Dietary flaxseed inhibits atherosclerosis in the LDL receptor-deficient mouse in part through antiproliferative and anti-inflammatory actions

Dietary flaxseed has been shown to have potent antiatherogenic effects in rabbits. The purpose of the present study was to investigate the antiatherogenic capacity of flaxseed in an animal model that more closely represents the human atherosclerotic condition, the LDL receptor-deficient mouse (LDLrKO), and to identify the cellular mechanisms for these effects. LDLrKO mice were administered a regular diet (RG), a 10% flaxseed-supplemented diet (FX), or an atherogenic diet containing 2% cholesterol alone (CH) or supplemented with 10% flaxseed (CF), 5% flaxseed (CF5), 1% flaxseed (CF1), or 5% coconut oil (CS) for 24 wk. LDLrKO mice fed a cholesterol-supplemented diet exhibited a rise in plasma cholesterol without a change in triglycerides and an increase in atherosclerotic plaque formation. The CS mice exhibited elevated levels of plasma cholesterol, triglycerides, and saturated fatty acids and an increase in plaque development. Supplementation of the cholesterol-enriched diet with 10% (wt/wt) ground flaxseed lowered plasma cholesterol and saturated fatty acids, increased plasma ALA, and inhibited plaque formation in the aorta and aortic sinus compared with mice fed a diet supplemented with only dietary cholesterol. The expression of proliferating cell nuclear antigen (PCNA) and the inflammatory markers IL-6, mac-3, and VCAM-1 was increased in aortic tissue from CH and CS mice. This expression was significantly reduced or normalized when flaxseed was included in the diet. Our results demonstrate that dietary flaxseed can inhibit atherosclerosis in the LDLrKO mouse through a reduction of circulating cholesterol levels and, at a cellular level, via antiproliferative and anti-inflammatory actions.     

Deficiency of PPARalpha disturbs the response of lipogenic flux and of lipogenic and cholesterogenic gene expression to dietary cholesterol in mouse white adipose tissue

PPARalpha-deficiency in mice fed a high-carbohydrate, low-cholesterol diet was associated with a decreased weight of epididymal adipose tissue and an increased concentration of adipose tissue cholesterol. Consumption of a high (2% w/w) cholesterol diet resulted in a further increase in the concentration of cholesterol and a further decrease in epididymal fat pad weight in PPARalpha-null mice, but had no effect in the wild-type. These reductions in fat pad weight were associated with an increase in hepatic triacylglycerol content, indicating that both PPARalpha-deficiency and cholesterol altered the distribution of triacylglycerol in the body. Adipose tissue de novo lipogenesis was increased in PPARalpha-null mice and was further enhanced when they were fed a cholesterol-rich diet; no such effect was observed in the wild-type mice. The increased lipogenesis in the chow-fed PPARalpha-null mice was accompanied paradoxically by lower mRNA expression of SREBP-1c and its target genes, acetyl-CoA carboxylase and fatty acid synthase. Consumption of a high-cholesterol diet increased the mRNA expression of these genes in the PPARalpha-deficient mice but not in the wild-type. De novo cholesterol synthesis was not detectable in the adipose tissue of either genotype despite a relatively high expression of the mRNA's encoding SREBP-2 and 3-hydroxy-3-methylglutaryl Coenzyme A reductase. The mRNA expression of these genes and of the LDL-receptor in adipose tissue of the PPARalpha-deficient mice was lower than that of the wild-type and was not downregulated by cholesterol feeding. The results suggest that PPARalpha plays a role in adipose tissue cholesterol and triacylglycerol homeostasis and prevents cholesterol-mediated changes in de novo lipogenesis.     

