SOAT1 promotes mevalonate pathway dependency in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. Altered metabolism is a cancer vulnerability, and several metabolic pathways have been shown to promote PDAC. However, the changes in cholesterol metabolism and their role during PDAC progression remain largely unknown. Here we used organoid and mouse models to determine the drivers of altered cholesterol metabolism in PDAC and the consequences of its disruption on tumor progression. We identified sterol O-acyltransferase 1 (SOAT1) as a key player in sustaining the mevalonate pathway by converting cholesterol to inert cholesterol esters, thereby preventing the negative feedback elicited by unesterified cholesterol. Genetic targeting of Soat1 impairs cell proliferation in vitro and tumor progression in vivo and reveals a mevalonate pathway dependency in p53 mutant PDAC cells that have undergone p53 loss of heterozygosity (LOH). In contrast, pancreatic organoids lacking p53 mutation and p53 LOH are insensitive to SOAT1 loss, indicating a potential therapeutic window for inhibiting SOAT1 in PDAC.

Novel lecithin: cholesterol acyltransferase-based therapeutic approaches.

PURPOSE OF REVIEW: To review recent lecithin:cholesterol acyltransferas (LCAT)-based therapeutic approaches for atherosclerosis, acute coronary syndrome, and LCAT deficiency disorders. RECENT FINDINGS: A wide variety of approaches to using LCAT as a novel therapeutic target have been proposed. Enzyme replacement therapy with recombinant human LCAT is the most clinically advanced therapy for atherosclerosis and familial LCAT deficiency (FLD), with Phase I and Phase 2A clinical trials recently completed. Liver-directed LCAT gene therapy and engineered cell therapies are also another promising approach. Peptide and small molecule activators have shown efficacy in early-stage preclinical studies. Finally, lifestyle modifications, such as fat-restricted diets, cessation of cigarette smoking, and a diet rich in antioxidants may potentially suppress lipoprotein abnormalities in FLD patients and help preserve LCAT activity and renal function but have not been adequately tested. SUMMARY: Preclinical and early-stage clinical trials demonstrate the promise of novel LCAT therapies as HDL-raising agents that may be used to treat not only FLD but potentially also atherosclerosis and other disorders with low or dysfunctional HDL.

Sterol O-acyltransferase 1 deficiency improves defective insulin signaling in the brains of mice fed a high-fat diet.

Insulin resistance induced by a high-fat diet (HFD) is related to metabolic diseases, and sterol O-acyltransferase 1 (SOAT1) is a key enzyme for the biosynthesis of cholesteryl ester. In the present study, wild-type (WT) mice and SOAT1-knockout (KO) mice with a C57BL6 background fed a HFD were used to explore the role of SOAT1 in the hypothalamus. The results show that the WT mice exhibited a significant increase in body weight as well as hepatic histologic changes; they also had a lower glucose and insulin tolerance than the WT mice fed a normal diet. However, the metabolic syndrome was attenuated in the SOAT1-KO HFD-fed mice. With regard to brain function, the SOAT1-KO HFD-fed mice showed improved cognitive function; they also manifested reduced levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6, which would otherwise be raised by a HFD. In addition, the HFD led to the overexpression of GFAP and phosphorylated NF-κB in the hypothalamus, changes that were reversed in the SOAT1-KO HFD-fed mice. Moreover, SOAT1-KO mice improved HFD-caused defective hypothalamic insulin resistance, as evidenced by the upregulation of p-insulin receptor (INSR), p-AKT and p-glycogen synthase kinase (GSK)-3ß, while the downregulation of p-AMP-activated protein kinase (AMPK)-α and p-acetyl-CoA carboxylase (ACC)-α. In addition, similar results were observed in high fructose (HFR)-stimulated astrocytes (ASTs) isolated from WT or KO mice. These results suggest that SOAT1 plays an important role in hypothalamic insulin sensitivity, linked to cognitive impairment, in HFD-fed mice.

Cholesterol and fatty acids regulate cysteine ubiquitylation of ACAT2 through competitive oxidation.

