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Abstract
The involvement of pancreatic cholesterol esterase (bile salt-stimulated lipase) in cholesterol absorption through the intestine has been controversial. We have addressed this issue by using homologous recombination in embryonic stem cells to produce mice lacking a functional cholesterol esterase gene. Cholesterol esterase knockout mice and their wild type counterparts were fed a bolus dose of [3H]cholesterol and a trace amount of [beta-14C]sitosterol by gavage. The ratio of the two radiolabels excreted in the feces over a 24-h period was found to be similar in the control and cholesterol esterase-null mice. Similar results were observed when the radiolabeled sterols were supplied in an emulsion with phospholipid and triolein or in lipid vesicles with phosphatidylcholine. Cholesterol absorption results were similar between the control and cholesterol esterase-null mice regardless of whether the animals were fed a low fat diet or a high fat/high cholesterol diet. The rate of [3H]cholesterol appearance in the serum of the gene-targeted mice paralleled that observed in control animals. In contrast to these results, when experiments were performed with [3H]cholesteryl oleate instead of [3H]cholesterol, a higher amount of the 3H radiolabel was found excreted in feces and dramatically less of the radiolabel was detected in the serum of the cholesterol esterase-null mice in comparison with that detected in control animals. Serum cholesterol levels were not significantly different between control and cholesterol esterase-null mice fed either control or an atherogenic diet. These results indicate that cholesterol esterase is responsible for mediating intestinal absorption of cholesteryl esters but does not play a primary role in free cholesterol absorption.
Highlights
Cholesterol esterase, called bile salt-stimulated lipase or carboxyl ester lipase,1 is a lipolytic enzyme capable of hydrolyzing triacylglycerol, phospholipid, lysophospholipid, and cholesteryl esters
The results of the current study show that disruption of the CEL gene has no significant effect on the ability of mice to absorb unesterified cholesterol from the gastrointestinal tract
Similar results were observed when cholesterol absorption was determined based on the amount of nonabsorbed cholesterol present in the feces or on the appearance of the radiolabeled cholesterol in the serum
Summary
Cloning of the Mouse Cholesterol Esterase Gene and Production of the Targeting Vector—A strain 129 mouse genomic library made in -DASH phage vector was obtained from Dr Thomas Doetschman at our institution and used to isolate the mouse CEL gene. Restriction mapping, Southern hybridization with various cholesterol esterase cDNA fragments, and partial nucleotide sequencing were performed to determine the intron and exon locations of the mouse CEL gene. A 4.7-kb SacI DNA fragment, encoding sequences from 540 bp upstream of exon 1 to intron 7 of the mouse cholesterol esterase gene, was subcloned into a -digested PTZ18U plasmid. After CsCl purification, the targeting vector was digested with SacI, and the 6.5-kb DNA fragment containing the disrupted CEL gene sequence was purified by agarose gel electrophoresis. Cholesterol Absorption Studies—The single-dose, dual-isotope feeding method, originally described by Zilversmit and Hughes [27] and validated for measuring cholesterol absorption in mouse by Dueland et al [28], was used to determine cholesterol absorption efficiency in control and CEL gene-targeted mice. Differences in cholesterol absorption between groups were evaluated for statistical significance by Mann-Whitney rank sum and Student’s t
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