Expression of human lecithin-cholesterol acyltransferase in transgenic mice. Effect of human apolipoprotein AI and human apolipoprotein all on plasma lipoprotein cholesterol metabolism.

Abstract

IntroductionHuman (Hu) lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol. To assess the effects of increased plasma levels of LCAT, four lines of transgenic mice were created expressing a Hu LCAT gene driven by either its natural or the mouse albumin enhancer promoter. Plasma LCAT activity increased from 1. The physiologic substrates for LCAT are nascent and mature HDL. Human (Hu) HDL is very heterogeneous both in particle size (5) and protein composition (6). HDL can be separated into two subpopulations on the basis of protein composition, one containing both apo AI and apo All (LpAI/AHI) and one containing apo AI but no apo All (LpAI). Both are heterogeneous in size (7). The size heterogeneity of Hu HDL distinguishes it from the monodisperse population of HDL particles present in the plasma of mice (8).LCAT is associated with transformation and remodeling of HDL. Plasma from LCAT-deficient patients (10, 11 ) contains small, discoidal HDL particles that probably represent nascent HDL (12). These particles undergo marked changes in composition and morphology when LCAT is added, suggesting an essential role of this enzyme in the biogenesis of circulating HDL.In Plasmid DNA was purified by alkaline hydrolysis (14) followed by two rounds of ultracentrifugation. Genomic DNA fragments were obtained by BamHI/NsiI or AatII/SspI digestion of clones pUCLCATBamNsi and pGEMAlbLCAT, respectively ( Fig. 1), separated from vector sequences by agarose gel electrophoresis, extracted from gels using the Geneclean kit (Bio 101, La Jolla, CA), and dialyzed in injection buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4).Creation of transgenic mice. Methods utilized in the creation of transgenic mice have been previously described (15). Fertilized embryos used for the microinjection were derived from matings of inbred FVB mice (Charles River Laboratories, Wilmington, MA). The Hu apo AI and Hu apo All transgenic mice used in these studies have previously been described (16,17). These transgenic lines were created and maintained in the C57BL/6 background. In all the studies involving combinations of Hu apo Al, Hu apo All, and LCAT transgenes, the genetic background of each of the transgenic and control mice was (FVB x C57BL/6) F1 hybrids.Preparation and analysis ofDNA and RNA by Southern and Northern blot. Tail tip DNA from 3-wk-old mice was screened for integration of Hu LCAT gene sequences by PCR. Primers were directed to synthesize the exon 6 of the Hu LCAT gene. Amplification reaction using nontransgenic mouse DNA as a template yielded no amplification products. In some experiments, integration of the full-length genomic construct was detected by Southern blot hybridization. 10 jig genomic DNA was digested overnight with 5 U Pstl/jig DNA, electrophoresed in a 1% agarose gel, and transferred to a nylon membrane (Sigma Chemical Co., S. Louis, MO). DNA was cross-linked to the membranes (Stratagene, La Jolla, CA) and hybridized with a 32P-radiolabeled full-length Hu LCAT cDNA as a pro...

Hypertriglyceridemia in Lecithin-cholesterol Acyltransferase-deficient Mice Is Associated with Hepatic Overproduction of Triglycerides, Increased Lipogenesis, and Improved Glucose Tolerance

“…In rodent models, there is an inverse relationship between fasting plasma TG levels and the murine lcat gene dose (6). Furthermore, transgenic mouse and rabbit models with overexpression of the human LCAT gene have been found to have a modest reduction in fasting TG (8,9). The mechanism by which LCAT modulates TG metabolism has not been fully addressed.…”

“…Gene-targeted mice deficient in LCAT activities are associated with HTG in a gene dose-dependent manner (6,7). Transgenic mice (8) and rabbits (9) overexpressing human LCAT gene have been reported to have a modest reduction in fasting plasma triglycerides (TG). These observations suggest that LCAT may play an important role in modulating plasma TG levels, but the underlying mechanisms have not been explored.…”

LCAT synthesized by primary astrocytes esterifies cholesterol on glia-derived lipoproteins

“…Previous studies identified the brain as one of three organs that synthesize LCAT, together with liver and testes (6,9,(32)(33)(34)(35). In situ hybridization experiments showed that LCAT mRNA is present in neuronal and nonneuronal cell types in rhesus monkeys (9).…”

Endothelial lipase

“…It is generally postulated that variation in plasma HDL-C levels are determined not by the rate at which they are produced, but rather by the rate of catabolism of their components. There are several known molecules that play a role in regulating plasma HDL-C levels (31), including HL (32)(33)(34)(35), LPL (36)(37)(38)(39)(40), lecithin:cholesterol acyl transferase (LCAT) (41,42), cholesterol ester transfer protein (CETP) (43), and phospholipid transfer protein (PLTP) (44). Intense study over the last few years has focused on the HDL particle in the context of reverse cholesterol transport and anti-oxidant properties, providing significant insights into the mechanism of HDL's antiatherogenic properties.…”