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    • PF-5274857 The translation of our rat findings to

    2022-08-17

    The translation of our rat findings to bovine and human lenses revealed that the expression of glucose transporters was more straightforward in bovine and human than in the rat lens, since only GLUT1 was detected in these lenses. Using the C-terminal GLUT1 antibody, we observed bands for GLUT1 in all regions of the lens in the bovine and human. In addition, using the N-terminal GLUT1 antibody revealed that in PF-5274857 to rat lenses, antibody labelling of the N-terminus of the GLUT1 protein remains intact throughout all areas of the bovine lens. In contrast, in human donor lenses, no bands were detected using the N-terminus GLUT1 antibody, indicating that the N-terminus of the proteins is modified in all regions of the human lens. This is similar to that observed in rat lenses, although the N-terminus modification in human lenses occurred in both epithelial and fiber cells. With increasing age in human lenses, the bands for GLUT1 using the C-terminus GLUT1 antibody remained intact, indicating that the C-terminus is preserved with increasing age. In the future, we intend to utilize immunohistochemistry to confirm these localization patterns and in particular examine more closely the distribution and organization of GLUT1 across the epithelium since it is known that the peripheral epithelium can exhibit different expression patterns to the central region (Li et al., 2010). The strong bands for GLUT1 in the center of bovine and human lenses are interesting given that this region of the lens is often regarded as a metabolically inactive area. If GLUT1 is revealed to be functionally active in the lens core, this indicates that the lens center is capable of direct accumulation of glucose. Previous studies have shown that the lens core contains enzymes involved in the metabolism of glucose. This includes glucose- 6-phosphate dehydrogenase, glutathione reductase (GR) and hexose monophosphate (HMP) activity which is used for the production of NADPH (Giblin et al., 1981, Giblin and Reddy, 1980). Although, the activities of these enzymes in the core are much lower relative to the cortex, it is possible that glucose metabolism and production of reducing equivalents can occur and potentially helps to maintain the redox status of lens proteins in the lens center. Finally, the absence of GLUT3 protein in the bovine and human lens was an intriguing finding given that GLUT3 expression was shown to be up-regulated in lenses from diabetic rats (Merriman-Smith et al., 2003). This suggests that glucose uptake pathways and changes in glucose transport may differ between species under normal and diabetic conditions. The absence of GLUT3 protein expression in young bovine and human lenses suggests one of two scenarios. The first scenario is that GLUT1 is exclusively involved in glucose uptake in bovine and human lens and under hyperglycemic conditions, GLUT1 is up-regulated to increase glucose uptake. The second scenario is that GLUT1 is responsible for glucose uptake, but that in the presence of excess glucose, GLUT3 expression, which normally exists at low levels, is stimulated to increase glucose uptake under these conditions. A search of the literature reveals studies which investigate changes in glucose transport function in response to diabetics. However, in the majority of these studies the streptozocin (STZ) rat has typically been used as a model (Bond et al., 1996, Chatzigeorgiou et al., 2009, Obrosova et al., 2010, Perry et al., 1987, Suryanarayana et al., 2005, Suryanarayana et al., 2007, Varsha et al., 2014, Wang et al., 2016). This raises the questions as to the suitability of the rat as a model since it expresses an array of glucose uptake transporters that are not expressed in bovine or human lenses. In these diabetic rat studies, there appears to be a mixed response with intestinal enterocytes shown to increase GLUT1 expression (Boyer et al., 1996), while GLUT1 mRNA and protein decrease in the kidney (Chin et al., 1997, Vestri et al., 2001) and retina (Badr et al., 2000). To distinguish between our two scenarios, more detailed work will be required to examine changes in GLUT mRNA and protein expression under normal and hyperglycemic/diabetic conditions in bovine and human lenses. This will be difficult to achieve in humans given the limited availability of human donor lenses. However, given the similar pattern of GLUT1 expression between bovine and human lenses, we suggest that the bovine lens may be a more relevant model, compared to the rat, for translating our work to humans. We also know that distinct biochemical differences exist between humans and rats with respect to aldose reductase (AR) activity and polyol accumulation. Adult rat lenses have very high levels of AR activity which is the opposite of human and bovine lenses which exhibit low AR activity (Jedziniak et al., 1981, Srivastava et al., 1982). As such, the amount of sorbitol present in human diabetic cataractous lenses is considerably less than that found in STZ injected rats (Chylack et al., 1979). As a first step, we are now focusing on developing an in vitro bovine model of hyperglycemia with which to assess glucose transporter expression, function and metabolism.