Effect of delta22-5beta-taurocholenic acid on hepatic cholesterol and fatty acid in gold thioglucose obese mice fed low- or high-fat diets

We previously demonstrated that hyperglycemic-obese (obob) mice fed a 1% corn oil diet accumulated 10 times as much hepatic cholesterol as did their non-obese (+/?) littermates fed this diet because of difficulty in removal of cholesterol from the liver rather than from increased synthesis. Furthermore, feeding the bile acid analog Delta(22)-5beta-taurocholenic acid completely prevented the accumulation of hepatic cholesterol in obob mice fed the 1% corn oil diet. The hypothesis to be tested in the current study is that these aspects of cholesterol metabolism in the obob mouse do not occur in the hyperinsulinemic and insulin-resistant gold thioglucose obese mouse. Gold thioglucose obese (gtgo) and non-obese (ngtgo) mice were fed diets containing either 1% corn oil or 40% lard each with or without added taurocholenic acid for 6 weeks and then given a 250 mg meal of [U-(14)C]-glucose with incorporation of label into hepatic cholesterol and fatty acid measured 2 hours later. Consistent with earlier results in the obob model, incorporation of labeled glucose was significantly increased in obese compared with non-obese mice fed 1% corn oil and significantly reduced either by feeding 40% lard or by adding taurocholenic acid to the diet. In addition, taurocholenic acid greatly increased incorporation of labeled glucose into hepatic cholesterol in obese or non-obese mice fed either diet. In contrast to obob mice, the percentage of fat in the liver of gtgo mice was increased only 50% compared with ngtgo mice. The comparable increase in obob mice was 480%. Hepatic cholesterol did not increase significantly in the liver of gtgo mice fed 1% corn oil when compared with the ngtgo controls. The comparable increase in obob mice fed 1% corn oil was 350%. Also in marked contrast to obob mice, feeding taurocholenic acid increased hepatic cholesterol compared with non-obese controls fed either diet. The results are discussed in the light of the presence of circulating leptin in gtgo but not in obob mice.     

Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia

Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P < 0.05) exhibited by CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.     

Effects of a Daisaikoto Extract on the Biliary Constituents in Mice and Rats

The effects of an extract of Daisaikoto (a traditional Chinese medicine) on biliary constituents was studied in mice fed with a lithogenic diet containing 0.5% cholesterol and 0.25% sodium cholate (the control diet) and in rats fed with a cholesterol-free diet. The Daisaikoto extract was added to the control diet at a level of 0.5%. A high incidence of cholesterol gallstones were found in the control mice, but not in the mice given the Daisaikoto extract. This difference was concluded to have been due to the absolute concentration of bile acid in the bile being significantly higher in the mice given the Daisaikoto extract than in the control mice. The result from rats fed with the cholesterol-free diet also demonstrated that the Daisaikoto extract caused an increase in the absolute concentration of bile acid in the bile.     

Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

Ecabet sodium inhibits Helicobacter pylori lipopolysaccharide-induced activation of NADPH oxidase 1 or apoptosis of guinea pig gastric mucosal cells

Cholestatic hepatitis is frequently found in Niemann-Pick C (NPC) disease. We studied the influence of diet and the low density lipoprotein receptor (LDLR, Ldlr in mice, known to be the source of most of the stored cholesterol) on liver disease in the mouse model of NPC. Npc1-/- mice of both sexes, with or without the Ldlr knockout, were fed a 18% fat, 1% cholesterol ("high-fat") diet and were evaluated by chemical and histological methods. Bile acid transporters [multidrug resistance protein (Mrps) 1-5; Ntcp, Bsep, and OatP1, 2, and 4] were quantitated by real-time RT-PCR. Many mice died prematurely (within 6 wk) with hepatomegaly. Histopathology showed an increase in macrophage and hepatocyte lipids independent of Ldlr genotype. Non-zone-dependent diffuse fibrosis was found in the surviving mice. Serum alanine aminotransferase was elevated in Npc1-/- mice on the regular diet and frequently became markedly elevated with the high-fat diet. Serum cholesterol was increased in the controls but not the Npc1-/- mice on the high-fat diet; it was massively increased in the Ldlr-/- mice. Esterified cholesterol was greatly increased by the high-fat diet, independent of Ldlr genotype. gamma-Glutamyltransferase was also elevated in Npc1-/- mice, more so on the high-fat diet. Mrps 1-5 were elevated in Npc1-/- liver and became more elevated with the high-fat diet; Ntcp, Bsep, and OatP2 were elevated in Npc1-/- liver and were suppressed by the high-fat diet. In conclusion, Npc1-/- mice on a high-fat diet provide an animal model of NPC cholestatic hepatitis and indicate a role for altered bile acid transport in its pathogenesis.     

