Glucose uptake is mediated by a family of glucose transporters known as GLUT. Insights into how the activity of these transporters is regulated are important for a better understanding of metabolic disorders such as diabetes and metabolic syndrome where glucose uptake is compromised. We propose to investigate the mechanism for the acute activation of GLUT1. Both published and additional preliminary evidence from our laboratory indicate that GLUT1-mediated glucose uptake in L929 fibroblast cells can be activated within minutes by two distinct pathways. Low concentrations of methylene blue and glucose deprivation each activate glucose uptake within minutes. The two maximal effects are additive and each has different kinetics of uptake. The focus of this proposal is to identify what happens to GLUT1 at the membrane as a result of activation by these two stimulants. We will specifically explore three possible activating membrane events: 1) Glucose transport activity changes when GLUT1 moves into or out of lipid rafts. We will investigate this possible mechanism by utilizing agents to disrupt lipid rafts to see if this also affects the activation of glucose uptake. We will also isolate lipid rafts by density centrifugation to determine if the concentration of GLUT1 in lipid rafts changes upon activation. Finally, we will determine if the FRAP signal of GLUT1-GFP changes upon activation or if the FRET signal between fluorescently tagged GLUT1 and fluorescently labeled raft and non-raft proteins changes when the transporter is activated. 2) Glucose transport activity is enhanced by palmitoylation of GLUT1. This will be explored by labeling GLUT1 with radioactive palmitate in the presence and absence of glucose uptake activators. We will also use inhibitors of palmitoylation to determine if we can inhibit both the radiolabeling and the activation of GLUT1 transport activity. And we will generate point mutations of GLUT1 substituting for cysteine to see how that affects activity. 3) Activation of glucose uptake is correlated with the oligomerization (tetramer formation) of GLUT1. We will transfect cells with both GLUT1-CFP and GLUT1-YFP to see if the FRET signal between these is enhanced when transport is activated. We will also use reversible crosslinkers to stabilize tetramers in both control and activated L929 cells. Isolation by immunoprecipitation and reversal of the crosslinked protein products may help us identify changes in GLUT1 structure that result from the activation of glucose uptake. The investigation of these three potential mechanisms will help identify similarities and differences in the activation of glucose uptake by glucose deprivation and methylene blue as well as provide valuable insights into understanding how membranes control transporter activity.
It is estimated that over 20 million Americans suffer from diabetes and 50 million more have a prediabetic condition known as metabolic syndrome. One hallmark feature of these two conditions is that glucose uptake is compromised. This proposal focuses on understanding the mechanism for the acute activation of one of the transporters responsible for glucose uptake-GLUT1. Understanding how GLUT1 is activated can contribute to a clearer understanding of these diseases and perhaps lead to the development of more effective treatments. In addition, this study should contribute to a greater understanding of how membranes regulate transporter activity.
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