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We present an assay to study cholesterol crystallization that is fast, facile, and highly reproducible. Cholesterol crystallization from metastable vesicles was induced upon addition of bile salts and depended on the hydrophobicity of the bile salt used and the cholesterol-to-phospholipid ratio of the vesicles. Bile salt-induced crystallization was stimulated upon addition of Con A-binding glycoproteins (CABGs), comparable to the results of the same CABGs in a crystal growth assay. The onset time and total measuring time, however, were much shorter. This assay might, moreover, provide a tool to study the mechanism of cholesterol crystallization in more detail.

Background & Aims Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO 4 precipitation were studied. Methods Binding of CBP to GBM was assessed by inhibition of the fluorescence of the complex of GBM with bis-1,8-anilinonaphthalene sulfonic acid (bisANS). The effects of the proteins on precipitation of CaHPO 4 were assessed by nephelometry and gravimetry. Precipitates were analyzed for calcium, phosphate, and protein. Results CBP and bis-ANS competitively displaced each other from 30 binding sites on mucin, with a 1:1 stoichiometry and similar affinity. The rate of precipitation of CaHPO 4 was retarded by mucin and CBP. Precipitate mass was unaffected by GBM alone but decreased with the addition of CBP. Complexing CBP with GBM abolished or moderated this latter effect, altered precipitate morphology, and changed the stoichiometric ratios of Ca to PO 4 in the precipitates from 1:1 to 3:2. Mucin and CBP were incorporated into the precipitates. Conclusions These studies suggest that the formation of calcium-containing gallstones is a biomineralization process regulated by both GBM and CBP.

Chronic cholangiopathies often lead to fibrosis, as a result of a perpetuated wound healing response, characterized by increased inflammation and excessive deposition of proteins of the extracellular matrix. Our previous studies have shown that food deprivation suppresses the immune response, which led us to postulate its beneficial effects on pathology in liver fibrosis driven by portal inflammation. We investigated the consequences of fasting on liver fibrosis in Abcb4(-/-) mice that spontaneously develop it due to a lack of phospholipids in bile. The effect of up to 48 h of food deprivation was studied by gene expression profiling, (immuno) histochemistry, and biochemical assessments of biliary output, and hepatic and plasma lipid composition. In contrast to increased biliary output in the wild type counterparts, bile composition in Abcb4(-/-) mice remained unchanged with fasting and did not influence the attenuation of fibrosis. Markers of inflammation, however, dramatically decreased in livers of Abcb4(-/-) mice already after 12 h of fasting. Reduced presence of activated hepatic stellate cells and actively increased tissue remodeling further propelled a decrease in parenchymal fibrosis in fasting. This study is the first to show that food deprivation positively influences liver pathology in a fibrotic mouse model for chronic cholangiopathies, opening a door for new strategies to improve liver regeneration in chronic disease. (C) 2013 Elsevier B.V. All rights reserved.

The iminosugar N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynoijirimycin (AMP-DNM), an inhibitor of glycosphingolipid (GSL) biosynthesis is known to ameliorate diabetes, insulin sensitivity and to prevent liver steatosis in ob/ob mice. Thus far the effect of GSL synthesis inhibition on pre-existing NASH has not yet been assessed. To investigate it, LDLR(-/-) mice were kept on a western-type diet for 12 weeks to induce NASH. Next, the diet was continued for 6 weeks in presence or not of AMP-DNM in the diet. AMP-DNM treated mice showed less liver steatosis, inflammation and fibrosis. Induction of fatty acid beta-oxydation was observed, as well as a reduction of plasma lipids. Our study demonstrates that AMP-DNM treatment is able to significantly correct pre-existing NASH, suggesting that inhibiting GSL synthesis may represent a novel strategy for the treatment of this pathology.

Alterations in intestinal microbiota are associated with obesity and insulin resistance. We studied the effects of infusing intestinal microbiota from lean donors to male recipients with metabolic syndrome on the recipients' microbiota composition and glucose metabolism. Subjects were assigned randomly to groups that were given small intestinal infusions of allogenic or autologous microbiota. Six weeks after infusion of microbiota from lean donors, insulin sensitivity of recipients increased (median rate of glucose disappearance changed from 26.2 to 45.3 mu mol/kg/min; P

Human bile contains cholesterol crystallization—stimulating proteins that can be isolated by concanavalin A—Sepharose chromatography. In the past few years an increasing number of different pronucleating proteins have been identified in the concanavalin A—binding fraction. In this study we attempted to estimate the relative contribution of a number of these proteins to total concanavalin A—binding pronucleating activity. For this purpose, concanavalin A—binding glycoproteins were isolated from gallbladder bile samples from 12 patients with gallstones. The role of IgA, IgG and IgM and α1-acid glycoprotein was investigated by means of immunoextraction. No decrease in crystallization-promoting activity was observed after precipitation of more than 98% of the different immunoglobulins. In addition, removal of more than 95% of α1-acid glycoprotein from different concanavalin A—binding fractions had no significant effect on cholesterol crystallization—promoting activity. The influence of fibronectin was estimated by addition of physiological concentrations to a model bile system. At these concentrations fibronectin did not promote crystallization. From these data we conclude that immunoglobulins, α1-acid glycoprotein and probably also fibronectin do not significantly contribute to total concanavalin A—binding activity. (HEPATOLOGY 1994;20:626–632).

The main disadvantage of endoscopic insertion of an endoprosthesis is the tendency of the stent to clog after a few months. In this study we determined the role of bile viscosity in stent clogging. Sixty patients were stented with 10 Fr 11-cm stents. The stents were electively removed after 2 months, and a bile sample was obtained simultaneously. Bile viscosity was measured with a coaxial rotation viscometer. The influence of treatment with antibiotics and a mucolytic agent on viscosity was assessed in a randomized trial. Bile viscosity correlated significantly with DNA and total protein concentration. After treatment with either N-acetylcysteine or co-trimoxazole a lower mean value of the viscosity was observed, but this was not statistically significant. There was no correlation between bile viscosity and the amount of sludge adhering to the stents. In most patients bile viscosity plays a limited role in stent clogging. Only in patients with excessively high viscosity do mucolytic agents or treatment with antibiotics seem to have a role