Ubiquitin linkage to cysteine is an unconventional modification targeting protein for degradation. However, the physiological regulation of cysteine ubiquitylation is still mysterious. Here we found that ACAT2, a cellular enzyme converting cholesterol and fatty acid to cholesteryl esters, was ubiquitylated on Cys277 for degradation when the lipid level was low. gp78-Insigs catalysed Lys48-linked polyubiquitylation on this Cys277. A high concentration of cholesterol and fatty acid, however, induced cellular reactive oxygen species (ROS) that oxidized Cys277, resulting in ACAT2 stabilization and subsequently elevated cholesteryl esters. Furthermore, ACAT2 knockout mice were more susceptible to high-fat diet-associated insulin resistance. By contrast, expression of a constitutively stable form of ACAT2 (C277A) resulted in higher insulin sensitivity. Together, these data indicate that lipid-induced stabilization of ACAT2 ameliorates lipotoxicity from excessive cholesterol and fatty acid. This unconventional cysteine ubiquitylation of ACAT2 constitutes an important mechanism for sensing lipid-overload-induced ROS and fine-tuning lipid homeostasis.

Evaluation of sterol transport from the endoplasmic reticulum to mitochondria using mitochondrially targeted bacterial sterol acyltransferase in Saccharomyces cerevisiae.

To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.

Baker's Yeast Deficient in Storage Lipid Synthesis Uses cis-Vaccenic Acid to Reduce Unsaturated Fatty Acid Toxicity.

The role of cis-vaccenic acid (18:1n-7) in the reduction of unsaturated fatty acids toxicity was investigated in baker's yeast Saccharomyces cerevisiae. The quadruple mutant (QM, dga1Δ lro1Δ are1Δ are2Δ) deficient in enzymes responsible for triacylglycerol and steryl ester synthesis has been previously shown to be highly sensitive to exogenous unsaturated fatty acids. We have found that cis-vaccenic acid accumulated during cultivation in the QM cells but not in the corresponding wild type strain. This accumulation was accompanied by a reduction in palmitoleic acid (16:1n-7) content in the QM cells that is consistent with the proposed formation of cis-vaccenic acid by elongation of palmitoleic acid. Fatty acid analysis of individual lipid classes from the QM strain revealed that cis-vaccenic acid was highly enriched in the free fatty acid pool. Furthermore, production of cis-vaccenic acid was arrested if the mechanism of fatty acids release to the medium was activated. We also showed that exogenous cis-vaccenic acid did not affect viability of the QM strain at concentrations toxic for palmitoleic or oleic acids. Moreover, addition of cis-vaccenic acid to the growth medium provided partial protection against the lipotoxic effects of exogenous oleic acid. Transformation of palmitoleic acid to cis-vaccenic acid is thus a rescue mechanism enabling S. cerevisiae cells to survive in the absence of triacylglycerol synthesis as the major mechanism for unsaturated fatty acid detoxification.

Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver.

Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal(-)(/)(-):Soat2(+)(/)(+) mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs 1.9mg in Lal(+/+):Soat2(+/+) littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal(-)(/)(-):Soat2(+)(/)(+) mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal(-)(/)(-):Soat2(-)(/)(-) littermates. The level of EC accumulation in the SI of the Lal(-)(/)(-):Soat2(-)(/)(-) mice was also much less than in their Lal(-)(/)(-):Soat2(+)(/)(+) littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal(-)(/)(-):Soat2(-)(/)(-) mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function.

Inhibiting ACAT1/SOAT1 in microglia stimulates autophagy-mediated lysosomal proteolysis and increases Aß1-42 clearance.

Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a resident endoplasmic reticulum enzyme that prevents the buildup of cholesterol in membranes by converting it to cholesterol esters. Blocking ACAT1 pharmacologically or by Acat1 gene knock-out (KO) decreases amyloidopathy in mouse models for Alzheimer's disease. However, the beneficial actions of ACAT1 blockage to treat Alzheimer's disease remained not well understood. Microglia play essential roles in the proteolytic clearance of amyloid ß (Aß) peptides. Here we show that Acat1 gene KO in mouse increases phagocytic uptake of oligomeric Aß1-42 and stimulates lysosomal Aß1-42 degradation in cultured microglia and in vivo. Additional results show that Acat1 gene KO or a specific ACAT1 inhibitor K604 stimulates autophagosome formation and transcription factor EB-mediated lysosomal proteolysis. Surprisingly, the effect of ACAT1 blockage does not alter mTOR signaling or endoplasmic reticulum stress response but can be modulated by agents that disrupt cholesterol biosynthesis. To our knowledge, our current study provides the first example that a small molecule (K604) can promote autophagy in an mTOR-independent manner to activate the coordinated lysosomal expression and regulation network. Autophagy is needed to degrade misfolded proteins/peptides. Our results implicate that blocking ACAT1 may provide a new way to benefit multiple neurodegenerative diseases.

Cholesterol esters (CE) derived from hepatic sterol O-acyltransferase 2 (SOAT2) are associated with more atherosclerosis than CE from intestinal SOAT2.

RATIONALE: Cholesterol esters (CE), especially cholesterol oleate, generated by hepatic and intestinal sterol O-acyltransferase 2 (SOAT2) play a critical role in cholesterol homeostasis. However, it is unknown whether the contribution of intestine-derived CE from SOAT2 would have similar effects in promoting atherosclerosis progression as for liver-derived CE. OBJECTIVE: To test whether, in low-density lipoprotein receptor null (LDLr(-/-)) mice, the conditional knockout of intestinal SOAT2 (SOAT2(SI-/SI-)) or hepatic SOAT2 (SOAT2(L-/L-)) would equally limit atherosclerosis development compared with the global deletion of SOAT2 (SOAT2(-/-)). METHODS AND RESULTS: SOAT2 conditional knockout mice were bred with LDLr(-/-) mice creating LDLr(-/-) mice with each of the specific SOAT2 gene deletions. All mice then were fed an atherogenic diet for 16 weeks. SOAT2(SI-/SI-)LDLr(-/-) and SOAT2(-/-)LDLr(-/-) mice had significantly lower levels of intestinal cholesterol absorption, more fecal sterol excretion, and lower biliary cholesterol levels. Analysis of plasma LDL showed that all mice with SOAT2 gene deletions had LDL CE with reduced percentages of cholesterol palmitate and cholesterol oleate. Each of the LDLr(-/-) mice with SOAT2 gene deletions had lower accumulations of total cholesterol and CE in the liver compared with control mice. Finally, aortic atherosclerosis development was significantly lower in all mice with global or tissue-restricted SOAT2 gene deletions. Nevertheless, SOAT2(-/-)LDLr(-/-) and SOAT2(L-/L-)LDLr(-/-) mice had less aortic CE accumulation and smaller aortic lesions than SOAT2(SI-/SI-)LDLr(-/-) mice. CONCLUSIONS: SOAT2-derived CE from both the intestine and liver significantly contribute to the development of atherosclerosis, although the CE from the hepatic enzyme appeared to promote more atherosclerosis development.

Acyl-CoA:cholesterol acyltransferase 1 mediates liver fibrosis by regulating free cholesterol accumulation in hepatic stellate cells.

BACKGROUND & AIMS: Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes the conversion of free cholesterol (FC) to cholesterol ester, which prevents excess accumulation of FC. We recently found that FC accumulation in hepatic stellate cells (HSCs) plays a role in progression of liver fibrosis, but the effect of ACAT1 on liver fibrosis has not been clarified. In this study, we aimed to define the role of ACAT1 in the pathogenesis of liver fibrosis. METHODS: ACAT1-deficient and wild-type mice, or Toll-like receptor 4 (TLR4)(-/-)ACAT1(+/+) and TLR4(-/-)ACAT1(-/-) mice were subjected to bile duct ligation (BDL) for 3 weeks or were given carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis. RESULTS: ACAT1 was the major isozyme in mice and human primary HSCs, and ACAT2 was the major isozyme in mouse primary hepatocytes and Kupffer cells. ACAT1 deficiency significantly exaggerated liver fibrosis in the mouse models of liver fibrosis, without affecting the degree of hepatocellular injury or liver inflammation, including hepatocyte apoptosis or Kupffer cell activation. ACAT1 deficiency significantly increased FC levels in HSCs, augmenting TLR4 protein and downregulating expression of transforming growth factor-ß (TGFß) pseudoreceptor Bambi (bone morphogenetic protein and activin membrane-bound inhibitor), leading to sensitization of HSCs to TGFß activation. Exacerbation of liver fibrosis by ACAT1 deficiency was dependent on FC accumulation-induced enhancement of TLR4 signaling. CONCLUSIONS: ACAT1 deficiency exaggerates liver fibrosis mainly through enhanced FC accumulation in HSCs. Regulation of ACAT1 activities in HSCs could be a target for treatment of liver fibrosis.

