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

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.;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.;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.