Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo

Abstract

Liver Kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich-cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking, and bile canalicular formation. In the present study, we used liver-specific (Albumin-Cre) LKB1 knockout mice (LKB1−/−) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy (TEM) revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1 −/− livers. Immunofluorescence (IF) studies showed that loss of LKB1 leads to longer and wider canalicular structures correlating with mis-localization of the junctional protein, cingulin. To test junctional function, we used Intravital Microscopy (IVM) to quantify the transport kinetics of 6-carboxy-fluorescein diacetate (6-CFDA), which is processed in hepatocytes into its fluorescent derivative 6-carboxyfluorescein (6-CF) and secreted into the canaliculi. In LKB1 −/− mice, 6-CF largely remained in hepatocytes, canalicular secretion was greatly delayed and 6-CF appeared in the blood. To test whether 6-CF was transported through permeable TJ, we intravenously injected a low molecular weight (3kDa) dextran in combination with 6-CFDA. In wild type mice, 3kDa dextran remained in the vasculature whereas it rapidly appeared in the abnormal bile canaliculi in LKB1 −/− mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. Conclusion LKB1 plays a critical role in regulating maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway, result in cholestasis.

Deletion of Lkb1 in adult mice results in body weight reduction and lethality

“…More recently, Porat-Shliom et al . directly demonstrated that Lkb1 plays a key role in maintaining the functional tight junction and paracellular permeability34, which may explain why there were more EB uptake in liver tissues of Rosa-Lkb1 mice compared to control mice. These findings combined with our results indicated that the lethality of Rosa-Lkb1 mice maybe mainly due to the ablation of Lkb1 in liver or haematopoietic stem cells.…”

A Predictive 3D Multi-Scale Model of Biliary Fluid Dynamics in the Liver Lobule

“…In contrast to humans, in rodents BSIF contributes substantially to the total bile flow (Boyer, 2013;Erlinger, 1996). Therefore, to derive an estimate of apical bile secretion and bile velocity in the bile canaliculi network, we performed IVM of the well-established fluorescent tracer 6-carboxyfluorescein diacetate (CFDA) (Babbey et al, 2012;Breeuwer et al, 1995;Liu et al, 2007;Porat-Shliom et al, 2016). CFDA secretion depends on the apical multidrug resistance-associated protein 2 (MRP2) (Zamek-Gliszczynski et al, 2003), which primarily transports organic anions (BSIF) (Paulusma et al, 1999;Wielandt et al, 1999) and only to a minor extent glucuronidated bile acids (BSDF) (Akita et al, 2001).…”

The role of microtubules in the regulation of epithelial junctions

“…70 Interestingly, in LKB1-KO mice the level of phosphorylated AMPK is highly reduced, and CGN junctional accumulation in hepatocytes is lost, correlating with changes in cell morphology, and TJ integrity and permeability. 71 This suggests that the LKB1-AMPK kinase axis controls the apical accumulation of cingulin through its phosphorylation.…”