Abstract-Atherosclerotic coronary heart disease is a common complication of the insulin resistance syndrome that can occur with or without diabetes mellitus. Thiazolidinediones (TZDs), which are insulin-sensitizing antidiabetic agents, can modulate the development of atherosclerosis not only by changing the systemic metabolic conditions associated with insulin resistance but also by exerting direct effects on vascular wall cells that express peroxisome proliferatoractivated receptor-␥ (PPAR-␥), a nuclear receptor for TZDs. Here we show that troglitazone, a TZD, significantly inhibited fatty streak lesion formation in apolipoprotein E-knockout mice fed a high-fat diet (en face aortic surface lesion areas were 6.9Ϯ2.5% vs 12.7Ϯ4.7%, PϽ0.05; cross-sectional lesion areas were 191 974Ϯ102 911 m 2 vs 351 738Ϯ175 597 m 2 , PϽ0.05; nϭ10). Troglitazone attenuated hyperinsulinemic hyperglycemia and increased high density lipoprotein cholesterol levels. In the aorta, troglitazone markedly increased the mRNA levels of CD36, a scavenger receptor for oxidized low density lipoprotein, presumably by upregulating its expression, at least in part, in the macrophage foam cells. These results indicate that troglitazone potently inhibits fatty streak lesion formation by modulating both metabolic extracellular environments and arterial wall cell functions.

Liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) are members of nuclear receptors that form obligate heterodimers with retinoid X receptors (RXRs). These nuclear receptors play crucial roles in the regulation of fatty acid metabolism: LXRs activate expression of sterol regulatory element-binding protein 1c (SREBP-1c), a dominant lipogenic gene regulator, whereas PPARalpha promotes fatty acid beta-oxidation genes. In the current study, effects of PPARs on the LXR-SREBP-1c pathway were investigated. Luciferase assays in human embryonic kidney 293 cells showed that overexpression of PPARalpha and gamma dose-dependently inhibited SREBP-1c promoter activity induced by LXR. Deletion and mutation studies demonstrated that the two LXR response elements (LXREs) in the SREBP-1c promoter region are responsible for this inhibitory effect of PPARs. Gel shift assays indicated that PPARs reduce binding of LXR/RXR to LXRE. PPARalpha-selective agonist enhanced these inhibitory effects. Supplementation with RXR attenuated these inhibitions by PPARs in luciferase and gel shift assays, implicating receptor interaction among LXR, PPAR, and RXR as a plausible mechanism. Competition of PPARalpha ligand with LXR ligand was observed in LXR/RXR binding to LXRE in gel shift assay, in LXR/RXR formation in nuclear extracts by coimmunoprecipitation, and in gene expression of SREBP-1c by Northern blot analysis of rat primary hepatocytes and mouse liver RNA. These data suggest that PPARalpha activation can suppress LXR-SREBP-1c pathway through reduction of LXR/RXR formation, proposing a novel transcription factor cross-talk between LXR and PPARalpha in hepatic lipid homeostasis.

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes esterification of cellular cholesterol. To investigate the role of ACAT-1 in atherosclerosis, we have generated ACAT-1 null (ACAT-1؊/؊) mice. ACAT activities were present in the liver and intestine but were completely absent in adrenal, testes, ovaries, and peritoneal macrophages in our ACAT-1؊/؊ mice. The ACAT-1؊/؊ mice had decreased openings of the eyes because of atrophy of the meibomian glands, a modified form of sebaceous glands normally expressing high ACAT activities. This phenotype is similar to dry eye syndrome in humans. To determine the role of ACAT-1 in atherogenesis, we crossed the ACAT-1؊/؊ mice with mice lacking apolipoprotein (apo) E or the low density lipoprotein receptor (LDLR), hyperlipidemic models susceptible to atherosclerosis. High fat feeding resulted in extensive cutaneous xanthomatosis with loss of hair in both ACAT-1؊/؊:apo E؊/؊ and ACAT-1؊/؊:LDLR؊/؊ mice. Free cholesterol content was significantly increased in their skin. Aortic fatty streak lesion size as well as cholesteryl ester content were moderately reduced in both double mutant mice compared with their respective controls. These results indicate that the local inhibition of ACAT activity in tissue macrophages is protective against cholesteryl ester accumulation but causes cutaneous xanthomatosis in mice that lack apo E or LDLR.