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 [(3)H]sitosterol and [(14)C]cholesterol tracers in lymph after thoracic lymph duct cannulation. The average percentage (± SEM) absorption of [(14)C]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. [(3)H]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.

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 (ACAT2(L-/L-)) and intestine-specific (ACAT2(SI-/SI-)) ACAT2 knockout mice and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. ACAT2(SI-/SI-) mice, in contrast to ACAT2(L-/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. ACAT2(L-/L-) and ACAT2(SI-/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.

Dietary fat-induced alterations in atherosclerosis are abolished by ACAT2-deficiency in ApoB100 only, LDLr-/- mice.

OBJECTIVES: The enzyme acyl-coenzymeA (CoA):cholesterol O-acyltransferase 2 (ACAT2) in the liver synthesizes cholesteryl esters (CE) from cholesterol and fatty acyl-CoA, which get incorporated into apoB-containing lipoproteins that are secreted into the bloodstream. Dietary fatty acid composition influences the amount and fatty acid composition of CE within apoB-containing lipoproteins. We hypothesized that when ACAT2 activity is removed by gene deletion, hepatic CE synthesis and secretion would be minimal and, as a result, dietary fat-related differences in atherosclerosis would be eliminated. METHODS AND RESULTS: Groups of female apoB100 only, LDLr-/- mice with and without ACAT2 were fed diets enriched in either omega-3 or omega-6 polyunsaturated fat, saturated fat, and cis or trans monounsaturated fat. After 20 weeks on diet, mice fed diets enriched in monounsaturated or saturated fat exhibited significantly higher amounts of plasma cholesterol, larger LDL particles enriched in monounsaturated CE, and more atherosclerosis than mice fed polyunsaturated fat. The dietary fat-induced shifts in plasma cholesterol, LDL size, LDL CE composition, and atherosclerosis were not observed in ACAT2-/- mice. Regardless of the diet fed, the ACAT2-/- mice were protected from atherosclerosis. CONCLUSIONS: The results indicate that in apoB100 only, LDLr-/- mice, ACAT2 plays an essential role in facilitating dietary fat type-specific atherosclerosis through its various effects on plasma lipoprotein concentration and composition.

Monounsaturated fatty acids and atherosclerosis: opposing views from epidemiology and experimental animal models.

A substantial body of epidemiologic data has shed light on the potential protective effects of the Mediterranean diet against atherosclerosis in humans. Many believe the reason the Mediterranean diet is atheroprotective is the elevated consumption of olive oil, an oil poor in saturated fatty acids (SFA) and highly enriched in monounsaturated fatty acids (MUFA). Based on human feeding studies, the American Heart Association and the US Food and Drug Administration have advocated for the consumption of MUFA as a more healthy replacement for SFA. However, using experimental animal models in which extent of atherosclerosis can be directly measured following dietary intervention, it has been demonstrated that MUFA-enriched diets are not atheroprotective when compared with SFA-enriched diets. Hence, the current body of experimental evidence refutes the idea that MUFAs per se are atheroprotective; therefore much additional work is needed to determine which aspects of the Mediterranean diet are indeed heart healthy.

