Kathryn L. Kelley

Plasma Cholesteryl Esters Provided by Lecithin:Cholesterol Acyltransferase and Acyl-Coenzyme A:Cholesterol Acyltransferase 2 Have Opposite Atherosclerotic Potential

Evidence suggests that ACAT2 is a proatherogenic enzyme that contributes cholesteryl esters (CEs) to apoB-containing lipoproteins, whereas LCAT is an antiatherogenic enzyme that facilitates reverse cholesterol transport by esterifying free cholesterol on HDL particles. We hypothesized that deletion of LCAT and ACAT2 would lead to absence of plasma CEs and reduced atherosclerosis. To test this hypothesis, ACAT2−/− LCAT−/− LDLr−/−, ACAT2−/− LDLr−/−, and LCAT−/− LDLr−/− mice were fed a 0.15% cholesterol diet for 20 weeks. In comparison to LDLr−/− mice, the total plasma cholesterol (TPC) of ACAT2−/− LCAT−/− LDLr−/− mice was 67% lower because of the complete absence of plasma CEs, leading to 94% less CE accumulation in the aorta. In the LCAT−/− LDLr−/− mice, TPC and atherosclerosis were significantly higher because of increased accumulations of ACAT2-derived CE. In ACAT2−/− LDLr−/− mice, again compared with LDLr−/− mice, TPC was 19% lower, whereas atherosclerosis was 88% lower. Therefore, the absence of ACAT2 led to a significant reduction in TPC although benefits in reduction of atherosclerosis were much more pronounced. Overall, the data suggest that ACAT2-derived CE is the predominant atherogenic lipid in blood, and that an important goal for prevention of atherosclerosis is to limit ACAT2-derived CE accumulation in lipoproteins.

Effects of peroxisome proliferator-activated receptor alpha/delta agonists on HDL-cholesterol in vervet monkeys.

The objective of this study was to demonstrate the efficacy of a novel peroxisome proliferator-activated receptor (PPAR) agonist and known PPARα and PPARδ agonists to increase HDL-cholesterol (HDL-C) in the St. Kitts vervet, a nonhuman primate model of atherosclerosis. Four groups (n = 6) were studied and each group was assigned one of the following “treatments”: a) vehicle only (vehicle); b) the PPARδ selective agonist GW501516 (GW); c) the PPARα/δ agonist T913659 (T659); and d) the PPARα agonist TriCor® (fenofibrate). No statistically significant changes were seen in body weight, total plasma cholesterol, plasma triglycerides, VLDL-C, LDL-C, or apolipoprotein B (apoB) concentrations. Each of the PPARα and PPARδ agonists investigated in this study increased plasma HDL-C, apoA-I, and apoA-II concentrations and increased HDL particle size in St. Kitts vervets. The maximum percentage increase in HDL-C from baseline for each group was as follows: vehicle, 5%; GW, 43%; T659, 43%; and fenofibrate, 20%. Treatment with GW and T659 resulted in an increase in medium-sized HDL particles, whereas fenofibrate showed increases in large HDL particles. These data provide additional evidence that PPARα and PPARδ agonists (both mixed and selective) have beneficial effects on HDL-C in these experimental primates.

Inhibition of acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) prevents dietary cholesterol-associated steatosis by enhancing hepatic triglyceride mobilization.

Acyl-CoA:cholesterol O-acyl transferase 2 (ACAT2) promotes cholesterol absorption by the intestine and the secretion of cholesteryl ester-enriched very low density lipoproteins by the liver. Paradoxically, mice lacking ACAT2 also exhibit mild hypertriglyceridemia. The present study addresses the unexpected role of ACAT2 in regulation of hepatic triglyceride (TG) metabolism. Mouse models of either complete genetic deficiency or pharmacological inhibition of ACAT2 were fed low fat diets containing various amounts of cholesterol to induce hepatic steatosis. Mice genetically lacking ACAT2 in both the intestine and the liver were dramatically protected against hepatic neutral lipid (TG and cholesteryl ester) accumulation, with the greatest differences occurring in situations where dietary cholesterol was elevated. Further studies demonstrated that liver-specific depletion of ACAT2 with antisense oligonucleotides prevents dietary cholesterol-associated hepatic steatosis both in an inbred mouse model of non-alcoholic fatty liver disease (SJL/J) and in a humanized hyperlipidemic mouse model (LDLr−/−, apoB100/100). All mouse models of diminished ACAT2 function showed lowered hepatic triglyceride concentrations and higher plasma triglycerides secondary to increased hepatic secretion of TG into nascent very low density lipoproteins. This work demonstrates that inhibition of hepatic ACAT2 can prevent dietary cholesterol-driven hepatic steatosis in mice. These data provide the first evidence to suggest that ACAT2-specific inhibitors may hold unexpected therapeutic potential to treat both atherosclerosis and non-alcoholic fatty liver disease.

Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation

The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2+/+, ACAT2+/−, and ACAT2−/− mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [3H]sitosterol and [14C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2−/− mice was 16% compared with 46–47% in ACAT2+/+ and ACAT2+/− mice. Chylomicrons from ACAT2+/+ and ACAT2+/− mice carried ∼80% of total sterol mass as CE, whereas ACAT2−/− chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2−/− mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.

Tissue-specific knockouts of ACAT2 reveal that intestinal depletion is sufficient to prevent diet-induced cholesterol accumulation in the liver and blood

Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) generates cholesterol esters (CE) for packaging into newly synthesized lipoproteins and thus is a major determinant of blood cholesterol levels. ACAT2 is expressed exclusively in the small intestine and liver, but the relative contributions of ACAT2 expression in these tissues to systemic cholesterol metabolism is unknown. We investigated whether CE derived from the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. We generated liver-specific (ACAT2L−/L−) and intestine-specific (ACAT2SI−/SI−) ACAT2 knockout mice and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. ACAT2SI−/SI− mice, in contrast to ACAT2L−/L− mice, had blunted cholesterol absorption. However, specific deletion of ACAT2 in the intestine generated essentially a phenocopy of the conditional knockout of ACAT2 in the liver, with reduced levels of plasma very low-density lipoprotein and hepatic CE, yet hepatic-free cholesterol does not build up after high cholesterol intake. ACAT2L−/L− and ACAT2SI−/SI− mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice and that depletion of intestinal ACAT2 is sufficient for most of the beneficial effects on cholesterol metabolism. Inhibitors of ACAT2 targeting either tissue likely would be beneficial for atheroprotection.

LDL particle core enrichment in cholesteryl oleate increases proteoglycan binding and promotes atherosclerosis.

Several studies in humans and animals suggest that LDL particle core enrichment in cholesteryl oleate (CO) is associated with increased atherosclerosis. Diet enrichment with MUFAs enhances LDL CO content. Steroyl O-acyltransferase 2 (SOAT2) is the enzyme that catalyzes the synthesis of much of the CO found in LDL, and gene deletion of SOAT2 minimizes CO in LDL and protects against atherosclerosis. The purpose of this study was to test the hypothesis that the increased atherosclerosis associated with LDL core enrichment in CO results from an increased affinity of the LDL particle for arterial proteoglycans. ApoB-100-only Ldlr−/− mice with and without Soat2 gene deletions were fed diets enriched in either cis-MUFA or n-3 PUFA, and LDL particles were isolated. LDL:proteogylcan binding was measured using surface plasmon resonance. Particles with higher CO content consistently bound with higher affinity to human biglycan and the amount of binding was shown to be proportional to the extent of atherosclerosis of the LDL donor mice. The data strongly support the thesis that atherosclerosis was induced through enhanced proteoglycan binding of LDL resulting from LDL core CO enrichment.

Phytosterol Feeding Causes Toxicity in ABCG5/G8 Knockout Mice

Plant sterols, or phytosterols, are very similar in structure to cholesterol and are abundant in typical diets. The reason for poor absorption of plant sterols by the body is still unknown. Mutations in the ABC transporters G5 and G8 are known to cause an accumulation of plant sterols in blood and tissues (sitosterolemia). To determine the significance of phytosterol exclusion from the body, we fed wild-type and ABCG5/G8 knockout mice a diet enriched with plant sterols. The high-phytosterol diet was extremely toxic to the ABCG5/G8 knockout mice but had no adverse effects on wild-type mice. ABCG5/G8 knockout mice died prematurely and developed a phenotype that included high levels of plant sterols in many tissues, liver abnormalities, and severe cardiac lesions. This study is the first to report such toxic effects of phytosterol accumulation in ABCG5/G8 knockout mice. We believe these new data support the conclusion that plant sterols are excluded from the body because they are toxic when present at high levels.

