Submitted to: Domestic Animal Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2006
Publication Date: 5/3/2006
Citation: Caperna, T.J., Shannon, A.E., Richards, M.P., Garrett, W.M., Talbot, N.C. 2006. Identification and characterization of aquaporin-9 (aqp9) in porcine hepatic tissue and hepatocytes in monolayer culture. Domestic Animal Endocrinology. 32:273-286. Interpretive Summary: One of the main limitations in studying the roles of specific proteins in metabolic processes in domestic animals such as pigs, is the lack of commercially -available antibody reagents. In this study we used available gene sequence data to design a peptide that would serve as an antigen to prepare a suitable antiserum for pig aquaporin-9. The aquaporins are channel forming proteins that reside within cell membranes and facilitate to movement of water and other molecules such as glycerol and urea into and out of cells. Synthesis and transport of glycerol and urea are key facets of the system that shuttles sugar, lipid and amino acid components between the liver and other tissues. The purpose of this study was to determine the regulation of Aquaporin-9 by hormones involved in energy metabolism. Initial characterization of the antiserum that was prepared, indicated that this would indeed make a valuable tool to investigate porcine aquaporin-9. We demonstrated that the aquaoprin-9 molecule resides within the membranes of hepatocytes, while bile duct, gall bladder and hepatic vascular tissues do not possess this protein. Glucagon, but not insulin increased the levels of aquaporin-9 protein in liver cell cultures prepared from adult pigs. Further, when the levels of aquaporin-9 mRNA levels were determined, glucagon and thyroid hormone enhanced the expression of this gene. These studies demonstrate that it is imperative to investigate cellular protein, as well as, gene expression levels to understand the influences of hormones and other regulatory molecules on metabolic processes. In addition, the antiserum that was prepared and characterized can now be used by other investigators interested in studying porcine aquaporin-9 regulation.
Technical Abstract: Aquaporins (AQPs) are a large family of integral membrane proteins involved in the rapid movement of water and neutral solutes across cell membranes. In this study, we have prepared an affinity-purified porcine-specific polyclonal antiserum to AQP9 and have investigated its distribution and expression in pig liver tissue and in hepatocytes in primary culture. Immunocytochemical analysis showed that AQP9 was primarily localized in the membrane structures of hepatocytes and was not associated with intrahepatic bile ducts or blood vessels. Western blot analysis indicated that AQP9 ranged in apparent molecular mass between 27 and 38 kD in whole liver and hepatocyte membrane fractions; minor components were also observed at approximately 34 kD in the cytosol compartment of hepatocytes, bile duct and gall bladder. A prominent immuno-reactive band at 44 kD was shown to be an artifact of western blot analysis. In primary cultures of porcine hepatocytes, glucagon enhanced absolute levels of AQP9 protein, while gene expression was enhanced by T3 and glucagon. Insulin alone had no discernable influence on AQP9 gene expression or its cellular protein levels. These data suggest that AQP9 is a major AQP in porcine hepatic tissue and appears to be primarily responsive to glucagon induction. Key words: pig hepatocytes, AQP9, glucagon, T3