We are pursuing an investigation of the molecular events that trigger the biochemical sequelae of infection. One of the clinical hallmarks of animals with chronic infections or tumors is the presence of a catabolic state which can proceed to cachexia, shock, and death. The biochemical basis for this phenomenon is not understood but presumably once triggered is of a universal nature. In order to gain insight into the mechanism of this process, we have selected endotoxemia as a model system and have applied cell culture techniques to its investigation. conditioned medium from the macrophage cell line, RAW 264.7, after exposure to endotoxin was the source of a mediator that when added to 3T3-L1 adipocytes markedly suppressed the activity of lipoprotein lipase at the level of synthesis. This mediator purified to homogeneity was termed cachectin and demonstrated to be identical to tumor necrosis factor (TNF). It is our objective to understand the mechanism by which TNF regulates cellular metabolism. To do this it is critical to first characterize the effect of TNF on lipoprotein lipase mRNA levels, the half-life of that message as well as rates of transcription. We will also determine if TNF affects the rate of secretion of the lipase. We have observed that TNF stimulates glucose uptake by 250% in 3T3-L1 fibroblasts while in the fully- differentiated adipocytes hexose uptake is suppressed by 50%. This regulation of glucose entry into the cell will be characterized by quantifying levels of transporter kinetically, as well as by equilibrium binding and photoaffinity labeling. We will also examine the effects on mRNA levels and rates of transcription. we have defined the presence of a second monokine in the conditioned medium of the RAW 264.7 cells that stimulates glycogen depletion and subsequently hexose uptake in L6 rat muscle myotubes in culture. We plan to purify this monokine to homogeneity and quantify its effects on: hexose uptake, cellular glycogen levels, lactate production, and examine changes in the levels of the glucose transporter by binding and photoaffinity labeling studies. We will also determine if changes in the number of transporters are due to changes in the levels of transporter mRNA and rates of transcription. These studies will aid in our understanding of how these monokines, produced when the immune system is exposed to invasive stimuli, are capable of regulating metabolism in energy storage tissues.
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