Acyl-coenzyme A:cholesterol acyltransferase family

The enzymes of the acyl-coenzyme A:cholesterol acyltransferase (ACAT) family are responsible for the in vivo synthesis of neutral lipids.They are potential drug targets for the intervention of atherosclerosis,hyperlipidemia,obesity,type Ⅱ diabetes and even Alzheimer's disease.ACAT family enzymes are integral endoplasmic reticulum (ER) membrane proteins and can be divided into ACAT branch and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGATI) branch according to their substrate specificity.The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates,while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates.In this review,we mainly focus on the recent progress in the structural research of ACAT family enzymes,including their disulfide linkage,membrane topology,subunit interaction and catalysis mechanism.     

Acyl-coenzyme A: cholesterol acyltransferase family

The enzymes of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) family are responsible for the in vivo synthesis of neutral lipids. They are potential drug targets for the intervention of atherosclerosis, hyperlipidemia, obesity, type II diabetes and even Alzheimer’s disease. ACAT family enzymes are integral endoplasmic reticulum (ER) membrane proteins and can be divided into ACAT branch and acyl-coenzyme A: diacylglycerol acyltransferase 1 (DGAT1) branch according to their substrate specificity. The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates, while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates. In this review, we mainly focus on the recent progress in the structural research of ACAT family enzymes, including their disulfide linkage, membrane topology, subunit interaction and catalysis mechanism.     

Effects of high-density lipoprotein(2) on cholesterol transport and acyl-coenzyme A:cholesterol acyltransferase activity in P388D1 macrophages

High-density lipoproteins are the putative vehicles for cholesterol removal from monocyte-derived macrophages, which are an important cell type in all stages of atherosclerosis. The role of HDL(2), an HDL subclass that accounts for most variation in plasma HDL-cholesterol concentration, in cholesterol metabolism in monocyte-derived macrophages is not known. In this study, the dose-dependent effects of HDL(2) on cellular cholesterol mass, efflux, and esterification, and on cellular cholesteryl ester (CE) hydrolysis using the mouse macrophage P388D1 cell line was investigated. HDL(2) at low concentrations (40 microg protein/ml) decreased CE content without affecting cellular free cholesterol content (FC), CE hydrolysis, or cholesterol biosynthesis. In addition, HDL(2) at low concentrations reduced cellular acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and increased FC efflux from macrophages. Thus, HDL(2) has two potential roles in reverse cholesterol transport. In one, HDL(2) is an acceptor of macrophage FC. In the other, more novel role, HDL(2) increases the availability of macrophage FC through the inhibition of ACAT. Elucidation of the mechanism by which HDL(2) inhibits ACAT could identify new therapeutic targets that enhance the transfer of cholesterol from macrophages to the liver.     

Importance of microbial defence systems to bile salts and mechanisms of serum cholesterol reduction

An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera (Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo. Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol.     

Cholagogic effect of trimethylglycine in normal animals of different ages and in experimental atherosclerosis

Trimethylglycine at a dose of 1.5 g/kg was found to produce marked bile secretory effect in young and old rats. In rabbits with experimental atherosclerosis, trimethylglycine increased the content of biliary acids in the bile and normalized the indexes of lipid metabolism in the blood serum. Apparently, the effect on cholesterol transformation into biliary acids and its excretion with the bile is one of the mechanisms of anti-atherosclerotic action of trimethylglycine.     

Effect of catecholamines on the hepatic rate-limiting enzymes of cholesterol metabolism in normally fed and cholesterol-fed rabbits

Adrenergic control of liver cholesterol metabolism was studied in the rabbit. The effects of noradrenaline (α₁, α₂, β₂ agonist) and isoprenaline (β₁, β₂ agonist) on 3-hydroxy-3-methylglutaryl coenzyme A reductase, acyl-coenzyme A: cholesterol-o-acyltransferase (cholesterol acyltransferase) and cholesterol 7α-hydroxylase, the rate-limiting enzymes of cholesterol biosynthesis and esterification and bile acid synthesis, respectively, were examined in the normally fed and cholesterol-fed male New Zealand White rabbit. Isoprenaline increased the activities of hydroxymethylglutaryl CoA reductase and cholesterol acyltransferase approx. 12-fold and 5-fold, respectively, in normally fed rabbits. Noradrenaline, by contrast, produced an effect only on hydroxymethylglutaryl CoA reductase, the activity of which was increased 3-fold in these animals. Neither catecholamine had an effect on hydroxymethylglutaryl CoA reductase in the cholesterol-fed rabbit. Isoprenaline decreased the activity of cholesterol acyltransferase by approx. 40% and increased the activity of cholesterol 7α-hydroxylase 2-fold in the cholesterol-fed rabbit compared to cholesterol-fed controls. Noradrenaline had no effect on either cholesterol acyltransferase or cholesterol 7α-hydroxylase in either the normally fed or the cholesterol-fed rabbit. We suggest that β₂-adrenergic stimulation by isoprenaline in the normally fed rabbit may enhance cholesterol synthesis and storage, but that in the cholesterol-fed rabbit, it facilitates the elimination of cholesterol from the body by increasing the rate of bile acid synthesis.     

