PGE RECEPTOR - LIVER AND FAT METABOLISM

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Our overall goal is to clarify the regulatory effects that prostaglandins of the E series have on liver and fat cell metabolism. The specific aims are to ascertain whether down-regulation of PGE receptors by in vivo treatment of Sprague-Dawley rats with a PGE2-analog is accompanied by heterologous regulatory changes in receptors for insulin and glucagon; to determine whether down-regulation of the PGE receptor is associated with changes in the G/F subunit of adenylate cyclase in fat cells such as we have recently demonstrated in liver cells; to assess whether changes in G/F subunit availability are specifically associated with changes in PGE receptor density on plasma membranes or whether changes in the G/F subunit are also associated with insulin and glucagon receptor down-regulation;and to ascertain the consequences of PGE receptor down-regulation on certain cAMP-related aspects of liver and fat cell metabolism. The general approach to studying down-regulation of receptors for PGE, insulin and glucagon will be to use conventional techniques for studying the binding of radiolabeled ligands for these receptors. Down-regulation will be induced in vivo in Sprague-Dawley rats by injection of a PGE2-analog, insulin and glucagon, respectively. Studies of adenylate cyclase activity will be performed to characterize dose-response curves using plasma membranes from control and down-regulated animals and stimulators such as PGE, glucagon, sodium fluoride, GppNHp and forskolin. The G/F subunit of adenylate cyclase will be studied more extensively through the use of cholera toxin activation and labeling of the G/F subunit with [32P]NAD and SDS gels. G/F subunit reconstitution studies using either red blood cells or cyc- cells will be performed to confirm changes in the subunit in down-regulated liver and fat cells. A major aspect of this proposal involves experiments to ascertain whether PGE exerts biologically meaningful alterations in liver and fat metabolism through interactions with the PGE receptor. This will be approached through extensive studies of metabolic processes related to cyclic AMP in fat and liver tissues. This will involve studies of glycogenolysi, gluconeogenesis, fatty acid synthesis, fatty acid oxidation, and lipolysis. The health-related nature of this project emanates from our prior observation in humans that PGE induces change in circulating hormones and substrates related to glucose homeostasis.