Elsevier

Metabolism

Volume 61, Issue 4, April 2012, Pages 470-481
Metabolism

Basic Science
Small molecule activation of lecithin cholesterol acyltransferase modulates lipoprotein metabolism in mice and hamsters

https://doi.org/10.1016/j.metabol.2011.08.006Get rights and content

Abstract

The objective was to assess whether pharmacological activation of lecithin cholesterol acyltransferase (LCAT) could exert beneficial effects on lipoprotein metabolism. A putative small molecule activator (compound A) was used as a tool compound in in vitro and in vivo studies. Compound A increased LCAT activity in vitro in plasma from mouse, hamster, rhesus monkey, and human. To assess the acute pharmacodynamic effects of compound A, C57Bl/6 mice and hamsters received a single dose (20 mg/kg) of compound A. Both species displayed a significant increase in high-density lipoprotein cholesterol (HDLc) and a significant decrease in non-HDLc and triglycerides acutely after dosing; these changes tracked with ex vivo plasma LCAT activity. To examine compound A's chronic effect on lipoprotein metabolism, hamsters received a daily dosing of vehicle or of 20 or 60 mg/kg of compound A for 2 weeks. At study termination, compound treatment resulted in a significant increase in HDLc, HDL particle size, plasma apolipoprotein A-I level, and plasma cholesteryl ester (CE) to free cholesterol ratio, and a significant reduction in very low-density lipoprotein cholesterol. The increase in plasma CE mirrored the increase in HDL CE. Triglycerides trended toward a dose-dependent decrease in very low-density lipoprotein and HDL, with multiple triglyceride species reaching statistical significance. Gallbladder bile acids content displayed a significant and more than 2-fold increase with the 60 mg/kg treatment. We characterized pharmacological activation of LCAT by a small molecule extensively for the first time, and our findings support the potential of this approach in treating dyslipidemia and atherosclerosis; our analyses also provide mechanistic insight on LCAT's role in lipoprotein metabolism.

Introduction

Lecithin cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies the 3β-hydroxyl group of free cholesterol (FC) with the fatty acyl chain from the sn-2 position of phosphatidylcholine (PC) [1]. The majority of circulating LCAT resides in high-density lipoprotein (HDL) particles and is responsible for cholesterol esterification in HDL and HDL maturation from small particles to large, cholesteryl ester (CE)–enriched particles [1], [2], [3]. It has therefore been hypothesized that increased LCAT activity can increase HDL cholesterol (HDLc) levels, promote reverse cholesterol transport (RCT), and exert beneficial effects on dyslipidemia and atherosclerosis [1], [4], [5], [6].

Human LCAT deficiency syndromes are associated with lipid disorders and pathological sequelae, and the full and partial loss-of-function mutations result in familial LCAT deficiency (FLD) and fish eye disease, respectively [7]. Major clinical findings for severe LCAT deficiency include corneal opacification, anemia, proteinuria, and glomerulosclerosis, which is marked by excessive deposition of lipids in the kidneys and often progression into renal failure [7], [8]. The lipid disorders associated with FLD and fish eye disease are highly variable but always include HDL deficiency [7], [9]. Lecithin cholesterol acyltransferase mutant allele carriers may also have elevated low-density lipoprotein (LDL) [10]. Phenotypes regarding atherosclerotic lesion development have appeared variable. For example, there have been cases where examination of arteries from FLD patients at autopsy revealed early or advanced atherosclerosis [11], [12], [13], but also cases in which FLD patients did not manifest premature atherosclerosis [14]. Controlled cohort studies where LCAT natural mutations were found to be associated [10] or not associated [15] with increased lesion development via imaging have both been reported. Possible reasons for the conflicting findings include small cohort size, variable confounding risk factors, and different methodologies for lesion assessment used.