1. Human gallbladder mucin has been implicated as playing a role in the pathogenesis of gallstones. In previous studies no differences have been found in the content or composition of mucins derived from control bile or cholesterol gallstone bile. Until now, no differences were also found between these two groups of mucins with regard to their ability to cause cholesterol nucleation. In the accompanying paper we have reported that there is a strong heterogeneity of gallbladder mucins derived from individual patients (M. J. A. van Wijland, J. H. Klinkspoor, L. Th. de Wit, R. P. J. Oude Elferink, G. N. J. Tytgat and A. K. Groen, Clin Sci 1994; 86: 67–74). In the present study we further investigated a possible patient to patient heterogeneity of mucin by means of immunological and functional characterization of mucins isolated from hepatic bile of six different patients with gallstones. 2. Considerable heterogeneity was found. Two of the mucins barely reacted with a polyclonal anti-mucin antibody, whereas the other four mucins reacted very strongly. Lectin-binding studies indicated that the glycans of these two mucins expressed less D-N-acetylgalactosamine residues than the other four mucins. This was confirmed by analyses of the glycan compositions. These studies furthermore indicated that the glycans were of the O-linked type, contained α-D-N-acetylglucosamine and were fucosylated, sialy-lated and sulphated to different extents. Except for a strong heterogeneity in the sugar composition of the mucins, heterogeneity was also found in the biological activity of the mucins. The two immunologically diverging mucins nucleated cholesterol from model bile 1–2 days earlier and also caused an almost threefold more rapid rupture of cholesterol/phospholipid vesicles than the other mucins. 3. We conclude that considerable differences exist between mucins derived from individual patients and that the heterogeneity in glycan composition might play a role in the pathogenesis of gallstone disease.

Objective: Regular physical activity decreases the risk for atherosclerosis but underlying mechanisms are not fully understood. We questioned whether voluntary wheel running provokes specific modulations in cholesterol turnover that translate into a decreased atherosclerotic burden in hypercholesterolemic mice.Methods: Male LDLR-deficient mice (8 weeks old) had either access to a voluntary running wheel for 12 weeks (RUN) or remained sedentary (CONTROL). Both groups were fed a western-type/high cholesterol diet. Running activity and food intake were recorded. At 12 weeks of intervention, feces, bile and plasma were collected to determine fecal, biliary and plasma parameters of cholesterol metabolism and plasma cytokines. Atherosclerotic lesion size was determined in the aortic root.Results: RUN weighed less (similar to 13%) while food consumption was increased by 17% (p = 0.004). Plasma cholesterol levels were decreased by 12% (p = 0.035) and plasma levels of pro-atherogenic lipoproteins decreased in RUN compared to control. Running modulated cholesterol catabolism by enhancing cholesterol turnover: RUN displayed an increased biliary bile acid secretion (68%, p = 0.007) and increased fecal bile acid (93%, p = 0.009) and neutral sterol (33%, p = 0.002) outputs compared to control indicating that reverse cholesterol transport was increased in RUN. Importantly, aortic lesion size was decreased by similar to 33% in RUN (p = 0.033).Conclusion: Voluntary wheel running reduces atherosclerotic burden in hypercholesterolemic mice. An increased cholesterol turnover, specifically its conversion into bile acids, may underlie the beneficial effect of voluntary exercise in mice. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Glycosphingolipids are structural membrane components, residing largely in the plasma membrane with their sugar-moieties exposed at the cell's surface. In recent times a crucial role for glycosphingolipids in insulin resistance has been proposed. A chronic state of insulin resistance is a rapidly increasing disease condition in Western and developing countries. It is considered to be the major underlying cause of the metabolic syndrome, a combination of metabolic abnormalities that increases the risk for an individual to develop Type 2 diabetes, obesity, cardiovascular disease, polycystic ovary syndrome and nonalcoholic fatty liver disease. As discussed in this chapter, the evidence for a direct regulatory interaction of glycosphingolipids with insulin signaling is still largely indirect. However, the recent finding in animal models that pharmacological reduction of glycosphingolipid biosynthesis ameliorates insulin resistance and prevents some manifestations of metabolic syndrome, supports the view that somehow glycosphingolipids act as critical regulators, Importantly, since reductions in glycosphingolipid biosynthesis have been found to be well tolerated, such approaches may have a therapeutic potential.

Dietary plant sterols (PS) reduce serum total and LDL-cholesterol in hyperlipidemic animal models and in humans. This hypocholesterolemic effect is generally ascribed to inhibition of cholesterol absorption. However, whether this effect fully explains the reported strong induction of neutral sterol excretion upon plant sterol feeding is not known. Recent data demonstrate that the intestine directly mediates plasma cholesterol excretion into feces, i.e., without involvement of the hepato-biliary route.Objective: Aim of this study was to determine whether stimulation of fecal neutral sterol loss during PS feeding is (partly) explained by increased intestinal cholesterol excretion and to assess the role of the cholesterol transporter Abcg5/Abcg8 herein.Methods and Results: Wild-type mice were fed a control diet or diets enriched with increasing amounts of PS (1%, 2%, 4% or 8%, wt/wt) for two weeks. In addition, Abcg5(-/-) mice were fed either control or 8% PS diet. PS feeding resulted in a dose-dependent decrease of fractional cholesterol absorption (similar to 2-7-fold reduction) in wild-type mice and similar to 80% reduction in Abcg5(-/-) mice. Furthermore, PS feeding led to a strong, dose-independent induction of neutral sterol excretion (3.4-fold in wild-types and 2.7-fold in Abcg5(-/-) mice) without changes in biliary cholesterol secretion. It was calculated that PS feeding stimulated intestinal cholesterol excretion by similar to 500% in wild-type mice and by similar to 250% in Abcg5(-/-).Conclusions: Our data indicate that in mice the cholesterol-lowering effects of PS are to a large extent attributable to stimulation of intestinal, non-bile derived, cholesterol excretion. The Abcg5/Abcg8 heterodimer is involved in facilitating this PS-induced flux of cholesterol.

Glycosphingolipids are structural membrane components, residing largely in the plasma membrane with their sugar-moieties exposed at the cell's surface. In recent times a crucial role for glycosphingolipids in insulin resistance has been proposed. A chronic state of insulin resistance is a rapidly increasing disease condition in Western and developing countries. It is considered to be the major underlying cause of the metabolic syndrome, a combination of metabolic abnormalities that increases the risk for an individual to develop Type 2 diabetes, obesity, cardiovascular disease, polycystic ovary syndrome and nonalcoholic fatty liver disease. As discussed in this chapter, the evidence for a direct regulatory interaction of glycosphingolipids with insulin signaling is still largely indirect. However, the recent finding in animal models that pharmacological reduction of glycosphingolipid biosynthesis ameliorates insulin resistance and prevents some manifestations of metabolic syndrome, supports the view that somehow glycosphingolipids act as critical regulators, Importantly, since reductions in glycosphingolipid biosynthesis have been found to be well tolerated, such approaches may have a therapeutic potential.

Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Universita‘di Parma, 43100 Parma, Italy Atherosclerosis Research Unit, Department of Medicine, Karolinska University Hospital, SE-171 76, Stockholm, Sweden Centre for Population and Health Sciences, University of Glasgow, Western Infirmary Glasgow Glasgow G11 6NT, United Kingdom Department of Pediatrics (Room Y2-145), University Medical Centre Groningen, 9713ZG Groningen, The Netherlands INSERM, U872, Equipe 4, Centre de Recherche des Cordeliers, F-75006 Paris, France Medical University of Graz, Center of Molecular Medicine, Institute of Molecular Biology and Biochemistry, A 8010 GRAZ, Austria Erasmus Medical Center, Department of Internal Medicine, Department of Pharmacology, Vascular and Metabolic Diseases, 3015 CE Rotterdam, The Netherlands University Medical Center Hamburg-Eppendorf, Department of Orthopaedics, D — 20246 Hamburg, Germany Division of Clinical Chemistry, Department of Laboratory Medicine, and Molecular Nutrition Unit, Department of Biosciences, Karolinska Institutet at Huddinge University Hospital, -141 86 Stockholm, Sweden Department Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen O, Denmark University Hospital Zurich, Institute of Clinical Chemistry, CH 8091 Zurich, Switzerland

Rationale: Macrophages cannot limit the uptake of lipids and rely on cholesterol efflux mechanisms for maintaining cellular cholesterol homeostasis. Important mediators of macrophage cholesterol efflux are ATP-binding cassette transporter 1 (ABCA1), which mediates the efflux of cholesterol to lipid-poor apolipoprotein AI, and scavenger receptor class B type I (SR-BI), which promotes efflux to mature high-density lipoprotein. Objective: The aim of the present study was to increase the insight into the putative synergistic roles of ABCA1 and SR-BI in foam cell formation and atherosclerosis. Methods and Results: Low-density lipoprotein receptor knockout (LDLr KO) mice were transplanted with bone marrow from ABCA1/SR-BI double knockout mice, the respective single knockouts, or wild-type littermates. Serum cholesterol levels were lower in ABCA1/SR-BI double knockout transplanted animals, as compared to the single knockout and wild-type transplanted animals on Western-type diet. Despite the lower serum cholesterol levels, massive foam cell formation was found in macrophages from spleen and the peritoneal cavity. Interestingly, ABCA1/SR-BI double knockout transplanted animals also showed a major increase in proinflammatory KC (murine interleukin-8) and interleukin-12p40 levels in the circulation. Furthermore, after 10 weeks of Western-type diet feeding, atherosclerotic lesion development in the aortic root was more extensive in the LDLr KO mice reconstituted with ABCA1/SR-BI double knockout bone marrow. Conclusions: Deletion of ABCA1 and SR-BI in bone marrow–derived cells enhances in vivo macrophage foam cell formation and atherosclerotic lesion development in LDLr KO mice on Western diet, indicating that under high dietary lipid conditions, both macrophage ABCA1 and SR-BI contribute significantly to cholesterol homeostasis in the macrophage in vivo and are essential for reducing the risk for atherosclerosis.

Objective— The iminosugar N -(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynoijirimycin (AMP-DNM), an inhibitor of the enzyme glucosylceramide synthase catalyzing glycosphingolipid (GSL) biosynthesis, ameliorates diabetes and reduces liver steatosis in ob/ob mice. Because an accumulation of sphingolipids, including sphingomyelin and GSLs, has been reported in atherosclerotic lesions in animal models and in humans, the objective of this study was to determine whether AMP-DNM also exerts beneficial effects on the development of atherosclerosis. Methods and Results— APOE*3 Leiden mice, maintained on a high-cholesterol diet, were treated for up to 18 weeks with AMP-DNM. The iminosugar prevented hyperlipidemia, generated a less atherogenic lipid profile, and induced a dramatic reduction in the development of atherosclerotic lesions. At the highest dose, no lesions were detectable. The effect of AMP-DNM was associated with a decrease in liver cholesterol, an increase in bile secretion, and enhanced excretion of cholesterol in the feces. Similar effects of AMP-DNM were observed in mice deficient for the low-density lipoprotein receptor. Conclusion— By lowering plasma cholesterol, the iminosugar AMP-DNM dramatically reduces the development of atherosclerosis in APOE*3 Leiden and low-density lipoprotein receptor −/− mice. Thus, targeting GSL synthesis may be a new treatment modality to prevent cardiovascular disease.

Purpose of review Regulation of cholesterol homeostasis is a complex interplay of a multitude of metabolic pathways situated in different organs. The liver plays a central role and has received most attention of the research community. In this review, we discuss recent progress in the understanding of the emerging role of the intestine in cholesterol transport. Recent findings In recent years, insight in the transport systems that mediate intestinal cholesterol excretion has deepened considerably. Evidence is emerging that the proximal part of the small intestine is able to secrete cholesterol actively, a pathway called transintestinal cholesterol efflux (TICE). In mice, TICE accounts for up to 70% of fecal neutral sterol excretion. Summary The small intestine plays a significant role in the regulation of body cholesterol homeostasis. Active processes control both absorption and excretion of the sterol and the pathways involved are being elucidated. TICE might provide an attractive target for therapy aiming at reduction of atherosclerosis.

MEISSNER, M., R. HAVINGA, R. BOVERHOF, I. KEMA, A. K. GROEN, and F. KUIPERS. Exercise Enhances Whole-Body Cholesterol Turnover in Mice. Med. Sci. Sports Exerc., Vol. 42, No. 8, pp. 1460-1468, 2010. Purpose: Regular exercise reduces cardiovascular risk in humans by reducing cholesterol levels, but the underlying mechanisms have not been fully explored. Exercise might provoke changes in cholesterol and bile acid metabolism and thereby reduce cardiovascular risk. We examined whether voluntary wheel running in mice modulates cholesterol and bile acid metabolism. Methods: Male mice (10 wk old) were randomly assigned to have access to a voluntary running wheel for 2 wk (RUN group) or remained sedentary (SED group). Running wheel activity was recorded daily. In a first experiment, fecal sterol outputs, fecal bile acid profiles, plasma parameters, and expression levels of genes involved in cholesterol and bile acid metabolism were determined. In a second experiment, bile flow, biliary bile acid profile, and biliary secretion rates of cholesterol, phospholipids, and bile acids were determined. Results: The RUN group ran an average of 10 km.d(-1) and displayed lower plasma cholesterol compared with SED (P = 0.030). Fecal bile acid loss was induced by similar to 30% in running mice compared with SED (P = 0.0012). A similar to 30% increase in fecal cholesterol output in RUN (P = 0.014) was consistent with changes in parameters of cholesterol absorption, such as reduced plasma plant sterol-cholesterol ratio (P = 0.044) and decreased jejunal expression of Npc1l1 (P = 0.013). Supportive of an increased cholesterol synthesis to compensate for fecal sterol loss were increased hepatic mRNA levels of HMGCoA reductase (P = 0.006) and an increased plasma lathosterol-cholesterol ratio (P = 0.0011) in RUN. Conclusions: Voluntary wheel running increased cholesterol turnover in healthy mice owing to an increased fecal bile acid excretion and a decreased intestinal cholesterol absorption. Enhanced cholesterol turnover may contribute to the established reduction of cardiovascular risk induced by regular exercise.

