Glucose Transporters in Diabetic Complications of the Lens

Abstract

Lens transparency is primarily maintained by the anaerobic metabolism of glucose. Glucose is transported from the aqueous humuor to the lens epithelial cells however, it has not yet been established how glucose penetrates to the inner part of the lens. The core of the lens is acidic, approximately pH 6.5, as an effect of the accumulation of lactate, the end-product of glycolysis. This confirms that glucose is drawn deep into the core of the lens. Until recently it was assumed that glucose was transported to the core via a gap junction-mediated route by cell-cell diffusion. However, passive diffusion is limited in capacity and is unlikely to be sufficient for nutrient transport especially for larger lenses. Instead, an active circulation system has been proposed that has the potential to transport glucose deep into the lens via an extracellular route. This would imply that fibre cells may have evolved their own glucose uptake system, yet no direct evidence to this effect has been available. My thesis describes new molecular evidence that both epithelial and fibre cells have evolved their own glucose uptake system. The rat lens expresses the facilitative glucose transporters GLUTI and GLUT3 differentially. GLUTI is predominantly expressed in the epithelium while GLUT3 is predominantly expressed in the fibre cells of the lens. In the normal lens, this makes good physiological sense. GLUTI has a high Km suitable for situations of high glucose concentrations, as is the case for the epithelium where the aqueous humour mirrors glucose concentrations found in blood. GLUT3 has a lower Km and is particularly suitable for the fibre cells, where the supply of glucose from the tortuous extracellular space is limited. The discovery of GLUT3 in the fibre cells lends strong support for the existence of an active circulation system in the lens and makes it seem unlikely that glucose is transported into the core via gap junction-mediated diffusion. In the diabetic state, sorbitol - a product of glucose metabolism, occurs at elevated levels of about 30 times more than that of the normal, suggesting a significant increase in glucose uptake. This imposes an osmotic stress on the lens, which can be countered by regulated cell volume decrease only in the outer but not in the inner cortex. As a consequence inner cortex tissue breaks down and opacities result. My studies of the diabetic rat lens shows that the situation is made worse by an apparent up-regulation of GLUT3 in the fibre cells. Quantitative RT-PCR shows that the GLUT3 transcript is up-regulated six-fold during the initial weeks of diabetic insult in the streptozotocin rat model. The up-regulated GLUT3 protein is detected in the region where maximum tissue damage occurs. These results suggest a new mechanism for the initial tissue damage in the diabetic lens, whereby increased uptake of glucose leads to an over-production of sorbitol which causes osmotic stress on the fibre cells that is beyond their defense capability of regulated cell volume decrease. While my results described above have revolutionized our view of nutrient transport in the lens and its potential role in the early stages of diabetic cataract, the picture is only complete when the functionality of GLUT3 can be demonstrated. For this purpose, vesicles were prepared from isolated lens fibre cells and subjected to quantitative uptake studies using a fluorescent glucose derivative. Uptake was greatly reduced using the GLUT-specific inhibitor phloretin demonstrating that GLUT3 of the rat lens fibre cells is indeed fully functional. In summary, my results add a new dimension to our understanding of how the lens maintains homeostasis and tissue transparency. The lens has evolved an ingenious system to supply, nutrients to the core region to compensate for the absence of a vasculature. However, by achieving this, it has rendered itself unable to defend itself against the adverse effects of high glucose. My results also contribute towards developing new strategies of rational drug design to prevent or delay cataract.