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The development of a metabolic disease phenotype in CTP:phosphoethanolamine cytidylyltransferase-deficient mice.

ABSTRACT: Phosphatidylethanolamine (PE) is an important inner membrane phospholipid mostly synthesized de novo via the PE-Kennedy pathway and by the decarboxylation of phosphatidylserine. CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the formation of CDP-ethanolamine, which is often the rate regulatory step in the PE-Kennedy pathway. In the current investigation, we show that the reduced CDP-ethanolamine formation in Pcyt2(+/-) mice limits the rate of PE synthesis and increases the availability of diacylglycerol. This results in the increased formation of triglycerides, which is facilitated by stimulated de novo fatty acid synthesis and increased uptake of pre-existing fatty acids. Pcyt2(+/-) mice progressively accumulate more diacylglycerol and triglycerides with age and have modified fatty acid composition, predominantly in PE and triglycerides. Pcyt2(+/-) additionally have an inherent blockage in fatty acid utilization as energy substrate and develop impaired tolerance to glucose and insulin at an older age. Accordingly, gene expression analyses demonstrated the up-regulation of the main lipogenic genes and down-regulation of mitochondrial fatty acid beta-oxidation genes. These data demonstrate for the first time that to preserve membrane PE phospholipids, Pcyt2 deficiency generates compensatory changes in triglyceride and energy substrate metabolism, resulting in a progressive development of liver steatosis, hypertriglyceridemia, obesity, and insulin resistance, the main features of the metabolic syndrome.

SUBMITTER: Fullerton MD

PROVIDER: S-EPMC2757972 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Male-Specific Cardiac Dysfunction in CTP:Phosphoethanolamine Cytidylyltransferase (Pcyt2)-Deficient Mice.

Project description:Phosphatidylethanolamine (PE) is the most abundant inner membrane phospholipid. PE synthesis from ethanolamine and diacylglycerol is regulated primarily by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2). Pcyt2(+/-) mice have reduced PE synthesis and, as a consequence, perturbed glucose and fatty acid metabolism, which gradually leads to the development of hyperlipidemia, obesity, and insulin resistance. Glucose and fatty acid uptake and the corresponding transporters Glut4 and Cd36 are similarly impaired in male and female Pcyt2(+/-) hearts. These mice also have similarly reduced phosphatidylinositol 3-kinase (PI3K)/Akt1 signaling and increased reactive oxygen species (ROS) production in the heart. However, only Pcyt2(+/-) males develop hypertension and cardiac hypertrophy. Pcyt2(+/-) males have upregulated heart AceI expression, heart phospholipids enriched in arachidonic acid and other n-6 polyunsaturated fatty acids, and dramatically increased ROS production in the aorta. In contrast, Pcyt2(+/-) females have unmodified heart phospholipids but have reduced heart triglyceride levels and altered expression of the structural genes Acta (low) and Myh7 (high). These changes together protect Pcyt2(+/-) females from cardiac dysfunction under conditions of reduced glucose and fatty acid uptake and heart insulin resistance. Our data identify Pcyt2 and membrane PE biogenesis as important determinants of gender-specific differences in cardiac lipids and heart function.

| S-EPMC4524120 | biostudies-literature

Regulation of Phosphatidylethanolamine Homeostasis—The Critical Role of CTP:Phosphoethanolamine Cytidylyltransferase (Pcyt2).

Project description:Phosphatidylethanolamine (PE) is the most abundant lipid on the protoplasmatic leaflet of cellular membranes. It has a pivotal role in cellular processes such as membrane fusion, cell cycle regulation, autophagy, and apoptosis. CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme in de novo biosynthesis of PE from ethanolamine and diacylglycerol by the CDP-ethanolamine Kennedy pathway. The following is a summary of the current state of knowledge on Pcyt2 and how splicing and isoform specific differences could lead to variations in functional properties in this family of enzymes. Results from the most recent studies on Pcyt2 transcriptional regulation, promoter function, autophagy, and cell growth regulation are highlighted. Recent data obtained from Pcyt2 knockout mouse models is also presented, demonstrating the essentiality of this gene in embryonic development as well as the major physiological consequences of deletion of one Pcyt2 allele. Those include development of symptoms of the metabolic syndrome such as elevated lipogenesis and lipoprotein secretion, hypertriglyceridemia, liver steatosis, obesity, and insulin resistance. The objective of this review is to elucidate the nature of Pcyt2 regulation by linking its catalytic function with the regulation of lipid and energy homeostasis.

| S-EPMC3588000 | biostudies-literature

Isoform-specific and protein kinase C-mediated regulation of CTP:phosphoethanolamine cytidylyltransferase phosphorylation.

