Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

Abstract

Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MbetaCD. The inhibition in ASBT activity by MbetaCD was blocked in the cells treated with MbetaCD-cholesterol complexes. Kinetic analysis revealed that MbetaCD treatment decreased the V(max) of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis.

Fig. 1

Expression of apical sodium-dependent bile acid transporter (ASBT)-V5 fusion protein in transfected human embryonic kidney (HEK)-293 cells. A: HEK-293 fibroblasts were transfected with pcDNA 2.3/V5 vector engineered to stably express ASBT protein fused to V5 tagged at the COOH terminus. Western blot analysis with anti-V5 antibodies demonstrates expression of ASBT-V5 fusion protein in HEK-293 cells stably transfected with ASBT-V5 (designated as 2BT cells) compared with wild-type (WT) cells. Actin expression from the same samples is shown in both WT and transfected cells. B: total membrane preparations were made from 2BT cells and lysed with 1% Triton X-100 at 4°C, as described in materials and methods. Detergent-soluble (DS) and detergent-insoluble (DI) fractions were isolated, and level ASBT-V5 fusion protein associated with these fractions was assessed by Western blot analyses utilizing anti-V5 antibodies.

Fig. 2

Association of ASBT-V5 fusion protein with lipid rafts in 2BT cells. 2BT cells were incubated with biotin-conjugated cholera toxin subunit B (CTxB) to label lipid rafts of plasma membrane. Labeled cells were incubated with methyl-β-cyclodextrin (MβCD) to remove cholesterol from plasma membrane or with vehicle alone. Cells were then lysed with 1% Triton X-100, and total membrane extracts were prepared as described in materials and methods. Samples were subjected to floatation on Optiprep density gradients, and fractions were collected from top of the gradients. Fractions were separated by electrophoresis and analyzed by Western blotting. A: Western blot results using anti-V5 antibodies. B: blots were probed with avidin peroxidase antibodies to label the biotinylated CTxB. C: blots were probed with actin antibodies.

Fig. 3

MβCD decreased ASBT function in 2BT cells. A: 2BT cells were treated with different concentrations of MβCD for 30 min at 37°C. Cells were washed and then incubated at 25°C with HEPES-buffered uptake solution (pH 7.4) containing 110 mM of NaCl or choline chloride along with 10 μM [³H]taurocholic acid (TC) for 5 min, and sodium-dependent [³H]TC uptake was determined. Results are presented as % of control and are means ± SE obtained from at least 3 separate experiments. *P < 0.05 compared with control. B: 2BT cells were treated with 10 mM MβCD alone or with 10 mM of MβCD + 1.25 mM of cholesterol for 30 min at 37°C, and Na⁺-dependent [³H]TC uptake was assessed. Data are expressed as % of control and are means ± SE obtained from at least 3 separate experiments. *P < 0.05 compared with control.

Fig. 4

Human ASBT (hASBT) is associated with DI fractions of human ileal brush-border membrane vesicles (BBMVs). A: human ileal BBMVs were treated with MβCD in the presence or the absence of 1.25 mM cholesterol or with vehicle alone and solubilized with Triton X-100, and then DS and DI fractions were isolated as described in materials and methods. Equal amounts of proteins (~80 μg) from DI and DS fractions were separated by electrophoresis on 10% polyacrylamide gel and then analyzed by Western blotting for ASBT and actin expression. B: blots were scanned, and the densities of the bands were determined by densitometric analysis. y-Axis indicates the density of DI fractions from the Optiprep gradient. Data are expressed as arbitrary units with control set as 100% and are means ± SE of 4 separate experiments. *P < 0.05 compared with control.

Fig. 5

ASBT is detected in the floating fractions of human ileal BBMVs treated with Triton X-100 on Optiprep density gradient. A: Human ileal BBMVs were treated with 1% Triton X-100 for 30 min at 4°C and then were layered at the bottom of a discontinuous density gradient of Optiprep as described in materials and methods. After high-speed centrifugation, fractions were collected from the top to the bottom of the gradient, and their proteins were separated on 2 separate SDS-PAGE gels and analyzed simultaneously by Western blotting utilizing ASBT antibodies. The blots shown are a representative of 3 separate experiments. B: specific activity of alkaline phosphatase (a marker of lipid rafts) was assessed in each fraction of the Optiprep floatation gradient of human ileal BBMVs. Alkaline phosphatase activity is expressed as nmol•mg protein⁻¹ •min⁻¹, and data are means ± SE of 3 separate measurements from different occasions.

Fig. 6

MβCD inhibits ASBT activity in human intestinal Caco-2 cells. A: postconfluent Caco-2 cells were incubated with either 10 mM MβCD alone or with 10 mM MβCD + 1.25 mM cholesterol for 1 h at 37°C. Na⁺-dependent [³H]TC uptake (10 μM) was then determined for 5 min. Results are presented as % of control and are means ± SE obtained from 5 separate experiments. *P < 0.05 compared with control. B: postconfluent Caco-2 cells were incubated with vehicle alone (●) or with 10 mM MβCD (◯) for 1 h at 37°C, and Na⁺-dependent TC uptake was performed in the presence of increasing concentrations of the substrate TC. Sodium-dependent TC uptake was expressed as pmol•mg protein⁻¹ •5 min⁻¹. Experiments were performed 4 times in triplicate in separate groups of cells. Michaelis-Menten plot of a representative experiment is shown. Results indicate a decrease in the V_max of the transport process from 63 ± 8 (control) to 35 ± 4 pmol•mg protein⁻¹ •5 min⁻¹ in MβCD-treated cells.

Fig. 7

Plasma membrane expression of ASBT-V5 fusion protein in Caco-2 cells. Caco-2 cells were transfected with ASBT-V5 by electroporation utilizing Amaxa Nucleofector System as described in materials and methods; 24 h posttransfection, monolayers grown on plastic support were treated with 10 mM MβCD or vehicle alone for 1 h at 37°C and subjected to biotinylation at 4°C using sulfo-NHS-SS-biotin. Cells were then lysed, and surface biotinylated proteins were precipitated with streptavidin-agarose from equal amounts of total cellular protein. Precipitated proteins (surface) were separated on SDS-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose blots. Western blotting analysis was performed with anti-V5. The relative abundance of ASBT-V5 fusion protein in the biotinylated fractions is shown and is expressed as the density of ASBT-V5 band normalized to the density of total hASBT-V5 (surface+intracellular). Data were obtained from 3 separate experiments.