Bacterial endotoxins (ETX), components of the cell wall of gram-negative bacteria, elicit a number of cardiovascular, inflammatory, and hormonal responses. ETX-mediated alterations in metabolism, including glycogen depletion and diminished gluconeogenesis, occur at a time when plasma glucocorticoid, glucogan and epinephrine levels are normal to elevated. Our overall objective is to determine the mechanism(s) by which ETX perturbs host carbohydrate metabolism during gram-negative speticemia. This study is based on the hypothesis that ETX interferes with the normal action of glucocorticoids at the molecular level. To examine this hypothesis, we propose to investigate the consequences of down regulation of glucocorticoid receptors during endotoxemia. We will extend preliminary studies by comparing glucocorticoid binding to liver, spleen, skeletal muscle, and kidney cytosolic receptors from ETX-treated mice. We will determine whether the effects of ETX on cytosolic binding are direct or mediated by adding ETX directly to isolated normal hepatocytes in vitro, and with the passive transfer of serum and peritoneal cells between ETX-normal responder mice and ETX-low responder mice. Soluble mediators that may regulate hepatic glucocorticoid receptors will be screened for, using cultured peritoneal exudate cells and isolated Kupffer cells. Purification and characterization of soluble factors found to regulate steroid receptors will be pursued. A secondary aim of this study is to examine other aspects of intracellular steroid action in liver during endotoxic shock. We will determine the magnitude of nuclear binding of steroid-protein complexes in liver (in vivo) and in hepatocyte (in vitro) preparations. RNA polymerase activity will be measured, as will the ability of mRNA isolated from endotoxin-treated and control mouse livers to direct protein synthesis in a cell-free translation system. Successful completion of this study will yield data describing whether interference with glucocorticoid action in liver ETX is direct or mediated. In addition, the project will ultimately lead to an understanding of the role of altered hepatic metabolism in bacterial sepsis and shock.