It has been hypothesized that bile acids and fatty acids promote colon cancer. A proposed mechanism is a lytic effect of these surfactants on colonic epithelium, resulting in a compensatory proliferation of colonic cells. To investigate the first step of this hypothesis, we studied the lytic activity of fatty acids and physiological mixtures of fatty acids and bile acids. Experiments were performed in both erythrocytes and cultured Caco-2 cells, a model system for intestinal epithelium. Fatty acids with a chain length of 10 C atoms or more were lytic, and the hemolytic activity increased in the order C10:0 less than C18:0 less than C16:0 less than C12:0 less than C14:0 much less than C18:1 approximately C18:2 but was not dependent on their critical micellar concentration. Addition of a sublytic, submicellar concentration of cholate resulted in the formation of highly lytic mixed micelles. Lytic activity of these mixed micelles was closely associated with their micellar aggregation as determined in parallel incubations using a fluorescent micellar probe. With use of identical concentrations of fatty acids and mixed micelles, lysis of erythrocytes was highly correlated (r greater than 0.95) with lysis of Caco-2 cells measured by either release of the apical membrane-marker alkaline phosphatase or the cytosolic marker lactate dehydrogenase. This indicates that the cytolytic activity of these surfactants is not cell-type dependent. Addition of bile acids in concentrations corresponding with the total bile acid concentration in human fecal water resulted in an increased lytic activity of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance, and type 2 diabetes. The hyperinsulinemia that occurs as a consequence of insulin resistance is thought to be an important contributor to the development of fatty liver. We have shown that the iminosugar N-(5'-adamantane-1'-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM), an inhibitor of the enzyme glucosylceramide synthase, is a potent enhancer of insulin signaling in rodent models for insulin resistance and type 2 diabetes. The present study was designed to assess the impact of AMP-DNM on insulin levels, liver triglyceride synthesis, and gene expression profile. Treatment of ob/ob mice with AMP-DNM restored insulin signaling in the liver, corrected blood glucose values to levels found in lean mice, and decreased insulin concentration. The expression of sterol regulatory element-binding protein 1c target genes involved in fatty acid synthesis normalized. AMP-DNM treatment significantly reduced liver to body weight ratio and reversed hepatic steatosis, comprising fat as well as inflammatory markers. In addition, AMP-DNM treatment corrected to a large extent the gene expression profile of ob/ob mice livers toward the profile of lean mice. Conclusion: Pharmacological lowering of glycosphingolipids with the iminosugar AMP-DNM is a promising approach to restore insulin signaling and improve glucose homeostasis as well as hepatic steatosis. (HEPATOLOGY 2009;50:1431-1441.)

Background: High-fat diets promote hepatic lipid accumulation. Paradoxically, these diets also induce lipogenic gene expression in rodent liver. Whether high expression of these genes actually results in an increased flux through the de novo lipogenic pathway in vivo has not been demonstrated.Methodology/Principal Findings: To interrogate this apparent paradox, we have quantified de novo lipogenesis in C57Bl/6J mice fed either chow, a high-fat or a n-3 polyunsaturated fatty acid (PUFA)-enriched high-fat diet. A novel approach based on mass isotopomer distribution analysis (MIDA) following 1-(13)C acetate infusion was applied to simultaneously determine de novo lipogenesis, fatty acid elongation as well as cholesterol synthesis. Furthermore, we measured very low density lipoprotein-triglyceride (VLDL-TG) production rates. High-fat feeding promoted hepatic lipid accumulation and induced the expression of lipogenic and cholesterogenic genes compared to chow-fed mice: induction of gene expression was found to translate into increased oleate synthesis. Interestingly, this higher lipogenic flux (+74 mu g/g/h for oleic acid) in mice fed the high-fat diet was mainly due to an increased hepatic elongation of unlabeled palmitate (+66 mu g/g/h) rather than to elongation of de novo synthesized palmitate. In addition, fractional cholesterol synthesis was increased, i.e. 5.8 +/- 0.4% vs. 8.1 +/- 0.6% for control and high fat-fed animals, respectively. Hepatic VLDL-TG production was not affected by high-fat feeding. Partial replacement of saturated fat by fish oil completely reversed the lipogenic effects of high-fat feeding: hepatic lipogenic and cholesterogenic gene expression levels as well as fatty acid and cholesterol synthesis rates were normalized.Conclusions/Significance: High-fat feeding induces hepatic fatty acid synthesis in mice, by chain elongation and subsequent desaturation rather than de novo synthesis, while VLDL-TG output remains unaffected. Suppression of lipogenic fluxes by fish oil prevents from high fat diet-induced hepatic steatosis in mice.

Recent studies have indicated that direct intestinal secretion of plasma cholesterol significantly contributes to fecal neutral sterol loss in mice. The physiological relevance of this novel route, which represents a part of the reverse cholesterol transport pathway, has not been directly established in vivo as yet. We have developed a method to quantify the fractional and absolute contributions of several cholesterol fluxes to total fecal neutral sterol loss in vivo in mice, by assessing the kinetics of orally and intravenously administered stable isotopically labeled cholesterol combined with an isotopic approach to assess the fate of de novo synthesized cholesterol. Our results show that trans-intestinal cholesterol excretion significantly contributes to removal of blood-derived free cholesterol in C57B16/J mice (33% of 231 mu mol/kg/day) and that pharmacological activation of LXR with T0901317 strongly stimulates this pathway (63% of 706 mu mol/kg/day). Trans-intestinal cholesterol excretion is impaired in mice lacking Abcg5 (-4%), suggesting that the cholesterol transporting Abcg5/Abcg8 heterodimer is involved in this pathway. Our data demonstrate that intestinal excretion represents a quantitatively important route for fecal removal of neutral sterols independent of biliary secretion in mice. This pathway is sensitive to pharmacological activation of the LXR system. These data support the concept that the intestine substantially contributes to reverse cholesterol transport.