Dietary fat saturation produces lipid modifications in peritoneal macrophages of mouse

We investigated the effects of a saturated fat diet on mice lipid metabolism in resident peritoneal macrophages. Male C57BL/6 mice were weaned at 21 days of age and assigned to either the experimental diet, containing coconut oil (COCO diet), or the control diet, containing soybean oil as fat source. Fat content of each diet was 15% (w/w). Mice were fed for 6 weeks until sacrifice. In plasma of mice fed the COCO diet, the concentration of triglyceride, total cholesterol, HLD- and (LDL+VLDL)-cholesterol, and thiobarbituric acid-reactive substances (TBARS) increased, without changes in phospholipid concentration, compared with the controls. In macrophages of COCO-fed mice, the concentration of total (TC), free and esterified cholesterol, triglyceride, phospholipid (P) and TBARS increased, while the TC/P ratio did not change. The phospholipid compositions showed an increase of phosphatidylcholine and phosphatidylserine + phosphadytilinositol, a decrease of phosphatidylethanolamine, and no change in phosphatidylglycerol. (3)H(2)O incorporation into triglyceride and phospholipid fractions of macrophages increased, while its incorporation into free cholesterol decreased. Incorporation of [(3)H]cholesterol into macrophages of COCO-fed mice and the fraction of [(3)H]cholesterol ester increased. COCO diet produced an increase in myrystic, palmitic and palmitoleic acids proportion, a decrease in linoleic and arachidonic acids and no changes in stearic and oleic acids, compared with the control. Also, a higher relative percentage of saturated fatty acid and a decrease in unsaturation index (p <0.001) were observed in macrophages of COCO-fed mice. These results indicate that the COCO-diet, high in saturated fatty acids, alters the lipid metabolism and fatty acid composition of macrophages and produces a significant degree of oxidative stress.     

Delineation of molecular changes in intrahepatic cholesterol metabolism resulting from diminished cholesterol absorption

The absorption of cholesterol by the small intestine is a major route for the net entry of cholesterol into the body and can therefore affect the plasma low density lipoprotein-cholesterol (LDL-C) concentration. These studies used ezetimibe, a potent inhibitor of cholesterol absorption, to delineate the biochemical and molecular changes in intrahepatic metabolism and biliary lipid secretion when there is a major reduction in chylomicron cholesterol delivery to the liver. In female LDL receptor (LDLR)-deficient (LDLR-/-) mice fed a basal diet containing ezetimibe (0-10 mg/day/kg body weight), cholesterol absorption was reduced up to 91%, fecal neutral sterol excretion was increased up to 4.7-fold, and plasma total cholesterol concentrations decreased by up to 18%. Blocking cholesterol absorption prevented the accumulation of very low density lipoproteins and LDL in the circulation of LDLR-/- mice fed a lipid-rich diet. In female LDLR+/+ mice fed the lipid-rich diet with ezetimibe, the relative mRNA level for the LDLR in the liver was 2-fold greater than in matching mice given the lipid-rich diet alone. We conclude that in the mouse the reduction in plasma LDL-C levels induced by blocking cholesterol absorption reflects both a diminished rate of LDL-C production and a modest increase in hepatic LDLR expression.     