ACAT1 deficiency increases cholesterol synthesis in mouse peritoneal macrophages.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) esterifies free cholesterol and stores cholesteryl esters in lipid droplets. Macrophage ACAT1 deficiency results in increased atherosclerotic lesion area in hyperlipidemic mice via disrupted cholesterol efflux, increased lipoprotein uptake, accumulation of intracellular vesicles, and accelerated apoptosis. The objective of this study was to determine whether lipid synthesis is affected by ACAT1. The synthesis, esterification, and efflux of new cholesterol were measured in peritoneal macrophages from ACAT1(-/-) mice. Cholesterol synthesis was increased by 134% (p=0.001) in ACAT1(-/-) macrophages compared to wildtype macrophages. Increased synthesis resulted in a proportional increase in the efflux of newly synthesized cholesterol. Although the esterification of new cholesterol was reduced by 93% (p<0.001) in ACAT1(-/-) macrophages, trace amounts of newly synthesized cholesteryl esters were detectable. Furthermore, the expression of SREBP1a mRNA was increased 6-fold in ACAT1(-/-) macrophages compared to wildtype macrophages, suggesting an up-regulation of cholesterol and fatty acid synthesis in ACAT1(-/-) macrophages. Increased cholesterol synthesis and up-regulation of SREBP in ACAT1(-/-) macrophages suggests that ACAT1 affects the regulation of lipid metabolism in macrophages. This change in cholesterol homeostasis may contribute to the atherogenic potential of ACAT1(-/-) macrophages.

Point mutations in apolipoprotein A-I mimic the phenotype observed in patients with classical lecithin:cholesterol acyltransferase deficiency.

We have analyzed the effect of charged to neutral amino acid substitutions around the kinks flanking helices 4 and 6 of apoA-I and of the deletion of helix 6 on the in vivo activity of LCAT and the biogenesis of HDL. The LCAT activation capacity of apoA-I in vitro was nearly abolished by the helix 6 point (helix 6P-apoA-I[R160V/H162A]) and deletion {helix 6Delta-apoA-I[Delta(144-165)]} mutants, but was reduced to 50% in the helix 4 point mutant (helix 4P-apoA-I[D102A/D103A]). Following adenovirus-mediated gene transfer in apoA-I deficient mice, the level of plasma HDL cholesterol was greatly reduced in helix 6P and helix 6Delta mutants. Electron microscopy and two-dimensional gel electrophoresis showed that the helix 6P mutant formed predominantly high levels of apoA-I containing discoidal particles and had an increased prebeta1-HDL/alpha-HDL ratio. The helix 6Delta mutant formed few spherical particles and had an increased prebeta1-HDL/alpha-HDL ratio. Mice infected with adenovirus expressing the helix 4P mutant or wild-type apoA-I had normal HDL cholesterol and formed spherical alpha-HDL particles. Coinfection of mice with adenoviruses expressing human LCAT and the helix 6P mutant dramatically increased plasma HDL and apoA-I levels and converted the discoidal into spherical HDL, indicating that the LCAT activity was rate-limiting for the biogenesis of HDL. The LCAT treatment caused only a small increase in HDL cholesterol and apoA-I levels and in alpha-HDL particle numbers in the helix 6Delta mutant. The findings indicate a critical contribution of residue 160 of apoA-I to the in vivo activity of LCAT and the subsequent maturation of HDL and explain the low HDL levels in heterozygous subjects carrying this mutation.

ACAT1 deficiency disrupts cholesterol efflux and alters cellular morphology in macrophages.

OBJECTIVE: Acyl-coenzyme A: cholesterol acyltransferase (ACAT) converts intracellular free cholesterol (FC) into cholesteryl esters (CE) for storage in lipid droplets. Recent studies in our laboratory have shown that the deletion of the macrophage ACAT1 gene results in apoptosis and increased atherosclerotic lesion area in the aortas of hyperlipidemic mice. The objective of the current study was to elucidate the mechanism of the increased atherosclerosis. METHODS AND RESULTS: CE storage and FC efflux were studied in ACAT1(-/-) peritoneal macrophages that were treated with acetylated low-density lipoprotein (acLDL). Our results show that efflux of cellular cholesterol was reduced by 25% in ACAT1-deficient cells compared with wild-type controls. This decrease occurred despite the upregulated expression of ABCA1, an important mediator of cholesterol efflux. In contrast, ACAT1 deficiency increased efflux of the cholesterol derived from acLDL by 32%. ACAT1-deficient macrophages also showed a 26% increase in the accumulation of FC derived from acLDL, which was associated with a 75% increase in the number of intracellular vesicles. CONCLUSIONS: Together, these data show that macrophage ACAT1 influences the efflux of both cellular and lipoprotein-derived cholesterol and propose a pathway for the pro-atherogenic transformation of ACAT1(-/-) macrophages.

