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Transintestinal cholesterol efflux (TICE) has been proposed to represent a non-hepatobiliary route of cholesterol secretion directly “from blood to gut” and to play a physiologically significant role in excretion of neutral sterols, but so far little is known about the proteins involved in the process. We have previously observed that apolipoprotein A-1 (apoA-1) synthesized by enterocytes of the small intestine is mainly secreted apically into the gut lumen during fasting where its assembly into chylomicrons and basolateral discharge is at a minimal level. In the present work we showed, both by immunomicroscopy and subcellular fractionation, that a fraction of the apically secreted apoA-1 in porcine small intestine was not released from the cell surface but instead deposited in the brush border. Cholesterol was detected in immunoisolated microvillar apoA-1, and it was partially associated with detergent resistant membranes (DRMs), indicative of localization in lipid raft microdomains. The apolipoprotein was not readily released from microvillar vesicles by high salt or by incubation with phosphatidylcholine-specific phospholipase C or trypsin, indicating a relatively firm attachment to the membrane bilayer. However, whole bile or taurocholate efficiently released apoA-1 at low concentrations that did not solubilize the transmembrane microvillar protein aminopeptidase N. Based on these findings and the well known role played by apoA-1 in extrahepatic cellular cholesterol removal and reverse cholesterol transport (RCT), we propose that brush border-deposited apoA-1 in the small intestine acts in TICE by mediating cholesterol efflux into the gut lumen.

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host's response to endotoxin and mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including the enterocyte-like cell line Caco-2. To study in closer detail the synthesis and storage of LBP in the intestinal mucosal epithelium, we performed an immunolocalization of LBP in mouse small intestine. By immunofluorescence microscopy, an antibody recognizing the 58-60 kDa protein of LBP distinctly labeled a small population of cells located deep into the crypts. This cell population was also positive for lysozyme and alpha-defensin 4, identifying Paneth cells as the main intestinal LBP-producing cells. By immunogold electron microscopy, intense labeling was observed in the secretory granules of these cells. We conclude that Paneth cells express LBP together with other proteins acting in the innate immune response of the gut, such as lysozyme, defensins and intelectin.

The fibroblast-like synoviocyte is a CD13-positive cell-type containing numerous caveolae, both single and interconnected clusters. In unstimulated cells, all single caveolae at the cell surface and the majority of those localized deeper into the cytoplasm were freely accessible from the medium, as judged from electron microscopy of synoviocytes exposed to the membrane impermeable marker Ruthenium Red. Caveolar internalization could be induced by a CD13 antibody or by cholera toxin B subunit (CTB). Thus, in experiments using sequential labeling with Alexa 488- and 594-conjugated CTB, about 50% of CTB-positive caveolae were internalized by 5 min of chase, and these remained inaccessible from the cell surface for periods up to 24 h. No colocalization with an endosomal marker, EEA1, or Lysotracker was observed, indicating that internalized caveolae clusters represent a static compartment. Vimentin was identified as the most abundant protein in detergent resistant membranes (DRM’s), and by immunogold electron microscopy caveolae were seen in intimate contact with intermediate-size filaments. These observations indicate that vimentin-based filaments are responsible for the spatio-temporal fixation of caveolae clusters. RECK, a glycosylphosphatidylinositol-anchored protein acting as a negative regulator of cell surface metalloproteinases, was also localized to the caveolae clusters. We propose that these clusters function as static reservoirs of specialized lipid raft domains where proteins involved in cell–cell interactions, such as CD13, can be sequestered by binding to RECK in a regulatory manner.

Leptin is a hormone that plays an important role in overall body energy homeostasis, and the obesity receptor, OB-R, is widely distributed in the organism. In the intestine, a multitude of leptin actions have been reported, but it is currently unclear to what extent the hormone affects the intestinal epithelial cells by an endocrine or exocrine signaling pathway. To elucidate this, the localization of endogenous porcine leptin and OB-R in enterocytes and colonocytes was studied. By immunofluorescence microscopy, both leptin and OB-R were mainly observed in the basolateral membrane of enterocytes and colonocytes but also in the apical microvillar membrane of the cells. By electron microscopy, coclustering of hormone and receptor in the plasma membrane and localization in endosomes was frequently detected at the basolateral surface of the epithelial cells, indicative of leptin signaling activity. In contrast, coclustering occurred less frequently at the apical cell surface, and subapical endosomal localization was hardly detectable. We conclude that leptin action in intestinal epithelial cells takes place at the basolateral plasma membrane, indicating that the hormone uses an endocrine pathway both in the jejunum and colon. In contrast, the data obtained did not provide evidence for an exocrine, lumenal action of the hormone in the intestine. (J Histochem Cytochem 56:677–685, 2008)