Hypocholesterolemic effects of fatty acid bile acid conjugates (FABACs) in mice

Fatty acid bile acid conjugates (FABACs) prevent and dissolve cholesterol gallstones and prevent diet induced fatty liver, in mice. The present studies aimed to test their hypocholesterolemic effects in mice. Gallstone susceptible (C57L/J) mice, on high fat (HFD) or regular diet (RD), were treated with the conjugate of cholic acid with arachidic acid (FABAC; Aramchol). FABAC reduced the elevated plasma cholesterol levels induced by the HFD. In C57L/J mice, FABAC reduced plasma cholesterol by 50% (p < 0.001). In mice fed HFD, hepatic cholesterol synthesis was reduced, whereas CYP7A1 activity and expression were increased by FABAC. The ratio of fecal bile acids/neutral sterols was increased, as was the total fecal sterol excretion. In conclusion, FABACs markedly reduce elevated plasma cholesterol in mice by reducing the hepatic synthesis of cholesterol, in conjunction with an increase of its catabolism and excretion from the body.     

Increased susceptibility to mouse hepatitis virus type 3 (MHV3) infection induced by a hypercholesterolaemic diet with increased adsorption of MHV3 to primary hepatocyte cultures

The administration of a hypercholesterolaemic (HC) diet rendered genetically resistant A/J mice susceptible to mouse hepatitis 3 (MHV3) infection. The animals died cf acute hepatitis with high viral titres in the liver accompanied by many necrotic foci and high serum transaminase levels. Resistance to virus was re-established by refeeding HC mice with a normal diet for 2 weeks. This of modification by pathogenesis was accompanied by an increase in the susceptibility of hepatocyte cultures from HC mice to MHV3 and could be explained by an enhancement in virus adsorption. We hypothesize that the incorporation of cholesterol into the plasma membranes of hepatocytes of HC mice, thereby decreasing the membrane fluidity, may lead to an increase in the availability of virus receptors.     

Lecithin:cholesterol acyltransferase deficiency increases atherosclerosis in the low density lipoprotein receptor and apolipoprotein E knockout mice.

The purpose of the present study was to test the hypothesis that lecithin:cholesterol acyltransferase (LCAT) deficiency would accelerate atherosclerosis development in low density lipoprotein (LDL) receptor (LDLr-/-) and apoE (apoE-/-) knockout mice. After 16 weeks of atherogenic diet (0.1% cholesterol, 10% calories from palm oil) consumption, LDLr-/- LCAT-/- double knockout mice, compared with LDLr-/- mice, had similar plasma concentrations of free (FC), esterified (EC), and apoB lipoprotein cholesterol, increased plasma concentrations of phospholipid and triglyceride, decreased HDL cholesterol, and 2-fold more aortic FC (142 +/- 28 versus 61 +/- 20 mg/g protein) and EC (102 +/- 27 versus 61+/- 27 mg/g). ApoE-/- LCAT-/- mice fed the atherogenic diet, compared with apoE-/- mice, had higher concentrations of plasma FC, EC, apoB lipoprotein cholesterol, and phospholipid, and significantly more aortic FC (149 +/- 62 versus 109 +/- 33 mg/g) and EC (101 +/- 23 versus 69 +/- 20 mg/g) than did the apoE-/- mice. LCAT deficiency resulted in a 12-fold increase in the ratio of saturated + monounsaturated to polyunsaturated cholesteryl esters in apoB lipoproteins in LDLr-/- mice and a 3-fold increase in the apoE-/- mice compared with their counterparts with active LCAT. We conclude that LCAT deficiency in LDLr-/- and apoE-/- mice fed an atherogenic diet resulted in increased aortic cholesterol deposition, likely due to a reduction in plasma HDL, an increased saturation of cholesteryl esters in apoB lipoproteins and, in the apoE-/- background, an increased plasma concentration of apoB lipoproteins.     

