Chronic hyperglycemia has been implicated in the pathogenesis and progression of complications in diabetes although the underlying mechanisms remain uncertain. Interestingly, inhibitors of aldose reductase (AR) have been shown to ameliorate some of the complications of diabetes. For example they preserve nerve conduction velocity nerve blood flow and promote nerve regeneration in human and experimental diabetic neuropathy. AR catalyzes the conversion of glucose to sorbitol and is thought to act as an osmotic stress response protein by compensating for hypertonic stress through intracellular accumulation of sorbitol. In addition to this mechanism, other stress response proteins such as the NA-taurine co-transporter (TT) and the Na-myo- inositol co-transporter accumulate other osmolytes such as taurine and myo-inositol. In isotonic hyperglycemic stress, inappropriate sorbitol accumulation causes compensatory depletion of intracellular osmolytes such as taurine and myo-inositol, rendering them rate limiting for normal intracellular metabolism. This application focuses on the regulation of the human TT gene expression and function by glucose and osmotic stress mediated by AR- related and AR-unrelated mechanisms. The overall hypothesis is that glucose induces specific metabolically and/or osmotically mediated alterations in transcriptional, post-transcriptional and/or post- translational regulation of the hTT. As a model system, human retinal pigment epithelial cells (RPE) that vary in their degree of AR expression will be used in conjunction with AR inhibitors. This will allow to differentiate the osmotic from the metabolic effects of glucose.
Three specific aims are proposed: (1) To characterize the glucose mediated AR-related and AR-unrelated changes in hTT mRNA abundance and correlate mRNA levels with peptide levels, activity and taurine content. (2) To determine whether changes in mRNA levels are due to changes in transcription or mRNA stability. (3) To determine the structure of the hTT gene and its 5' regulator region and identify cis elements that are necessary for glucose mediated changes in transcription and/or changes in mRNA stability. As an alternative, if in aim 1 little correlation between hTT mRNA abundance and hTT activity is found, then glucose mediated post-translational modifications will be examined.
