The extract of Ilex kudingcha inhibits atherosclerosis in apoE-deficient mice by suppressing cholesterol accumulation in macrophages

It was previously reported that oleanolic acid and ursolic acid, triterpenoid compounds occurring in Ilex kudingcha, ameliorate hyperlipidemia and atherosclerosis in apoE-deficient mice. In the present study, we investigated whether Ilex kudingcha extract exerts similar inhibitory effects on cholesterol accumulation in human monocyte-derived macrophages (HMDMs) and atherogenesis in apoE-deficient mice. Ilex kudingcha extract significantly inhibited cholesterol ester (CE) accumulation induced by acetyl-LDL (acetylated LDL) in HMDMs; however, it generated no effect on cell viability in HMDMs. Ilex kudingcha extract also suppressed CE accumulation in acyl-CoA: cholesterol acyl-transferase (ACAT)-overexpressing CHO (Chinese hamster ovary) cells, thereby indicating that it inhibits ACAT activity. Furthermore, the oral administration of Ilex kudingcha extract to apoE-deficient mice significantly decreased the levels of serum cholesterol, triglyceride, sLOX-1, as well as the regions of atherosclerotic lesions in the mice. Our study reveals crucial new-found evidence that Ilex kudingcha extract significantly inhibits ACAT activity and suppresses atherogenesis.

Triterpenic Acids Present in Hawthorn Lower Plasma Cholesterol by Inhibiting Intestinal ACAT Activity in Hamsters

Hawthorn (Crataegus pinnatifida) is an edible fruit used in traditional Chinese medicine to lower plasma lipids. This study explored lipid-lowering compounds and underlying mechanisms of action of hawthorn. Hawthorn powder extracts inhibited acylCoA:cholesterol acyltransferase (ACAT) activity in Caco-2 cells. The inhibitory activity was positively associated with triterpenic acid (i.e., oleanolic acid (OA) and ursolic acid (UA)) contents in the extracts. Cholesterol lowering effects of hawthorn and its potential additive effect in combination with plant sterol esters (PSE) were further studied in hamsters. Animals were fed a semi-synthetic diet containing 0.08% (w/w) cholesterol (control) or the same diet supplemented with (i) 0.37% hawthorn dichloromethane extract, (ii) 0.24% PSE, (iii) hawthorn dichloromethane extract (0.37%) plus PSE (0.24%) or (iv) OA/UA mixture (0.01%) for 4 weeks. Compared to the control diet, hawthorn, PSE, hawthorn plus PSE and OA/UA significantly lowered plasma non-HDL (VLDL + LDL) cholesterol concentrations by 8%, 9%, 21% and 6% and decreased hepatic cholesterol ester content by 9%, 23%, 46% and 22%, respectively. The cholesterol lowering effects of these ingredients were conversely associated with their capacities in increasing fecal neutral sterol excretion. In conclusion, OA and UA are responsible for the cholesterol lowering effect of hawthorn by inhibiting intestinal ACAT activity. In addition, hawthorn and particularly its bioactive compounds (OA and UA) enhanced the cholesterol lowering effect of plant sterols.

Altered hepatic cholesterol metabolism compensates for disruption of phosphatidylcholine transfer protein in mice

Phosphatidylcholine transfer protein (PC-TP) is a member of the steroidogenic acute regulatory transfer protein-related domain superfamily and is enriched in liver. To explore a role for PC-TP in hepatic cholesterol metabolism, Pctp −/− and wild-type C57BL/6J mice were fed a standard chow diet or a high-fat, high-cholesterol lithogenic diet. In chow-fed Pctp−/− mice, acyl CoA:cholesterol acyltransferase (Acat) activity was markedly increased, 3-hydroxy-3-methylglutaryl-CoA reductase activity was unchanged, and cholesterol 7α-hydroxylase activity was reduced. Consistent with increased Acat activity, esterified cholesterol concentrations in livers of Pctp−/− mice were increased, whereas unesterified cholesterol concentrations were reduced. Hepatic phospholipid concentrations were also decreased in the absence of PC-TP and consequently, unesterified cholesterol-to-phospholipid ratios in liver remained unchanged. The lithogenic diet downregulated 3-hydroxy-3-methylglutaryl-CoA reductase in wild-type and Pctp−/− mice, whereas Acat was increased only in wild-type mice. In response to the lithogenic diet, a greater reduction in cholesterol 7α-hydroxylase activity in Pctp−/− mice could be attributed to increased size and hydrophobicity of the bile salt pool. Despite higher hepatic phospholipid concentrations, the unesterified cholesterol-to-phospholipid ratio increased. The lack of Acat upregulation suggests that, in the setting of the dietary challenge, the capacity for esterification to defend against hepatic accumulation of unesterified cholesterol was exceeded in the absence of PC-TP expression. We speculate that regulation of cholesterol homeostasis is a physiological function of PC-TP in liver, which can be overcome with a cholesterol-rich lithogenic diet.

