They facilitate lipid absorption, inhibit microbe growth in the biliary tract and intestine, and function as signaling molecules that regulate energy expenditure and carbohy-

Journal of Lipid Research Volume 53, 2012 1535 Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc. They facilitate lipid absorption, inhibit microbe growth in the biliary tract and intestine, and function as signaling molecules that regulate energy expenditure and carbohydrate and lipid metabolism ( 2 ). The bile salt pool is maintained in an enterohepatic circulation by bile salt transporters in the distal ileum and the liver ( 3 ). Central to this process is the apical sodium (Na + )-dependent bile salt transporter (ASBT/SLC10A2) located on the luminal membrane in the distal ileum and proximal tubule of the kidney in humans and rodents ( 4 ). ASBT maintains the enterohepatic and renal-hepatic circulation of bile salts by facilitating their reabsorption from the intestinal lumen and renal tubules. Dysfunction of ASBT/Asbt interrupts the bile salt enterohepatic circulation, reduces the bile salt pool size by 80% in mice, and leads to bile salt malabsorption, diarrhea, and steatorrhea in humans, where reduced plasma levels of cholesterol are also observed ( 5, 6 ). Thus, there is considerable pharmaceutical interest in ASBT inhibition as a potential target for drug discovery for the treatment of hypercholesterolemia and diabetes mellitus type 2 ( 7, 8 ). Furthermore, an ASBT inhibitor has been demonstrated to be benefi cial for patients with chronic idiopathic constipation ( 9, 10 ). In contrast to what is known for mammalian species, very little is known about the presence or function of Asbt in other vertebrates. Bile salts demonstrate considerable structural variation across vertebrate classes ( 1, 11 ) ( Fig. 1 ). The enzymatic pathway that converts cholesterol into bile salts is complex and requires a minimum of fi ve enzymes in primitive vertebrates and up to 16 enzymes in humans ( 12–14 ). The most primitive vertebrates (agnathans or jawless fi sh) use early evolving C27 sulfated bile alcohols with a C-5 hydrogen at  confi guration (i.e., 5 ), which is an overall planar Abstract The apical Na + -dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5 -bile alcohols and the skate that utilizes structurally different 5 -bile alcohols, and compared substrate specifi city with ASBT from humans who utilize modern 5 -bile acids. Everted gut sacs of skate but not the more primitive lamprey transported 3 H-taurocholic acid (TCA), a modern 5 -bile acid. However, molecular cloning identifi ed ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt’s indicate that lamprey Asbt has high affi nity for 5 -bile alcohols, low affi nity for 5 -bile alcohols, and lacks affi nity for TCA, whereas skate Asbt showed high affi nity for 5 and 5 -bile alcohols but low affi nity for TCA. In contrast, human ASBT demonstrated high affi nity for all three bile salt types. These fi ndings suggest that ASBT evolved from the earliest vertebrates by gaining affi nity for modern bile salts while retaining affi nity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution. —Lionarons, D. A., J. L. Boyer, and S-Y. Cai. Evolution of substrate specifi city for the bile salt transporter ABST (SLC10A2). J. Lipid Res. 2012. 53: 1535–1542.