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.

SummaryMitochondria are dynamic organelles undergoing fission, fusion, and translocation. These processes have been studied in cultured cells; however, little is known about their regulation in cells within tissues in vivo. We applied four-dimensional intravital microscopy to address this in secretory cells of the salivary gland. We found that mitochondria are organized in two populations: one juxtaposed to the basolateral plasma membrane and the other dispersed in the cytosol. Under basal conditions, central mitochondria exhibit microtubule-dependent motility and low fusion rate, whereas basolateral mitochondria are static and display high fusion rate. Increasing cellular energy demand by β-adrenergic stimulation of regulated exocytosis selectively enhanced motility and fusion of central mitochondria. Inhibition of microtubule polymerization led to inhibition of central mitochondrial motility and fusion and a marked reduction in exocytosis. This study reveals a conserved heterogeneity in mitochondrial positioning and dynamics in exocrine tissues that may have fundamental implications in organ pathophysiology.