Jun Kusunoki

Acyl-CoA:Cholesterol Acyltransferase Inhibition Reduces Atherosclerosis in Apolipoprotein E–Deficient Mice

Background—Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters. The form of ACAT in macrophages, ACAT1, contributes to foam cell formation in the arterial wall and the development of atherosclerosis. Recent studies in a mouse model of atherosclerosis (the apolipoprotein E [apoE]-deficient mouse), however, have suggested that complete deficiency of ACAT1 activity is not antiatherogenic, in part because of toxicity resulting from adverse effects on tissue cholesterol homeostasis. We have tested whether partial inhibition of ACAT1 and ACAT2 (expressed in liver and intestine) activities reduces atherosclerosis development in apoE-deficient mice and avoids toxicity. Methods and Results—ApoE-deficient mice were maintained for 17 weeks on a Western-type diet without (control) or with the ACAT inhibitor F-1394 (effective against ACAT1 and ACAT2) at doses of either 300 (low) or 900 (high) mg/kg. Intimal lesion area at the aortic sinus in controls was 0.69±0.06 mm2. F-1394 treatment significantly decreased lesional area by 39% (low) or 45% (high). F-1394 treatment also reduced lesional immunostaining for macrophages by 61% (low) or 83% (high). En face analysis showed that surface lipid staining in control aortas was 20.0±2.8%; F-1394 treatment reduced this by 46% (low) or 62% (high). There were no obvious signs of systemic or vessel wall toxicity associated with F-1394 treatment. Conclusions—Partial ACAT inhibition by F-1394 had antiatherogenic effects in apoE-deficient mice that were achieved without obvious toxicity. Partial ACAT inhibition may have therapeutic potential in the clinical treatment of atherosclerosis.

Hepatic De Novo Lipogenesis Is Present in Liver-Specific ACC1-Deficient Mice

ABSTRACT Acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) catalyzes carboxylation of acetyl-CoA to form malonyl-CoA. In mammals, two isozymes exist with distinct physiological roles: cytosolic ACC1 participates in de novo lipogenesis (DNL), and mitochondrial ACC2 is involved in negative regulation of mitochondrial β-oxidation. Since systemic ACC1 null mice were embryonic lethal, to clarify the physiological role of ACC1 in hepatic DNL, we generated the liver-specific ACC1 null mouse by crossbreeding of an Acc1lox(ex46) mouse, in which exon 46 of Acc1 was flanked by two loxP sequences and the liver-specific Cre transgenic mouse. In liver-specific ACC1 null mice, neither hepatic Acc1 mRNA nor protein was detected. However, to compensate for ACC1 function, hepatic ACC2 protein and activity were induced 1.4 and 2.2 times, respectively. Surprisingly, hepatic DNL and malonyl-CoA were maintained at the same physiological levels as in wild-type mice. Furthermore, hepatic DNL was completely inhibited by an ACC1/2 dual inhibitor, 5-tetradecyloxyl-2-furancarboxylic acid. These results strongly demonstrate that malonyl-CoA from ACC2 can access fatty acid synthase and become the substrate for the DNL pathway under the unphysiological circumstances that result with ACC1 disruption. Therefore, there does not appear to be strict compartmentalization of malonyl-CoA from either of the ACC isozymes in the liver.

A Novel Mosaic Protein Containing LDL Receptor Elements Is Highly Conserved in Humans and Chickens

Certain receptors belonging to the LDL receptor (LDLR) gene family appear to constitute a newly identified branch whose members are expressed in brain, in addition to other tissues. In support of this concept, we have now discovered the expression and delineated the molecular structures of a representative of this emerging branch from two such diverse species as human and chicken. This membrane receptor, called LR11 and thus far only known to exist in the rabbit, is a complex seven-domain mosaic protein containing, among other structural elements, a cluster of 11 LDLR ligand-binding repeats and a domain with homology to VPS10, a yeast receptor for vacuolar protein sorting. Cytoplasmic signature sequences define the receptor as competent for endocytosis. The most striking properties of LR11s are their (1) high degree of structural conservation (>80% identity among mammals and birds), with 100% identity in the membrane-spanning and cytoplasmic domains of rabbit and human; (2) lack of regulation by cholesterol and estrogen; and (3) expression in brain. The features of LR11 suggest important roles in intercellular and intracellular ligand transport processes, some of which it may share with other brain-specific LDLR family members.