Upregulation of acyl-CoA:cholesterol acyltransferase in chronic renal failure

Chronic renal failure (CRF) is associated with profound abnormalities of lipid metabolism and accelerated arteriosclerotic cardiovascular disease. In a recent study, we found marked downregulation of hepatic lecithin-cholesterol acyltransferase, or LCAT, expression, which can account for impaired HDL maturation and depressed HDL cholesterol concentration in CRF. Here, we report on the effect of CRF on acyl-CoA:cholesterol acyltransferase (ACAT) expression. ACAT is an intracellular enzyme that catalyzes esterification of free cholesterol to cholesterol ester for storage or secretion. ACAT plays a major role in hepatic production and release of VLDL, intestinal absorption of cholesterol, foam cell formation, and atherogenesis. We examined hepatic expression of ACAT-1 and ACAT-2 mRNA (Northern blot) and protein (Western blot) abundance and total ACAT activity in male CRF rats (6 wk after 5/6 nephrectomy) and sham-operated controls. The CRF animals showed a significant reduction in creatinine clearance, marked hypertriglyceridemia, modest hypercholesterolemia, and significant upregulation of hepatic tissue ACAT-2 protein and mRNA abundance. In contrast, hepatic ACAT-1 mRNA and protein abundance were unaffected by CRF. Upregulation of ACAT-2 expression was accompanied by a significant increase in hepatic ACAT activity and a significant decrease in hepatic microsomal and whole liver free cholesterol concentration. Thus CRF results in significant upregulation of hepatic ACAT-2 (but not ACAT-1) expression and ACAT activity, which may, in part, contribute to the associated lipid disorders.

Effects of dietary oxysterols on coronary arteries in hyperlipidaemic hamsters

The aim of this study was to evaluate the effect of dietary oxysterols on coronary atherosclerosis and vasospasm. Golden Syrian hamsters were fed three diets with different lipid contents for 3 months: (1) a normolipidaemic diet containing 25 g corn oil–fish oil (4:1, w/w)/kg (group Low L); (2) a hyperlipidaemic diet composed of the normolipidaemic diet supplemented with 150 g lard+30 g cholesterol/kg (group High L); (3) a third diet, similar to the hyperlipidaemic diet, in which 4 g cholesterol/kg was replaced by a mixture of oxysterols (group High L+OS). The oxysterol mixture contained (g/kg): 5,6α-epoxycholesterol 211, 5,6β-epoxycholesterol 179, 7α-hydroxycholesterol 67, 7β-hydroxycholesterol (7βOH) 185, 7-ketocholesterol (7 K) 235; and trace amounts of 7-hydroperoxycholesterols (approximately 30 g/kg). Atherosclerosis was evaluated by measuring myocardial Ca, oxysterols and acyl-CoA cholesterol acyl transferase (ACAT) activity; furthermore, coronary reactivity to sodium nitroprusside (5×10-6 m) was measured and the morphology of coronary arteries was visualized by transmission electron microscopy. Coronary spasm was determined by evaluating reactivity to serotonin (5×10-6 m). Feeding the high-lipid diet (group High L) increased the plasma level of 7βOH, 7 K and cholestanetriol. The presence of oxysterols in the diet (group High L+OS) further increased the concentrations of 7βOH and 7 K in the plasma. However, as evidenced by myocardial Ca, ACAT activity and coronary reactivity to sodium nitroprusside, severe atherosclerosis did not develop during the 3-month diet. 7 K was increased in myocardial lipids of groups High L and High L+OS. Electron microscopy did not show the development of atherosclerosis in group High L, whereas vascular wall thickening, endothelial damage and smooth muscle cell proliferation and migration occurred when oxysterols were present in the food. Serotonin (5×10-6 m) induced exacerbated coronary vasoconstriction in group High L that was completely reversed by dietary oxysterols. In conclusion, dietary oxysterols exhibit anti-spasmodic properties, but they cannot be used as agents against excess dietary lipid-induced coronary spasm because of their atherogenic properties.

Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis

The initial and rate-limiting step in the classic pathway of bile acid biosynthesis is 7α-hydroxylation of cholesterol, a reaction catalyzed by cholesterol 7α-hydroxylase (CYP7A1). The effect of CYP7A1 overexpression on cholesterol homeostasis in human liver cells has not been examined. The specific aim of this study was to determine the effects of overexpression of CYP7A1 on key regulatory steps involved in hepatocellular cholesterol homeostasis, using primary human hepatocytes (PHH) and HepG2 cells. Overexpression of CYP7A1 in HepG2 cells and PHH was accomplished by using a recombinant adenovirus encoding a CYP7A1 cDNA (AdCMV-CYP7A1). CYP7A1 overexpression resulted in a marked activation of the classic pathway of bile acid biosynthesis in both PHH and HepG2 cells. In response, there was decreased HMG-CoA-reductase (HMGR) activity, decreased acyl CoA:cholesterol acyltransferase (ACAT) activity, increased cholesteryl ester hydrolase (CEH) activity, and increased low-density lipoprotein receptor (LDLR) mRNA expression. Changes observed in HMGR, ACAT, and CEH mRNA levels paralleled changes in enzyme specific activities. More specifically, LDLR expression, ACAT activity, and CEH activity appeared responsive to an increase in cholesterol degradation after increased CYP7A1 expression. Conversely, accumulation of the oxysterol 7α-hydroxycholesterol in the microsomes after CYP7A1 overexpression was correlated with a decrease in HMGR activity.