HDL revisited: new opportunities for managing dyslipoproteinaemia and cardiovascular disease

Low concentrations of high-density lipoprotein (HDL) cholesterol constitute a risk factor for coronary heart disease (CHD). There is increasing evidence that increasing HDL-cholesterol levels reduces cardiovascular risk. The phenotype of low HDL cholesterol with or without elevated triglycerides is common and it is characteristic of patients with central obesity, insulin resistance, hypertension and type 2 diabetes mellitus; conditions associated with increased cardiovascular risk and are part of the rubric of the metabolic syndrome. Epidemiological, experimental and clinical trial evidence suggests that there is a good rationale for raising HDL-cholesterol in these and other high-risk patients. The protective effect of HDL-cholesterol against atherosclerosis and cardiovascular disease is mediated by both enhanced reverse cholesterol transport (RCT) and by direct anti-atherosclerotic mechanisms. Recent studies have elucidated mechanisms whereby HDL acts to reduce cardiovascular risk, supporting the rationale for targeting of HDL with lipid-modifying therapy. Ongoing investigation of mechanisms by which HDL acts to reduce the risk of atherosclerosis will provide opportunities for the development of new therapeutic strategies to decrease the risk of atherosclerosis.     

Effects of high-density lipoprotein2 on cholesterol transport and acyl-coenzyme A:cholesterol acyltransferase activity in P388D1 macrophages

High-density lipoproteins are the putative vehicles for cholesterol removal from monocyte-derived macrophages, which are an important cell type in all stages of atherosclerosis. The role of HDL₂, an HDL subclass that accounts for most variation in plasma HDL-cholesterol concentration, in cholesterol metabolism in monocyte-derived macrophages is not known. In this study, the dose-dependent effects of HDL₂ on cellular cholesterol mass, efflux, and esterification, and on cellular cholesteryl ester (CE) hydrolysis using the mouse macrophage P388D1 cell line was investigated. HDL₂ at low concentrations (40 μg protein/ml) decreased CE content without affecting cellular free cholesterol content (FC), CE hydrolysis, or cholesterol biosynthesis. In addition, HDL₂ at low concentrations reduced cellular acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and increased FC efflux from macrophages. Thus, HDL₂ has two potential roles in reverse cholesterol transport. In one, HDL₂ is an acceptor of macrophage FC. In the other, more novel role, HDL₂ increases the availability of macrophage FC through the inhibition of ACAT. Elucidation of the mechanism by which HDL₂ inhibits ACAT could identify new therapeutic targets that enhance the transfer of cholesterol from macrophages to the liver.     

Role of naturally-occurring plant sterols on intestinal cholesterol absorption and plasmatic levels

Cardiovascular disease is a major health problem in developed countries although its incidence is relatively lower in Mediterranean countries which is partly ascribed to dietary habits. Epidemiologic evidence shows that elevated serum cholesterol, specifically low-density lipoprotein cholesterol (c-LDL), increases cardiovascular disease. Phytosterols are bioactive compounds, found in all vegetable foods, which inhibit intestinal cholesterol absorption and, therefore, have a serum cholesterollowering effect. Intestinal cholesterol absorption is a multistep process where, plant sterols and stanols may act:a) attenuating the NPC1L1 gene expression, which may result in a lower cholesterol uptake from the lumen;b) lowering the cholesterol esterification rate by the ACAT2 (acyl-CoA cholesterol acyltransferase) and, consequently, the amount of cholesterol secreted via the chylomicrons andc) upregulating the expression of ABC-transporters ABCG5 and ABCG8 in intestinal cells, which may result in an increased excretion of cholesterol by the enterocyte back in the lumen. Many clinical trials proved that commercial products enriched with phytosterols reduce cholesterol levels. Likewise, recent studies show that phytosterols present in natural food matrices are also effective and could be an important component of cardioprotective dietary patterns such as the Mediterranean diet.     

