The molecular and metabolic characterization of the CTP : phosphoethanolamine cytidylyltransferase knockout mouse

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Fullerton, Morgan D.
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University of Guelph

Phosphatidylethanolamine (PE) is a critical inner membrane phospholipid, important for various cellular functions. CTP:phosphoethanolamine cytidylyltransferase ('Pcyt2'), catalyzes the formation of CDP-ethanolamine, which is then combined onto diacylglycerol (DAG) to form PE via the 'de novo ' PE-Kennedy pathway. This thesis aims to elucidate the role of ' Pcyt2' and therefore the 'de novo' pathway in a ' Pcyt2' knockout model. Homozygous disruption of 'Pcyt2' results in embryonal lethality prior to 8 days of development. Pcyt2+/- mice were phenotypically indistinguishable from wildtype littermate controls during the first months of development. The 'Pcyt2' mRNA and protein content as well as the 'in vitro' enzyme activity in heterozygous liver, heart, brain and kidney were ~65% of wildtype levels and therefore up-regulated from the expected 50%, given the single allele. Despite the alteration in 'Pcyt2' expression, there were no changes in PE tissue content. Phospholipid homeostasis was preserved without compensatory increases in phosphatidylserine decarboxylation and in spite of a decreased rate of PE synthesis via that PE-Kennedy pathway, due to a corresponding decrease in the PE degradation. Although normal after birth, 'Pcyt2'+/- mice became significantly heavier than littermate controls at ~5-6 months. This phenotype was accompanied by an array of metabolic disturbances including hepatic and skeletal muscle triglyceride (TG) accumulation, increased adiposity, hypertriglyceridemia, decreased fatty acid oxidation and diminished insulin sensitivity. Metabolic radiolabeling experiments revealed that in ' Pcyt2'+/- primary hepatocytes, DAG synthesis and degradation as well as TG formation were increased. Oleate uptake was increased in heterozygous hepatocytes, while no increase in total content of the main fatty acid transporters were established and 'in vivo' labeling indicated that hepatic fatty acid synthesis was also increased. 'Pcyt2'+/- mice had increased mRNA expression of various lipogenic transcription factors as well as their downstream targets in both liver and skeletal muscle. These data suggest that a diminished PE synthesis caused a redirection of DAG toward TG, which was facilitated by alterations in fatty acid metabolism. To further investigate the role of 'Pcyt2' in the progression of the heterozygous metabolic phenotype, a wildtype or mutant 'Pcyt2 ' cDNA construct was transiently transfected into primary hepatocytes. Expression of 'Pcyt2' completely restored the diminished rates of PE synthesis and degradation via the PE-Kennedy pathway, where the mutant construct (~40% decreased activity) was unable to fully compensate. In addition, the rates of DAG and TG synthesis from [3H]glycerol were normalized and 'de novo' fatty acid synthesis was decreased to wildtype levels with 'Pcyt2' expression. These data demonstrate that the genetic disruption of 'Pcyt2' causes a chronic state of decreased PE production and turnover, altered DAG and TG synthesis and defects in fatty add metabolism in 'Pcyt2'+/- mice.

phosphatidylethanolamine, CTP: phosphoethanolamine cytidylyltransferase, CDP-ethanolamine, diacylglycerol, de novo PE-Kennedy pathway, Pcyt2 knockout model