Postprandial Hyperlipidemia in Streptozotocin-Induced Diabetic Rats Is Due to Abnormal Increase in Intestinal Acyl Coenzyme A:Cholesterol Acyltransferase Activity

Postprandial hyperlipidemia (PH) is recognized as a significant risk factor for cardiovascular disease. The present study, involving rats with streptozotocin (STZ)-induced diabetes, was performed to establish a PH model and to examine the relation between small intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and serum lipid levels in the postprandial state. The small intestinal ACAT activities in normal rats during the experimental period were 4 to 5 pmol/mg protein per minute. In contrast, in the diabetic rats, the ACAT activities were 2 to 3 times higher than activities seen in normal rats from 7 to 21 days after the STZ injection in the absence of a high fat diet and hyperplasia in the gut. In an oral fat-loading test that used diabetic rats that had been injected with STZ (60 mg/kg) intravenously 14 days previously, the postloading changes in the serum concentrations of total cholesterol (TC) and triglyceride (TG) were significantly greater in the diabetic rats than in normal rats. Single oral administration of (1s,2s)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]cyclohexane- 1-yl 3-[(4R)-N-(2,2,5,5-tetramethyl-1, 3-dioxane-4-carbonyl)amino]propionate (F-1394, 3 to 30 mg/kg), a potent ACAT inhibitor, suppressed the post-fat-loading elevation of serum TC levels in the diabetic rats in a dose-dependent manner without affecting serum glucose levels. Furthermore, the small intestinal ACAT activity, serum TG levels, and lymphatic absorption of TC and TG in the rats that were administered F-1394 (30 mg/kg) were reduced by approximately 90%, 70%, 30%, and 15%, respectively. This is the first evidence that elevated ACAT activity in the gut, unlike hyperplasia and hyperphagia, induces PH in rats. Our results strongly suggest that F-1394 may be a potential treatment for PH in humans.

Direct effect of an acyl-CoA:cholesterol acyltransferase inhibitor, F-1394, on atherosclerosis in apolipoprotein E and low density lipoprotein receptor double knockout mice.

The acyl‐CoA:cholesterol acyltransferase (ACAT) enzyme is thought to be responsible for foam cell formation and the subsequent progression of atherosclerosis. The apolipoprotein E and low density lipoprotein receptor double knockout (apoE/LDLr‐DKO) mouse is an animal model that develops severe hyperlipidaemia and atherosclerosis. Here we have examined the effect of oral administration of an ACAT inhibitor, F‐1394, on atherosclerosis in apoE/LDLr‐DKO mice fed a regular chow diet. In en face analysis, a dose of 10, 30, or 100 mg kg−1 day−1 F‐1394 for 10 weeks reduced the extent of lesions visible in the aorta by 24, 28 and 38%, respectively, as detected by staining with oil red O, without affecting serum cholesterol level in these mice. At the highest dose 100 mg kg−1 day−1 of F‐1394, the reduction was statistically significant. For quantitative analysis of the cellular and non‐cellular components comprising the lesions at the aortic sinus, the effects of an oral dose of 100 mg kg−1 day−1 F‐1394 for 15 weeks were studied. There was a significant reduction (31.9%) in the oil‐red O‐stained area in cross‐sections of the aortic sinus. In addition, the neointimal area, as well as levels of ACAT‐1 protein tended to be decreased (15.2 and 25.8%, respectively, not significant). However, the areas containing macrophages, smooth muscle cells, and collagen were not affected by F‐1394. In vitro, F‐1394 attenuated foam cell formation in mouse peritoneal macrophages. These results indicate that ACAT may be primarily responsible for lipid accumulation in atherosclerotic lesions, and that its inhibition diminishes the lipid deposition via a direct effect on macrophages in the arterial wall.

ACC2 Is Expressed at High Levels Human White Adipose and Has an Isoform with a Novel N-Terminus

Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2) that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis. The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.