Inhibition of nuclear factor-kappaB attenuates artherosclerosis in apoE/LDLR - double knockout mice.

Nuclear factor - kappaB (NF-kappaB) is a good therapeutic target for cardiovascular disease and numerous efforts are being made to develop safe NF-kappaB inhibitors. Nowadays many authors address NF-kappaB as a major therapeutic target in atherosclerosis, especially for preventive measures, in the light of two main hypothesis of atherosclerosis: oxidation and inflammation. We hypothesized that ammonium pyrrolidinedithioocarbamate (PDTC) - a well-known inhibitor of NF-kappaB could inhibit the development of atherosclerosis in this experimental model. We used apoE/LDLR - DKO mouse model, which is considered as a one of the best models to study the anti-atherosclerotic effect of drugs. In this model PDTC inhibited atherogenesis, measured both by "en face" method (25,15+/-2,9% vs. 15,63+/-0,6%) and "cross-section" method (565867+/-39764 microm2 vs. 291695+/-30384 microm2). Moreover, PDTC did not change the profile of cholesterol and triglycerides in blood. To our knowledge, this is the first report that shows the effect of PDTC on atherogenesis in gene-targeted apoE/LDLR - double knockout mice.     

Synthesis and coenzyme A-dependent esterification of cholesterol in rat intestinal epithelium. Differences in cellular localization and mechanisms of regulation

The current studies were undertaken to characterize the localization and regulation of cholesterol synthesis and acyl-CoA:cholesterol acyltransferase activity in rat intestinal crypt and villus cells. Both parameters were determined in groups of animals with widely varying sterol fluxes across the intestinal mucosa. In animals on control diet the rates of cholesterol synthesis, measured by the incorporation of [3H]water per mg of protein, were similar along the villus/crypt axis in the jejunum, whereas in the ileum, villus cells were significantly more active than crypt cells. In both areas, however, the majority of total synthetic activity was found in cells from the crypts and lower villi. In contrast, the highest specific and total acyl-CoA:cholesterol acyltransferase activity was recovered in the villus cells of the jejunum and ileum. Dietary cholesterol did not affect sterol synthesis in any of the cell fractions but increased acyl-CoA:cholesterol acyltransferase activity approximately 2-fold in jejunal cell fractions. Inhibition of cholesterol absorption or sequestration of intestinal bile acids stimulated sterol synthetic activity up to 7-fold, and this occurred mainly in the lower villus and crypt cells in both jejunum and ileum. An increased demand for lipoprotein cholesterol, generated by triglyceride feeding, similarly was associated with enhanced synthetic rates. However, unlike cholesterol feeding, these manipulations did not increase acyl-CoA:cholesterol acyltransferase activity in any of the villus cell fractions. These studies suggest, therefore, that the intracellular pools of cholesterol that regulate the rate of cholesterol synthesis and the rate of cholesterol esterification are functionally distinct.     

Liver X receptors: Xcreting Xol to combat atherosclerosis

Liver X receptors (LXRs) are nuclear receptors that act as metabolic sensors for cellular cholesterol (Xol) and oxysterol content. Increased oxysterol levels activate LXRs, which then induce: the removal of cholesterol out of peripheral cells; transport of this cholesterol to the liver; excretion of cholesterol through production of bile acids; and inhibition of intestinal cholesterol absorption. Recent evidence indicates that LXRs are not only master regulators of cholesterol homeostasis, but also decrease the development of atherosclerosis - a disease intimately linked with abnormal cholesterol homeostasis. This evidence shows that LXRs are promising drug development targets for atherosclerosis.     

