keeping with a stimulation of the CT-catalyzed reaction. The CDP-choline pool increased 12-fold suggesting that the conversion of CDP-choline to PC, catalyzed by cholinesphosphotransferase, could not keep pace with the CT-catalyzed reaction. This could account for the discrepancy between the increases in the amount of active (membrane-bound) CT and the rate of PC synthesis. Incubation of CT-transfected cells with sodium oleate to increase the supply of cellular diacylglycerol resulted in a further 2-fold increase in the rate of PC synthesis. This suggests that the diacylglycerol supply may be a limiting factor in the degree of stimulation of PC synthesis in CT-transfected COS cells. Despite the increased rate of PC synthesis, the total cellular PC mass increased only 17%, due to a 3-fold acceleration of the PC degradation rate. To determine which degradative pathway for PC was accelerated in the CT-transfected cells, we measured the pool sizes of several catabolites. Neither diacylglycerol nor phosphatidic acid mass was altered. The pool of glycerophosphocholine (GPC) was increased approximately 4-fold, and there was elevated release of GPC from the CT-transfected cells. The turnover of choline in GPC and lyso-PC was very slow compared with that of choline, phosphocholine, or CDP-choline, suggesting that GPC and lyso-PC were derived from slowly degraded choline-labeled PC. The metabolism of GPC and lyso-PC was stimulated in the cells over-expressing CT. These data suggest that PC synthesis and degradation are coordinated and that PC catabolism involving PC-->lyso-PC-->GPC is accelerated in COS cells overexpressing CT.
Animals↗
Comparison of the lipid regulation of yeast and rat CTP: phosphocholine cytidylyltransferase expressed in COS cells.
The CTP: phosphocholine cytidylyltransferase (CT) gene from yeast and cDNA from rat liver were over-expressed 20-30-fold in COS cells. Most of the CT activities were found in the cytosolic fraction. The regulation of the yeast CT activity (Y-CT) by lipids was characterized for the first time in comparison with the regulation of the well-studied rat CT (R-CT). Sonicated vesicles composed of egg phosphatidylcholine (PC) or 1-stearoyl-2-oleoyl PC had no effect on Y-CT and only slightly stimulated R-CT activity. Both CTs were activated 10-50-fold by the anionic lipids cardiolipin, phosphatidyl-glycerol, phosphatidylinositol and oleic acid. The effects of varying the vesicle concentration and the mol% of anionic lipid in PC vesicles were tested. The concentration optima for the activation of Y-CT by oleic acid or anionic phospholipids were 5-10-fold lower than those for R-CT. For example, the stimulation of Y-CT activity by phosphatidylglycerol vesicles was optimal between 5 and 15 microM and declined at higher concentrations, but R-CT activation by these vesicles saturated at approximately 25 microM. The positively charged aminolipid sphingosine antagonized the stimulation by oleic acid of both Y-CT and R-CT. Y-CT activity was insensitive to PC vesicles containing the neutral lipids diacylglycerol, monoacylglycerol or oleyl alcohol. However, R-CT was stimulated 10-20-fold by vesicles containing these neutral lipids. Translocation of the CTs to microsomal membranes enriched with anionic or neutral lipids was compared. Oleic acid enrichment promoted translocation of Y-CT and R-CT, whereas diacylglycerol promoted only R-CT translocation. These data show that the activity of Y-CT is lipid-sensitive. Y-CT is affected only by charged lipids, whereas R-CT responds to charged and neutral lipid activators. The data are consistent with different modes of interaction of the two CTs with lipids.
Animals↗