Bile acids are synthesized from cholesterol and have long been thought to be just a degradation product with an additional function in food digestion. During the past decade many new functions of bile acids emerged and, instead of functioning at the interphase of the outside world and the body, bile acids turned out to be extremely important signal transduction molecules which play an important role in balancing flux through diverse metabolic pathways. In this chapter we focus on the function of bile acids in regulation of cholesterol homeostasis at both the cellular and organismal level.

Department of Epidemiology, Nutrition Toxicology and Environment Research Institute Maastricht, Maastricht University, The Netherlands.We explored the association between serum level of alpha-1-acid glycoprotein (AGP, an acute phase protein) and the risk of gallstones. AGP was determined in stored serum samples from a case-control study (cases: 113 patients who underwent cholecystectomy for gallstone disease; controls: 184 surgery patients screened by ultrasound, showing no gallstones). Serum AGP correlated negatively with HDL-cholesterol and positively with triglycerides. Presence of gallstones was positively associated with serum AGP, more strongly so for cholesterol gallstones than for pigment gallstones, but not differently between solitary and multiple cholesterol stones. Asymptomatic gallstones (13 surgery patients with gallstones found by screening) were also associated with serum AGP levels. A causal role of elevation of serum AGP (or underlying immune activation) in gallstone formation is discussed.

Adipose tissue is a critical mediator in obesity-induced insulin resistance. Previously we have demonstrated that pharmacological lowering of glycosphingolipids and subsequently GM3 by using the iminosugar AMP-DNM, strikingly improves glycemic control. Here we studied the effects of AMP-DNM on adipose tissue function and inflammation in detail to provide an explanation for the observed improved glucose homeostasis. Leptin-deficient obese (Lep(Ob)) mice were fed AMP-DNM and its effects on insulin signalling, adipogenesis and inflammation were monitored in fat tissue. We show that reduction of glycosphingolipid biosynthesis in adipose tissue of Lep(Ob) mice restores insulin signalling in isolated ex vivo insulin-stimulated adipocytes. We observed improved adipogenesis as the number of larger adipocytes was reduced and expression of genes like peroxisome proliferator-activated receptor (PPAR) gamma, insulin responsive glucose transporter (GLUT)-4 and adipsin increased. In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM. As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN). In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals.

Biliary secretion is generally considered to be an obligate step in the pathway of excess cholesterol excretion from the body. We have recently shown that an alternative route exists. Direct transintestinal cholesterol efflux (TICE) contributes significantly to cholesterol removal in mice. Our aim was to investigate whether the activity of this novel pathway can be influenced by dietary factors. In addition, we studied the role of cholesterol acceptors at the luminal side of the enterocyte. Mice were fed a Western-type diet (0.25% wt/wt cholesterol; 16% wt/wt fat), a high-fat diet (no cholesterol; 24% wt/wt fat), or high-cholesterol diet (2% wt/wt), and TICE was measured by isolated intestinal perfusion. Bile salt-phospholipid mixtures served as cholesterol acceptor. Western-type and high-fat diet increased TICE by 50 and 100%, respectively. In contrast, the high-cholesterol diet did not influence TICE. Intestinal scavenger receptor class B type 1 (Sr-B1) mRNA and protein levels correlated with the rate of TICE. Unexpectedly, although confirming a role for Sr-B1, TICE was significantly increased in Sr-B1-deficient mice. Apart from the long-term effect of diets on TICE, acute effects by luminal cholesterol acceptors were also investigated. The phospholipid content of perfusate was the most important regulator of TICE; bile salt concentration or hydrophobicity of bile salts had little effect. In conclusion, TICE can be manipulated by dietary intervention. Specific dietary modifications might provide means to stimulate TICE and, thereby, to enhance total cholesterol turnover.

Previous studies have shown that mice deficient in Scd1 have a dramatically reduced level of liver triglyceride and an improvement in insulin sensitivity. The mice are lean and partially protected from obesity induced by leptin deficiency or high fat diets. These results predicted that Scd1(-/-) mice would be protected from the increased serum triglyceride levels that result from a lipogenic diet and might also might protect a diabetes-susceptible obese mouse strain from diabetes. We studied Scd1(-/-) mice on a very low-fat high-carbohydrate lipogenic diet. The animals were almost entirely devoid of high density lipoprotein (HDL). Nonetheless, they were hypercholesterolaemic due to a large increase in low density lipoprotein (LDL). They had a high level of serum bilirubin and bile acids, and the appearance of lipoprotein-X, indicative of cholestasis. These changes were reversible with oil containing both mono- and polyunsaturated fat, but not entirely reversible with just triolein, suggesting that Scd1 deficiency increased the requirement for polyunsaturated fat. We performed hyperinsulinemic-euglycemic clamp experiments and found that the Scd1(-/-) mice on a normal chow diet had dramatically improved insulin sensitivity. But, surprisingly, leptin(ob/ob) Scd1(-/-) mice had worse diabetes than leptin(ob/ob) Scd1(wt/wt) mice. Thus, despite its effects on insulin sensitivity, Scdl deficiency worsens diabetes in leptin-deficient obese mice.

Gallstone disease occurs more frequently in subjects with the metabolic syndrome and type 2 diabetes. Findings in a mouse model suggest that the forkhead transcription factor FoxO1 lies behind this association ( pages 778–782 ).

Apolipoprotein E2 (apoE2)-associated hyperlipidemia is characterized by a disturbed clearance of apoE2-enriched VLDL remnants. Because excess apoE2 inhibits LPL-mediated triglyceride (TG) hydrolysis in vitro, we investigated whether direct or indirect stimulation of LPL activity in vivo reduces the apoE2-associated hypertriglyceridemia. Here, we studied the role of LPL and two potent modifiers, the LPL inhibitor apoC-III and the LPL activator apoA-V, in APOE2-knockin (APOE2) mice. Injection of heparin in APOE2 mice reduced plasma TG by 53% and plasma total cholesterol (TC) by 18%. Adenovirus-mediated overexpression of LPL reduced plasma TG by 85% and TC by 40%. Both experiments indicate that the TG in apoE2-enriched particles is a suitable substrate for LPL. Indirect activation of LPL activity via deletion of Apoc3 in APOE2 mice did not affect plasma TG levels, whereas overexpression of Apoa5 in APOE2 mice did reduce plasma TG by 81% and plasma TC by 41%. In conclusion, the hypertriglyceridemia in APOE2 mice can be ameliorated by the direct activation of LPL activity. Indirect activation of LPL via overexpression of apoA-V does, whereas deletion of apoC-III does not, affect the plasma TGs in APOE2 mice. These data indicate that changes in apoA-V levels have a dominant effect over changes in apoC-III levels in the improvement of APOE2-associated hypertriglyceridemia.