Cholesterol and vitamins: revisited study

The link between low density lipoprotein and coronary heart disease has been widely studied. Oxidized LDL damages the artery wall, and a diet rich in vitamins and low in saturated fat and cholesterol may reduce this risk. Not only hypercholesterolemia but also low levels of high density lipoprotein cholesterol are critical risk factors for atherosclerosis and related diseases. It has been reported that high doses of B complex vitamin may be useful in lowering blood cholesterol and triglyceride levels in the body, however the use of this compound has been limited by an annoying flush and concern for toxicity. Niacin is a B-complex vitamin with anti-atherosclerotic properties and is an effective medication for raising high density lipoprotein. The combination of niacin with other lipid-lowering drugs, such as statins, reduces the dynamic of atherosclerosis disease. In addition, vitamin E is one of the most important lipid soluble anti-oxidants in humans, and reduces atherosclerosis plaque, coronary artery diseases and myocardial infarction. Vitamin E protects the integrity of membranes by inhibiting lipid peroxidation. In this study we revisited the interrelationship between cholesterol, low density lipoproteins and vitamins.     

Cholesterol, bile acid, and lipoprotein metabolism in two strains of hamster, one resistant, the other sensitive (LPN) to sucrose-induced cholelithiasis

A comprehensive study of cholesterol, bile acid, and lipoprotein metabolism was undertaken in two strains of hamster that differed markedly in their response to a sucrose-rich/low fat diet. Under basal conditions, hamsters from the LPN strain differed from Janvier hamsters by a lower cholesterolemia, a higher postprandial insulinemia, a more active cholesterogenesis in both liver [3- to 4-fold higher 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoAR) activity and mRNA] and small intestine, and a lower hepatic acyl-coenzyme A:cholesterol acyltransferase activity. Cholesterol saturation indices in the gallbladder bile were similar for both strains, but the lipid concentration was 2-fold higher in LPN than in Janvier hamsters. LPN hamsters had a lower capacity to transform cholesterol into bile acids, shown by the smaller fraction of endogenous cholesterol converted into bile acids prior to fecal excretion (0.34 vs. 0.77). In LPN hamsters, the activities of cholesterol 7alpha-hydroxylase (C7OHase) and sterol 27-hydroxylase (S27OHase), the two rate-limiting enzymes of bile acid synthesis, were disproportionably lower (by 2-fold) to that of HMG-CoAR. When fed a sucrose-rich diet, plasma lipids increased, dietary cholesterol absorption improved, hepatic activities of HMG-CoA reductase, C7Ohase, and S27OHase were reduced, and intestinal S27OHase was inhibited in both strains. Despite a similar increase in the biliary hydrophobicity index due to the bile acid enrichment in chenodeoxycholic acid and derivatives, only LPN hamsters had an increased lithogenic index and developed cholesterol gallstones (75% incidence), whereas Janvier hamsters formed pigment gallstones (79% incidence).These studies indicate that LPN hamsters have a genetic predisposition to sucrose-induced cholesterol gallstone formation related to differences in cholesterol and bile acid metabolism.     

Intestinal fatty acid‐binding protein mediates atherosclerotic progress through increasing intestinal inflammation and permeability

Atherosclerosis is one of leading phenotypes of cardiovascular diseases, featured with increased vascular intima‐media thickness (IMT) and unstable plaques. The interaction between gastrointestinal system and cardiovascular homeostasis is emerging as a hot topic. Therefore, the present study aimed to explore the role of an intestinal protein, intestinal fatty acid‐binding protein (I‐FABP/FABP2) in the atherosclerotic progress. In western diet–fed ApoE^?/? mice, FABP2 was highly expressed in intestine. Silence of intestinal Fabp2 attenuated western diet–induced atherosclerotic phenotypes, including decreasing toxic lipid accumulation, vascular fibrosis and inflammatory response. Mechanistically, intestinal Fabp2 knockdown improved intestinal permeability through increasing the expression of tight junction proteins. Meanwhile, intestinal Fabp2 knockdown mice exhibited down‐regulation of intestinal inflammation in western diet–fed ApoE^?/? mice. In clinical patients, the circulating level of FABP2 was obviously increased in patients with cardiovascular disease and positively correlated with the value of carotid intima‐media thickness, total cholesterol and triglyceride. In conclusion, FABP2‐induced intestinal permeability could address a potential role of gastrointestinal system in the development of atherosclerosis, and targeting on intestinal FABP2 might provide a therapeutic approach to protect against atherosclerosis.     

