This study describes a unique function of taurocholate in bile canalicular formation involving signaling through a cAMP-Epac-MEK-Rap1-LKB1-AMPK pathway. In rat hepatocyte sandwich cultures, polarization was manifested by sequential progression of bile canaliculi from small structures to a fully branched network. Taurocholate accelerated canalicular network formation and concomitantly increased cAMP, which were prevented by adenyl cyclase inhibitor. The cAMP-dependent PKA inhibitor did not prevent the taurocholate effect. In contrast, activation of Epac, another cAMP downstream kinase, accelerated canalicular network formation similar to the effect of taurocholate. Inhibition of Epac downstream targets, Rap1 and MEK, blocked the taurocholate effect. Taurocholate rapidly activated MEK, LKB1, and AMPK, which were prevented by inhibition of adenyl cyclase or MEK. Our previous study showed that activated-LKB1 and AMPK participate in canalicular network formation. Linkage between bile acid synthesis, hepatocyte polarization, and regulation of energy metabolism is likely important in normal hepatocyte development and disease.primary hepatocytes | occludin | P-glycoprotein H epatocytes, the major epithelial cells in the liver, are polarized. Tight junction proteins, including occludin, claudin, and ZO-1, seal the canalicular lumen, thereby separating apical and basolateral membrane domains and forming the bile canaliculus (1). Polarization is essential for biliary secretion. The mechanisms of polarization are complex and include cytoskeletal, tight junctional, and intracellular trafficking components (2-5). Loss of polarity causes bile secretory failure (cholestasis) and liver damage (6).Bile acids are synthesized from cholesterol in hepatocytes, secreted by ABCB11 (Bsep) into the bile canaliculus, and then mostly absorbed into the enterohepatic circulation (7). The major bile acids in mammals are tauro or glycine conjugates of cholic, deoxycholic, and chenodeoxycholic acids (8). In addition to their traditional function in emulsification of dietary fat (8), recent studies reveal that bile acids function as signaling molecules (9), which increase calcium mobilization (10) and cellular cAMP (9); translocate and activate protein kinase C (11), nuclear farnesoid X receptor (FXR), and pregnane X receptors (PXR) (7, 9); activate PI3K/AKT/glycogen synthase kinase 3 (GSK3) (12); and enhance liver regeneration (13).During embryonic development, early fetal hepatocytes are not polarized (14-16). In fetal mice and rats, infrequent small canaliculi are present, but do not attain an adult appearance until several days postpartum (17). Bile acid synthesis, turnover, and secretion are sparse in fetal liver, and rapidly increase postnatally (18,19), concomitant with hepatocyte polarization and development of a branched canalicular network. Based on these events, we postulated that bile acids may regulate hepatocyte polarization and canalicular formation. Using collagen sandwich cultures of rat primary hepatocytes, we confirmed this hy...