Biliary cholesterol/phospholipid vesicles play an important role in the pathogenesis of gallstone disease. A prerequisite for the study of the lipid composition and stability of these vesicles is a reliable method to quantify the amount of vesicular lipid. In the present report we show that NMR can be used to determine the distribution of biliary lecithin between the micellar and vesicular phases. The relatively large size of the vesicles leads to such a broadening of the lipid resonances that they are no longer visible in high resolution 1H-NMR spectra. Since micelles are much smaller, lipid present in the micellar phase does give rise to sharp peaks in 1H-NMR spectra. Micellar lecithin can easily be quantified in these spectra. The resonances of cholesterol are masked by the closely related bile acid that is present in a much higher concentration. By determining the difference between chemically and NMR estimated lecithin, the distribution of this phospholipid between the micellar phase and vesicular phase can be assessed. We have compared the results of NMR with gel permeation and density gradient ultracentrifugation. Using standard fractionation conditions, both gel permeation and density gradient ultracentrifugation lead to an underestimation of vesicular lecithin, the difference being minor at relatively high total lipid concentrations (10 g/dl) but large in diluted model bile. We conclude that 1H-NMR can be used to determine the distribution of lecithin in model bile.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies showed that mice deficient in Scd1 had a reduced level of liver triglyceride and an improvement in insulin sensitivity. We studied Scd1 –/– mice on a very low-fat, high-carbohydrate lipogenic diet. The animals were almost entirely devoid of high-density lipoprotein (HDL). Nonetheless, they were hypercholesterolemic and had cholestasis. These changes were reversible with oil containing both mono- and polyunsaturated fat, but not entirely reversible with just triolein, suggesting that Scd1 deficiency increased the requirement for polyunsaturated fat. We also found that the Scd1 ‐/‐ mice on a normal chow diet had dramatically improved insulin sensitivity. However, leptin ob/ob Scd1 –/– mice had worse diabetes than leptin ob/ob Scd1 wt/wt mice.

A recent population-based study showed that cholesteryl ester transfer protein (CETP) gene variations, which relate to lower plasma CETP, may predict increased cardiovascular risk, in spite of higher HDL cholesterol. Among other functions, CETP activity contributes to cellular cholesterol efflux, an early step in the anti-atherogenic reverse cholesterol transport (RCT) process. We hypothesized that cellular cholesterol efflux stimulating capacity of plasma could be associated with CETP gene variation. In this study, we tested the extent to which the ability of plasma to promote cholesterol efflux from cultured human fibroblasts is associated with CETP gene variation. In 223 men, the -629C -> A CETP promoter polymorphism, plasma lipids, CETP mass, cholesteryl ester transfer (CET), lecithin: cholesterol acyltransferase (LCAT) activity and the ability of plasma to promote cholesterol efflux from human skin fibroblasts, obtained from a single normolipidemic donor, were determined. In -629CC homozygotes (n=52), cholesterol efflux, plasma CETP mass, CET and LCAT activity were higher, whereas HDL cholesterol was lower compared to -629 AA homozygotes (n = 62) and - 629CA+ AA carriers (n = 17 1) (P A CETP polymorphism, possibly also involving LCAT activity, could provide a mechanism explaining why CETP gene variation, which relates to lower plasma CETP, does not confer diminished cardiovascular risk. (c) 2007 Elsevier B.V. All rights reserved.

The oxysterol-activated nuclear receptor liver X receptor alpha (LXR alpha) has been implicated in the control of both cholesterol and fatty acid metabolism. In this study, we have evaluated the effects of excess dietary cholesterol on hepatic cholesterol metabolism, lipogenesis, and VLDL production in homozygous (Lxr alpha(-/-)), heterozygous (Lxr alpha(+/-)), and wild-type mice. Mice were fed either chow or a cholesterol-enriched diet (1%, w/w) for 2 weeks. On the high-cholesterol diet, fractional cholesterol absorption was higher in Lxr alpha(-/-) mice than in controls, leading to delivery of more dietary cholesterol to the liver. Lxr alpha(-/-) mice were not able to induce expression of hepatic Abcg5/Abcg8, and massive accumulation of free cholesterol and cholesteryl esters (CEs) occurred. Interestingly, despite the inability to upregulate Abcg5/Abcg8, the highly increased hepatic free cholesterol content did stimulate biliary cholesterol output in Lxr alpha(-/-) mice. Hepatic cholesterol accumulation was accompanied by decreased hepatic expression of lipogenic genes, probably caused by impaired sterol-regulatory element binding protein 1c processing, lower hepatic triglyceride (TG) contents, strongly reduced plasma TG concentrations (290%), and reduced VLDL-TG production rates (-60%) in Lxr alpha(-/-) mice. VLDL particles were smaller and CE-enriched under these conditions. Lxr alpha deficiency did not affect VLDL formation under chow-fed conditions. Hepatic stearyl coenzyme A desaturase 1 expression was decreased dramatically in Lxr alpha(-/-) mice and did not respond to cholesterol feeding, but fatty acid profiles of liver and VLDL were only slightly different between Lxr alpha(-/-) and wild-type mice. Our data indicate that displacement of TGs by CEs during the VLDL assembly process underlies hypotriglyceridemia in cholesterol-fed Lxr alpha(-/-) mice. - van der Veen, J. N., R. Havinga, V. W. Bloks, A. K. Groen, and F. Kuipers. Cholesterol feeding strongly reduces hepatic VLDL-triglyceride production in mice lacking the liver X receptor a.

The ATP binding cassette transporters ABCG5 and ABCG8 are indispensable for hepatobiliary cholesterol transport. In this study, we investigated the specificity of the heterodimer for cholesterol acceptors. Dog gallbladder epithelial cells were mono- or double-transfected with lentiviral mouse Abcg5 and Abcg8 vectors. Double-transfected cells showed increased efflux to different bile salt (BS) species, while mono-transfected cells did not show enhanced efflux. The efflux was initiated at micellar concentrations and addition of phosphatidylcholine increased efflux. Cholesterol secretion was highly BS dependent, whereas other cholesterol acceptors such as ApoAI, HDL or methyl-β-cyclodextrin did not elicit Abcg5/g8 dependent cholesterol secretion.