Absorption of dietary fat in the small intestine is accompanied by a rise of intestinal alkaline phosphatase (IAP) in the serum and of secretion of IAP-containing surfactant-like particles from the enterocytes. In the present work, fat absorption was studied in organ cultured mouse intestinal explants. By immunofluorescence microscopy, fat absorption caused a translocation of IAP from the enterocyte brush border to the interior of the cell, whereas other brush-border enzymes were unaffected. By electron microscopy, the translocation occurred by a rapid (5 min) induction of endocytosis via clathrin-coated pits. By 60 min, IAP was seen in subapical endosomes and along membranes surrounding fat droplets. IAP is a well-known lipid raft-associated protein, and fat absorption was accompanied by a marked change in the density and morphology of the detergent-resistant membranes harboring IAP. A lipid analysis revealed that fat absorption caused a marked increase in the microvillar membrane contents of free fatty acids. In conclusion, fat absorption rapidly induces a transient clathrin-dependent endocytosis via coated pits from the enterocyte brush border. The process selectively internalizes IAP and may contribute to the appearance of the enzyme in serum and surfactant-like particles.

The pig small intestinal brush border is a glycoprotein- and glycolipid-rich membrane that functions as a digestive/absorptive surface for dietary nutrients as well as a permeability barrier for pathogens. The present work was performed to identify carbohydrate-binding (lectinlike) proteins associated with the brush border. Chromatography on lactose-agarose was used to isolate such proteins, and their localization was studied biochemically and by immunofluorescence microscopy and immunogold electron microscopy. IgG and IgM were the two major proteins isolated, indicating that naturally occurring anti-glycosyl antibodies are among the major lectinlike proteins in the gut. IgG and IgM as well as IgA were localized to the enterocyte brush border, and a brief lactose wash partially released all three immunoglobulins from the membrane, indicating that anti-glycosyl antibodies constitute a major part of the immunoglobulins at the lumenal surface of the gut. The antibodies were associated with lipid rafts at the brush border, and they frequently (52%) coclustered with the raft marker galectin 4. A lactose wash increased the susceptibility of the brush border toward lectin peanut agglutin and cholera toxin B, suggesting that anti-glycosyl antibodies compete with other carbohydrate-binding proteins at the lumenal surface of the gut. Thus anti-glycosyl antibodies constitute a major group of proteins associated with the enterocyte brush border membrane. We propose they function by protecting the lipid raft microdomains of the brush border against pathogens.

The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization. CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation. By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C. By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis. In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.

The brush border of small intestinal enterocytes is highly enriched in cholesterol- and glycosphingolipid-containing membrane microdomains, commonly termed as lipid ‘rafts’. Functionally, transcytosis of IgA and exocytosis of newly made brush-border proteins in enterocytes occur through apical lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized by the non-permeable surface marker Ruthenium Red in the brush-border region of the cells. The surface-connected tubules were labelled by antibodies to caveolin-1 and the glycolipid asialo GM1, and they were sensitive to cholesterol depletion by methyl-β-cyclodextrin, indicating the presence of raft microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50–100 nm, and penetrated up to 1 μm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion suggests that deep-apical tubules function as a cell-surface membrane reservoir for cholesterol and for rapid adaptive changes in the size of microvilli at the brush border.

Lipid rafts (glycosphingolipid/cholesterol-enriched membrane microdomains) have been isolated as low temperature, detergent-resistant membranes from many cell types, but despite their presumed importance as lateral sorting and signaling platforms, fundamental questions persist concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at physiological temperature to compare with rafts, obtained by “conventional” extraction using Triton X-100 at low temperature. Microvillar rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 °C and are not “low temperature artifacts.” We also employed a novel method of sequential detergent extraction at increasing temperature to define a fraction of highly detergent-resistant “superrafts.” These were enriched in galectin-4, a β-galactoside-recognizing lectin residing on the extracellular side of the membrane. Superrafts also harbored the glycosylphosphatidylinositol-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane microdomain systems.

