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 ␤-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.

Mechanism of hypertriglyceridemia in CTP:phosphoethanolamine cytidylyltransferase-deficient mice

“…Complete deletion of Pcyt2 in mice is embryonic lethal ( 8 ), which confi rms the essentiality of this gene for animal growth and development. Interestingly, heterozygous mice ( Pcyt2 Ϫ /+ ) mice ( 16,17 ), as well as Pcyt2 liver-specifi c knockout mice ( 18 ), develop liver steatosis, which establishes a strong physiological connection between PE and TAG syntheses through the common intermediate DAG. To eliminate the excess DAG formed by Pcyt2 gene deletion, both knockout models synthesize additional FAs from glucose by lipogenesis, and animals inevitably accumulate TAG ( 16,18 -/ -mice also have unmodifi ed plasma lipids (even slightly reduced plasma TAG), suggesting impairments in the liver lipoprotein secretion ( 18 ).…”

“…To eliminate the excess DAG formed by Pcyt2 gene deletion, both knockout models synthesize additional FAs from glucose by lipogenesis, and animals inevitably accumulate TAG ( 16,18 -/ -mice also have unmodifi ed plasma lipids (even slightly reduced plasma TAG), suggesting impairments in the liver lipoprotein secretion ( 18 ). In the heterozygous Pcyt2 +/ Ϫ state, mice have reduced PE synthesis in all tissues, which manifests as a chronic development of metabolic syndrome: the appearance of hepatic steatosis, hypertriglyceridemia, and peripheral insulin resistance at adult stage ( 16 ).…”

“…The Pcyt2 +/ Ϫ mice of a mixed genetic background (C57Bl/6 × 129/Sv) were generated as described previously ( 16 ). Mice were housed under standard conditions with a 12 h light cycle (7.00 AM-7.00 PM), were fed a regular chow diet (Harlan Teklad S-2335), and were given free access to food and water.…”

“…Lipids were extracted from the small intestinal mucus at 30 min and 3 h, and from 1 h plasma, according to the method of Bligh and Dyer ( 21 ). [ 3 H] radiolabeled TAG, DAG, FFAs, total cholesterol, and total phospholipids were separated and characterized by LSC as we have previously described ( 16,17 ).…”

Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity

“…Mouse primary hepatocytes were obtained from C57bl/6 mice by collagenase digestion, as previously described [6,15]. For human primary hepatocyte experiments, cryopreserved primary human hepatocytes (Triangle Research Labs) were thawed, centrifuged and resuspended in complete William's E medium (11 mM glucose) containing 10 % FBS and plated in 24-well collagen coated plates at 0.35 × 10 6 cells/well and allowed to adhere for 4 h. Donors of cryopreserved human hepatocytes were male [age 46, body mass index (BMI) 23.5] and female (age 59, BMI 29), were non-drug users (including alcohol), non-smokers.…”