As a common complication of diabetes, sugar cataract represents a significant public health problem with no known effective treatment other than surgery. Hyperglycemia has been implicated in the etiology of sugar cataract formation, but the metabolic pathways and their modulation by genetic and biochemical factors have yet to be determined. The conversion of tissue glucose to sorbitol by the enzyme aldose reductase (AR), has been implicated as a potential mechanism leading to sugar cataract in the lens. Over the last six years, studies from this laboratory have defined metabolic links between hyperglycemia, polyol pathway activation and myo- inositol (MI) depletion. Recent studies from this laboratory have shown that hypertonic stress stimulates gene expression of the Na+ /M1 cotransporter and AR, increases protein synthesis and elevates both M1 uptake activity and polyol formation. The intracellular accumulation of these compatible, reciprocal, organic osmolytes operate to maintain osmotic balance and protect the lens against the perturbing effects of high intracellular concentrations of electrolytes. However, if polyol accumulation continues over a protracted period of time, it divests the lens of essential M1 by restricting M1 uptake activity via multiple inhibitory mechanisms; (1) polyol-mediated suppression of m1 carrier protein uptake activity, (2) polyol-mediated downregulation of the Na+ /M1 cotransporter mRNA and (3) polyol-activated efflux of Mi from cell to medium. In this regard hypertonicity is analogous to hyperglycemia, in that the former (by virtue of induction of AR mRNA), like the latter (by virtue of increased substrate availability) elicits an increase in intracellular polyol. The direct impairment of the M1 transport system by either means represents a plausible common mechanism that could account for the depletion of lenticular MI. This competing renewal application proposed detailed study of the mechanisms, implications and consequences of polyol- and non-polyol-induced M1 depletion in lens cell culture and lens organ culture. The depletion of intracellular M1 without alteration of intracellular polyol content will be achieved by employing a feeding regimen with L-glucose, a potent competitive inhibitor of M1 uptake, a novel technique shown by us to promote the depletion of intracellular M1 in lens cell culture without raising polyol content. In the first specific aim the molecular mechanisms which regulate the expression of the Na+/M1 cotransporter in hypertonicity and hyperosmotic diabetic conditions will be examined. In the second specific aim the interrelationship of polyol and M1 osmoregulatory mechanisms will be carefully defined. The third specific aim will identify and characterize the regulatory mechanism which modulates M1 efflux, currently the least understood mechanism of polyol-induced M1 depletion. The fourth specific aim will define the kinetics of M1 uptake in intact bovine lenses and correlate these results with known kinetic parameters from lens cell cultures. The final specific aim will utilize immunoelectron microscopy to localize the Na+/M1 cotransporter tot eh basolateral or apical aspect of lens epithelial cells in the intact lens. The studies proposed in this competitive renewal grant application will yield new information on the molecular and cellular regulation of the Na+/myo-inositol cotransporter and M1 uptake activity and provide new insight into a mechanism we believe contributes to the formation of sugar cataract.
