Sepsis is that critical condition of the patient which, if uncorrected, leads to death. The initial hyperglycemia seen in sepsis is followed by hypoglycemia with concomitant hyperlacticacidemia. Glucose-6-phosphatase (Glu-6-Pase) is a key enzyme in the homeostatic regulation of blood glucose concentration, which catalyses the final step in gluconeogenesis and glycogenolysis. Another important enzyme for regulation of gluconeogenesis is 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (6PF2K/FBPase), which is responsible for the production and hydrolysis of fructose-2,6-bisphosphate (Fru-2,6- P2), a modulator of glycolytic to gluconeogenic flux. We have demonstrated that gene expression of Glu-6-Pase and 6PF2K/FBPase are reciprocally modulated during hyperglycemic phase of hemorrhagic shock. In this proposal, we are focusing on the regulation of expression of these two genes as models of hormonal counter-regulation during sepsis. In addition to studying the hormonal regulation of these genes in sepsis, we also propose to determine the hormonal response elements of these genes by promoter analysis. Sepsis will be induced in fasted, anesthetized rats by cecal ligation and puncture (CLP) method and will be observed for 0.5 h, 3 h, and 20 h periods. Control rats will undergo sham operation and will be observed for the same time. The liver and kidney from control and CLP rats will be freeze-clamped in liquid nitrogen and stored at -70 degrees C for future assay of glucose-6- phosphate, fructose-6-phosphate, glucose-6-phosphate dehydrogenase activity, Glu-6-Pase and 6PF2K/FBPase enzyme activity and gene expression. Blood samples will be collected at the same time periods to assay glucose, corticosterone, insulin and glucagon concentration in the plasma. Relatively selective pharmacologic antagonists of hormones and cytokines will be administered prior to CLP to observe their effects on Glu-6-Pase and 6PF2K/FBPase gene expression. Hepatocytes will be isolated from control and CLP rats and will be incubated with agonists to observe their effects on Glu-6-Pase and 6PF2K/FBPase gene expression. Freshly prepared, primary cultures of hepatocytes will be transfected and then incubated with hormones and hormonal response element will be confirmed by DNA footprinting. The results of this study are essential to understand the molecular basis of deranged metabolism in sepsis. Understanding the mechanism might be useful for developing appropriate therapeutic intervention during sepsis.