Glycosphingolipid/cholesterol-rich membranes ("rafts")can be isolated from many types of cells, but their existence as stable microdomains in the cell membrane has been elusive. Addressing this problem, we studied the distribution of galectin-4, a raft marker, and lactase, a protein excluded from rafts, on microvillar vesicles from the enterocyte brush border membrane. Magnetic beads coated with either anti-galectin-4 or anti-lactase antibodies were used for immunoisolation of vesicles followed by double immunogold labeling of the two proteins. A morphometric analysis revealed subpopulations of raft-rich and raft-poor vesicles by the following criteria: 1) the lactase/galectin-4 labeling ratio/vesicle captured by the anti-lactase beads was significantly higher (por = 0.01) than that of vesicles captured by anti-galectin-4 beads, 2) subpopulations of vesicles labeled by only one of the two antibodies were preferentially captured by beads coated with the respective antibody (por = 0.01), 3) the average diameter of "galectin-4 positive only" vesicles was smaller than that of vesicles labeled for lactase. Surprisingly, pretreatment with methyl-beta-cyclodextrin, which removed70% of microvillar cholesterol, did not affect the microdomain localization of galectin-4. We conclude that stable, cholesterol-independent raft microdomains exist in the enterocyte brush border.

Membrane peptidases play important roles in cell activation, proliferation and communication. Human fibroblast-like synoviocytes express considerable amounts of aminopeptidase N/CD13, dipeptidyl peptidase IV/CD26, and neprilysin/CD10, transmembrane proteins previously proposed to be involved in the regulation of intra-articular levels of neuropeptides and chemotactic mediators as well as in adhesion and cell–cell interactions. Here, we report these peptidases in synoviocytes to be localized predominantly in glycolipid- and cholesterol-rich membrane microdomains known as ‘rafts’. At the ultrastructural level, aminopeptidase N/CD13 and dipeptidyl peptidase IV/CD26 were found in caveolae, in particular in intracellular yet surface-connected vesicle-like structures and ‘rosettes’ made up of several caveolae. In addition, clusters of peptidases were seen at the cell surface in flat patches ranging in size from about 60 to 160nm. Cholesterol depletion of synoviocytes by methyl-β-cyclodextrin disrupted > 90% of the caveolae and reduced the raft localization of aminopeptidase N/CD13 without affecting Ala-p-nitroanilide-cleaving activity of confluent cell cultures. In co-culture experiments with T-lymphocytes, cholesterol depletion of synoviocytes greatly reduced their capability to induce an early lymphocytic expression of aminopeptidase N/CD13. We propose caveolae/rafts to be peptidase-rich ‘hot-spot’ regions of the synoviocyte plasma membrane required for functional cell–cell interactions with lymphocytes. The peptidases may act in concert with other types of proteins such as receptors and signal transducers localized in these specialized membrane domains.

Intestinal brush border enzymes, including aminopeptidase N and sucrase-isomaltase, are associated with “rafts” (membrane microdomains rich in cholesterol and sphingoglycolipids). To assess the functional role of rafts in the present work, we studied the effect of cholesterol depletion on apical membrane trafficking in enterocytes. Cultured mucosal explants of pig small intestine were treated for 2 h with the cholesterol sequestering agent methyl-β-cyclodextrin and lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase. The treatment reduced the cholesterol content >50%. Morphologically, the Golgi complex/trans-Golgi network was partially transformed into numerous 100–200 nm vesicles. By immunogold electron microscopy, aminopeptidase N was localized in these Golgi-derived vesicles as well as at the basolateral cell surface, indicating a partial missorting. Biochemically, the rates of the Golgi-associated complex glycosylation and association with rafts of newly synthesized aminopeptidase N were reduced, and less of the enzyme had reached the brush border membrane after 2 h of labeling. In contrast, the basolateral Na+/K+-ATPase was neither missorted nor raft-associated. Our results implicate the Golgi complex/trans-Golgi network in raft formation and suggest a close relationship between this event and apical membrane trafficking.

