Biochemical Studies on Uncoupling Proteins and Glucose Metabolism in Skeletal Muscle
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El-Ella, Ghada Abou
Uncoupling protein (UCP) is an inner-mitochondrial membrane protein that uncouples the production of ATP from the electron transport chain, thereby inducing thermogenesis. Recently, a family of UCP homologues was identified, two of which (UCP-2 and UCP-3) are highly expressed in skeletal muscle. The physiological function of these UCPs is not established, but evidence suggests that they are involved in the regulation of metabolism rather than thermogenesis. The aim of my project was to investigate the relationship between uncoupling protein expression and alterations in glucose metabolism in different pathological conditions. In the first part of these studies, the expression of uncoupling proteins in pancreatic cancer patients was determined. A significant increase in UCP-3 and UCP-2 protein and mRNA expression was detected. This increase was associated with impaired insulin response and decreased ATP and phosphocreatine contents, indicating loss of energy. To further explore the relationship between UCP and insulin resistance, expression of the UCPs and glucose metabolism was examined in skeletal muscle samples from a post-traumatic rat model. A significant increase of UCP-2 and UCP-3 expression was observed, indicates a strong relationship between UCP expression and alterations in glucose metabolism and substrate utilization. The reported involvement of TNF-a and IL-ip in the pathogenesis of cachexia and insulin resistance triggered investigation of the effect of these inflammatory cytokines on the expression of the UCPs in skeletal muscle cells. TNF-a and IL-ip induced an increase in the expression of UCP-2 and UCP-3 that was positively associated with an increase in glucose uptake in Glut-4 transfected L6 myotubes. These findings relate increased glucose uptake to an increase in UCP expression. Finally, the signal pathway through which TNF-a induces its effect was investigated. The results of this study show that TNF-a stimulates an increase in UCP expression and giucose uptake through a SMase-ceramidedependant pathway which inciudes activation of MAPK and translocation of NF-xB to the nucleus. Experiments were then designed to try to uncover whether uncoupling caused an increase in glucose uptake or vice versa. Treatment of Glut-4 transfected L6 myotubes with the uncoupling agent, dinitrophenol resulted in an increase in glucose uptake, which was presumably a response to mitochondrial uncoupling. The glucose analogue, 2-deoxyglose is taken up into cells and phosphorylated but is not further metabolized. When 80% of the glucose in the media was replaced with 2-deoxyglucose, the stimulatory effect of TNF-a on the UCP expression was abolished, suggesting that the increase in UCP expression is dependent on the increase in glucose uptake. Taken together, these results support our hypothesis that there is a close relationship between an increase in UCP expression and glucose uptake. However, one study shows that glucose uptake increases in response to mitochondrial uncoupling, while the other shows that, when the TNF-a stimulated increase in glucose uptake is prevented this, in turn, prevents the increase of UCP expression. Thus, a question still remains as to whether increased glucose uptake induces UCP expression or the reverse pertains.
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