Numerous animal models for LCAT over- or under-expression have been generated, and results from mouse models have been inconsistent. For example, an LCAT knockout (KO) mouse generated on a background of either apolipoprotein (apo) E KO or LDL receptor KO displayed reduced HDLc but paradoxically reduced atherosclerosis, possibly due to concomitant reduction in non-HDLc [16]. Transgenic overexpression of LCAT in mice resulted in an increase in large, CE-enriched HDL but paradoxically accelerated atherosclerosis [17], which was ameliorated when the cholesteryl ester transfer protein (CETP) transgene was introduced [18]. In comparison, in higher species such as rabbit, transgenic overexpression of LCAT resulted in not only a significant increase in HDLc, but also a marked decrease in non-HDLc, triglyceride, and lesion development [19]. Overexpression of LCAT via somatic viral delivery also resulted in an antiatherogenic lipid profile characterized by increased HDLc and decreased apoB and triglycerides (TG) in monkeys [20]. In hamsters, LCAT overexpression resulted in increased HDLc, biliary cholesterol excretion, and hepatic Cyp7a1 messenger RNA (mRNA) [21]. Taken together, animal studies suggest that effects of LCAT on lipid profile and lesion development are highly dependent on animal models and the presence of additional key lipid metabolizing enzymes such as CETP. Studies conducted in hamsters, rabbits, and monkeys, which more closely resemble humans in their lipoprotein metabolism, suggest that increased LCAT functionality likely is beneficial for lipid metabolism and atherosclerosis.

Recombinant LCAT infusion as a potential therapeutic approach for treating dyslipidemia and atherosclerosis has gained attention in recent years. Examples include recombinant LCAT infusion studies in mice [22] and rabbits [23]. In an effort to identify therapeutically relevant strategies to stimulate LCAT activity, recent reports have described a class of small-molecule activators of LCAT [24], [25]. The therapeutic implications of a small-molecule activator are promising; however, information regarding the pharmacology and mechanism of action of these putative LCAT activators (including compound A, Fig. 1) are lacking. The purpose of the current study was to characterize the pharmacology and mechanism of action of compound A using both in vitro and in vivo models of LCAT biology.

Section snippets

Compound A

Compound A was prepared as described previously [25]. Briefly, to a cooled (0°C) solution of 5-(ethylthio)-1,3,4-thiadiazole-2-thiol (2.56 g, 14.3 mmol) in benzene (24 mL) and DMF (N,N-dimethylformamide) (24 mL) was added sodium hydride (0.631 g, 60% by weight, 15.8 mmol). After 15 minutes, 3-chloropyrazine-2-carbonitrile (2.00 g, 14.3 mmol) was added; and the resulting mixture was heated at 80°C. After 3 hours, the reaction mixture was allowed to cool to ambient temperature and then was poured

Compound A activated plasma LCAT from multiple species in vitro

Compound A was spiked into plasma from C57Bl/6 mouse, hamster, rhesus monkey, and human at different concentrations. The final concentration of the vehicle (dimethyl sulfoxide) in all plasma samples was 2%. Lecithin cholesterol acyltransferase activity in each sample was then analyzed by an internally developed LC/MS assay that uses stable isotope (six deuterium [d6])–labeled proteoliposome as substrate. Compound A stimulated LCAT activity for all major CE products (d6-CE 22:6, d6-CE 20:4,

Discussion

In models of genetic or somatic LCAT overexpression, including those in higher animal species (rabbit, hamster, and nonhuman primate), LCAT gain of function is associated with changes in lipoprotein metabolism that are consistent with an antiatherosclerotic benefit (eg, increased HDLc) [19], [20], [21]. Although studies describing beneficial effects of LCAT overexpression highlight LCAT as a possible target for therapeutic intervention, the notion that small molecule activation of LCAT might

Funding

None.

Conflict of Interest

All authors are (or were) employees of Merck Sharp & Dohme and potentially own stock and/or hold stock options in the company.

Acknowledgment

The authors thank Lei Zhu for assistance in mouse liver mRNA analysis.

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