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  • The simple carrier model for GLUT transport activity propose

    2022-06-23

    The simple carrier model for GLUT1 transport activity proposes that the transporter alternates between an outward facing glucose binding site and an inward, cytoplasmic, facing glucose binding site. Cytochalasin B is an endofacial inhibitor and appears to preferentially bind to the open, inward facing conformation [32,48,49]. The mutual enhancement of quercetin and cytochalasin B binding suggests that both compounds preferentially bind to GLUT1 in the inward facing glucose binding site conformation. Thus, WZB-117, which binds to outward facing conformation, would be expected to reduce the preferential binding of quercetin to the inward facing conformation, which is exactly what we observe. This is in precise agreement with the model for quercetin inhibition of GLUT1 proposed from kinetic and binding studies in erythrocytes [10]. The observation that BAY-876 does not affect quercetin binding indicates that further studies are needed to map its binding or interactions with GLUT1.
    Conclusions
    Acknowledgements This research was supported by a NIH R15 grant (DK08193-1A1).
    Introduction Glucose is the primary energy substrate of many tissues including the avascular ocular lens. The uptake of glucose into TG101209 is regulated by facilitated and sodium-coupled glucose transporters whose expression pattern and subcellular distribution vary in a developmental and tissue specific manner. Facilitated glucose transporters are encoded by the Slc2 gene family and sodium-coupled glucose transporters are encoded by the Slc5 gene family (Wood and Trayhurn, 2003). GLUT1, encoded by Slc2a1, is the major facilitative glucose transporter expressed in the eye (Kumagai et al., 1994) (Takata, 1996) (Merriman-Smith et al., 2003). In mammals, the lens is derived from surface ectoderm and the adult lens is comprised of lens epithelium and lens fiber cells. In the equatorial region of the lens, epithelial cells proliferate then differentiate into elongated fiber cells. Differentiation into mature secondary fiber cells is accompanied by loss of organelles, including nuclei and mitochondria. The lens epithelium has mitochondria that can utilize oxidative phosphorylation for the generation of ATP, while the fully differentiated lens fiber cells are reliant on glycolytic metabolism of glucose to support their energy demands. In the lens, glucose uptake is mediated by glucose transporters, however across different species the specific transporters used and their localization differs (Merriman-Smith et al., 1999) (Merriman-Smith et al., 2003) (Mantych et al., 2015) (Lim et al., 2017). In bovine and human lenses, GLUT1 is the only glucose transporter expressed and is found throughout the lens including lens epithelium and fiber cells. The lens epithelium and fiber cells of the rat expressed GLUT1 while GLUT3, SGLT1 and SGLT2 were only expressed in fiber cells in the inner cortex and core regions (Lim et al., 2017). The lens is an avascular tissue and relies on the aqueous humor for supply of oxygen and nutrients including glucose (Dahm et al., 2011). Within the lens, the lens epithelium has been identified as the more metabolically active compartment (Bhat, 2001). Previous studies have shown that in the presence of glucose, both the lens epithelium and fiber cells from freshly isolated rabbit lenses were able to maintain ATP levels under aerobic and anaerobic conditions. In the absence of glucose the rabbit lens epithelium and fiber cells rapidly lost ATP (Winkler and Riley, 1991). In another study, rat lenses were incubated ex vivo in decreasing concentrations of glucose, which resulted in decreased ATP and cataract formation when glucose fell below 2 mM. Cells were unable to maintain the osmotic gradient, leading to an increase in Na+ and a decrease in K+ levels in these lenses. (Chylack, 1975). The osmotic cataracts formed in these lenses ex vivo mimicked the cataracts in vivo and also mimicked hypoglycemic lamellar cataracts reported in children (Merin and Crawford, 1971). In previous studies, glucose was the only substrate provided in the buffer and other metabolic fuels found in the aqueous humor such as amino acids and lipids were not tested to see if they could support ATP production in the lens epithelium.