Acyl coenzyme A:cholesterol acyltransferase inhibitors as hypolipidemic and antiatherosclerotic drugs.

Acyl coenzyme A:cholesterol acyltransferase (ACAT) is the enzyme that catalyzes the conversion of intracellular cholesterol into cholesteryl esters. Two ACAT isoforms, termed ACAT1 and ACAT2, have been described. ACAT1 is ubiquitously found, with high expression levels in macrophages, adrenals, sebaceous glands and foam cells from human atherosclerotic lesions. In contrast, ACAT2 expression is restricted to the intestine and the liver of mice and non-human primates. The reaction catalyzed by ACAT is essential for intestinal cholesterol absorption, synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins, and intracellular storage of cholesterol. Therefore, ACAT inhibitors would theoretically reduce plasma cholesterol levels by blocking cholesterol absorption from the diet and by reducing hepatic VLDL synthesis. Moreover, ACAT inhibition could limit the accumulation of cholesteryl esters in the cytoplasm of macrophages, thus reducing the formation of foam cells. In view of these attractive possibilities, a great deal of molecules with ACAT inhibitory properties have been synthesized in the last 20 years. However, only a few of them have reached clinical studies, mainly due to unexpected side effects. On the other hand, most of the compounds assayed in humans have not shown substantial hypolipidemic efficacy. The present article focuses on the current knowledge of the pharmacology of ACAT inhibitors, and, specifically, on the different pharmacological approaches used to evaluate these compounds as hypolipidemic and antiatherosclerotic agents.

ACAT2 deficiency limits cholesterol absorption in the cholesterol-fed mouse: impact on hepatic cholesterol homeostasis.

Acyl CoA:cholesterol acyltransferase (ACAT) 2 is the major cholesterol-esterifying enzyme in mouse enterocytes and hepatocytes. Male ACAT2(+/+) and ACAT2(-/ -) mice were fed chow containing added cholesterol (0%-0.500% w/w) for 24 days. Over this range, fractional cholesterol absorption in the ACAT2(+/+) mice fell from 41.4% +/- 6.6% to 21.0% +/- 5.2%, and in their ACAT2(-/-) counterparts it fell from 35.1% +/- 4.5% to 7.9% +/- 0.8%. The mass of dietary cholesterol absorbed (mg/d per 100 g body weight) increased from 1.2 +/- 0.2 to 14.7 +/- 4.4 in the ACAT2(+/+) mice and from 1.0 +/- 0.2 to 5.5 +/- 0.6 in those without ACAT2. In the ACAT2(+/+) mice, hepatic cholesterol concentrations increased as a function of intake despite compensatory changes in cholesterol and bile acid synthesis and in the expression of adenosine triphosphate-binding cassette transporter G5 (ABCG5) and ABC transporter G8 (ABCG8). In contrast, in ACAT2(-/-) mice in which the amount of cholesterol absorbed at the highest intake was only 37% of that in the ACAT2(+/+) mice, suppression of synthesis was a sufficient adaptive response; there was no change in bile acid synthesis, ABCG5/G8 expression, or hepatic cholesterol concentration. The expression of adenosine triphosphate-binding cassette transporter A1 (ABCA1) in the jejunum was markedly elevated in the ACAT2(-/-) mice, irrespective of dietary cholesterol level. In conclusion, although ACAT2 deficiency limits cholesterol absorption, the extent to which it impacts hepatic cholesterol homeostasis depends on cholesterol intake. Loss of ACAT2 activity may result in unesterified cholesterol being absorbed via an ABCA1-mediated basolateral efflux pathway.