Improved lipid profile through liver-specific knockdown of liver X receptor a in KKAy diabetic mice

Nuclear hormone receptors liver X receptor (LXRa and LXRb) ligands are attractive approaches for the treatment of dyslipidemia and atherosclerosis. To further elucidate the function of LXRa in liver lipid metabolism in a disease-relevant animal model, the KKAy mouse, we used adenoviral vectors to selectively knock down LXRa gene expression. Out of five different short hairpin RNAs (shRNAs) that were tested in vitro, one construct was selected for detailed analysis of LXRa knockdown in vivo. Reduction of LXRa transcript levels to 48 6 13% compared with control virus transduction resulted in a significant downregulation of the LXRa-regulated lipogenic genes sterol-regulatory element binding protein-1c (SREBP1c) and stearoyl CoA desaturase 1 in vivo. Interestingly, ABCA1 and phoshoenolpyruvate carboxykinase 1 expression was not affected, whereas lipoprotein lipase (LPL) expression was found to be increased. In addition, 8 days after virus transduction, both plasma and liver triglycerides (TGs) were reduced by about 50%. Changes in TG levels were not due to reduced food intake in virustreated animals, because pair-fed mice showed unchanged TG levels. Taken together, liver-specific knockdown of LXRa in vivo by shRNA reduced expression of lipogenic master genes, like SREBP1c, and improved the lipid profile of hypertriglyceridemic KKAy mice.—Rippmann, J. F., C. Schoelch, T. Nolte, H. Pavliska, A. van Marle, H. van Es, and J. Prestle. Improved lipid profile through liver-specific knockdown of liver X receptor a in KKA y diabetic mice. J. Lipid Res. 2009. 50: 22–31. Supplementary key words nuclear hormone receptor • hypertriglyceridemia • short hairpin RNA • adenoviral gene transfer • triglycerides • gene expression Disorders in lipid metabolism still represent a major unmet medical need. Lipid disorders are main risk factors for cardiovascular diseases like atherosclerosis, which is the leading cause of death in industrialized countries. Statins, drugs that inhibit HMG-CoA reductase, are effective in lowering LDL cholesterol levels, but they have only moderate effects on plasma triglyceride (TG) and HDLcholesterol levels. The nuclear hormone receptors liver X receptors LXRa and LXRb are important cellular cholesterol sensors. Activated by oxygenated sterols, they form obligate heterodimers with retinoid X receptor and regulate expression of genes involved in cholesterol and lipid metabolism, as well as glucose homeostasis and inflammation (as reviewed in Refs. 1, 2). LXRa and LXRb have both complementary and autonomous functions (3, 4). This is also reflected by differential tissue expression of both isoforms. In the adult mouse, LXRa is the predominant isoform in liver, adipose tissue, and macrophages, whereas LXRb is expressed ubiquitously at lower levels (5). Small-molecule LXR agonists have gained attention owing to their putative anti-atherosclerotic effects: LXR agonists promote reverse cholesterol transport in vivo, that is, the elimination of cholesterol from foam cells in the vascular wall and transport to the liver via HDL particles for secretion as bile acids (6). The downside of unspecific pharmacological LXR activation is an induction of lipogenesis in the liver, which causes hypertriglyceridemia and liver steatosis (7, 8). At the molecular level, the positive therapeutic effect is exemplified by gene induction of members of the ABC superfamily of membrane transporters driving the reverse cholesterol transport, for example, ABCA1. Upregulation of the transcription factor sterol-regulatory element binding Manuscript received 6 December 2007 and in revised form 4 June 2008 and in re-revised form 21 August 2008. Published, JLR Papers in Press, September 3, 2008. DOI 10.1194/jlr.M700571-JLR200 Abbreviations: ALP, alkaline phosphatase; AST, aspartate aminotransferase; ChREBP, carbohydrate response element binding protein; CYP7a, cholesterol 7a-hydroxylase; FASN, fatty acid synthase; LDH, lactate dehydrogenase; LPL, lipoprotein lipase; LXR, liver X receptor; PCK1, phosphoenolpyruvate carboxykinase 1; SCD1, stearoyl CoA desaturase 1; shRNA, short hairpin RNA; SREBP1c, sterol-regulatory element binding protein-1c; TC, total cholesterol; TG, triglyceride; 22-SHC, 22-S-hydroxycholesterol. 1 Present address of H. van Es: Stem Cell Innovations, 2333 BZ Leiden, The Netherlands. 2 J. F. Rippmann and C. Schoelch contributed equally to this work. 3 To whom correspondence should be addressed. e-mail: [email protected] Copyright © 2009 by the American Society for Biochemistry and Molecular Biology, Inc. 22 Journal of Lipid Research Volume 50, 2009 This article is available online at http://www.jlr.org by gest, on N ovem er 7, 2017 w w w .j.org D ow nladed fom protein-1c (SREBP1c) and carbohydrate response element binding protein (ChREBP), on the other hand, drives the lipogenic gene program (7–10). Transgenic mice with a targeted deletion of LXRa and LXRb, as well as double knockouts, have been generated (11, 12). LXRb-deficient mice, unlike LXRa-deficient mice, do not display a hepatic phenotype, confirming the prominent role of LXRa in liver. Transgenic mice with a liverspecific overexpression of LXRa have been described as well (13). Moreover, an interesting combinatorial approach using different synthetic LXR modulators in LXR subtypespecific knockout animals has recently been published (14). Double knockouts of LXRa and apoE have recently been shown to exhibit massive cholesterol accumulation in peripheral tissues and accelerated atherosclerosis. This phenotype could be overcome by pharmacological activation of LXRb, demonstrating that LXRb could compensate for the loss of LXRa in that respect (15). Altogether, genetic and pharmacological evidence not only supports the concept of subtype or tissue-specific LXRb agonists for the treatment of atherosclerosis, but conceptually opens the possibility of developing LXRa-specific antagonists for the treatment of hypertriglyceridemia. Transgenic animals, however, hamper the limitation of possible developmental adaptation. In the present study, we describe an interventional genetic approach in a disease-relevant animal model. For the first time, Ad5mediated gene transfer was used to specifically knock down LXRa mRNA in vivo using short hairpin RNA (shRNA) (Ad5-shRNA-LXRa). KKAy mice were chosen as an established animal model characterized by hyperglycemia, hypercholesterolemia, and severe hypertriglyceridemia (16). We analyzed plasma lipid parameters and measured gene expression of established LXR target genes.