Coordinated function of different hepatic cells (Kupffer, endothelial and parenchymal) is essential for the metabolic support and nonspecific immune response of the host to trauma and infections. Endotoxemia activates hepatic nonparenchymal cells (Kupffer and endothelial) and is associated with an elevated glucose utilization by these cells. The exact role and functional importance of augmented glucose metabolism during infection in the different hepatic cells are poorly understood. The long term objective of this project is to elucidate the role and regulation of glucose metabolism through the pentose cycle in various hepatic cells during bacterial infections. The proposed study focuses on the effect of bacterial endotoxin on the pentose cycle as a primary regulatory site for the support of augmented scavenger function, bacterial killing and protective mechanisms against oxidative damage in Kupffer, hepatic endothelial and parenchymal cells. Three hypotheses will be tested: (1) The elevated glucose utilization of macrophages during infection subserves the enhanced activity of the pentose cycle, which is a primary regulatory site for the development of primed and fully activated functional state of the mononuclear phagocytes. (2) The upregulation of pentose cycle during infection in hepatic endothelial cells is an important protective mechanism against oxidative tissue injury of the liver. (3) The effect of endotoxin is mediated by glucoregulatory hormones and cytokines which upregulate the pentose cycle in a cell specific manner. To address the first two hypotheses, following in vivo experimental endotoxemia, glucose uptake, phosphorylation and entry in the pentose cycle will be determined in vivo and in vitro in isolated Kupffer, hepatic endothelial, and parenchymal cells, using isotope tracer techniques. The targets of endotoxin on the flux generating enzymes (primarily glucose-6-phosphate dehydrogenase and hexokinase) will be determined by measuring the maximal activities and kinetic parameters, enzyme synthesis rates, posttranslational modifications expression of mRNA. The activity of the pentose cycle will be correlated with superoxide production, elimination of oxygen radicals and macromolecule synthesis in various hepatic cells. To test the third hypothesis, different hormones and cytokines will be administered in vivo to rats, or in vitro to cultures of Kupffer, hepatic endothelial and parenchymal cells, and the activity of pentose cycle will be assessed. Our primary goal is to elucidate the biochemical characteristics of the primed state of the pentose cycle and its regulation by the endocrine and paracrine system in different hepatic cells. This study will provide valuable information for new therapeutic approaches for the support of host defense related hepatic function in severe infection and trauma, and will eventually contribute to our ability to reverse the multiorgan failure syndrome.