Project description:CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, -? and -?; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2? and -? in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform-specific as well as shared phosphorylations. Pcyt2? was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2? was phosphorylated within the ?-specific motif that is spliced out in Pcyt2? and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2? phosphorylation, activity, and phosphatidylethanolamine synthesis by 50-90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2? was phosphorylated by PKC?, PKC?I, and PKC?II. Pcyt2? Ser-215 was also directly phosphorylated with PKC?. Mapping of the Pcyt2?- and -?-phosphorylated sites to the solved structure of a human Pcyt2? showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation.

| S-EPMC3979399 | biostudies-literature

Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP:phosphoethanolamine cytidylyltransferase.

Project description:CTP:phosphoethanolamine cytidylyltransferase (ECT) is considered to be the regulatory enzyme in the CDP-ethanolamine pathway of phosphatidylethanolamine (PE) biosynthesis. The ECT cDNA of Chlamydomonas reinhardtii encodes a protein of 443 amino acid residues, which is longer than the same protein in yeast, rat or human. The translated product of cloned cDNA was expressed as a fusion protein in Escherichia coli, and was shown to have ECT activity. The deduced amino acid sequence has 41% identity with that of human or rat, and 30% with yeast. The ECT protein has a repetitive internal sequence in its N- and C-terminal halves and a signature peptide sequence, RTXGVSTT, typical of the cytidylyltransferase family. The first 70 amino acid residues do not match the N-terminal part of the cytidylyltransferases from other organisms, and we hypothesize that it is a subcellular targeting signal to mitochondria. ECT and organelle marker enzyme assays showed that the total activity of ECT correlates well with that of fumarase, a marker enzyme for mitochondria. Northern blots showed an increase in mRNA abundance during reflagellation, indicating a possibility of transcriptional regulation. A notable change in the enzyme activity in C. reinhardtii cells was observed during the cell cycle, increasing during the dark and then decreasing during the light period, while the mRNA level did not alter, providing evidence for post-translational regulation.

| S-EPMC1133914 | biostudies-literature

Cloning and expression of CTP:phosphoethanolamine cytidylyltransferase cDNA from rat liver.

Project description:CTP:phosphoethanolamine cytidylyltransferase (ET) is a key regulatory enzyme in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. As a first step in the elucidation of the structure-function relationship and the regulation of ET, an ET cDNA was cloned from rat liver. The cloned cDNA encodes a protein of 404 amino acid residues with a calculated molecular mass of 45.2 kDa. The deduced amino acid sequence is very similar to that of human ET (89% identity). Furthermore, it shows less, but significant, similarity to yeast ET as well as to other cytidylyltransferases, including rat CTP:phosphocholine cytidylyltransferase and Bacillus subtilis glycerol-3-phosphate cytidylyltransferase. Like human and yeast ET, rat ET has a large repetitive internal sequence in the N- and C-terminal halves of the protein. Both parts of the repeat contain the HXGH motif, the most conserved region in the N-terminal active domain of other cytidylyltransferases, indicating the existence of two catalytic domains in ET. The hydropathy profile revealed that rat ET is largely hydrophilic and lacks a hydrophobic stretch long enough to span a bilayer membrane. There was no prediction for an amphipathic alpha-helix. Transfection of COS cells with the cDNA clone resulted in an 11-fold increase in ET activity, corresponding to an increase in the amount of ET protein as detected on a Western blot. Determination of the ET activity during liver development showed a 2. 5-fold increase between day 17 of gestation and birth (day 22) and the amount of ET protein changed accordingly. Northern blot analysis showed that this was accompanied by an increase in the amount of ET mRNA. Between day 17 of gestation and birth, the amount of mRNA in fetal rat liver increased approx. 6-fold, suggesting the regulation of ET at both pretranslational and post-translational levels during rat liver development.

| S-EPMC1220530 | biostudies-other

Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase.

Project description:Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40°C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line.

| S-EPMC4470507 | biostudies-literature

Early embryonic lethality in mice with targeted deletion of the CTP:phosphocholine cytidylyltransferase alpha gene (Pcyt1a).

Project description:CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-controlling step in the biosynthesis of phosphatidylcholine (PtdCho). Multiple CCT isoforms, CCTalpha, CCTbeta2, and CCTbeta3, are encoded by two genes, Pcyt1a and Pcyt1b. The importance of CCTalpha in mice was investigated by deleting exons 5 and 6 in the Pcyt1a gene using the Cre-lox system. Pcyt1a-/- zygotes failed to form blastocysts, did not develop past embryonic day 3.5 (E3.5), and failed to implant. In situ hybridization in E11.5 embryos showed that Pcyt1a is expressed ubiquitously, with the highest level in fetal liver, and CCTalpha transcripts are significantly more abundant than transcripts encoding CCTbeta or phosphatidylethanolamine (PtdEtn) N-methyl transferase, two other enzymes capable of producing PtdCho. Reduction of the CCTalpha transcripts in heterozygous E11.5 embryos was accompanied by upregulation of CCTbeta and PtdEtn N-methyltransferase transcripts. In contrast, enzymatic and real-time PCR data revealed that CCTbeta (Pcyt1b) expression is not upregulated to compensate for the reduction in CCTalpha expression in adult liver and other tissues from Pcyt1a+/- heterozygous mice. PtdCho biosynthesis measured by choline incorporation into isolated hepatocytes was not compromised in the Pcyt1a+/- mice. Liver PtdCho mass was the same in Pcyt1a+/+ and Pcyt1a+/- adult animals, but lung PtdCho mass decreased in the heterozygous mice. These data show that CCTalpha expression is required for early embryonic development, but that a 50% reduction in enzyme activity has little detectable impact on the operation of the CDP-choline metabolic pathway in adult tissues.

| S-EPMC1069620 | biostudies-literature

Transcriptional repression of the CTP:phosphocholine cytidylyltransferase gene by sphingosine.

