Bile acid stimulates hepatocyte polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway

Abstract

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...

Hepatic biliary epithelial cells acquire epithelial integrity but lose plasticity to differentiate into hepatocytes in vitro during development

“…2D). Finally, to confirm whether BC-like structures were generated, we added fluorescein diacetate (FDA) to the culture medium after augmenting formation of BC-like structures in the presence of taurocholate (Fu et al, 2011). We found that metabolized fluorescein was excreted into BC-like structures (Fig.…”

Coordinated elevation of mitochondrial oxidative phosphorylation and autophagy help drive hepatocyte polarization

“…This enhances mitochondrial catabolic activities (including fatty acid β-oxidation, which provides fuel for driving the tricarboxylate acid cycle) and also increases mitochondrial bioenergetics, whose respiratory oxidative phosphorylation (OXPHOS) mechanism is more efficient in generating cellular ATP compared with glycolysis (14, 15). Thus, by switching on pathways that can mobilize energy and cause cell growth, AMPK allows cells to overcome nutrient deprivation and stress conditions (16).AMPK becomes active during cell polarization (17)(18)(19), and its activity is required for this process (17). Indeed, AMPK activators accelerate polarization, whereas AMPK inhibition (by overexpression of a dominant-negative form) blocks polarization (17, 18).…”

Cytokinesis defines a spatial landmark for hepatocyte polarization and apical lumen formation

“…However, a recent paper reports that, in regenerating mouse livers, weaned rat livers, and HepG2 cells, the spindle axis is largely perpendicular to the bile canaliculus lumen, such that cell division results in asymmetric inheritance of the existing bile canaliculus by the daughter cells (Slim et al, 2013). The authors hypothesize that this mode of oriented cell division, coupled with de novo bile canaliculus formation at the site of abscission and further elongation and fusion of individual bile canaliculi, leads to the formation of a complex bile canaliculus network, as seen previously in primary hepatocytes that had been cultured at a high density, sandwiched between two layers of collagen or other ECM (Fu et al, 2010;Fu et al, 2011). Taking these observations together, it is possible that both modes of oriented cell division are involved in bile canaliculus biogenesis in vivo.…”