Regulation of bile canalicular network formation and maintenance by AMP-activated protein kinase and LKB1

Abstract

AMP-activated protein kinase (AMPK), a cellular metabolic sensor, is essential in energy regulation and metabolism. Hepatocyte polarization during liver development and regeneration parallels increased metabolism. The current study investigates the effects of AMPK and its upstream activator LKB1 on polarity and bile canalicular network formation and maintenance in collagen sandwich cultures of rat hepatocytes. Immunostaining for the apical protein ABCB1 and the tight junction marker occludin demonstrated that canalicular network formation is sequential and is associated with activation of AMPK and LKB1. AMPK and LKB1 activators accelerated canalicular network formation. Inhibition of AMPK or LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs blocked canalicular network formation. AICAR and 2-deoxyglucose, which activate AMPK, circumvented the inhibitory effect of kinase-dead LKB1 on canalicular formation, indicating that AMPK directly affects canalicular network formation. After the canalicular network was formed, inhibition of AMPK and LKB1 by dominant-negative AMPK or kinase-dead LKB1 constructs resulted in loss of canalicular network, indicating that AMPK and LKB1 also participate in network maintenance. In addition, activation of AMPK and LKB1 prevented low-Ca(2+)-mediated disruption of the canalicular network and tight junctions. These studies reveal that AMPK and its upstream kinase, LKB1, regulate canalicular network formation and maintenance.

Fig. 1.

Progressive canalicular network formation in sandwich cultures of rat primary hepatocytes. Immunofluorescence of the tight junction marker occludin (green) and the apical marker ABCB1 (red) was used to study canalicular structure in day 1 to day 6 cultures. Projection images are from z-series of confocal images. (A) Daily progress of canalicular network formation in sandwich culture. Scale bars: 15 μm. (B) Diagram of canalicular network formation. (C) Mean canalicular length (±s.d.) from three individual experiments (n, cell number; **P<0.01, ***P<0.001).

Fig. 2.

AMPK activation in day 1 to day 6 cultures. Total proteins were extracted from day 1 to day 6 cultures. (A) Western blot for AMPK and phosphorylated AMPK (Thr172). (B) Mean densitometry measurements (±s.d.) from four individual experiments (*P<0.05, **P<0.01, ***P<0.001). (C) Ratio (mean ± s.d.) of phosphorylated to total AMPK (*P<0.05). (D) Western blot for total and phosphorylated ACC (Ser79) in day 1 to day 6 cultures. (E) Densitometry measurements for ratio of phosphorylated to total ACC (from three individual experiments; mean ± s.d.; **P<0.01). (F) Inhibitory effect of dominant-negative AMPK on canalicular network formation. In day 1 cultures, hepatocytes were infected with GFP or Myc-DN-AMPK adenoviruses. 48 hours after infection, immunostaining was performed for control cells (occludin and ABCB1), GFP cells (ABCB1) and Myc-DN-AMPK cells (Myc and ABCB1). Projection images are from z-series of confocal images. (G) Mean canalicular length per cell (± s.d. from three individual experiments; n, cell number; ***P<0.001).

Fig. 3.

LKB1 activation in day 1 to day 6 cultures. Total proteins were extracted from Day1 to day 6 cultures. (A) Western blot for LKB1 and phosphorylated LKB1 (Ser431). (B) Mean densitometry measurements (±s.d. from four individual experiments; *P<0.05, **P<0.01, ***P<0.001). (C) Mean ratio (±s.d.) of phosphorylated to total LKB1 (*P<0.05). (D) Inhibitory effect of kinase-dead LKB1 on canalicular network formation. In day 1 cultures, hepatocytes were infected with GFP or KD-LKB1 adenoviruses. One day later, cells were treated with or without 2-DG (25 mM) or AICAR for 24 hours. Immunostaining was performed using control cells (occludin and ABCB1), GFP cells (ABCB1), KD-LKB1 cells (LKB1 and ABCB1), KD-LKB1 + 2-DG cells (LKB1, ABCB1) and KD-LKB1 + AICAR cells (LKB1, ABCB1). Projection images are from z-series of confocal images. Arrows indicate loss of canalicular structure between KD-LKB1 overexpressed cells (bottom left image) or prevention of loss of canalicular structure after treatment with 2-DG or AICAR (bottom middle and right images, respectively). (E) Mean canalicular length per cell (±s.d. from three individual experiments; n, cell number; ***P<0.001).

Fig. 4.

Effect of AMPK and LKB1 activators on total and phosphorylated LKB1 and AMPK. (A) In day 2 cultures, hepatocytes were treated with forskolin (100 μM), metformin (500 μM), 2-DG (100 mM) and AICAR (500 μM) for 24 hours, respectively. Total proteins were extracted from the cells and western blots for LKB1, AMPK, phosphorylate-LKB1 (Ser431) and phosphorylated-AMPK (Thr172) were performed. (B,C) Mean densitometry measurements (±s.d. from three individual experiments; *P<0.05, **P<0.01).

Fig. 5.

Effect of AMPK and LKB1 activators on bile canalicular network formation. (A) Day 2 cultures were treated with forskolin (100 μM), metformin (500 μM), 2-DG (100 mM) or AICAR (500 μM) for 24 hours. Immunofluorescence of the tight junction marker occludin (green) and the apical marker, ABCB1 (red) was used to study canalicular structure. Projection images are from z-series of confocal images. Scale bars: 15 μm. (B) Mean canalicular length per cell (±s.d from three individual experiments; n, cell number; *P<0.05, **P<0.01, ***P<0.001).

Fig. 6.

Inhibitory effects of dominant-negative AMPK and kinase-dead LKB1 on canalicular network maintenance. On day 6, hepatocytes were infected with GFP, Myc-DN-AMPK and KD-LKB1 adenoviruses respectively. 72 hours after infection, immunostaining was performed for control cells (occludin and ABCB1), GFP cells (ABCB1), Myc-DN-AMPK cells (Myc and ABCB1) and KD-LKB1 cells (LKB1 and ABCB1). (A) Projection images from z-series of confocal images. (B) Mean canalicular length per cell ± s.d. (n, cell number; ***P<0.001).

Fig. 7.

AMPK and LKB1 activators prevent low-Ca²⁺-mediated canalicular network disruption. (A) Low-Ca²⁺ treatment on day 6 culture for 24 hours. Cells were restored to normal medium for 24 hours. (B) Low-Ca²⁺ medium in the presence of forskolin (100 μM), metformin (500 μM), 2-DG (100 mM) and AICAR (500 μM) for 24 hours. Immunostaining for occludin and ABCB1. Projection images are from z-series of confocal images. Scale bars: 15 μm. (C) Mean canalicular length per cell (±s.d. from four individual experiments; n, cell number; ***P<0.001).