The broad objective of this research project is to study the regulation of the altered glutamine (GLN) transport that occurs in the liver of tumor- bearing rats. The liver plays a central role in maintaining glutamine homeostasis and our preliminary data indicate that the liver switches from net glutamine release to uptake as the tumor grows. The transport of GLN into the hepatocyte is mediated by the unique Na+-dependent System N while its transport out of the hepatocyte is mediated by a novel Na+-independent pathway (System n) that we recently described. Since the activities of these two carriers working in concert are the major determinants of the net uptake or release of GLN by the liver, we will study the regulation of their activities in the liver as the tumor grows. The regulation of glutamine transport across the hepatocyte plasma membrane of tumor-bearing rats will be studied using 2 complimentary models: hepatic plasma membrane vesicles (MPMVs) and cultured rat hepatocytes. MPMVs reflect transport in vivo while isolated hepatocytes allow us to study glutamine transport in vitro. First, we will examine the effects of progressive tumor growth on glutamine transport in HPMVs. We will investigate the time it takes changes in transport to occur as well as their recovery after surgical removal of the tumor. In vivo flux studies will be done to determine whether the changes in transport we observe translate into functional changes in glutamine flux at the organ level. Since Na+-dependent and Na+-independent glutamine transport is differentially expressed in periportal and perivenous cells, vesicles will be prepared from livers in which these cell populations have been preferentially destroyed. We will investigate whether the increase in System N activity we have observed can be attenuated in vivo with a TNF mAb or an lL-6 mAb. We will test the hypothesis that TNF stimulates hepatocyte glutamine transport in vivo indirectly by stimulating IL-6 the principal cytokine regulator of GLN transport. We will also feed tumor- bearing rats GLN-enriched diets to maintain plasma GLN concentrations; this will allow us to determine if changes in transport represent a response to low blood GLN levels. We will study GLN transport in hepatocytes isolated from tumor-bearing rats and in normal hepatocytes that are cultured with serum from tumor- bearing and control rats. We will assay GLN transport in periportal and perivenous hepatocytes isolated from the livers at different stages of tumor growth. We will add neutralizing antibodies to cytokines to learn which of these mediators play the major role in regulating transport. We will determine whether the increase in GLN transport we have observed is dependent on de novo RNA and protein synthesis. We will also examine the effects of cytokines on GLN transport in isolated hepatocytes. Our preliminary data indicate that lL-6 directly stimulates a 3-4 fold increase in glutamine transport in cultured hepatocytes. Finally, we will study the selectivity of this response by examining the effects of the growing tumor on other amino acid carriers in the hepatocyte plasma membrane. Glutamine appears to be an essential amino acid in the host with cancer and understanding its handling during cancer may lead to improved nutritional and metabolic therapies (e.g. GLN-enriched diets) targeted to support the tumor-bearing host in a beneficial manner.
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