Choline Ethanolamine Phosphotransferase Research Articles

Abstract 1051: Endothelial CEPT1 Impacts Aortic Atherosclerosis And Hepatic Lipoprotein Metabolism

Background: Circulating lipids that contribute to the development of atherosclerosis are derived from very low-density lipoprotein (VLDL), produced in liver by a process requiring microsomal triglyceride transfer protein (MTTP). Since choline ethanolamine phosphotransferase 1 (CEPT1) generates phospholipids critical for membrane composition and signaling, we hypothesized that Cept1 deficiency reduces hepatic MTTP and aortic atherosclerosis. Methods: Conditional endothelial cell (EC)-specific knockdown of Cept1 ( Cept1 fl/fl Cre+ ) was performed in ApoE -/- mice maintained on a 42% high-fat diet for 12 weeks, serum lipids were measured, and aorta, aortic roots, and liver tissue were harvested (Figure, A). In addition, EC-hepatocyte (HepG2 cells) signaling was evaluated using an in vitro co-culture assay, and Mttp gene expression was assessed in HepG2 cells, cultured with and without ECs treated with Cept1 siRNA (Figure, B and C). Results: Compared to ApoE -/- mice, Cept1 fl/fl Cre+ ApoE -/- mice had significantly reduced serum cholesterol (P < .05; Figure, D) and triglyceride (P < .01; Figure, E), and reduced atherosclerotic lesions of the aorta and aortic root (P < .01 and P < .05, respectively; Figure, F-I). Cept1 fl/fl Cre+ ApoE -/- mice also demonstrated more severe hepatic steatosis and numerically greater content of liver lipid droplets (Figure, J and K). Cept1 knockdown in ECs co-cultured with HepG2 cells resulted in a significant decrease in Mttp expression (P < .01; Figure, L). Conclusion: Our findings suggest that deficiency of endothelial Cept1 significantly reduces aortic atheroprogression. This may, in part, be due to a reduction in serum lipid levels via inhibition of hepatocyte MTTP driven by signals from endothelial cells. These findings open a new avenue of exploration regarding the influence of EC signaling on hepatic lipid metabolism and its impact on hyperlipidemia and atherosclerotic cardiovascular diseases.

Integrative Metabolomics and Proteomics Detected Hepatotoxicity in Mice Associated with Alkaloids from Eupatorium fortunei Turcz.

The traditional Chinese herbal medicine Eupatorium fortunei Turcz. (E. fortunei) has been widely adopted to treat nausea, diabetes, siriasis, and poor appetite. However, E. fortunei contains multiple pyrrolizidine alkaloids (PAs). This study aimed to investigate the hepatotoxicity of total alkaloids in E. fortunei (EFTAs) and identify the toxic mechanisms of EFTAs on hepatocytes. Liquid chromatography with a tandem mass spectrometry assay with reference standards indicated that EFTAs mainly consisted of eight PAs whose content accounted for 92.38% of EFTAs. EFTAs markedly decreased mouse body and liver weights and increased the contents of AST and ALT. The histopathological assays demonstrated that, after exposition to EFTAs, the structures of hepatocytes were damaged and the fibrosis and apoptosis in hepatocytes were accelerated. Moreover, EFTAs increased the serum level of inflammatory cytokines and aggravated circulating oxidative stress. A combination of hepatic proteomics and metabolomics was used to investigate the toxic mechanisms of EFTAs. The study revealed that EFTAs seriously disrupted glycerophospholipid metabolism by upregulating the contents of lysophosphatidylglycerol acyltransferase 1 and phosphatidylinositol and downregulating the contents of choline/ethanolamine kinase beta, choline-ethanolamine phosphotransferase 1, phospholipase D4, 1-acylglycerophosphocholine, phosphatidylcholine, and dihydroxyacetone phosphate in the liver, resulting in detrimental inflammation, fibrosis, and apoptosis. This study revealed that EFTAs induced severe hepatotoxicity by disrupting glycerophospholipid metabolism.

De Novo Synthesis of Phosphatidylcholine Is Essential for the Promastigote But Not Amastigote Stage in Leishmania major.

Phosphatidylcholine (PC) is the most abundant type of phospholipids in eukaryotes constituting ~30% of total lipids in Leishmania. PC synthesis mainly occurs via the choline branch of the Kennedy pathway (choline ⇒ choline-phosphate ⇒ CDP-choline ⇒ PC) and the N-methylation of phosphatidylethanolamine (PE). In addition, Leishmania parasites can acquire PC and other lipids from the host or culture medium. In this study, we assessed the function and essentiality of choline ethanolamine phosphotransferase (CEPT) in Leishmania major which is responsible for the final step of the de novo synthesis of PC and PE. Our data indicate that CEPT is localized in the endoplasmic reticulum and possesses the activity to generate PC from CDP-choline and diacylglycerol. Targeted deletion of CEPT is only possible in the presence of an episomal CEPT gene in the promastigote stage of L. major. These chromosomal null parasites require the episomal expression of CEPT to survive in culture, confirming its essentiality during the promastigote stage. In contrast, during in vivo infection of BALB/c mice, these chromosomal null parasites appeared to lose the episomal copy of CEPT while maintaining normal levels of virulence, replication and cellular PC. Therefore, while the de novo synthesis of PC/PE is indispensable for the proliferation of promastigotes, intracellular amastigotes appear to acquire most of their lipids through salvage and remodeling.

