ONC201 In the present study we characterized the expression

In the present study, we characterized the expression of glucose transporters in the developing and adult mouse lens and showed that GLUT1 is the primary transporter expressed in the lens epithelium. We found that mice with a homozygous deletion of Slc2a1 from the lens epithelium developed cataracts, which were not observed in heterozygous mice expressing a single GLUT1 allele. These studies thus establish a critical role of GLUT1 expression in the mouse lens epithelium for the maintenance of lens transparency.
Materials and methods
Results
Discussion Previous studies performed on isolated lenses established an essential role for glycolysis in generating ATP required to maintain osmotic gradients and lens transparency (Winkler and Riley, 1991) (Chylack, 1975). In these ex vivo studies glucose was the only carbon source provided in the incubation media. In the present study, we demonstrated in an in vivo mouse model that glucose uptake by GLUT1 is required for maintaining metabolic homeostasis and transparency in the lens even when other metabolic substrates are available. In the mature mouse lens, we found by immunofluorescence localization and immunoblot analysis that GLUT1 was most highly expressed in the basolateral membrane of the lens epithelium, which is bathed in the nutrient rich aqueous humor. Low levels of expression were detected in the fiber ONC201 in the cortical region when images were overexposed (Fig. 1). These results are consistent with mouse lens proteome data showing that GLUT1 comprises only 1% of the membrane protein in the fiber cells (Bassnett et al., 2009). During lens development, hyaloid vessels surround the growing lens providing a supply of nutrients and GLUT1 was detected in the primary fiber cells to facilitate glucose uptake. However, as the lens grows in size, the hyaloid vessels regress and ciliary epithelium transports glucose into the aqueous humor to support lens metabolism (Ito and Yoshioka, 1999). As GLUT1 was only detected at very low levels in the apical membrane of the mouse lens epithelium, glucose may diffuse into lens fiber cells via gap junctions (Goodenough et al., 1980). A similar system has been described for movement of glucose between pigmented and non-pigmented epithelium in the ciliary body and at the interface of maternal-fetal circulation (Kumagai, 1999). The flow of glucose from the lens epithelium to lens fiber cells is essential since, like red blood cells, lens fiber cells lose their organelles as they mature and rely solely on glycolysis for ATP production (Winkler and Riley, 1991). In contrast, the lens epithelium and fiber cells in the outer cortex have mitochondria and can oxidize other substrates to support their anabolic and catabolic needs (Kinoshita, 1965). The distribution of glucose transporters in the mouse lens differs from that observed in larger lenses including rat, bovine, and human lenses where GLUT1 or GLUT3 are both detected in fiber cells (Lim et al., 2017). In these larger lenses the observed expression of glucose transporters in differentiating and mature fiber cells suggests glucose diffuses through the extracellular space and that fiber cells directly uptake the glucose delivered to them via the extracellular space. To determine the importance of GLUT1 in maintaining metabolic homeostasis and transparency in the lens, we used a genetic approach to delete Slc2a1. GLUT1f/f mice were crossed with GFAP-Cre mice to generate LensΔGlut1het and LensΔGlut1 mouse lines. GFAP is expressed in the lens epithelium at E18 so the early development of the lens was not affected. The LensΔGlut1het mice did not develop cataracts even though the levels of GLUT1 expression were significantly reduced. This is consistent with the observation that patients with GLUT1 deficiency syndrome do not generally develop cataracts (Winkler et al., 2015). LensΔGlut1 mice do not develop cataracts until about three months of age. The late onset of cataract formation in the LensΔGlut1 mice correlates with the time required for complete loss of GLUT1 mRNA and protein. While GLUT1 expression levels were much lower in the 2-month-old LensΔGlut1 mice relative to controls, it was adequate to maintain glycolysis and generate sufficient ATP to maintain transparency. At 3 months of age, when GLUT1 was no longer detectable, the levels of lactate and ATP were decreased compared to control mice. Mature fiber cells in the organelle free zone became vacuolated and disorganized while the lens epithelium and fiber cells that still had organelles retained their structural integrity. Lenses of the LensΔGlut1 mice continued to grow in size demonstrating that glucose was not required for proliferation and differentiation into lens fiber cells. This suggests that other carbon sources can be used to support anabolic and catabolic processes in the lens epithelium and cortical fiber cells. Consistent with this observation, we found that while glucose was increased in the aqueous humor of LensΔGlut1 mice compared to controls, the levels of several amino acids were decreased (Table 2). The decrease in amino acids in the aqueous humor of the LensΔGlut1 mice compared to the controls, suggests an increase in oxidation of amino acids may be used to support the metabolic needs of the epithelium and differentiating fiber cells in mice (Waley, 1964) (Trayhurn and Van Heyningen, 1973).