Mitochondria are remarkably plastic organelles constantly changing their shape to fulfil their various functional activities. Although the osmotic movement of water into and out of the mitochondrion is central for its morphology and activity, the molecular mechanisms and the pathways for water transport across the inner mitochondrial membrane (IMM), the main barrier for molecules moving into and out of the organelle, are completely unknown. Here, we show the presence of a member of the aquaporin family of water channels, AQP8, and demonstrate the strikingly high water permeability (P f ) characterizing the rat liver IMM. Immunoblotting, electron microscopy, and biophysical studies show that the largest mitochondria feature the highest AQP8 expression and IMM P f . AQP8 was also found in the mitochondria of other organs, whereas no other known aquaporins were seen. The osmotic water transport of liver IMM was partially inhibited by the aquaporin blocker Hg 2؉ , while the related activation energy remained low, suggesting the presence of a Hg 2؉ -insensitive facilitated pathway in addition to AQP8. It is suggested that AQP8-mediated water transport may be particularly important for rapid expansions of mitochondrial volume such as those occurring during active oxidative phosphorylation and those following apoptotic signals.Mitochondrial volume is of pivotal importance for the activity of the electron transport chain (1) and a control point of apoptosis (2). Changes in mitochondrial volume occur in many other physiological and patho-physiological conditions, including intracellular signal transduction, liver regeneration, ischemia/reperfusion-induced damage, and anoxia (3-5). Mitochondria are well behaved osmometers, and swelling and contraction of the mitochondrial matrix and related changes to mitochondrial morphology are the consequence of the water movement that osmotically accompanies the net transport of solutes into and out of the mitochondrion (6), respectively. Mitochondrial volume changes are modulated by the net movement of solutes including K ϩ and Ca 2ϩ ions across the IMM 1 (7,8). The inner mitochondrial membrane acts as a major barrier for the solutes and water moving between the cytoplasm and the mitochondrial matrix, the outer membrane being freely permeable to molecules of up to 1.5 kDa due to the presence of the exceedingly large pores formed by VDAC, the voltage-dependent anion channel (9). However, although a number of IMM transport systems have been cloned and characterized for their ability to transport solutes across the IMM (10), the molecular pathway for the movement of water remains obscure. Important clues for understanding the molecular bases of the mitochondrial osmotic properties were recently provided by the identification of an aquaporin water channel (11), AQP8, in rat hepatocyte mitochondria (12). AQP8 was also found in intracellular vesicles that are shuttled to the hepatocyte apical membrane under choleretic stimuli such as those brought about by glucagon (13). This led us to hyp...

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Overall, these results experimentally prove major functional significance for AQP9 in maximising liver glycerol import during states requiring increased glucose production. If any, alternative facilitated pathways would be of minor importance in transporting glucogenetic glycerol into hepatocytes during starvation. Refining the understanding of liver AQP9 in metabolic and energy homeostasis may reveal helpful for therapeutic purposes.

AQP9 downregulation together with the subsequent reduction in hepatic glycerol permeability in insulin-resistant states emerges as a compensatory mechanism whereby the liver counteracts further triacylglycerol accumulation within its parenchyma as well as reduces hepatic gluconeogenesis in patients with NAFLD.

Rat hepatocytes express aquaporin-9 (AQP9), a basolateral channel permeable to water, glycerol, and other small neutral solutes. Although liver AQP9 is known for mediating the uptake of sinusoidal blood glycerol, its relevance in bile secretion physiology and pathophysiology remains elusive. Here, we evaluated whether defective expression of AQP9 is associated to secretory dysfunction of rat hepatocytes following bile duct ligation (BDL). By immunoblotting, 1-day BDL resulted in a slight decrease of AQP9 protein in basolateral membranes and a simultaneous increase of AQP9 in intracellular membranes. This pattern was steadily accentuated in the subsequent days of BDL since at 7 days BDL basolateral membrane AQP9 decreased by 85% whereas intracellular AQP9 increased by 115%. However, the AQP9 immunoreactivity of the total liver membranes from day 7 of BDL rats was reduced by 49% compared with the sham counterpart. Results were confirmed by immunofluorescence and immunogold electron microscopy and consistent with biophysical studies showing considerable decrease of the basolateral membrane water and glycerol permeabilities of cholestatic hepatocytes. The AQP9 mRNA was slightly reduced only at day 7 of BDL, indicating that the dysregulation was mainly occurring at a posttranslational level. The altered expression of liver AQP9 during BDL was not dependent on insulin, a hormone known to negatively regulate AQP9 at a transcriptional level, since insulinemia was unchanged in 7-day BDL rats. Overall, these results suggest that extrahepatic cholestasis leads to downregulation of AQP9 in the hepatocyte basolateral plasma membrane and dysregulated aquaporin channels contribute to bile flow dysfunction of cholestatic hepatocyte.