Project description:We examined the effects of the bioactive lipid, sphingosine, on the expression of the rate-limiting enzyme involved in surfactant phosphatidylcholine synthesis, CCTalpha (CTP:phosphocholine cytidylyltransferase alpha). Sphingosine decreased phosphatidylcholine synthesis by inhibiting CCT activity in primary alveolar type II epithelia. Sphingosine decreased CCTalpha protein and mRNA levels by approx. 50% compared with control. The bioactive lipid did not alter CCTalpha mRNA stability, but significantly inhibited its transcriptional rate. In murine lung epithelia, sphingosine selectively reduced CCTalpha promoter-reporter activity when transfected with a 2 kb CCTalpha promoter/luciferase gene construct. Sphingosine also decreased transgene expression in murine type II epithelia isolated from CCTalpha promoter-reporter transgenic mice harbouring this 2 kb proximal 5'-flanking sequence. Deletional analysis revealed that sphingosine responsiveness was mapped to a negative regulatory element contained within 814 bp upstream of the coding region. The results indicate that bioactive sphingolipid metabolites suppress surfactant lipid synthesis by inhibiting gene transcription of a key surfactant biosynthetic enzyme.

| S-EPMC1133833 | biostudies-literature

Control of the CDPethanolamine pathway in mammalian cells: effect of CTP:phosphoethanolamine cytidylyltransferase overexpression and the amount of intracellular diacylglycerol.

Project description:For an insight regarding the control of PtdEtn (phosphatidylethanolamine) synthesis via the CDPethanolamine pathway, rat liver cDNA encoding ECT (CTP:phosphoethanolamine cytidylyltransferase) was transiently or stably transfected in Chinese-hamster ovary cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26- and 4-fold increase in specific activity of ECT respectively. However, no effect of ECT overexpression on the rate of [3H]ethanolamine incorporation into PtdEtn was detected in both cell lines. This was explored further in cells overexpressing four times ECT activity (McA-ECT1). The rate of PtdEtn breakdown and PtdEtn mass were not changed in McA-ECT1 cells in comparison with control-transfected cells. Instead, an accumulation of CDPethanolamine (label and mass) was observed, suggesting that in McA-ECT1 cells the ethanolaminephosphotransferase-catalysed reaction became rate-limiting. However, overexpression of the human choline/ethanolaminephosphotransferase in McA-ECT1 and control-transfected cells had no effect on PtdEtn synthesis. To investigate whether the availability of DAG (diacylglycerol) limited PtdEtn synthesis in these cells, intracellular DAG levels were increased using PMA or phospholipase C. Exposure of cells to PMA or phospholipase C stimulated PtdEtn synthesis and this effect was much more pronounced in McA-ECT1 than in control-transfected cells. In line with this, the DAG produced after PMA exposure was consumed more rapidly in McA-ECT1 cells and the CDPethanolamine level decreased accordingly. In conclusion, our results suggest that the supply of CDPethanolamine, via the expression level of ECT, is an important factor governing the rate of PtdEtn biosynthesis in mammalian cells, under the condition that the amount of DAG is not limiting.

| S-EPMC1224125 | biostudies-other

Heterologous expression of CTP:phosphocholine cytidylyltransferase from Plasmodium falciparum rescues Chinese Hamster Ovary cells deficient in the Kennedy phosphatidylcholine biosynthesis pathway.

Project description:The plasmodial CTP:phosphocholine cytidylyltransferase (PfCCT) is a promising antimalarial target, which can be inhibited to exploit the need for increased lipid biosynthesis during the erythrocytic life stage of Plasmodium falciparum. Notable structural and regulatory differences of plasmodial and mammalian CCTs offer the possibility to develop species-specific inhibitors. The aim of this study was to use CHO-MT58 cells expressing a temperature-sensitive mutant CCT for the functional characterization of PfCCT. We show that heterologous expression of wild type PfCCT restores the viability of CHO-MT58 cells at non-permissive (40 °C) temperatures, whereas catalytically perturbed or structurally destabilized PfCCT variants fail to provide rescue. Detailed in vitro characterization indicates that the H630N mutation diminishes the catalytic rate constant of PfCCT. The flow cytometry-based rescue assay provides a quantitative readout of the PfCCT function opening the possibility for the functional analysis of PfCCT and the high throughput screening of antimalarial compounds targeting plasmodial CCT.

| S-EPMC5997628 | biostudies-literature

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