Streptozotocin-induced diabetes in human apolipoprotein B transgenic mice. Effects on lipoproteins and atherosclerosis

The effects of diabetes and lipoprotein lipase (LpL) on plasma lipids were studied in mice expressing human apolipoprotein B (HuBTg). Our overall objective was to produce a diabetic mouse model in which the sole effects of blood glucose elevation on atherosclerosis could be assessed. Mice were made diabetic by intraperitoneal injection of streptozotocin, which led to a 2- to 2. 5-fold increase in plasma glucose. Lipids were assessed in mice on chow and on an atherogenic Western type diet (WTD), consisting of 21% (wt/wt) fat and 0.15% (wt/wt) cholesterol. Plasma triglyceride and cholesterol were the same in diabetic and non-diabetic mice on the chow diet. On the WTD, male diabetic HuBTg mice had a >50% increase in plasma cholesterol and more very low density lipoprotein (VLDL) cholesterol and triglyceride as assessed by FPLC analysis. A Triton study showed no increase in triglyceride or apolipoprotein B production, suggesting that the accumulation of VLDL was due to a decrease in lipoprotein clearance. Surprisingly, the VLDL increase in these mice was not due to a decrease in LpL activity in postheparin plasma. To test whether LpL overexpression would alter these diabetes-induced lipoprotein changes, HuBTg mice were crossed with mice expressing human LpL in muscle. LpL overexpression reduced plasma triglyceride, but not cholesterol, in male mice on WTD. Aortic root atherosclerosis assessed in 32-week-old mice on the WTD was not greater in diabetic mice. In summary, diabetes primarily increased plasma VLDL in HuBTg mice. LpL activity was not decreased in these animals. However, additional LpL expression eliminated the diabetic lipoprotein changes. These mice did not have more atherosclerosis with diabetes.     

ApoA-II expression in CETP transgenic mice increases VLDL production and impairs VLDL clearance

Apolipoprotein (apo)A-II is a major high density lipoprotein (HDL) protein; however, its role in lipoprotein metabolism is largely unknown. Transgenic (Tg) mice that overexpress human apoA-II present functional lecithin: cholesterol acyltransferase deficiency, HDL deficiency, hypertriglyceridemia and, when fed an atherogenic diet, increased non-HDL cholesterol and increased susceptibility to atherosclerosis. In contrast to humans, mice do not present cholesteryl ester transfer protein (CETP) activity in plasma. To study the in vivo interaction of these two proteins, we crossbred human apoA-II and CETP-Tg mice. CETP x apoA-II-Tg mice fed an atherogenic diet, compared with CETP-Tg mice presented a 2-fold decrease in HDL cholesterol and a quantitatively similar increase in total plasma cholesterol and percentage of free cholesterol, non-HDL cholesterol, and free fatty acids, together with a remarkable 112-fold increase in plasma triglycerides. Plasma triglycerides in CETP x apoA-II-Tg mice were mainly associated with very low density lipoproteins (VLDL), which were also enriched in protein content, and resulted from a combination of higher production rate compared with both of their progenitors and non-Tg control mice, and decreased catabolism compared only with CETP-Tg mice. These results show CETP x apoA-II-Tg mice to be a good model with which to study mechanisms leading to VLDL overproduction and suggest that CETP and, in particular apoA-II, may play a role in the regulation of VLDL metabolism.     

Skeletal effects of cholesterol feeding.

The effect of a 4% supplement of cholesterol to a standard diet on knee joints and vertebral columns was investigated in male mice of strain C57B1. The experimental diet was fed from the time of weaning through the 18-month period of observation or from the age of 1 year to the end of the experiment at 18 months of age. The incidence of osteoarthrosis was increased in mice fed cholesterol from the age of 12 months on. The incidence of spondylosis was increased after lifelong feeding as well as after feeding of cholesterol during the second year of life. This increase involved both, spondylosis associated with or uncomplicated by prolapse of intervertebral discs. The incidence of simple disc prolapse was not affected by the experimental diet.     

