Lipolytic glycerol, released from adipocytes, flows through bloodstream to liver where it is transported into hepatocytes to supply gluconeogenesis. AQP9, an aquaporin membrane channel, has been often linked to the uptake of glycerol in hepatocytes; however, no direct experimental evidence has been provided, yet. Here, we address the question by employing a stopped flow light scattering approach using vesicles of hepatocyte basolateral plasma membrane (BLPM) prepared from mouse livers in various metabolic states.The movement of glycerol across the hepatocyte BLPM vesicles occurred by facilitated diffusion as indicated by the low Arrhenius activation energy (3.5 kcal/mol) characterizing the transport and the strong (>60%) inhibition seen after incubation with phloretin or HgCl₂, two known AQP9 blockers. Fasting markedly increased both AQP9 expression and glycerol membrane permeability (P_gly) of BLPM in a time-dependent manner. In line with these results, the plasma glycerol levels of the examined animals changed accordingly to the extents of P_gly and AQP9 expression values. This was also in line with additional experiments where the P_gly of the liver vesicles of AQP9 knockout mice resulted markedly lower than the P_gly of wild type mice counterpart (5.43±0.2 vs. 10.03±0.6 x 10^­3 mm/s, respectively; P<0.01).

Overall, these results prove major functional relevance for AQP9 in hepatocyte glycerol uptake indicating AQP9 as a new player in metabolic and energy homeostasis.