N-glycosylation is essential for ileal ASBT function and protection against proteases.

The bile acid transporter ASBT is a glycoprotein responsible for active absorption of bile acids. Inhibiting ASBT function and bile acid absorption is an attractive approach to lower plasma cholesterol and improve glucose imbalance in diabetic patients. Deglycosylation of ASBT was shown to decrease its function. However, the exact roles of N-glycosylation of ASBT, and how it affects its function, is not known. Current studies investigated the roles of N-glycosylation in ASBT protein stability and protection against proteases utilizing HEK-293 cells stably transfected with ASBT-V5 fusion protein. ASBT-V5 protein was detected as two bands with molecular mass of ~41 and ~35 kDa. Inhibition of glycosylation by tunicamycin significantly decreased ASBT activity and shifted ASBT bands to ~30 kDa, representing a deglycosylated protein. Treatment of total cellular lysates with PNGase F or Endo H glycosidases showed that the upper 41-kDa band represents a fully mature N-acetylglucosamine-rich glycoprotein and the lower 35-kDa band represents a mannose-rich core glycoprotein. Studies with the glycosylation deficient ASBT mutant (N10Q) showed that the N-glycosylation is not essential for ASBT targeting to plasma membrane. However, mature glycosylation significantly increased the half-life and protected ASBT protein from digestion with trypsin. Incubating the cells with high glucose (25 mM) for 48 h increased mature glycosylated ASBT along with an increase in its function. These results unravel novel roles for N-glycosylation of ASBT and suggest that high levels of glucose alter the composition of the glycan and may contribute to the increase in ASBT function in diabetes mellitus.

Ileal apical Na+-dependent bile acid transporter ASBT is upregulated in rats with diabetes mellitus induced by low doses of streptozotocin.

Increased intestinal bile acid absorption and expansion of the bile acid pool has been implicated in the hypercholesterolemia associated with diabetes mellitus. However, the molecular basis of the increase in bile acid absorption in diabetes mellitus is not fully understood. The ileal apical Na(+)-dependent bile acid transporter (ASBT) is primarily responsible for active reabsorption of the majority of bile acids. Current studies were designed to investigate the modulation of ASBT function and expression in streptozotocin (STZ)-induced diabetes mellitus in rats and to examine the effect of insulin on rat ASBT promoter by insulin. Diabetes mellitus was induced in Sprague-Dawley rats by intraperitoneal injection of low doses of STZ (20 mg/kg body wt) on five consecutive days. Human insulin (10 U/day) was given to a group of diabetic rats for 3 days before euthanasia. RNA and protein were extracted from mucosa isolated from the small intestine and ASBT expression was assessed by real-time quantitative RT-PCR and Western blotting. Our data showed that ASBT mRNA and protein expression were significantly elevated in diabetic rats. Insulin treatment of diabetic rats reversed the increase in ASBT protein expression to control levels. Consistently, ileal Na(+)-dependent [(3)H]taurocholic uptake in isolated intestinal epithelial cells was significantly increased in diabetic rats. In vitro studies utilizing intestinal epithelial Caco-2 cells demonstrated that ASBT expression and promoter activity were significantly decreased by insulin. These studies demonstrated that insulin directly influences ASBT expression and promoter activity and that ASBT function and expression are increased in rats with STZ-induced diabetes mellitus. The increase in ASBT expression may contribute to disturbances in cholesterol homeostasis associated with diabetes mellitus.

Green tea catechin EGCG inhibits ileal apical sodium bile acid transporter ASBT.

Green tea catechins exhibit hypocholesterolemic effects probably via their inhibitory effects on intestinal bile acid absorption. Ileal apical sodium-dependent bile acid transporter (ASBT) is responsible for reabsorption of bile acids. The present studies were, therefore, designed to investigate the modulation of ASBT function and membrane expression by green tea catechins in human embryonic kidney HEK-293 cells stably transfected with ASBT-V5 fusion protein and intestinal Caco-2 monolayers. Our data showed that ASBT activity was significantly decreased by (-)-epigallocatechin-3-gallate (EGCG) but not other green tea catechins. Inhibition of PKC, phosphatidylinositol 3-kinase, and MAPK-dependent pathways failed to block the reduction in ASBT activity by EGCG. Kinetics studies showed a significant decrease in the V(max) of the transporter, whereas total ASBT content on the plasma membrane was unaltered by EGCG. Concomitant with the decrease in ASBT function, EGCG significantly reduced ASBT pool in the detergent-insoluble fraction, while increasing its presence in the detergent-soluble fraction of plasma membrane. Furthermore, EGCG decreased the association of ASBT with floating lipid raft fractions of cellular membrane on Optiprep density gradient. In conclusion, our data demonstrate a novel role of lipid rafts in the modulation of ASBT function by the dietary component EGCG, which may underlie the hypocholesterolemic effects of green tea.