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  • Due to the requirement of high

    2022-06-24

    Due to the requirement of high-end methods such as total internal reflection fluorescence (TIRF) microscopy to visualize lipid rafts, isolation procedures are employed to study the protein content of lipid rafts [29]. The different methods utilized to isolated lipid rafts and other low density microdomains, the varying lipid composition of rafts, the dynamic nature of lipid rafts and differences among cell types Silydianin all contribute to some confusion concerning the specific role that lipid rafts and other low density domains play in protein activity. However, given these limitations, we conclude that, in contrast to other cell types, GLUT1 in L929 fibroblast Silydianin is neither found in cholesterol-rich domains nor does its activity depend on cholesterol-based lipid rafts.
    Conclusions
    Contributors All authors approve and have contributed to the work reported here. Specifically, Lauren Rylaarsdam collected most of the data reported in this study and wrote a first draft of the paper. Grace Johnecheck performed the solubility studies reported in Fig. 4A. Brendan Looyenga provided experimental advice and generated the final versions of the figures. Larry Louters provided experimental advice and was the primary writer of the paper.
    Acknowledgments This research was supported by a NIH R15 grant (DK08193-1A1). Additional funding for undergraduate research was provided by Ken and Marcia Wierda.
    Introduction The flavone quercetin (3,3′,4′5,7-pentahydroxyflavone) is a natural polyphenol found in high concentrations in many fruits, vegetables, and wines [1,2]. The average daily intake of flavonoids in a normal diet is approximately 23 mg of which quercetin makes up 60–70% [2]. Quercetin has a wide variety of physiological effects and has received significant research attention with over 15,000 studies reported in the literature including 650+ reviews and 230+ clinical trials. Much of the work, as illustrated by recent reviews, has focused on the potential of quercetin as a therapeutic in disease states such as diabetes, obesity and cancer [1,[3], [4], [5], [6], [7], [8], [9]]. Of potential relevance to its anticancer and antidiabetic activity is the documented direct interaction of quercetin with the GLUT family of proteins, particularly GLUT1 [[10], [11], [12], [13], [14], [15], [16]]. The facilitated glucose transporter, GLUT1 (SLC2A1), is expressed in a wide variety of cell types and, while generally responsible for basal uptake of glucose, it also responds to changing metabolic conditions [17]. Chronic exposure to cell stressors—such as hypoxia, hypoglycemia, and AMP kinase activation—increase GLUT1 protein expression [[18], [19], [20]]. In addition, this transporter appears to be overexpressed in a number of cancers, especially those driven by KRAS mutations, thereby accelerating glucose uptake in support of glycolytic metabolism [[21], [22], [23], [24], [25]]. Given the dependence of many cancers on GLUT1 transport activity, there is strong interest in small molecule inhibitors of GLUT1 as potential therapeutics. Potential inhibitors include a number of natural products such as quercetin [13,14], curcumin [26,27], and caffeine [[28], [29], [30]], as well as newly synthesized high affinity inhibitors such as WZB-117 [31,32] and BAY-876 [33]. Quercetin is a competitive inhibitor of glucose entry via GLUT1 and a noncompetitive inhibitor of glucose exit. These studies suggest that quercetin binds to the exofacial surface of GLUT1 [10,14]. There is also evidence that suggests that quercetin itself is transported into cells via GLUT1 [11,12] and GLUT4 [13]. The purposes of this study was: 1) to verify the inhibitory effects of quercetin on 2-deoxyglucose uptake in L929 cells, which exclusively express GLUT1 [34]; 2) to measure quercetin binding in L929 cells; and 3) to measure the effects of other GLUT1 inhibitors on quercetin binding to help map the quercetin binding site on GLUT1 and to determine if quercetin is transported by GLUT1 into cells. We hypothesized that if quercetin was taken up by GLUT1 in a fashion similar to glucose, inhibitors of glucose uptake should also inhibit quercetin uptake.