Transgenic Mice Expressing Lipoprotein Lipase in Adipose Tissue: ABSENCE OF THE PROXIMAL 3′-UNTRANSLATED REGION CAUSES TRANSLATIONAL UPREGULATION

Lori L Hensley,Gouri Ranganathan,Elke M Wagner,Brian D Wells,Joseph C Daniel,Diane Vu,Clay F Semenkovich,Rudolf Zechner,Philip A Kern

doi:10.1074/jbc.m304200200

Abstract

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein and adipocyte metabolism. Defects in LPL can lead to hypertriglyceridemia and the subsequent development of atherosclerosis. The mechanisms of regulation of this enzyme are complex and may occur at multiple levels of gene expression. Because the 3'-untranslated region (UTR) is involved in LPL translational regulation, transgenic mice were generated with adipose tissue expression of an LPL construct either with or without the proximal 3'-UTR and driven by the aP2 promoter. Both transgenic mouse colonies were viable and expressed the transgene, resulting in a 2-fold increase in LPL activity in white adipose tissue. Neither mouse colony exhibited any obvious phenotype in terms of body weight, plasma lipids, glucose, and non-esterified fatty acid levels. In the mice expressing hLPL with an intact 3'-UTR, hLPL mRNA expression approximately paralleled hLPL activity. However in the mice without the proximal 3'-UTR, hLPL mRNA was low in the setting of large amounts of hLPL protein and LPL activity. In previous studies, the 3'-UTR of LPL was critical for the inhibitory effects of constitutively expressed hormones, such as thyroid hormone and catecholamines. Therefore, these data suggest that the absence of the 3'-UTR results in a translationally unrepressed LPL, resulting in a moderate overexpression of adipose LPL activity.

Highlights

From the ‡The Central Arkansas Veterans HealthCare System, and the Department of Medicine, Division of Endocrinology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, the §Institute of Molecular Biology, Biochemistry, and Microbiology, Karl-Franzens-University, Graz, Austria, and the ¶Department of Medicine, Washington University School of Medicine, St
Based on Lipoprotein lipase (LPL)’s putative role as an adipocyte “gatekeeper,” one might expect that transgenic mice would become obese if LPL were overexpressed in adipose tissue, and lean if LPL were overexpressed in muscle
By using a human-specific antibody, we found that the increase in LPL activity (LPLa) above that of control mice was entirely due to the transgene expression of hLPL

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction and Generation of Transgenic Mice—Two different human LPL transgenes, with or without the proximal 3Ј-UTR of the human LPL promoter were constructed. To generate transgenic mice expressing a human LPL gene deficient in the proximal 3Ј-untranslated region in adipose tissue, we utilized elements of two existing hLPL sequence constructs. Southern Blotting—For Southern blotting of hLPLe mice, 10 –15 ␮g of tail tip DNA, isolated according to manufacturer’s recommendations (Puregene), was digested with EcoRI, fractionated by agarose gel electrophoresis, and blotted on nylon membranes. RNA Analysis: Northern Blotting of hLPL3Ј Mice—Total RNA was isolated from tissues of fed mice, 10 ␮g were separated on a formaldehyde-agarose gel, blotted onto HybondTM-XL membrane (Amersham Biosciences) and hybridized with a radioactively labeled 1.1-kb EcoRI cDNA fragment of exon 10 of the hLPL. The extracts were immunoprecipitated using the monoclonal anti-HA antibody (Covance) according to manufacturer’s recommendations, followed by immunoprecipitation with a polyclonal affinity purified anti-LPL antibody as previously described (36). Statistical significance was set at the p Ͻ 0.05 level, and all comparisons used the Student’s t test

RESULTS

67 Ϯ 5 65 Ϯ 4 65 Ϯ 11

DISCUSSION