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K. Xing, J. M. Randazzo, M. F. Lou, P. F. Kador; Signaling in in vitro Cultured Rat Lens Treated With High Glucose. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2284.
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Excess glucose has been linked to osmotic and oxidative stress that stem from sugar alcohol production, prolonged hexose toxicity associated with glycation end products and redox changes associated with increased flux through the polyol pathway. Stress also regulates growth factors that, in turn, regulate aldose reductase (AR) activity which subsequently has been linked to signal transduction changes, cytotoxic signals and activation of apoptosis. The purpose of this study was to investigate the links between AR activity, sorbitol formation, and stress-related gene expression changes.
Lenses from young (100 g) Sprague Dawley lenses were cultured for up to 48 hrs in TC-199 - bicarbonate buffer containing either 30 mM fructose (control) or 30 mM glucose with/without 10 µM of the AR inhibitor AL1576 or tolrestat or the sorbitol inhibitor CP-470,711 (SDI). In addition, lenses were also cultured with 30 mM glucose and 30 mM mannitol to simulate iso-osmotic conditions where sorbitol accumulates but osmotic changes are minimized. Sorbitol level were determined by HPLC. GSH was spectrophotometrically measured using the DTNB method. Western blots were conducted using the commercially available antibodies P-c-Raf, P-Erk1/2, P-JNK, P-Akt.
Compared to control rat lenses cultured in vitro for 48 hrs with 30% fructose, similar lenses cultured under high glucose conditions had a 2.6-fold increase in sorbitol, an 8% increase in wet weight due to sorbitol-associated hydration, and a 40% reduction in GSH levels. Inhibition of AR with AL1576 and tolrestat resulted in a complete inhibition and 0.7-fold increase in sorbitol formation, respectively, no change in wet weight, and a 17% and 27% reduction of GSH, respectively. SDI treatment resulted in a 6-fold increase in sorbitol, a 13% increase in wet weight and a 27% reduction in GSH. Iso-osmotic culture resulted in 4.2-fold increase in sorbitol, a slight (3%) increase in wet weight, and a 42% reduction in GSH. P-Akt was increased in lenses incubated in high glucose with/without SDI and under iso-osmotic conditions; however no group demonstrated obvious changes in either P-Erk1/2, P-JNK and P-c-Raf.
P-Akt associated stress appears to be directly linked to AR activity rather than osmotic changed resulting from sorbitol accumulation.
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