Increased Hepatic Lipogenesis Elevates Liver Cholesterol Content

Cardiovascular diseases (CVDs) are the most common cause of death in patients with nonalcoholic fatty liver disease (NAFLD) and dyslipidemia is considered at least partially responsible for the increased CVD risk in NAFLD patients. The aim of the present study is to understand how hepatic de novo lipogenesis influences hepatic cholesterol content as well as its effects on the plasma lipid levels. Hepatic lipogenesis was induced in mice by feeding a fat-free/high-sucrose (FF/HS) diet and the metabolic pathways associated with cholesterol were then analyzed. Both liver triglyceride and cholesterol contents were significantly increased in mice fed an FF/HS diet. Activation of fatty acid synthesis driven by the activation of sterol regulatory element binding protein (SREBP)-1c resulted in the increased liver triglycerides. The augmented cholesterol content in the liver could not be explained by an increased cholesterol synthesis, which was decreased by the FF/HS diet. HMG-CoA reductase protein level was decreased in mice fed an FF/HS diet. We found that the liver retained more cholesterol through a reduced excretion of bile acids, a reduced fecal cholesterol excretion, and an increased cholesterol uptake from plasma lipoproteins. Very low-density lipoprotein-triglyceride and -cholesterol secretion were increased in mice fed an FF/HS diet, which led to hypertriglyceridemia and hypercholesterolemia in Ldlr^-/- mice, a model that exhibits a more human like lipoprotein profile. These findings suggest that dietary cholesterol intake and cholesterol synthesis rates cannot only explain the hypercholesterolemia associated with NAFLD, and that the control of fatty acid synthesis should be considered for the management of dyslipidemia.     

Alteration of a macrophages inflammatory protein-related protein-2 (MRP-2) response by high fat and cholesterol diet in mice

Macrophage inflammatory protein-related protein-2 (MRP-2) is a new member of the CC chemokine family that is recently identified in murine macrophages. MRP-2 is involved in leukocyte trafficking and activation, which can be implicated in inflammatory diseases including atherosclerosis. Little is known about the involvement of this novel chemokine MRP-2 in the pathogenesis of atherosclerosis. To explore the possible association of the MRP-2 with atherosclerosis, we investigated the effects of atherogenic diet on MRP-2 expression in mice. Male C57BL/6 mice were fed a high fat and cholesterol diet (20% fat and 1.5% cholesterol) or a control diet based on AIN-76 for 5, 10, or 14 weeks. The levels of total cholesterol, LDL cholesterol, and F2-isoprostanes in plasma were measured using appropriate enzymatic assays. Tumor necrosis factor alpha (TNF alpha) and MCP-1 release by peritoneal macrophages was determined by ELISA. The mRNA expression level of the MRP-2 was measured by RT-PCR. The levels of total cholesterol, LDL-cholesterol, and 8-iso-prostaglandin F2 alpha in plasma, well-known indexes of atherosclerosis, were significantly increased in the high fat and cholesterol diet group compared to those in the control. A significant increase in the TNF alpha and MCP-1 production by macrophages was also observed in the group fed high fat and cholesterol diet. The mRNA expression of MRP-2 was upregulated by oxLDL treatment in vitro and feeding a high fat and cholesterol diet in vivo at the late stage of atherosclerosis. These results suggest that MRP-2 may be an important contributing factor in the development of atherosclerosis.     

Inflammation and skin cholesterol in LDLr−/−, apoA-I−/− mice: link between cholesterol homeostasis and self-tolerance?

Diet-fed low density lipoprotein receptor-deficient/apolipoprotein A-I-deficient (LDLr-/-, apoA-I-/-) mice accumulate a 10-fold greater mass of cholesterol in their skin despite a 1.5- to 2-fold lower plasma cholesterol concentration compared with diet-fed LDLr-/- mice. The accumulation of cholesterol predominantly in the skin has been shown to occur in a growing number of other hypercholesterolemic double knockout mouse models sharing deficits in genes regulating cellular cholesterol homeostasis. Exploring the relationship between cholesterol balance and inflammation, we have examined the time course of cholesterol accumulation in a number of extrahepatic tissues and correlated with the onset of inflammation in diet-fed LDLr-/-, apoA-I-/- mice. After 4 weeks of diet, LDLr-/-, apoA-I-/- mice showed a significant increase in skin cholesterol mass compared with LDLr-/- mice. In addition, after 4 weeks on the diet, cholesterol accumulation in the skin was also found to be associated with macrophage infiltration and accompanied by increases in tumor necrosis factor-alpha, cyclooxygenase-2, and langerin mRNA, which were not seen in the liver. Overall, these data suggest that as early as 4 weeks after starting the diet, the accumulation of skin cholesterol and the onset of inflammation occur concurrently. In summary, the use of hypercholesterolemic LDLr-/-, apoA-I-/- mice may provide a useful tool to investigate the role that apoA-I plays in maintaining cholesterol homeostasis and its relationship to inflammation.     

Control of cholesterol biosynthesis,uptake and storage in hepatocytes by Cideb

Cideb, a member of CIDE family proteins, has emerged as an important regulator in the development of obesity and diabetes by controlling fatty acid synthesis and VLDL secretion in hepatocytes. Here, we investigated the role of Cideb in cholesterol biosynthesis, uptake and storage in the liver by using Cideb-null mice as a model system. Cideb-null mice and wild-type mice were treated with normal diet (ND) or high cholesterol diet (HCD) for one month. The metabolic parameters of cholesterol metabolism and expression profiles of genes in cholesterol biosynthesis and storage were measured. Cideb-null mice had lower levels of plasma cholesterol and LDL when fed with both ND and HCD and increased rate of cholesterol absorption. Furthermore, the liver of Cideb-null mice has lower rates of cholesterol biosynthesis and reduced expression levels of sterol response element-binding protein (SREBP) cleavage-activation protein (SCAP), and lower levels of nuclear form of SREBP2 and its downstream target genes in cholesterol biosynthesis pathway under a normal diet treatment. On the contrary, hepatic cholesterol biosynthesis rate between wild-type and Cideb-null mice was similar after high cholesterol diet treatment. Interestingly, hepatic cholesterol storage in the liver of Cideb-null mice was significantly increased due to its increased LDL receptor (LDLR) and acyl-CoA cholesterol acyltransferase (ACAT) expression. Finally, we observed drastically reduced cholesterol levels in the heart of Cideb-null mice fed with a high cholesterol diet. Overall, our data suggest that Cideb is a novel regulator in controlling cholesterol homeostasis in the liver. Therefore, Cideb could serve as an important therapeutical target for the treatment of atherosclerosis and cardiovascular diseases.     

Resistance of SHP-null mice to bile acid-induced liver damage

The orphan nuclear hormone receptor SHP (gene designation NROB2) is an important component of a negative regulatory cascade by which high levels of bile acids repress bile acid biosynthesis. Short term studies in SHP null animals confirm this function and also reveal the existence of additional pathways for bile acid negative feedback regulation. We have used long term dietary treatments to test the role of SHP in response to chronic elevation of bile acids, cholesterol, or both. In contrast to the increased sensitivity predicted from the loss of negative feedback regulation, the SHP null mice were relatively resistant to the hepatotoxicity associated with a diet containing 0.5% cholic acid and the much more severe effects of a diet containing both 0.5% cholic acid and 2% cholesterol. This was associated with decreased hepatic accumulation of cholesterol and triglycerides in the SHP null mice. There were also alterations in the expression of a number of genes involved in cholesterol and bile acid homeostasis, notably cholesterol 12alpha-hydroxylase (CYP8B1), which was strongly reexpressed in the SHP null mice, but not the wild type mice fed either bile acid containing diet. This contrasts with the strong repression of CYP8B1 observed with short term bile acid feeding, as well as the effects of long term feeding on other bile acid biosynthetic enzymes such as cholesterol 7alpha-hydroxylase (CYP7A1). CYP8B1 expression could contribute to the decreased toxicity of the chronic bile acid treatment by increasing the hydrophilicity of the bile acid pool. These results identify an unexpected role for SHP in hepatotoxicity and suggest new approaches to modulating effects of chronically elevated bile acids in cholestasis.