Overexpression of hepatic lipase in transgenic rabbits leads to a marked reduction of plasma high density lipoproteins and intermediate density lipoproteins.

Abstract

To elucidate the precise metabolic roles of hepatic lipase (HL), a human HL cDNA in a liver-specific expression vector was used to generate transgenic lines in the rabbit, an animal that normally expresses low levels of this enzyme. HL was detected in the plasma of all rabbits only after the aministration ofheparin; HL activity in transgenic rabbits was found at levels up to 80-fold greater than that in nontransgenic littermates. This increase in enzyme activity was associated with as much as a 5-fold decrease in total plasma cholesterol levels. Expression of the transgene resulted in a dramatic reduction in the level of large high density lipoproteins (HDL1 (6).Studies in vitro indicate that HL possesses both triglyceride hydrolase and phospholipase activities, and it has a high affinity for HDL particles (1). Administration of specific antibodies against HL in cats, rats, and monkeys indicated a role for this enzyme in the conversion of very low density lipoprotein (VLDL) remnants to low density lipoproteins (LDL) (7-9), in the metabolism of apolipoprotein (apo) B48-containing lipoproteins (10-12), and in the conversion of HDL2 to HDL3 (13,14). These findings are consistent with the phenotype of HL deficiency in human subjects. This disorder, in which the premature development of atherosclerosis is a cardinal feature, is characterized by elevated total plasma triglycerides and cholesterol corresponding to an increase in the amount of triglyceride-rich . However, the precise role of HL in lipoprotein metabolism remains unclear.To elucidate the physiological roles of HL, we generated transgenic rabbits that express human HL only in the liver, a major site of metabolic activity. The rabbit was selected as a model for studying HL functions because it has naturally decreased activity of this enzyme; it has about 1/10th as much activity as that of the rat (18, 19). Our results demonstrate that HL plays a key rate-limiting role in both IDL andThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8724HDL metabolism; it has dramatic effects on the distribution and composition of these particles in plasma, with concomitant effects on plasma cholesterol levels. To our knowledge, this study is the first report of transgenic rabbits that overexpress an enzyme involved in lipid metabolism. MATERIALS AND METHODSCreation of the Human HL Constructs. Vectors designed for liver-specific expression contained sequences from the human apoE gene: 3 kb (plivhHL1) or 5 kb (plivhHL2) of 5'-flanking sequence, the first exon, first intron, the first six nucleotides (a nontranslated portion) of the second exon, a polylinker for cDNA insertion, the distal 92 nucleotides in the noncoding region of the fourth exon, the proximal 114 nucleotides of 3'-flanking sequence (20), and the hepatic control region (21, 22) of the apoE/C

The Ligand-binding Function of Hepatic Lipase Modulates the Development of Atherosclerosis in Transgenic Mice

“…A 1.6-kb fragment of human HL cDNA was excised from the pCMV vector and subcloned into the expression plasmid pLIV.11 (39) modified by the addition of NotI linkers. After digestion with SalI and SpeI, a fragment containing the human apoE promoter, the human HL cDNA, the poly(A) signal from the human apoE gene, and the hepatic and macrophage control regions of the apoE/apoC-I locus was isolated and injected into the male pronucleus of fertilized eggs from superovulating C57BL/6N females (Charles River Laboratories, Wilmington, MA).…”

“…HL overexpression beneficially alters the plasma lipid profile of mice and rabbits by reducing the amount of cholesterol present in apoB-Lp (26,27,39). In addition, overexpression of human HL reduces the aortic cholesterol content in cholesterol-fed mice (40).…”

Overexpression of Lipoprotein Lipase in Transgenic Rabbits Inhibits Diet-induced Hypercholesterolemia and Atherosclerosis

“…All animal experiments were performed with the approval of the Animal Research Committee of the University of Tsukuba. The presence of the transgene in founder transgenic rabbits was examined by Southern blotting using a human LPL cDNA probe (19).…”

Transgenic rabbits with increased VEGF expression develop hemangiomas in the liver: a new model for Kasabach–Merritt syndrome

“…Hybridization was performed with a 32 P-labeled hVEGF cDNA probe synthesized by using a Prime-It II random primer labeling kit. 22 Northern Blotting and Reverse Transcriptase-Polymerase Chain Reaction Analysis Total RNA from various tissues was rapidly isolated using Trizol reagent (Invitrogen, CA, USA). RNA (10 mg) was then subjected to electrophoresis in a 1.2% agarose gel and transferred to a Nytran nylon membrane.…”