The liver is considered the major "control center" for maintenance of whole body cholesterol homeostasis. This organ is the main site for de novo cholesterol synthesis, clears cholesterol-containing chylomicron remnants and low density lipoprotein particles from plasma and is the major contributor to high density lipoprotein (HDL; good cholesterol) formation. The liver has a central position in the classical definition of the reverse cholesterol transport pathway by taking up periphery-derived cholesterol from lipoprotein particles followed by conversion into bile acids or its direct secretion into bile for eventual removal via the feces. During the past couple of years, however, an additional important role of the intestine in maintenance of cholesterol homeostasis and regulation of plasma cholesterol levels has become apparent. Firstly, molecular mechanisms of cholesterol absorption have been elucidated and novel pharmacological compounds have been identified that interfere with the process and positively impact plasma cholesterol levels. Secondly, it is now evident that the intestine itself contributes to fecal neutral sterol loss as a cholesterol-secreting organ. Finally, very recent work has unequivocally demonstrated that the intestine contributes significantly to plasma HDL cholesterol levels. Thus, the intestine is a potential target for novel anti-atherosclerotic treatment strategies that, in addition to interference with cholesterol absorption, modulate direct cholesterol excretion and plasma HDL cholesterol levels. (C) 2006 The WJG Press. All rights reserved.

The relevance of apolipoprotein A-V (apoA-V) for human lipid homeostasis is underscored by genetic association studies and the identification of truncation-causing mutations in the APOA5 gene as a cause of type V hyperlipidemia, compatible with an LPL-activating role of apoA-V. An inverse correlation between plasma apoA-V and triglyceride (TG) levels has been surmised from animal data. Recent studies in human subjects using (semi)quantitative immunoassays, however, do not provide unambiguous support for such a relationship. Here, we used a novel, validated ELISA to measure plasma apoA-V levels in patients (n = 28) with hypertriglyceridemia (HTG; 1.8–78.7 mmol TG/l) and normolipidemic controls (n = 42). Unexpectedly, plasma apoA-V levels were markedly increased in the HTG subjects compared with controls (1,987 vs. 258 ng/ml; P < 0.001). In the HTG group, apoA-V and TG were positively correlated (r = +0.44, P = 0.02). In addition, we noted an increased level of the LPL-inhibitory protein apoC-III in the HTG group (45.8 vs. 10.6 mg/dl in controls; P < 0.001). The correlation between apoA-V and TG levels in the HTG group disappeared (partial r = +0.09, P = 0.65) when controlling for apoC-III levels. In contrast, apoC-III and TG remained positively correlated in this group when controlling for apoA-V (partial r = +0.43, P = 0.025). Our findings suggest that in HTG patients, increased TG levels are accompanied by high plasma levels of apoA-V and apoC-III, apolipoproteins with opposite modes of action. This study provides evidence for a complex interaction between apoA-V and apoC-III in patients with severe HTG.

Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Watanabe M, Houten SM, Mataki C, Christoffolete MA, Kim BW, Sato H, Messaddeq N, Harney JW, Ezaki O, Kodama T, Schoonjans K, Bianco AC, Auwerx J. While bile acids (BAs) have long been known to be essential in dietary lipid absorption and cholesterol catabolism, in recent years an important role for BAs as signalling molecules has emerged. BAs activate mitogen-activated protein kinase pathways, are ligands for the G-protein-coupled receptor (GPCR) TGR5 and activate nuclear hormone receptors such as farnesoid X receptor-α (FXR-α; NR1H4). FXR-α regulates the enterohepatic recycling and biosynthesis of BAs by controlling the expression of genes such as the short heterodimer partner (SHP; NR0B2) that inhibits the activity of other nuclear receptors. The FXR-α-mediated SHP induction also underlies the downregulation of the hepatic fatty acid and triglyceride biosynthesis and very-low-density lipoprotein production mediated by sterol-regulatory-element-binding protein 1c. This indicates that BAs might be able to function beyond the control of BA homeostasis as general metabolic integrators. Here we show that the administration of BAs to mice increases energy expenditure in brown adipose tissue, preventing obesity and resistance to insulin. This novel metabolic effect of BAs is critically dependent on induction of the cyclic-AMP-dependent thyroid hormone activating enzyme type 2 iodothyronine deiodinase (D2) because it is lost in D2−/− mice. Treatment of brown adipocytes and human skeletal myocytes with BA increases D2 activity and oxygen consumption. These effects are independent of FXR-α, and instead are mediated by increased cAMP production that stems from the binding of BAs with the G-protein-coupled receptor TGR5. In both rodents and humans, the most thermogenically important tissues are specifically targeted by this mechanism because they coexpress D2 and TGR5. The BA-TGR5-cAMP-D2 signalling pathway is therefore a crucial mechanism for fine-tuning energy homeostasis that can be targeted to improve metabolic control. [Abstract reproduced by permission of Nature 2006;439:484–489]

Plasma HDL cholesterol levels are inversely related to risk for atherosclerosis. The ATP-binding cassette, subfamily A, member 1 (ABCA1) mediates the rate-controlling step in HDL particle formation, the assembly of free cholesterol and phospholipids with apoA-I. ABCA1 is expressed in many tissues; however, the physiological functions of ABCA1 in specific tissues and organs are still elusive. The liver is known to be the major source of plasma HDL, but it is likely that there are other important sites of HDL biogenesis. To assess the contribution of intestinal ABCA1 to plasma HDL levels in vivo, we generated mice that specifically lack ABCA1 in the intestine. Our results indicate that approximately 30% of the steady-state plasma HDL pool is contributed by intestinal ABCA1 in mice. In addition, our data suggest that HDL derived from intestinal ABCA1 is secreted directly into the circulation and that HDL in lymph is predominantly derived from the plasma compartment. These data establish a critical role for intestinal ABCA1 in plasma HDL biogenesis in vivo.