ACAT2 and ABCG5/G8 are both required for efficient cholesterol absorption in mice: Evidence from thoracic lymph duct cannulation

The metabolic fate of newly absorbed cholesterol and phytosterol is orchestrated through adenosine triphosphate-binding cassette transporter G5 and G8 heterodimer (G5G8), and acyl CoA:cholesterol acyltransferase 2 (ACAT2). We hypothesized that intestinal G5G8 limits sterol absorption by reducing substrate availability for ACAT2 esterification and have attempted to define the roles of these two factors using gene deletion studies in mice. Male ACAT2−/−, G5G8−/−, ACAT2−/−G5G8−/− (DKO), and wild-type (WT) control mice were fed a diet with 20% of energy as palm oil and 0.2% (w/w) cholesterol. Sterol absorption efficiency was directly measured by monitoring the appearance of [3H]sitosterol and [14C]cholesterol tracers in lymph after thoracic lymph duct cannulation. The average percentage (± SEM) absorption of [14C]cholesterol after 8 h of lymph collection was 40.55 ± 0.76%, 19.41 ± 1.52%, 32.13 ± 1.60%, and 21.27 ± 1.35% for WT, ACAT2−/−, G5G8−/−, and DKO mice, respectively. [3H]sitosterol absorption was <2% in WT and ACAT2−/− mice, whereas it was up to 6.8% in G5G8−/− and DKO mice. G5G8−/− mice also produced chylomicrons with ∼70% less cholesterol ester mass than WT mice. In contrast to expectations, the data demonstrated that the absence of G5G8 led to decreased intestinal cholesterol esterification and reduced cholesterol transport efficiency. Intestinal G5G8 appeared to limit the absorption of phytosterols; ACAT2 more efficiently esterified cholesterol than phytosterols. The data indicate that handling of sterols by the intestine involves both G5G8 and ACAT2 but that an additional factor (possibly Niemann-Pick C1-like 1) may be key in determining absorption efficiency.

Dietary n-3 LCPUFA from fish oil but not α-linolenic acid-derived LCPUFA confers atheroprotection in mice

The atheroprotective potential of n-3 α-linolenic acid (ALA) has not yet been fully determined, even in murine models of atherosclerosis. We tested whether ALA-derived, n-3 long chain polyunsaturated fatty acids (LCPUFA) could offer atheroprotection in a dose-dependent manner. Apolipoprotein B (ApoB)100/100LDLr−/− mice were fed with diets containing two levels of ALA from flaxseed oil for 16 weeks. Fish oil- and cis-monounsaturated-fat-enriched diets were used as positive and negative controls, respectively. The mice fed cis-monounsaturated fat and ALA-enriched diets exhibited equivalent plasma total cholesterol (TPC) and LDL-cholesterol (LDL-c) levels; only mice fed the fish-oil diet had lower TPC and LDL-c concentrations. Plasma LDL-CE fatty acid composition analysis showed that ALA-enriched diets lowered the percentage of atherogenic cholesteryl oleate compared with cis-monounsaturated-fat diet (44% versus 55.6%) but not as efficiently as the fish-oil diet (32.4%). Although both ALA and fish-oil diets equally enriched hepatic phospholipids with eicosapentaenoic acid (EPA) and ALA-enriched diets lowered hepatic cholesteryl ester (CE) levels compared with cis-monounsaturated-fat diet, only fish oil strongly protected from atherosclerosis. These outcomes indicate that dietary n-3 LCPUFA from fish oil and n-3 LCPUFA (mostly EPA) synthesized endogenously from ALA were not equally atheroprotective in these mice.

Reduction of VLDL Secretion Decreases Cholesterol Excretion in Niemann-Pick C1-Like 1 Hepatic Transgenic Mice

An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE) is unknown. To test the hypothesis that hepatic very low-density lipoproteins (VLDL) support TICE, antisense oligonucleotides (ASO) were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP), which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg) mice, which predominantly excrete cholesterol via TICE, and wild type (WT) littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG) and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE.