Hepatic cholesterol and bile acid metabolism and intestinal cholesterol absorption in scavenger receptor class B type I-deficient mice

The scavenger receptor class B type I (SR-BI), which is expressed in the liver and intestine, plays a critical role in cholesterol metabolism in rodents. While hepatic SR-BI expression controls high density lipoprotein (HDL) cholesterol metabolism, intestinal SR-BI has been proposed to facilitate cholesterol absorption. To evaluate further the relevance of SR-BI in the enterohepatic circulation of cholesterol and bile salts, we studied biliary lipid secretion, hepatic sterol content and synthesis, bile acid metabolism, fecal neutral sterol excretion, and intestinal cholesterol absorption in SR-BI knockout mice. SR-BI deficiency selectively impaired biliary cholesterol secretion, without concomitant changes in either biliary bile acid or phospholipid secretion. Hepatic total and unesterified cholesterol contents were slightly increased in SR-BI-deficient mice, while sterol synthesis was not significantly changed. Bile acid pool size and composition, as well as fecal bile acid excretion, were not altered in SR-BI knockout mice. Intestinal cholesterol absorption was somewhat increased and fecal sterol excretion was slightly decreased in SR-BI knockout mice relative to controls. These findings establish the critical role of hepatic SR-BI expression in selectively controlling the utilization of HDL cholesterol for biliary secretion. In contrast, SR-BI expression is not essential for intestinal cholesterol absorption.     

Class B scavenger receptor-mediated intestinal absorption of dietary beta-carotene and cholesterol

There is now a general consensus that the intestinal absorption of water-insoluble, dietary lipids is protein-mediated, but the assignment of protein(s) to this function is still a matter of debate. To address this issue, we measured beta-carotene and cholesterol absorption in wild-type and SR-BI knockout mice and the uptake of these lipids in vitro using brush border membrane (BBM) vesicles. From the comparison of the in vivo and in vitro results we conclude that both BBM-resident class B scavenger receptors, SR-BI and CD36, can facilitate the absorption of beta-carotene and cholesterol. SR-BI is essential for beta-carotene absorption, at least in mice on a high fat diet. This is due to the fact that the absorption of beta-carotene is restricted to the duodenum and SR-BI is the predominant receptor in the mouse duodenum. In contrast, SR-BI may be involved but is not essential for cholesterol absorption in the small intestine. The question of whether SR-BI contributes to cholesterol absorption in vivo is still unresolved. Transfection of COS-7 cells with SR-BI or CD36 confers on these cells lipid uptake properties closely resembling those of enterocytes and BBM vesicles. Both scavenger receptors facilitate the uptake of dietary lipids such as beta-carotene, free and esterified cholesterol, phospholipids, and fatty acids into COS-7 cells. This lipid uptake is effected from three different lipid donor particles: mixed bile salt micelles, phospholipid small unilamellar vesicles, and trioleoylglycerol emulsions which are all likely to be present in the small intestine. Ezetimibe, a representative of a new class of drugs that inhibit intestinal cholesterol absorption, blocks SR-BI- and CD36-facilitated uptake of cholesterol into COS-7 cells.     

Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport

Neutral cholesteryl ester hydrolase (CEH)-mediated hydrolysis of cellular cholesteryl esters (CEs) is required not only to generate free cholesterol (FC) for efflux from macrophages but also to release FC from lipoprotein-delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH would regulate the hydrolysis of lipoprotein-derived CE and enhance reverse cholesterol transport (RCT). Adenoviral-mediated CEH overexpression led to a significant increase in bile acid output. To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol-loaded and [(3)H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice and the appearance of [(3)H]cholesterol in gallbladder bile and feces over 48 h was quantified. Mice overexpressing CEH had significantly higher [(3)H]cholesterol radiolabel in bile and feces, and it was associated with bile acids. This CEH-mediated increased movement of [(3)H]cholesterol from macrophages to bile acids and feces was significantly attenuated in SR-BI(-/-) mice. These studies demonstrate that similar to macrophage CEH that rate-limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output.     

Susceptibility to murine cholesterol gallstone formation is not affected by partial disruption of the HDL receptor SR-BI

High density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver where its cholesterol is secreted preferentially into bile. The scavenger receptor class B type I (SR-BI) is believed to play a pivotal role in unloading HDL cholesterol and its ester to hepatocytes. Here, using male SR-BI "att" mice with a dysfunctional mutation in the Sr-b1 promoter, we studied whether approximately 50% of normal SR-BI expression influences gallstone susceptibility in these mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid and 15% butterfat. Our results showed that the disruption of SR-BI expression reduced cholesterol secretion by 37% in the chow-fed state and 10% on the lithogenic diet, and while delaying incidence slightly, did not influence cumulative susceptibility to cholesterol gallstones. The lithogenic diet induced marked increases in biliary cholesterol and phospholipid secretion rates but not of bile salts. Basal expression of hepatic SR-BI protein was dissimilar in both wild-type and SR-BI mice, and remained unaltered in response to the lithogenic diet. By two independent dual isotope methods, intestinal cholesterol absorption was unimpaired by attenuation of the SR-BI which also displays low-density expression on small intestinal enterocytes. We conclude that although HDL cholesterol is a principal source of biliary cholesterol in the basal state, uptake of cholesterol from chylomicron remnants appears to be the major contributor to biliary cholesterol hypersecretion during diet-induced cholelithogenesis in the mouse.     

Hepatic scavenger receptor BI promotes rapid clearance of high density lipoprotein free cholesterol and its transport into bile

The clearance of free cholesterol from plasma lipoproteins by tissues is of major quantitative importance, but it is not known whether this is passive or receptor-mediated. Based on our finding that scavenger receptor BI (SR-BI) promotes free cholesterol (FC) exchange between high density lipoprotein (HDL) and cells, we tested whether SR-BI would effect FC movement in vivo using [(14)C]FC- and [(3)H]cholesteryl ester (CE)-labeled HDL in mice with increased (SR-BI transgenic (Tg)) or decreased (SR-BI attenuated (att)) hepatic SR-BI expression. The initial clearance of HDL FC was increased in SR-BI Tg mice by 72% and decreased in SR-BI att mice by 53%, but was unchanged in apoA-I knockout mice compared with wild-type mice. Transfer of FC to non-HDL and esterification of FC were minor and could not explain differences. The hepatic uptake of FC was increased in SR-BI Tg mice by 34% and decreased in SR-BI att mice by 22%. CE clearance and uptake gave similar results, but with much slower rates. The uptake of HDL FC and CE by SR-BI Tg primary hepatocytes was increased by 2.2- and 2.6-fold (1-h incubation), respectively, compared with control hepatocytes. In SR-BI Tg mice, the initial biliary secretion of [(14)C]FC was markedly increased, whereas increased [(3)H]FC appeared after a slight delay. Thus, in the mouse, a major portion of the clearance of HDL FC from plasma is mediated by SR-BI.     

The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption

The molecular mechanisms of cholesterol absorption in the intestine are poorly understood. With the goal of defining candidate genes involved in these processes a fluorescence-activated cell sorter-based, retroviral-mediated expression cloning strategy has been devised. SCH354909, a fluorescent derivative of ezetimibe, a compound which blocks intestinal cholesterol absorption but whose mechanism of action is unknown, was synthesized and shown to block intestinal cholesterol absorption in rats. Pools of cDNAs prepared from rat intestinal cells enriched in enterocytes were introduced into BW5147 cells and screened for SCH354909 binding. Several independent clones were isolated and all found to encode the scavenger receptor class B, type I (SR-BI), a protein suggested by others to play a role in cholesterol absorption. SCH354909 bound to Chinese hamster ovary (CHO) cells expressing SR-BI in specific and saturable fashion and with high affinity (K(d) approximately 18 nM). Overexpression of SR-BI in CHO cells resulted in increased cholesterol uptake that was blocked by micromolar concentrations of ezetimibe. Analysis of rat intestinal sections by in situ hybridization demonstrated that SR-BI expression was restricted to enterocytes. Cholesterol absorption was determined in SR-B1 knockout mice using both an acute, 2-h, assay and a more chronic fecal dual isotope ratio method. The level of intestinal cholesterol uptake and absorption was similar to that seen in wild-type mice. When assayed in the SR-B1 knockout mice, the dose of ezetimibe required to inhibit hepatic cholesterol accumulation induced by a cholesterol-containing 'western' diet was similar to wild-type mice. Thus, the binding of ezetimibe to cells expressing SR-B1 and the functional blockade of SR-B1-mediated cholesterol absorption in vitro suggest that SR-B1 plays a role in intestinal cholesterol metabolism and the inhibitory activity of ezetimibe. In contrast studies with SR-B1 knockout mice suggest that SR-B1 is not essential for intestinal cholesterol absorption or the activity of ezetimibe.     

A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro.

The fluorescent cholesterol analog 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (fluoresterol) was characterized as a tool for exploring the biochemistry and cell biology of intestinal cholesterol absorption. Hamsters absorbed fluoresterol in a concentration- and time-dependent manner, with an efficiency of about 15-30% that of cholesterol. Fluoresterol absorption was blocked by compounds known to inhibit cholesterol absorption, implying that fluoresterol interacts with those elements of the normal pathway for cholesterol absorption on which the inhibitors act. Confocal microscopy of small intestinal tissue demonstrated that fluoresterol was taken up by absorptive epithelial cells and packaged into lipoprotein particles, suggesting a normal route of intracellular trafficking. Uptake of fluoresterol was confirmed by biochemical analysis of intestinal tissue, and a comparison of [(3)H] cholesterol and fluoresterol content in the mucosa suggested that fluoresterol moved through the enterocytes more rapidly than did cholesterol. This interpretation was supported by measurements of fluoresterol esterification in the mucosa. Four hours after hamsters were given fluoresterol and [(3)H]cholesterol orally, 44% of the fluoresterol in the intestinal mucosa was esterified, compared to 8% of the [(3)H]cholesterol. Caco-2 cells took up 2- to 5-fold more [(3)H]cholesterol than fluoresterol from bile acid micelles, and esterified 21-24% of the fluoresterol but only 1-4% of the [(3)H]cholesterol. Thus fluoresterol apparently interacts with the proteins required for cholesterol uptake, trafficking, and processing in the small intestine.     

Scavenger receptor class B type I reduces cholesterol absorption in cultured enterocyte CaCo-2 cells

Scavenger receptor class B type I (SR-BI) mediates selective uptake of cholesteryl esters from HDL as well as efflux of cellular free cholesterol to HDL. It is unclear whether the receptor is involved in intestinal cholesterol absorption. We addressed this issue by studying [3H]cholesterol flux in differentiated CaCo-2 cells incubated at their apical side with mixed taurocholate/phosphatidylcholine/cholesterol micelles. Biotinylation and HDL binding experiments showed predominant apical expression of endogenous and overexpressed SR-BI. Mixed micellar cholesterol saturation affected the magnitude and direction of cholesterol flux with significant net uptake only from supersaturated micelles and net efflux from unsaturated micelles. Incubation with micelles that depleted cellular cholesterol resulted in a decrease of SR-BI protein, whereas incubation with cholesterol-loading micelles resulted in a significant increase of SR-BI protein. Apical cholesterol uptake by CaCo-2 cells was increased in the presence of a SR-BI-blocking antibody and by partial inhibition of SR-BI expression with small inhibitory RNA. Adenovirus-mediated overexpression of apical SR-BI did not affect cholesterol uptake but stimulated apical cholesterol efflux, even to supersaturated mixed micelles. Partial inhibition of SR-BI with small inhibitory RNA reduced apical cholesterol efflux. Our data argue against a direct role for SR-BI in micellar cholesterol uptake. However, SR-BI might be involved in cholesterol absorption by facilitating cholesterol efflux to micelles.     

ACAT2 and ABCG5/G8 are both required for efficient cholesterol absorption in mice: evidence from thoracic lymph duct cannulation

The metabolic fate of newly absorbed cholesterol and phytosterol is orchestrated through adenosine triphosphate-binding cassette transporter G5 and G8 heterodimer (G5G8), and acyl CoA:cholesterol acyltransferase 2 (ACAT2). We hypothesized that intestinal G5G8 limits sterol absorption by reducing substrate availability for ACAT2 esterification and have attempted to define the roles of these two factors using gene deletion studies in mice. Male ACAT2(-/-), G5G8(-/-), ACAT2(-/-)G5G8(-/-) (DKO), and wild-type (WT) control mice were fed a diet with 20% of energy as palm oil and 0.2% (w/w) cholesterol. Sterol absorption efficiency was directly measured by monitoring the appearance of [(3)H]sitosterol and [(14)C]cholesterol tracers in lymph after thoracic lymph duct cannulation. The average percentage (± SEM) absorption of [(14)C]cholesterol after 8 h of lymph collection was 40.55 ± 0.76%, 19.41 ± 1.52%, 32.13 ± 1.60%, and 21.27 ± 1.35% for WT, ACAT2(-/-), G5G8(-/-), and DKO mice, respectively. [(3)H]sitosterol absorption was <2% in WT and ACAT2(-/-) mice, whereas it was up to 6.8% in G5G8(-/-) and DKO mice. G5G8(-/-) mice also produced chylomicrons with ～70% less cholesterol ester mass than WT mice. In contrast to expectations, the data demonstrated that the absence of G5G8 led to decreased intestinal cholesterol esterification and reduced cholesterol transport efficiency. Intestinal G5G8 appeared to limit the absorption of phytosterols; ACAT2 more efficiently esterified cholesterol than phytosterols. The data indicate that handling of sterols by the intestine involves both G5G8 and ACAT2 but that an additional factor (possibly Niemann-Pick C1-like 1) may be key in determining absorption efficiency.     

Cholesteryl ester transfer protein expression partially attenuates the adverse effects of SR-BI receptor deficiency on cholesterol metabolism and atherosclerosis

Scavenger receptor SR-BI significantly contributes to HDL cholesterol metabolism and atherogenesis in mice. However, the role of SR-BI may not be as pronounced in humans due to cholesteryl ester transfer protein (CETP) activity. To address the impact of CETP expression on the adverse effects associated with SR-BI deficiency, we cross-bred our SR-BI conditional knock-out mouse model with CETP transgenic mice. CETP almost completely restored the abnormal HDL-C distribution in SR-BI-deficient mice. However, it did not normalize the elevated plasma free to total cholesterol ratio characteristic of hepatic SR-BI deficiency. Red blood cell and platelet count abnormalities observed in mice liver deficient for SR-BI were partially restored by CETP, but the elevated erythrocyte cholesterol to phospholipid ratio remained unchanged. Complete deletion of SR-BI was associated with diminished adrenal cholesterol stores, whereas hepatic SR-BI deficiency resulted in a significant increase in adrenal gland cholesterol content. In both mouse models, CETP had no impact on adrenal cholesterol metabolism. In diet-induced atherosclerosis studies, hepatic SR-BI deficiency accelerated aortic lipid lesion formation in both CETP-expressing (4-fold) and non-CETP-expressing (8-fold) mice when compared with controls. Impaired macrophage to feces reverse cholesterol transport in mice deficient for SR-BI in liver, which was not corrected by CETP, most likely contributed by such an increase in atherosclerosis susceptibility. Finally, comparison of the atherosclerosis burden in SR-BI liver-deficient and fully deficient mice demonstrated that SR-BI exerted an atheroprotective activity in extra-hepatic tissues whether CETP was present or not. These findings support the contention that the SR-BI pathway contributes in unique ways to cholesterol metabolism and atherosclerosis susceptibility even in the presence of CETP.     

Differentiation-dependent expression and localization of the class B type I scavenger receptor in intestine

The current study used the human Caco-2 cell line and mouse intestine to explore the topology of expression of the class B type I scavenger receptor (SR-BI) in intestinal cells. Results showed that intestinal cells expressed only the SR-BI isoform with little or no expression of the SR-BII variant. The expression of SR-BI in Caco-2 cells is differentiation dependent, with little or no expression in preconfluent undifferentiated cells. Analysis of Caco-2 cells cultured in Transwell porous membranes revealed the presence of SR-BI on both the apical and basolateral cell surface. Immunoblot analysis of mouse intestinal cell extracts demonstrated a gradation of SR-BI expression along the gastrocolic axis of the intestine, with the highest level of expression in the proximal intestine and decreasing to minimal expression levels in the distal intestine. Immunofluorescence studies with SR-BI-specific antibodies also confirmed this expression pattern. Importantly, the immunofluorescence studies also revealed that SR-BI immunoreactivity was most intense in the apical membrane of the brush border in the duodenum. The crypt cells did not show any reactivity with SR-BI antibodies. The localization of SR-BI in the jejunum was found to be different from that observed in the duodenum. SR-BI was present on both apical and basolateral surfaces of the jejunum villus. Localization of SR-BI in the ileum was also different, with little SR-BI detectable on either apical or basolateral membranes.Taken together, these results suggest that SR-BI has the potential to serve several functions in the intestine. The localization of SR-BI on the apical surface of the proximal intestine is consistent with the hypothesis of its possible role in dietary cholesterol absorption, whereas SR-BI present on the basolateral surface of the distal intestine suggests its possible involvement in intestinal lipoprotein uptake.     

Effect of wheat bran and wheat germ on the intestinal uptake of oleic acid, monoolein, and cholesterol in the rat

The effects of fiber-rich wheat bran and wheat germ on the intestinal absorption of dietary cholesterol, free fatty acids, and monoglycerides were studied. Rats were given a test meal containing [(14)C]oleic acid, [(14)C]monoolein, and [(3)H]cholesterol. After a 1-hour digestion period, wheat bran or wheat germ (10% of meal solids) did not significantly modify the gastric emptying of lipids. No effect of wheat bran was evidenced on the amounts of lipids and cholesterol in the intestinal content or the mucosal segments, whereas wheat germ significantly increased the cholesterol in the small intestine content, decreasing its intestinal absorption. Both fractions only slightly influenced the levels of absorbed lipids and cholesterol in the plasma and liver. In vitro binding measurements showed that the wheat fractions bind only 7% to 15% of both lipids and cholesterol. Results indicate that wheat bran has no direct effect on the mucosal uptake process, whereas wheat germ might decrease the uptake of dietary cholesterol by an as yet unknown mechanism.     

Mechanisms of HDL deficiency in mice overexpressing human apoA-II

To ascertain the mechanisms underlying the hypoalphalipoproteinemia present in mice overexpressing human apolipoprotein A-II (apoA-II) (line 11.1), radiolabeled HDL or apoA-I were injected into mice. Fractional catabolic rate of [(3)H]cholesteryl oleoyl ether HDL ([(3)H]HDL) was 2-fold increased in 11.1 transgenic mice compared with control mice and this was concomitant with increased radioactivity in liver, gonads, and adrenals. However, scavenger receptor class B, type I (SR-BI) was increased only in adrenals. [(3)H]HDL of 11.1 transgenic mice presented greater binding but decreased uptake compared with control mice when Chinese hamster ovary cells transfected with SR-BI were used, thereby pointing to unknown but SR-BI-independent mechanisms as being responsible for the increased (3)H-radioactivity seen in liver and gonads. Synthesis rate (SR) of plasma [(3)H]HDL was 2-fold decreased in 11.1 transgenic mice. Mouse (125)I-apoA-I was 2-fold more rapidly catabolized (mainly by the kidney) in transgenic mice. Mouse apoA-I displacement from HDL by the addition of isolated human apoA-II was reproduced ex vivo; thus, this mechanism may be involved in the increased renal catabolism of apoA-I. ApoA-I SR was 2-fold decreased in 11.1 transgenic mice and this was concomitant with a 2.3-fold decrease in hepatic apoA-I mRNA abundance. Our findings show that multiple mechanisms are involved in the HDL deficiency presented by mice overexpressing human apoA-II.     

Carboxyl ester lipase expression in macrophages increases cholesteryl ester accumulation and promotes atherosclerosis

Carboxyl ester lipase (CEL, also called cholesterol esterase or bile salt-dependent lipase) is a lipolytic enzyme capable of hydrolyzing cholesteryl esters, triacylglycerols, and phospholipids in a trihydroxy bile salt-dependent manner but hydrolyzes ceramides and lysophospholipids via bile salt-independent mechanisms. Although CEL is synthesized predominantly in the pancreas, a low level of CEL expression was reported in human macrophages. This study used transgenic mice with macrophage CEL expression at levels comparable with that observed in human macrophages to explore the functional role and physiological significance of macrophage CEL expression. Peritoneal macrophages from CEL transgenic mice displayed a 4-fold increase in [(3)H]oleate incorporation into cholesteryl [(3)H]oleate compared with CEL-negative macrophages when the cells were incubated under basal conditions in vitro. When challenged with acetylated low density lipoprotein, cholesteryl ester accumulation was 2.5-fold higher in macrophages expressing the CEL transgene. The differences in cholesteryl ester accumulation were attributed to the lower levels of ceramide and lysophosphatidylcholine in CEL-expressing cells than in CEL-negative cells. CEL transgenic mice bred to an atherosclerosis susceptible apoE(-/-) background displayed an approximate 4-fold higher atherosclerotic lesion area than apoE(-/-) mice without the CEL transgene when both were fed a high fat/cholesterol diet. Plasma level of the atherogenic lysophosphatidylcholine was lower in the CEL transgenic mice, but plasma cholesterol level and lipoprotein profile were similar between the two groups. These studies documented that CEL expression in macrophages is pro-atherogenic and that the mechanism is because of its hydrolysis of ceramide and lysophosphatidylcholine in promoting cholesterol esterification and decreasing cholesterol efflux.     

Intestinal Scavenger Receptors Are Involved in Vitamin K1 Absorption

Vitamin K₁ (phylloquinone) intestinal absorption is thought to be mediated by a carrier protein that still remains to be identified. Apical transport of vitamin K₁ was examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium and in transfected HEK cells. Phylloquinone uptake was then measured ex vivo using mouse intestinal explants. Finally, vitamin K₁ absorption was compared between wild-type mice and mice overexpressing scavenger receptor class B type I (SR-BI) in the intestine and mice deficient in cluster determinant 36 (CD36). Phylloquinone uptake by Caco-2 cells was saturable and was significantly impaired by co-incubation with α-tocopherol (and vice versa). Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 85% of vitamin K₁ uptake. BLT1 also decreased phylloquinone apical efflux by ∼80%. Transfection of HEK cells with SR-BI and CD36 significantly enhanced vitamin K₁ uptake, which was subsequently decreased by the addition of BLT1 or sulfo-N-succinimidyl oleate (CD36 inhibitor), respectively. Similar results were obtained in mouse intestinal explants. In vivo, the phylloquinone postprandial response was significantly higher, and the proximal intestine mucosa phylloquinone content 4 h after gavage was increased in mice overexpressing SR-BI compared with controls. Phylloquinone postprandial response was also significantly increased in CD36-deficient mice compared with wild-type mice, but their vitamin K₁ intestinal content remained unchanged. Overall, the present data demonstrate for the first time that intestinal scavenger receptors participate in the absorption of dietary phylloquinone.     

Hepatic ABCG5 and ABCG8 overexpression increases hepatobiliary sterol transport but does not alter aortic atherosclerosis in transgenic mice

The individual roles of hepatic versus intestinal ABCG5 and ABCG8 in sterol transport have not yet been investigated. To determine the specific contribution of liver ABCG5/G8 to sterol transport and atherosclerosis, we generated transgenic mice that overexpress human ABCG5 and ABCG8 in the liver but not intestine (liver G5/G8-Tg) in three different genetic backgrounds: C57Bl/6, apoE-KO, and low density lipoprotein receptor (LDLr)-KO. Hepatic overexpression of ABCG5/G8 enhanced hepatobiliary secretion of cholesterol and plant sterols by 1.5-2-fold, increased the amount of intestinal cholesterol available for absorption and fecal excretion by up to 27%, and decreased the accumulation of plant sterols in plasma by approximately 25%. However, it did not alter fractional intestinal cholesterol absorption, fecal neutral sterol excretion, hepatic cholesterol concentrations, or hepatic cholesterol synthesis. Consequently, overexpression of ABCG5/G8 in only the liver had no effect on the plasma lipid profile, including cholesterol, HDL-C, and non-HDL-C, or on the development of proximal aortic atherosclerosis in C57Bl/6, apoE-KO, or LDLr-KO mice. Thus, liver ABCG5/G8 facilitate the secretion of liver sterols into bile and serve as an alternative mechanism, independent of intestinal ABCG5/G8, to protect against the accumulation of dietary plant sterols in plasma. However, in the absence of changes in fractional intestinal cholesterol absorption, increased secretion of sterols into bile induced by hepatic overexpression of ABCG5/G8 was not sufficient to alter hepatic cholesterol balance, enhance cholesterol removal from the body or to alter atherogenic risk in liver G5/G8-Tg mice. These findings demonstrate that overexpression of ABCG5/G8 in the liver profoundly alters hepatic but not intestinal sterol transport, identifying distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism.     

Use of NBD-cholesterol to identify a minor but NPC1L1-independent cholesterol absorption pathway in mouse intestine

The importance of Niemann-Pick C1 Like-1 (NPC1L1) protein in intestinal absorption of dietary sterols, including both cholesterol and phytosterols, is well documented. However, the exact mechanism by which NPC1L1 facilitates cholesterol transport remains controversial. This study administered 22-(N(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol) and [(3)H]cholesterol to Npc1l1(+/+) and Npc1l1(-/-) mice to determine whether NPC1L1 facilitates dietary sterol uptake by enterocytes and/or participates in intracellular sterol delivery to the endoplasmic reticulum (ER) for lipoprotein assembly before secretion into plasma circulation. Results showed that [(3)H]cholesterol absorption was reduced but not abolished in Npc1l1(-/-) mice compared with Npc1l1(+/+) mice. In the presence of Pluronic L-81 to block pre-chylomicron exit from the ER, significant amounts of [(3)H]cholesterol were found to be associated with lipid droplets in the intestinal mucosa of both Npc1l1(+/+) and Npc1l1(-/-) mice, and the intracellular [(3)H]cholesterol can be esterified to cholesteryl esters. These results provided evidence indicating that the main function of NPC1L1 is to promote cholesterol uptake from the intestinal lumen but that it is not necessary for intracellular cholesterol transport to the ER. Surprisingly, NBD-cholesterol was taken up by intestinal mucosa, esterified to NBD-cholesteryl esters, and transported to plasma circulation to similar extent between Npc1l1(+/+) and Npc1l1(-/-) mice. Ezetimibe treatment also had no impact on NBD-cholesterol absorption by Npc1l1(+/+) mice. Thus, NBD-cholesterol absorption proceeds through an NPC1L1-independent and ezetimibe-insensitive sterol absorption mechanism. Taken together, these results indicate that NBD-cholesterol can be used to trace the alternative cholesterol absorption pathway but is not suitable for tracking NPC1L1-mediated cholesterol absorption.     

Secretory phospholipase A2 increases SR-BI-mediated selective uptake from HDL but not biliary cholesterol secretion

High density lipoprotein cholesterol represents a major source of biliary cholesterol. Secretory phospholipase A2 (sPLA2) is an acute phase enzyme mediating decreased plasma HDL cholesterol levels. Clinical studies reported a link between increased sPLA2 expression and the presence of cholesterol gallstones. The aim of our study was to investigate whether the overexpression of human sPLA2 in transgenic mice affects biliary cholesterol secretion and gallstone formation. Liver weight (P < 0.01) and hepatic cholesterol content (P < 0.01) were significantly increased in sPLA2 transgenic mice compared with controls as a result of increased scavenger receptor class B type I (SR-BI)-mediated hepatic selective uptake of HDL cholesterol (P < 0.01), whereas hepatic SR-BI expression remained unchanged. However, biliary cholesterol secretion as well as fecal neutral sterol and fecal bile salt excretion remained unchanged in sPLA2 transgenic mice. Furthermore, gallstone prevalence in response to a lithogenic diet was identical in both groups. These data demonstrate that i) increased flux of cholesterol from HDL into the liver via SR-BI as a result of phospholipase modification of the HDL particle translates neither into increased biliary and fecal sterol output nor into increased gallstone formation, and ii) increased sPLA2 expression in patients with cholesterol gallstones might be a consequence rather than the underlying cause of the disease.     

Bile salt-stimulated carboxyl ester lipase influences lipoprotein assembly and secretion in intestine: a process mediated via ceramide hydrolysis.

Bile salt-stimulated carboxyl ester lipase (CEL), also called cholesterol esterase, is one of the major proteins secreted by the pancreas. The physiological role of CEL was originally thought to be its mediation of dietary cholesterol absorption. However, recent studies showed no difference between wild type and CEL knockout mice in the total amount of cholesterol absorbed in a single meal. The current study tests the hypothesis that CEL in the intestinal lumen may influence the type of lipoproteins produced. A lipid emulsion containing 4 mm phospholipid, 13.33 mm [(3)H]triolein, and 2.6 mm [(14)C]cholesterol in 19 mm taurocholate was infused into the duodenum of lymph fistula CEL(+/+) and CEL(-/-) mice at a rate of 0.3 ml/h. Results showed no difference between CEL(+/+) and CEL(-/-) mice in the rate of cholesterol and triglyceride transport from the intestinal lumen to the lymph. However, CEL(-/-) mice produced predominantly smaller lipoproteins, whereas the CEL(+/+) mice produced primarily large chylomicrons and very low density lipoprotein. The proximal intestine of CEL(-/-) mice was also found to possess significantly less ceramide hydrolytic activity than that present in CEL(+/+) mice. By using Caco2 cells grown on Transwell membranes as a model, sphingomyelinase treatment inhibited the secretion of larger chylomicron-like lipoproteins without affecting total cholesterol secretion. In contrast, the addition of CEL to the apical medium increased the amount of large lipoproteins produced and alleviated the inhibition induced by sphingomyelinase. Taken together, this study identified a novel and physiologically significant role for CEL, namely the promotion of large chylomicron production in the intestine. The mechanism appears to be mediated through CEL hydrolysis of ceramide generated during the lipid absorption process.