The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr؉/؊) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr؉/؊ embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr؉/؊ mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr؉/؊ and Hmgcr؉/؉ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.The mevalonate pathway produces isoprenoids that are essential for diverse cellular functions, ranging from cholesterol synthesis to growth control. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 1 reductase (EC 1.1.1.34), which catalyzes the conversion of HMG-CoA to mevalonate, is the rate-limiting enzyme in the mevalonate pathway (1). Because of its major role in cholesterol biosynthesis, the regulation of HMG-CoA reductase has been intensely studied. To ensure a steady mevalonate supply, the non-sterol and sterol end-products of mevalonate metabolism exert feedback regulation on the activity of this enzyme through multivalent mechanisms, including inhibition of transcription of the HMG-CoA reductase mRNA, blocking of translation, and acceleration of protein degradation, thus regulating the amount of reductase protein over a several hundred-fold range (reviewed in Refs.

Abstract-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 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 Ϸ90%, 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. C oronary heart disease (CHD) resulting from premature atherosclerosis is a major cause of death in insulindependent and non-insulin-dependent diabetic patients. 1,2 Abnormalities in the metabolism of plasma lipoproteins and lipids in the postprandial state have been reported in diabetic patients, 3-9 and now evidence is being accumulated indicating that postprandial increases in triglyceride (TG)-rich lipoprotein, 10,11 which is known as one of the atherogenic remnant lipoproteins, 12,13 TG, 14,15 and cholesterol 16 remarkably contribute to the occurrence of CHD in such patients.A number of studies have reported that rats with streptozotocin (STZ)-induced diabetes (STZ-diabetic rats) shows severe hyperlipidemia after exogenous fat loading. [17][18][19] It has been suggested that the severe hyperlipidemia occurring in STZ-diabetic rats fed a high fat diet could be attributable to a marked increase in fat absorption via the gut, which in turn could be due to an abnormal increase in small intestinal acylcoenzyme A:cholesterol acyltransferase (ACAT) activity. 20 Indeed, repeated administration of an ACAT inhibitor, such as 21 CL-277082, 22,23 or FR 145237, 24 markedly decreases both the serum total cholesterol (TC) level and enhanced small intestinal ACAT activity in STZ-diabetic rats fed a high fat diet. However, previous investigators using diabetic mo...

Cholesterol ester (CE)-laden foam cells are a hallmark of atherosclerosis. To determine whether stimulation of the hydrolysis of cytosolic CE can be used as a novel therapeutic modality of atherosclerosis, we overexpressed hormone-sensitive lipase (HSL) in THP-1 macrophage-like cells by adenovirus-mediated gene delivery, and we examined its effects on the cellular cholesterol trafficking. We show here that the overexpression of HSL robustly increased neutral CE hydrolase activity and completely eliminated CE in the cells that had been preloaded with CE by incubation with acetylated low density lipoprotein. In these cells, cholesterol efflux was stimulated in the absence or presence of high density lipoproteins, which might be at least partially explained by the increase in the expression of ABCA1. Importantly, these effects were achieved without the addition of acyl-CoA:cholesterol acyltransferase inhibitor, cAMP, or even high density lipoproteins. Furthermore, the uptake and degradation of acetylated low density lipoprotein was significantly reduced probably by decreased expression of scavenger receptor A and CD36. Notably, the cells with stimulated CE hydrolysis did not exhibit either buildup of free cholesterol or cytotoxicity. In conclusion, increased hydrolysis of CE by the overexpression of HSL leads to complete elimination of CE from THP-1 foam cells not only by increasing efflux but also by decreasing influx of cholesterol. Cholesterol ester (CE)1 -laden macrophage foam cells are a hallmark of fatty streak lesions in atherosclerotic plaques.Besides cleaning up extracellularly deposited atherogenic lipoproteins, macrophage foam cells secrete a wide variety of substances, which include inflammatory cytokines, chemokines, growth factors, and proteases (1). Furthermore, lipidrich lesions that are characterized by a plethora of macrophage foam cells are associated with massive infiltration with activated T lymphocytes and prone to rupture, thereby leading to thrombotic coronary occlusion (2). Thus, elimination of CE from foam cells is potentially a promising therapeutic strategy to stabilize rupture-prone atherosclerotic plaques.Foam cells are generated by the uptake of modified lipoproteins through scavenger receptors, such as scavenger receptor A (SR-A) and CD36 (3). Hydrolysis of the lipoprotein-associated CE by lysosomal acid lipase liberates free cholesterol (FC), which is subsequently re-esterified by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) to form CE for storage in the cytoplasmic lipid droplets (4, 5). The cytoplasmic CE is in turn hydrolyzed by neutral CE hydrolase (NCEH) to generate FC, which is transported to a compartment for re-esterification by ACAT1. The rest of the FC is released out of the cells primarily through ATP-binding cassette transporter A1 (ABCA1) (6, 7). Thus, the balance between synthesis and hydrolysis of CE conceivably governs the level of CE in macrophages.Inhibition of ACAT activity is shown to suppress CE accumulation with concomitant promotion of the net hydrolysis of ...

