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  • br Introduction Most organisms require glucose as

    2022-08-09


    Introduction Most organisms require glucose beta amyloid as a key metabolite, and it is critical that mammalian cells regulate glucose levels properly to maintain bioenergetic homeostasis. The major transporters responsible for the uptake of glucose from systemic circulation into cells are the GLUTs, members of the SLC2A superfamily of transporters featuring 12 α-helical transmembrane segments [1,2]. GLUT1 and GLUT4 are the best-studied members of this family. GLUT4 is primarily located in muscle and adipose tissue and is acutely activated by either exercise or insulin, which trigger a translocation of GLUT4 to the membrane surface [3,4]. In contrast, GLUT1 is widely expressed in most cell types and is responsible for basal glucose uptake [5]. However, GLUT1 can also be acutely activated by a variety of conditions and mechanisms that are not well understood and appear dependent on cell type [[6], [7], [8], [9], [10], [11], [12]]. Abnormal regulation of GLUT1-mediated glucose transport is linked to several serious and prevalent human diseases. The GLUT1 transporter appears to be overexpressed in a number of cancers, especially those driven by oncogenic KRAS mutations or loss of the p53 tumor suppressor [13,14]. The resulting enhancement in glucose uptake supports an aerobic fermentation process, known as the Warburg effect, that helps drive increased cellular proliferation [[15], [16], [17]]. It has been observed that higher GLUT1 levels are associated with a poor prognosis in a variety of human cancers [16,18,19]. In contrast, some Alzheimer's patients display reduced GLUT1 expression at the blood beta amyloid barrier, implicating a potential metabolic role in the etiology of neurodegenerative disease [20]. It is clear that proper GLUT1 regulation is essential to the health of an organism, and a better understanding of GLUT1 activity is essential for developing potential therapeutic strategies for patients with glucose imbalance-linked disorders. In L929 fibroblast cells, where GLUT1 is the only expressed member of the family, the activity of the transporter can by acutely activated by a variety of reagents or conditions [[9], [10], [11], [12],21]. These activations occur within minutes without a change in the membrane concentration of the transporter and demonstrate a variety of kinetic patterns. Therefore, it appears that there may be multiple mechanisms to alter the activity of GLUT1 in L929 fibroblast cells. One possibility, and the focus of this study, is that the activity of GLUT1 depends on its location within ordered lipid microdomains known as lipid rafts. The classically defined lipid raft is a planar membrane structure in which proteins and phospholipids are held together by the dynamic clustering of sphingolipids and cholesterol in association with the underlying cortical actin cytoskeleton [22]. However, due to the dynamic and transient nature of lipid rafts, the varied techniques utilized to isolate these structures, and the inability to directly image or visualize these structures, the actual structures and physiological importance of lipid rafts are not entirely clear. These regions can be isolated by Triton X-100 detergent, leading to the description of these domains as “detergent resistant” membrane microdomains [[23], [24], [25], [26], [27]]. According to single-particle tracking experiments, they are up to 260–330 nm in diameter, although a recent study describes them as <100 nm in a resting state [23]. Because these structures are extremely dynamic, few proteins reside exclusively in rafts. However, proteins involved in membrane-mediated processes such as cell signaling or cholesterol recycling [26,28] are often found primarily in these structures. Lipid modifications such as GPI anchors, palmitoylation or myristoylation can target proteins to lipid rafts [[29], [30], [31]]. Certain proteins such as CD44 and flotillin usually reside within raft structures, and are therefore commonly used in immunoblots as controls for the presence of lipid raft domains [32]. Caveolin-1 is a marker for caveolae, which also isolate as low density domains and are sometimes considered a lipid raft subtype. The few studies that have investigated the dependence of GLUT1 activity on its location within a lipid raft were conflicting, reporting either an increase or a decrease in GLUT1 activity when associated with lipid rafts [[33], [34], [35]]. Given the conflicting data, the purpose of this study was to better understand the role lipid rafts play in determining the activity of GLUT1 in L929 fibroblast cells.