CEPT1-Mediated Phospholipogenesis Regulates Endothelial Cell Function and Ischemia-Induced Angiogenesis Through PPARα.

De novo phospholipogenesis, mediated by choline-ethanolamine phosphotransferase 1 (CEPT1), is essential for phospholipid activation of transcription factors such as peroxisome proliferator-activated receptor α (PPARα) in the liver. Fenofibrate, a PPARα agonist and lipid-lowering agent, decreases amputation incidence in patients with diabetes. Because we previously observed that CEPT1 is elevated in carotid plaque of patients with diabetes, we evaluated the role of CEPT1 in peripheral arteries and PPARα phosphorylation (Ser12). CEPT1 was found to be elevated in diseased lower-extremity arterial intima of individuals with peripheral arterial disease and diabetes. To evaluate the role of Cept1 in the endothelium, we engineered a conditional endothelial cell (EC)-specific deletion of Cept1 via induced VE-cadherin-CreERT2-mediated recombination (Cept1Lp/LpCre +). Cept1Lp/LpCre + ECs demonstrated decreased proliferation, migration, and tubule formation, and Cept1Lp/LpCre + mice had reduced perfusion and angiogenesis in ischemic hind limbs. Peripheral ischemic recovery and PPARα signaling were further compromised by streptozotocin-induced diabetes and ameliorated by feeding fenofibrate. Cept1 endoribonuclease-prepared siRNA decreased PPARα phosphorylation in ECs, which was rescued with fenofibrate but not PC16:0/18:1. Unlike Cept1Lp/LpCre + mice, Cept1Lp/LpCre + Ppara -/- mice did not demonstrate hind-paw perfusion recovery after feeding fenofibrate. Therefore, we demonstrate that CEPT1 is essential for EC function and tissue recovery after ischemia and that fenofibrate rescues CEPT1-mediated activation of PPARα.

Membrane phospholipids remodeling upon imbibition in Brassica napus L. seeds

Successful seed germination depends on the rapid repair of cell membrane damaged by dry storage. However, little is known about the reorganization of lipids during this process. In this study, the changes of intracellular redox environment, cell membrane integrity, lipid composition, and expression of genes related to phospholipid metabolism were assessed during imbibition of Brassica napus seeds. A total number of 443 lipids belonging to 7 categories were detected by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). In the 24 h-imbibed seeds, the relative content of triacylglycerol was lower than in dry seeds, while the relative content of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), especially PC (36:2, number of carbons in the acyl chains: number of double bonds), PC (36:3), and PE (36:3) were higher than those in dry seeds. Meanwhile, the content and unsaturation levels of phospholipids increased, indicating membrane lipids remodeling during seed imbibition. The plasma membrane integrity, which was measured by the relative electrolyte leakage (REL) of the membrane and FM4-64 fluorescent dye, was improved upon imbibition, confirming that cell membrane was repaired after 24 h-imbibition. The reduction of H2O2 content, redox potential, and malondialdehyde (MDA) content indicated that the degree of membrane lipid peroxidation was significantly decreased upon imbibition. Gene expression analysis showed that the differential expression of genes for key enzymes occurred in the plateau phase of the imbibition curve, i.e. after 8 h-to 24 h-imbibition. Moreover, the differential expression of genes such as those encoding phospholipase C (PLC), phospholipase D (PLD), triacylglycerol lipase (TAG lipase), choline/ethanolamine phosphotransferase (CEPT), and phosphatidylserine synthase (PTDSS2) during imbibition indicated that membrane lipid remodeling was related to complex metabolic pathways, among which the degradation of triacylglycerol and the synthesis of phospholipids using diacylglycerol might play an important role during membrane remodeling.

