Scavenger receptor class B type I (SR-BI) in pig enterocytes: trafficking from the brush border to lipid droplets during fat absorption

Abstract

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.

Figure 1

Localisation of scavenger receptor class B type I (SR-BI) in the enterocyte in the fasting state. In the brush border, immunogold labelling was seen over the microvilli as well as at the base of the microvilli (MM) and in invaginations between adjacent microvilli (A). In the cytoplasm, labelling was seen in an apical tubulovesicular compartment (TVC) (B), and in small lipid droplets (LD) (C, D). Multivesicular bodies/lysosomes (MB) and mitochondria (MI) were not labelled (D). Bars 0.5 μm.

Figure 2

Localisation of scavenger receptor class B type I (SR-BI) in large cytoplasmic lipid droplets during fat absorption. (A) Electron micrograph of an Epon section showing numerous lipid droplets in the cytoplasm. Two morphologically distinct types of lipid droplets were seen: some relatively large and dark (*) and some relatively small and light, often seen in clusters (#). (B) Immunogold labelling of an Epon section for SR-BI, showing that only the large and dark lipid droplets were labelled. Note that labelling was not confined to the rim but distributed over the entire droplet. (C) Double immunogold labelling of an ultracryosection for apolipoprotein A-1 (10 nm particles) and SR-BI (5 nm particles) of a cluster of the small and light type of lipid droplets. Apolipoprotein A-1 was localised to the rim of the droplets, thereby identifying them as nascent chylomicrons. SR-BI labelling was not seen in chylomicrons. Bars 2 μm (A); 1 μm (B); 0.2 μm (C).

Figure 3

Scavenger receptor class B type I (SR-BI) localisation in coated pits during the early phase of lipid absorption. (A) Electron micrograph of the brush border region of an enterocyte of an explant, cultured for 30 minutes in the presence of lipid, as described in the methods section. Many coated pits (CP) are present between adjacent microvilli (MM) in the terminal web region. (B) Immunogold labelling of an ultracryosection for clathrin, showing labelling of an apical coated pit (CP) and a coated vesicle (CV). (C, D) Double immunogold labelling of ultracryosections for SR-BI (5 nm particles, indicated by arrowheads) and clathrin (10 nm particles, indicated by arrows), showing colocalisation in coated pits at the apical surface. Bars 0.5 μm (A); 0.2 μm (B–D).

Figure 4

Scavenger receptor class B type I (SR-BI) localisation in nascent lipid droplets during the early phase of lipid absorption. (A) SR-BI immunogold labelling of an Epon section from a mucosal explant, cultured for 60 minutes in the presence of lipid. Labelling was seen in nascent lipid droplets (LD), associated with the endoplasmic reticulum (ER), as well as in a large cytoplasmic lipid droplet (*). (B) Immunogold labelling for aminopeptidase N in a similar Epon section. The enzyme was seen at the microvillar surface (MM) and in the apical tubulovesicular compartment (TVC), but the lipid droplets (*) were devoid of labelling. Bars 0.5 μm.

Figure 5

Subcellular localisation of scavenger receptor class B type I (SR-BI) in enterocytes. (A) Small intestinal mucosa was fractionated into Mg²⁺ precipitated membranes (Mg), microvillar membranes (Mic), and soluble protein (Sol), as described in the methods section. Samples of the three subcellular fractions proportional to their relative amount in the homogenate (about 50–200 μg of protein) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis. After electrophoresis and transfer onto Immobilon, the 82 kDa band of SR-BI was visualised by immunoblotting. After blotting, total protein was stained by Coomassie brilliant blue. (B) Immunopurification of SR-BI. Microvillar membranes (1) were solubilised by 1% Triton X-100 at 20°C (both raft and non-raft membranes are solubilised at this temperature), and preincubated with protein A-Sepharose, followed either by anti-SR-BI antibodies coupled to protein A-Sepharose (2) or free protein A-Sepharose (3), as described in the methods section. Samples were analysed by immunoblotting as described above. (The broad band of 50 kDa in lane 2 represents the SR-BI antibody. In lane 3, protein A-Sepharose bound some endogeneous immunoglobulin and secretory component.)

Figure 6

Lipid raft analysis of scavenger receptor class B type I (SR-BI). Sucrose gradient centrifugation of Mg²⁺ precipitated (A) and microvillar (B) membranes prepared from the same mucosal homogenate. The membranes were prepared, extracted with 1% ice cold Triton X-100, and subjected to sucrose gradient centrifugation, as described in the methods section. Samples of the gradient fractions were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting for the distribution of SR-BI and, in the case of the microvillar gradient, for the lipid raft marker galectin-4 (Gal-4) and the “non-raft” marker lactase-phlorizin hydrolase. Gradient fractions containing lipid raft membranes and soluble (“non-raft”) proteins are indicated by arrows.

Figure 7

“Non-raft” localisation of microvillar scavenger receptor class B type I (SR-BI) is not cholesterol dependent. Microvillar membranes were resuspended in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1, in the presence or absence of 2% methyl-β-cyclodextrin (MβCD) for 30 minutes at room temperature before cooling on ice and solubilisation by 1% Triton X-100 for 10 minutes. After centrifugation at 20 000 g, 30 minutes, the supernatant (NR, the “non-raft” fraction) was collected, and the pellet was resuspended in the above buffer and solubilised by 1% Triton X-100 for 10 minutes at 37°C. After centrifugation as above, the resulting supernatant (R, the lipid raft fraction) and pellet (P, insoluble cytoskeletal proteins) were collected. NR, R, and P samples were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis. After electrophoresis, SR-BI, lactase, and galectin-4 (Gal-4) were detected by immunoblotting.