FABACs (fatty acid–bile acid conjugates) are synthetic molecules that are designed to treat a range of lipid disorders. The compounds prevent cholesterol gallstone formation and diet-induced fatty liver, and increase reverse cholesterol transport in rodents. The aim of the present study was to investigate the effect of FABACs on cholesterol efflux in human cells. Aramchol (3β-arachidylamido-7α,12α,5β-cholan-24-oic acid) increased cholesterol efflux from human skin fibroblasts in a dose-dependent manner in the absence of known efflux mediators such as apoA-I (apolipoprotein A-I), but had little effect on phospholipid efflux. An LXR (liver X receptor) agonist strongly increased Aramchol-induced cholesterol efflux; however, in ABCA1 (ATP-binding cassette transporter A1)-deficient cells from Tangier disease patients, the Aramchol effect was absent, indicating that activity of ABCA1 was required. Aramchol did not affect ABCA1 expression, but plasma membrane levels of the transporter increased 2-fold. Aramchol is the first small molecule that induces ABCA1-dependent cholesterol efflux without affecting transcriptional control. These findings may explain the beneficial effect of the compound on atherosclerosis.

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8+/+, Abcg8+/−, and Abcg8−/− mice with hydrophilic and hydrophobic bile salts. In Abcg8−/− mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8−/− mice at a much lower infusion rate compared with Abc8−/− and Abcg8+/− mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8−/− mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.—Kosters, A., C. Kunne, N. Looije, S. B. Patel, R. P. J. Oude Elferink, and A. K. Groen. The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice.

Dietary cholesterol absorption contributes to a large part of the circulating cholesterol. However, the mechanism of sterol intestinal uptake is not clearly elucidated. Scavenger receptor class B type I (SR-BI), major component in the control of cholesterol homeostasis, is expressed in the intestine, but its role in this organ remains unclear. We have generated transgenic mice over-expressing SR-BI primarily in the intestine by using the mouse SR-BI gene under the control of intestinal specific “apolipoprotein (apo) C-III enhancer coupled with apo A-IV promoter”. We found SR-BI overexpression with respect to the natural protein along the intestine and at the top of the villosities. After feeding a meal containing [14C] cholesterol and [3H] triolein, SR-BI transgenic mice presented a rise of intestinal absorption of both lipids that was not due to a defect in chylomicron clearance nor to a change in the bile flow or the bile acid content. Nevertheless, SR-BI transgenic mice showed a decrease of total cholesterol, but an increase of triglyceride content in plasma without any change in the HDL apo A-I level. Thus, we describe for the first time a functional role in vivo for SR-BI in cholesterol but also in triglyceride intestinal absorption.

The aim of this study was to evaluate the effect of a low-saturated-fat, low-cholesterol diet on plasma lipopoproteins, pre beta-high density lipoprotein (HDL) formation, lecithin: cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activities, as well as on the ability of plasma to stimulate cellular cholesterol efflux. Twelve male type 1 diabetic patients with plasma cholesterol >5.0 mmol/L were studied while consuming their usual diet and after 6 weeks of a low-fat, low-cholesterol diet. Pre beta-HDL formation was measured using crossed immuno-electrophoresis. Plasma LCAT, CETP and PLTP activities were assayed by exogenous substrate methods. The ability of plasma to promote cellular cholesterol efflux out of Fu5AH rat hepatoma cells and out of human skin fibroblasts was also determined. Saturated fat intake was lowered (p = 0.001) due to replacement with carbohydrates, while mono- and polyunsaturated fat intake remained unchanged. Cholesterol intake decreased as well (p = 0.003). The changes in plasma total cholesterol, very low and low-density lipoprotein (VLDL+LDL) cholesterol, HDL cholesterol, HDL phospholipids, apolipoprotein (apo) A-I, plasma LCAT activity and PLTP activity were not significant. Plasma CETP activity (p = 0.008) and pre beta-HDL formation (p = 0.008) decreased. The ability of plasma to promote cholesterol efflux out of fibroblasts and Fu5AH cells remained unchanged. Reduction in dietary saturated fat and cholesterol intake does not adversely affect cellular cholesterol efflux to plasma from type 1 diabetic patients, despite a drop in pre beta-HDL formation.

Background & Aims: Reverse cholesterol transport (RCT) is defined as high-density lipoprotein (HDL)-mediated flux of excess cholesterol from peripheral cells to liver, followed by secretion into bile and disposal via the feces. Various steps of this pathway are controlled by the liver X receptor (LXR). We addressed the role of the intestine in LXR-dependent stimulation of fecal cholesterol excretion. Methods: To segregate biliary from intestine-derived cholesterol, wild-type and Mdr2 P-glycoprotein-deficient mice (Mdr2(-/-)), which are unable to secrete cholesterol into bile, were treated with the LXR agonist GW3965. Results: Treatment with GW3965 increased biliary cholesterol secretion by 74% in wild-type mice but had no effect in Mdr2(-/-) mice. LXR activation increased fecal neutral sterol excretion 2.1-fold in wild-type mice. Surprisingly, an identical increase was observed in Mdr2(-/-) mice. Fractional cholesterol absorption was reduced on LXR activation in both strains but was more pronounced in Mdr2(-/-) mice, coinciding with reduced Npc111 expression. Intestinal gene expression of ATPbinding cassette transporters (Abc) Abca1, Abcg1, Abcg5, and Abcg8 was strongly induced upon LXR activation in both strains, whereas expression of HMGCoA reductase, controlling cholesterol synthesis, remained unaffected. Additionally, LXR activation stimulated the excretion of plasma-derived [H-3]cholesterol into the fecal neutral sterol fraction in Mdr2(-/-) mice. Conclusions: Increased fecal cholesterol loss upon LXR activation is independent of biliary cholesterol secretion in mice. An important part of excess cholesterol is excreted directly via the intestine, supporting the existence of an alternative, quantitatively important route for cholesterol disposal.

Hepatic expression of the scavenger receptor class B type I (SR-BI) promotes selective uptake of HDL cholesterol by the liver and is believed to play a role in the process of reverse cholesterol transport (RCT). We hypothesized that hepatic SR-BI expression is a regulator of the rate of integrated macrophage-to-feces RCT and used an in vivo model to test this hypothesis. Cholesterol-loaded and [3H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice, after which the appearance of the [3H]cholesterol in the plasma, liver, and feces over 48 hours was quantitated. Mice overexpressing SR-BI in the liver had significantly reduced [3H]cholesterol in the plasma but markedly increased [3H] tracer excretion in the feces over 48 hours. Conversely, mice deficient in SR-BI had significantly increased [3H]cholesterol in the plasma but markedly reduced [3H] tracer excretion in the feces over 48 hours. These studies demonstrate that hepatic SR-BI expression, despite its inverse effects on steady-state plasma HDL cholesterol concentrations, is an important positive regulator of the rate of macrophage RCT.