Abstract 283: Fenofibrate Induces Endothelial Cell Tubule Formation Independent of Phospholipogenesis

Fenofibrate, a proliferator-activated receptor (PPAR)α agonist, is the only oral medication demonstrated to prevent lower extremity amputations in diabetic patients. Phosphatidylcholines, generated by choline-ethanolamine phosphotransferase 1 (CEPT1) via the Kennedy Pathway, also induce PPARα activation, but their metabolism is altered in the setting of diabetes. It is unknown whether CEPT1 is essential for fenofibrate-mediated endothelial cell (EC) function. To evaluate this, we generated a murine model for conditional knockdown of Cept1 in the endothelium ( Cept1EC KO). Heart ECs (MHECs) were harvested from 6wk old Cept1EC KO and wildtype (WT) littermates, and cultured in vitro on growth factor-reduced Matrigel. Cultures were then supplemented with VEGF (50ng/mL), bFGF (50ng/mL), and fenofibrate (25uM), then assessed longitudinally at 0, 4, and 6 hours. We observed that compared to WT, Cept1EC KO MHECs had significantly less tubule formation (p < 0.0001). VEGF and bFGF failed to rescue Cept1EC KO MHECs, but demonstrated a robust agonist response in WT MHECs (bFGF: p=0.003; VEGF: p=0.0002). Interestingly, fenofibrate demonstrated complete rescue of Cept1EC KO MHECs at 4 and 6 hours of culture. This finding demonstrates that fenofibrate restores EC function even in the setting of impaired phospholipid biosynthesis. This observation may partially explain how fenofibrate confers added benefits in subjects with diabetic and peripheral arterial disease. Future work will further elucidate this mechanism of action in diabetic subjects.

Diabetes adversely affects phospholipid profiles in human carotid artery endarterectomy plaques

Patients with diabetes are at higher risk of developing carotid artery stenosis and resultant stroke. Arachidonoyl phospholipids affect plaque inflammation and vulnerability, but whether diabetic patients have unique carotid artery phospholipidomic profiles is unknown. We performed a comprehensive paired analysis of phospholipids in extracranial carotid endarterectomy (CEA) plaques of matched diabetic and nondiabetic patients and analyzed mass spectrometry-derived profiles of three phospholipids, plasmenyl-phosphatidylethanolamine (pPE), phosphatidylserine (PS), and phosphatidylinositol (PI), in maximally (MAX) and minimally (MIN) diseased CEA segments. We also measured levels of arachidonic acid (AA), produced by pPE hydrolysis, and choline-ethanolamine phosphotransferase 1 (CEPT1), responsible for most pPE de novo biosynthesis. In paired analysis, MIN CEA segments had higher levels than MAX segments of pPE (P < 0.001), PS (P < 0.001), and PI (P < 0.03). MIN diabetic plaques contained higher levels than MAX diabetic plaques of arachidonoyl pPE38:4 and pPE38:5 and CEPT1 was upregulated in diabetic versus nondiabetic plaques. AA levels were relatively greater in MIN versus MAX segments of all CEA segments, and were higher in diabetic than nondiabetic plaques. Our findings suggest that arachidonoyl phospholipids are more likely to be abundant in the extracranial carotid artery plaque of diabetic rather than nondiabetic patients.

Human Lysophosphatidylcholine Acyltransferases 1 and 2 Are Located in Lipid Droplets Where They Catalyze the Formation of Phosphatidylcholine

Phosphatidylcholine (PC) is synthesized by two different pathways, the Lands cycle and the Kennedy pathway. The recently identified key enzymes of the Lands cycle, lysophosphatidylcholine acyltransferase 1 and 2 (LPCAT1 and -2), were reported to localize to the endoplasmic reticulum and to function in lung surfactant production and in inflammation response. Here, we show in various mammalian cell lines that both enzymes additionally localize to lipid droplets (LDs), which consist of a core of neutral lipids surrounded by a monolayer of phospholipid, mainly PC. This dual localization is enabled by the monotopic topology of these enzymes demonstrated in this study. Furthermore, we show that LDs have the ability to locally synthesize PC and that this activity correlates with the LPCAT1 and -2 expression level. This suggests that LPCAT1 and -2 have, in addition to their known function in specialized cells, a ubiquitous role in LD-associated lipid metabolism.

Identification and characterization of human ethanolaminephosphotransferase1

CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EPT) catalyzes the transfer of phosphoethanolamine from CDP-ethanolamine to diacylglycerol to produce phosphatidylethanolamine (PE). To date, the dual specificity of choline/ethanolaminephosphotransferase (CEPT) has been recognized as the total activity responsible for the synthesis of PE via the CDP-ethanolamine pathway in human. We report here the identification and characterization of another human cDNA that encodes CDP-ethanolamine-specific human EPT (hEPT1). Through homology search, we found that human selenoprotein I contained the CDP-alcohol phosphatidyltransferase signature, a common motif conserved in phospholipid synthases. Bacterial expression of the cDNA in Escherichia coli demonstrated that the product specifically used CDP-ethanolamine as the phosphobase donor to produce PE with the activation by both Mn(2+) and Mg(2+). RT-PCR and Northern blot analysis revealed that hEPT1 was ubiquitously expressed in multiple tissues, but in brain it was highly expressed in cerebellum. Here, we propose that in addition to previously identified CEPT, hEPT1 is involved in the biosynthesis of PE via the Kennedy pathway.