Squalene synthase (SS) is the first committed enzyme for cholesterol biosynthesis, located at a branch point in the mevalonate pathway. To examine the role of SS in the overall cholesterol metabolism, we transiently overexpressed mouse SS in the livers of mice using adenovirusmediated gene transfer. Overexpression of SS increased de novo cholesterol biosynthesis with increased 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase activity, in spite of the downregulation of its own mRNA expression. Furthermore, overexpression of SS increased plasma concentrations of LDL, irrespective of the presence of functional LDL receptor (LDLR). Thus, the hypercholesterolemia is primarily caused by increased hepatic production of cholesterol-rich VLDL, as demonstrated by the increases in plasma cholesterol levels after intravenous injection of Triton WR1339. mRNA expression of LDLR was decreased, suggesting that defective LDL clearance contributed to the development of hypercholesterolemia. Curiously, the liver was enlarged, with a larger number of Ki-67-positive cells. These results demonstrate that transient upregulation of SS stimulates cholesterol biosynthesis as well as lipoprotein production, providing the first in vivo evidence that SS plays a regulatory role in cholesterol metabolism through modulation of HMG-CoA reductase activity and cholesterol biosynthesis. Cholesterol biosynthesis is subject to tight regulation by a multivalent feedback mechanism at both the transcriptional and the posttranscriptional level (1). The transcriptional regulation is mediated through the action of sterol-regulatory element binding proteins (SREBPs), membrane-bound transcription factors that enhance transcription of genes encoding cholesterol biosynthetic enzymes and the LDL receptor (LDLR) (2). The translational regulation of 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase (EC1.1.1.34), the rate-limiting enzyme in cholesterol biosynthesis, is mediated by nonsterol mevalonate-derived isoprenoids, which act by an undefined mechanism (3). Its degradation is regulated by both sterols and nonsterol end products of mevalonate metabolism (4, 5). The sterolregulated degradation of HMG-CoA reductase is mediated Abbreviations: Ad-SS, recombinant adenovirus carrying SS cDNA under the control of cytomegalovirus promoter; apoB, apolipoprotein B; E, embryonic day; ER, endoplasmic reticulum; LDLR, LDL receptor; m.o.i., multiplicity of infection; SREBP, sterol-regulatory element binding protein; SS, squalene synthase; TC total cholesterol; TG, triglyceride; TUNEL, terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling.

Mammals have developed sophisticated and complex systems to maintain cellular content of cholesterol, an essential component of cellular membranes and a precursor of bile acids and steroid hormones ( 1 ). In addition to dietary intake, cholesterol is supplied by de novo synthesis from acetate. Squalene synthase (SS; farnesyl-diphosphate farnesyltransferase, EC2.5.1.21) catalyzes the reductive head-to-head condensation of two molecules of farnesyl diphosphate (FPP) to form squalene, the fi rst committed intermediate in the cholesterol biosynthetic pathway ( 2, 3 ). SS contains ‫ف‬ 416 amino acids and is anchored to endoplasmic reticulum by a short C-terminal membrane-spanning domain, with its large N-terminal catalytic domain facing the cytosol, where water-soluble FPP and NADPH are present. Hepatic SS is highly regulated at the transcriptional level, not only by cellular cholesterol content ( 4 ) but also by proinfl ammatory cytokines: TNF-␣ and interleukin 1 ␤ ( 5 ). This enzyme has been an attractive target for cholesterol-lowering therapy because the inhibition of this step theoretically may not perturb the nonsterol pathway, which is a potential problem in the use of statins, inhibitors of HMG-CoA reductase Abstract Squalene synthase (SS) catalyzes the biosynthesis of squalene, the fi rst specifi c intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifi cally knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of suffi cient centripetal fl ow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were signifi cantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor ␣ target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specifi c ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing signifi cant mortality. -Nagashima, S