Background & Aims: Glycolipid "rafts" have been shown to play a role in apical membrane trafficking in the enterocyte. The present study characterized the membrane compartments of the enterocyte involved in transepithelial transport of small intestinal immunoglobulin A (IgA). Methods: Immunogold electron microscopy and radioactive labeling of mouse small intestinal explants were performed. Results: IgA and the polymeric immunoglobulin receptor/secretory component were present in a raft compartment. Raft association occurred posttranslationally within 30 minutes, preceding secretion into the culture medium. IgA labeling was seen primarily in enterocytes along the basolateral plasma membrane and over endosomes and small vesicles in the basolateral and apical regions of the cytoplasm. IgA and a brush border enzyme, aminopeptidase N, were colocalized in apical endosomes and small vesicles and were also frequently seen associated with the same vesicular profiles of glycolipid rafts. Colocalization of IgA and rab17, a small guanosine triphosphatase involved in transcytosis, was seen mainly along the basolateral plasma membrane and over basolateral endosomes and vesicles, but also in the apical region of the cytoplasm. Conclusions: IgA is transcytosed through a raft-containing compartment, most likely the apical endosomes. Our data also support the notion that rab17 is involved in transcytotic membrane traffic. GASTROENTEROLOGY 1999;116:610-622

Absorption of dietary fat in the small intestine involves epithelial exposure to potentially harmful molecules such as bile salts and free fatty acids. We used organ culture of porcine jejunal explants incubated with a pre-digested mixture of fat (plant oil), bile and pancreatin to mimick the physiological process of dietary fat absorption, and short exposures to the fat mixture caused fat droplet accumulation within villus enterocytes. Lucifer yellow (LY), a fluorescent membrane-impermeable polar tracer was included to monitor epithelial integrity. Both in controls and during fat absorption LY penetrated the epithelium and accumulated in the basal lamina and the lamina propria. LY was also seen in the paracellular space, whereas villus enterocytes were generally only weakly labeled except for small amounts taken up by apical endocytosis. In the crypts, however, fat absorption induced cell permeabilization with LY accumulating in the cytosol and nucleus. Morphologically, both apical and basolateral membranes appeared intact, indicating that the leakiness was caused by minor lesions in the membrane. Albeit to a lesser extent, bile alone was capable of permeabilizing crypt cells, implying that the surfactant properties of bile salts are involved in the process. In addition to LY, crypt enterocytes also became permeable for albumin, ovalbumin and insulin. In conclusion, during fat absorption the permeability of the gut epithelium is increased mainly in the crypts. A possible explanation is that cell membranes of immature crypt cells, lacking detergent-resistant lipid raft microdomains, are less resistant to the deleterious effects of bile salts and free fatty acids.

Background & Aims: Little is known about the expression of brush border enzymes in fetal enterocytes. The aim of this study was to describe the localization and biosynthesis of porcine fetal aminopeptidase N. Methods: This study was performed using histochemistry and immunoelectron microscopy and [ 35 S]methionine labeling of cultured mucosal explants. Results: Enzyme activity was present in the brush border membrane and extended into the apical cytoplasm. The protein was colocalized with cationized ferritin at the surface of endocytic structures including coated pits, vesicles, tubules, and large vacuoles in the apical cytoplasm. The transient high mannose-glycosylated form of fetal aminopeptidase N was processed to the mature complex-glycosylated form at a markedly slower rate than the enzyme in adult intestine. Likewise, dimerization occurred slowly compared with the adult form of aminopeptidase N, and it took place mainly after the Golgi-associated complex glycosylation. The enzyme had a biphasic appearance in the Mg 2+ -precipitated and microvillar fractions, indicating that the bulk of newly made aminopeptidase N is transported to the brush border membrane before appearing in the apical endocytic structures. Conclusions: In comparison with the adult enzyme, fetal aminopeptidase N has a more widespread subcellular distribution with substantial amounts present in apical endocytic compartments characteristic of the fetal enterocyte.

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) and KO mice were fed 3H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested 3H-SM in the intestinal content increased by 6-fold and the formation of 3H-ceramide decreased markedly, resulting in 98% reduction of 3H-ceramide/3H-SM ratio 1 h after gavage. The absorbed 3H-palmitic acid portion was decreased by 95%. After 3 h, a small increase in 3H-ceramide was identified in distal intestine in KO mice. In feces, 3H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.

The small intestinal brush border functions as the body's main portal for uptake of dietary nutrients and simultaneously acts as the largest permeability barrier against pathogens. To enable this, the digestive enzymes of the brush border are organized in lipid raft microdomains stabilized by cross-linking galectins and intelectin, but little is known about the dynamic properties of this highly specialized membrane. Here, we probed the endocytic membrane trafficking from the brush border of organ-cultured pig intestinal mucosal explants by use of a fixable, lipophilic FM dye. The fluorescent dye readily incorporated into the brush border, and by 15 min faint but distinct punctae were detectable ∼1 μm beneath the brush border, indicative of a constitutive endocytosis. The punctae represented a subpopulation of early endosomes confined to the actomyosin-rich terminal web region, and their number and intensity increased by 1 h, but trafficking further into the enterocyte was not observed except in immature epithelial cells of the crypts. A powerful ligand for receptor-mediated endocytosis, cholera toxin B subunit, increased apical endocytosis and caused membrane trafficking to proceed to compartments localized deeper into the cytoplasm of the enterocytes. Two major raft-associated brush border enzymes, alkaline phosphatase and aminopeptidase N, were excluded from endocytosis. We propose that the terminal web cytoskeleton, by inhibiting traffic from apical early endosomes further into the cell, contributes to the overall permeability barrier of the gut.