Cholesteryl Ester Transfer Protein (CETP) Deficiency and CETP Inhibitors

Epidemiologic studies have shown that low-density lipoprotein cholesterol (LDL-C) is a strong risk factor, whilst high-density lipoprotein cholesterol (HDL-C) reduces the risk of coronary heart disease (CHD). Therefore, strategies to manage dyslipidemia in an effort to prevent or treat CHD have primarily attempted at decreasing LDL-C and raising HDL-C levels. Cholesteryl ester transfer protein (CETP) mediates the exchange of cholesteryl ester for triglycerides between HDL and VLDL and LDL. We have published the first report indicating that a group of Japanese patients who were lacking CETP had extremely high HDL-C levels, low LDL-C levels and a low incidence of CHD. Animal studies, as well as clinical and epidemiologic evidences, have suggested that inhibition of CETP provides an effective strategy to raise HDL-C and reduce LDL-C levels. Four CETP inhibitors have substantially increased HDL-C levels in dyslipidemic patients. This review will discuss the current status and future prospects of CETP inhibitors in the treatment of CHD. At present anacetrapib by Merck and evacetrapib by Eli Lilly are under development. By 100mg of anacetrapib HDL-C increased by 138%, and LDL-C decreased by 40%. Evacetrapib 500 mg also showed dramatic 132% increase of HDL-C, while LDL-C decreased by 40%. If larger, long-term, randomized, clinical end point trials could corroborate other findings in reducing atherosclerosis, CETP inhibitors could have a significant impact in the management of dyslipidemic CHD patients. Inhibition of CETP synthesis by antisense oligonucleotide or small molecules will produce more similar conditions to human CETP deficiency and may be effective in reducing atherosclerosis and cardiovascular events. We are expecting the final data of prospective clinical trials by CETP inhibitors in 2015.     

Clinical pharmacology of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

In this article, de novo cholesterol synthesis, its inhibition by HMG-CoA reductase inhibitors (statins) and clinical pharmacology aspects of the statins have been reviewed. Statins are available in both active and pro-drug forms. Their affinity to bind and subsequently to inhibit HMG-CoA reductase activity is approximately 3 orders of magnitude higher than that of natural substrate (HMG-CoA). All members of this group of lipid-lowering agents are, to a varying degree, absorbed from the gut. However, their bioavailability depends on their lipophobicity and their concomitant use with meals. The interaction between HMG-CoA reductase inhibitors and other lipid-lowering agents has been reviewed in more detail. One major side-effect of lipid-lowering combination therapy is myopathy with or without rhabdomyolysis. Combination of statins with gemfibrozil seems to increase risk of this adverse event, particularly in patients with renal impairment, more than combination with other lipid-lowering agents. Combination therapy with other agents including anticoagulants, antihypertensive, anti-inflammatory, oral hypoglycemic and antifungal agents as well as beta-blockers, H2 blockers, cyclosporine and digoxin has been also reviewed. The pleiotropic non-lipid lowering properties of statins and their effects on the quality of lipoprotein particles, the activities of cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase as well as their possible synergistic effects with n-3 fatty acids, phytosterols, vitamin E and aspirin in reducing cardiovascular events warrant further investigation.     

Leptin promotes biliary cholesterol elimination during weight loss in ob/ob mice by regulating the enterohepatic circulation of bile salts

Leptin administration to obese C57BL/6J (ob/ob) mice results in weight loss by reducing body fat. Because adipose tissue is an important storage depot for cholesterol, we explored evidence that leptin-induced weight loss in ob/ob mice was accompanied by transport of cholesterol to the liver and its elimination via bile. Consistent with mobilization of stored cholesterol, cholesterol concentrations in adipose tissue remained unchanged during weight loss. Plasma cholesterol levels fell sharply, and microscopic analyses of gallbladder bile revealed cholesterol crystals as well as cholesterol gallstones. Surprisingly, leptin reduced biliary cholesterol secretion rates without affecting secretion rates of bile salts or phospholipids. Instead, cholesterol supersaturation of gallbladder bile was due to marked decreases in bile salt hydrophobicity and not to hypersecretion of biliary cholesterol per se, such as occurs in humans during weight loss. In addition to regulating bile salt composition, leptin treatment decreased bile salt pool size. The smaller, more hydrophilic bile salt pool was associated with substantial decreases in intestinal cholesterol absorption. Within the liver, leptin treatment reduced the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, but it did not change activities of cholesterol 7alpha-hydroxylase or acyl-CoA:cholesterol acyltransferase. These data suggest that leptin regulates biliary lipid metabolism to promote efficient elimination of excess cholesterol stored in adipose tissue. Cholesterol gallstone formation during weight loss in ob/ob mice appears to represent a pathologic consequence of an adaptive response that prevents absorption of biliary and dietary cholesterol.