Acyl-CoenzymeA (CoA):Cholesterol Acyltransferase Inhibition in Rat and Human Aortic Smooth Muscle Cells Is Nontoxic and Retards Foam Cell Formation

Objective— Studies in vitro and in vivo of macrophage foam cells have shown evidence of cytotoxicity after acyl-CoA:cholesterol acyltransferase (ACAT) inhibition. Foam cells of smooth muscle origin are also found in human and animal atherosclerotic lesions. Methods and Results— To study whether cytotoxicity from ACAT inhibition is independent of cell type, we first established a protocol to conveniently induce aortic smooth muscle foam cell formation using cholesterol–cyclodextrin complexes (CCC). Rat aortic smooth muscle cells (ASMCs) treated for 48 hours with CCC (20 &mgr;g/mL) became foam cells by morphological (oil-red-O staining) and biochemical (≈1200% and ≈180% increase in cellular esterified and free cholesterol, respectively) criteria. ACAT activity increased 500% (P<0.01 versus untreated). Similar results were obtained in human ASMC, but ACAT activity increased to an even greater extent (3200%; P<0.01 versus untreated). Western blots indicated that CCC treatment increased human (to 380±20% of untreated, P<0.001), but not rat, ACAT protein expression. ACAT inhibition by Fujirebio compound F1394 suppressed CCC-induced foam cell formation in rat and human ASMC, but, notably, did not induce significant cytotoxicity. Conclusion— ASMC might be more resistant to FC-induced adverse effects than are macrophages.

ACAT Inhibition Reduces the Progression of Preexisting, Advanced Atherosclerotic Mouse Lesions Without Plaque or Systemic Toxicity

Objective—Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E–deficient mice. Methods and Results—Apolipoprotein-E–deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels. Conclusion—Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E–deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.

Effect of F-1394, an acyl-CoA:cholesterol acyltransferase inhibitor, on atherosclerosis induced by high cholesterol diet in rabbits

Cholesterol-fed rabbits were used to study the anti-atherosclerotic effect of (1S,2S)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]cyclohexane-1-yl 3-[(4R)-N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate (F-1394), an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor. To test its effect on the development of atherosclerosis, rabbits were fed a high-cholesterol diet (HCD) for 6 weeks, followed by regular chow (RC) for 12 weeks plus 0 or 100 mg/kg per day F-1394. Serum total cholesterol (TC) rose to approximately 2000 mg/dl on HCD and then declined gradually after the change in diet in both groups. F-1394 significantly reduced the extent of the atherosclerotic lesions and the total and esterified cholesterol contents of the aorta (by 57,38, and 59%, respectively), without affecting the serum TC level. To clarify whether F-1394 accelerates the regression of preexisting atherosclerosis, rabbits were fed HCD for the first 6 weeks and then RC for the next 6 weeks. Then, the rabbits were given 0 or 100 x 2 mg/kg per day F-1394 for another 12 weeks while on RC. F-1394 significantly reduced the extent of the atherosclerotic lesions and the total and esterified cholesterol content in the aorta (by 31, 31, and 43%, respectively), without affecting the serum TC level. These results demonstrate that F-1394 both prevents the formation of atherosclerosis and accelerates its regression without affecting the serum TC level, indicating that F-1394 acts directly on the arterial wall.

ヒト腸上皮細胞株Caco-2のacyl-CoA:cholesterol acyltransferase(ACAT)活性とコレステロールエステル分泌に対するACAT阻害薬,F-1394の作用

The present study was conducted to investigate the inhibitory effect of F-1394, a potent and selective inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), on incorporation of 14C-oleic acid into cholesteryl ester in cultured Caco-2 cells, a human intestinal cell line, and compare its effect to those of other ACAT inhibitors and hypolipidemic agents. The cholesterol esterification in Caco-2 cells was strongly inhibited by F-1394 in a concentration-dependent manner with the estimated IC50 value of 71 nM. In contrast, the estimated IC50 values of the other ACAT inhibitors such as YM-17E, CI-976, CL-277,082 and DL-melinamide are 121 nM, 702 nM, 21.5 microM and 20.9 microM, respectively. Simvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, also inhibited the ACAT activity in Caco-2 cells with an IC50 value of 22.5 microM, whereas pravastatin Na, probucol and clofibrate did not affect the activity. Furthermore, F-1394 at a concentration of 100 nM inhibited the basolateral secretion of cholesteryl ester by 90% from differentiated Caco-2 cells that were cultured on a membrane filter. These results demonstrate that F-1394 strongly inhibits human intestinal ACAT activity and basolateral secretion of cholesterol from Caco-2 cells. Therefore, F-1394 may have a therapeutic potential for dietary hyperlipidemic subjects.