Synthesis and deposition of immunoglobulins in the brush border was studied in organ-cultured pig small intestinal mucosal explants. Surprisingly, comparable amounts of IgM and IgA were synthesized during a 6-h pulse, and also newly made IgG was detected in media and explants, including the microvillar fraction. For IgA and IgM, this subcellular distribution is consistent with basolateral-to-apical transcytosis, mediated by the polymeric immunoglobulin receptor. IgG is a ligand for the Fc receptor FcRn, and β2-microglobulin, the light chain of FcRn, coclustered in immunogold double labeling with IgG in subapical endosomes and in the basolateral membrane of enterocytes. In addition, β2-microglobulin was copurified with IgG on protein G-Sepharose. Apical endocytosis of IgG, as judged by internalization of fluorescent protein G, was not detectable except in a few isolated cells. This suggests that IgG in the adult small intestine is transported across the enterocyte mainly in the basolateral to apical direction. Significant fractions of all immunoglobulins bound to lactoseagarose, indicating that “anti-glycosyl” antibodies, raised against commensal gut bacteria, are synthesized locally in the small intestine. By partial deposition in the brush border, these antibodies therefore may have a protective function by preventing lectin-like pathogens from gaining access to the brush border surface.

The microdomain localization of the GABA(A) receptor in rat cerebellar granule cells was studied by subcellular fractionation and fluorescence- and immunogold electron microscopy. The receptor resided in lipid rafts, prepared at 37 degrees C by extraction with the nonionic detergent Brij 98, but the raft fraction, defined by the marker ganglioside GM(1) in the floating fractions following density gradient centrifugation, was heterogeneous in density and protein composition. Thus, another major raft-associated membrane protein, the Na(+), K(+)-ATPase, was found in discrete rafts of lower density, reflecting clustering of the two proteins in separate membrane microdomains. Both proteins were observed in patchy "hot spots" at the cell surface as well as in isolated lipid rafts. Their insolubility in Brij 98 was only marginally affected by methyl-beta-cyclodextrin. In contrast, both the GABA(A) receptor and Na(+), K(+)-ATPase were largely soluble in ice cold Triton X-100. This indicates that Brij 98 extraction defines an unusual type of cholesterol-independent lipid rafts that harbour membrane proteins also associated with underlying scaffolding/cytoskeletal proteins such as gephyrin (GABA(A) receptor) and ankyrin G (Na(+), K(+)-ATPase). By providing an ordered membrane microenvironment, lipid rafts may contribute to the clustering of the GABA(A) receptor and the Na(+), K(+)-ATPase at distinct functional locations on the cell surface.

Background: Scavenger receptor class B type I (SR-BI) is known to mediate cellular uptake of cholesterol from high density lipoprotein particles and is particularly abundant in liver and steroidogenic tissues. In addition, SR-BI expression in the enterocyte brush border has also been reported but its role in the small intestine remains unclear. Aim and methods: To gain insight into the possible function of pig SR-BI during uptake of dietary fat, its localisation in enterocytes was studied in the fasting state and during fat absorption by immunogold electron microscopy and subcellular fractionation. Results: In the fasting state, SR-BI was mainly localised in the microvillar membrane and in apical invaginations/pits between adjacent microvilli. In addition, a subapical compartment and small cytoplasmic lipid droplets were distinctly labelled. During lipid absorption, the receptor was found in clathrin positive apical coated pits and vesicles. In addition, cytoplasmic lipid droplets that greatly increased in size and number were strongly labelled by the SR-BI antibody whereas apolipoprotein A-1 positive chylomicrons were largely devoid of the receptor. Conclusion: During absorption of dietary fat, SR-BI is endocytosed from the enterocyte brush border and accumulates in cytoplasmic lipid droplets. Internalisation of the receptor occurs mainly by clathrin coated pits rather than by a caveolae/lipid raft based mechanism.