PURPOSE: The present study was aimed at evaluating early changes in glycolysis, the redox state of free cytosolic NAD(P)-couples, and the adenine nucleotide system in lens in both control and 50% galactose-fed rats, with the possibility of preventing these with an aldose reductase inhibitor (ARI). METHODS: Experiments were performed on male Sprague-Dawley rats fed the galactose diet for 2-14 days. The levels of glucose, galactose, glycolytic intermediates, alpha-glycerophosphate, malate, NAD, ATP, ADP, AMP were assayed spectrofluorometrically in individual lenses by enzymatic procedures, while galactitol and myo-inositol were quantified by GC-MS. Free cytosolic NAD+/NADH, NADP+/NADPH, and ATP/ADP x P(i) (phosphate potential) were estimated from lactate dehydrogenase, malic enzyme, and triose phosphate isomerase-glyceraldehyde 3-phosphate dehydrogenase-3-phosphoglycerate kinase systems. Lactate and pyruvate production by lenses of both control and galactose-fed rats was measured in a set of in vitro incubation studies (2 hr, 37 degrees C, Krebs bicarbonate-Hepes buffer, pH 7.45, with 5mM glucose or 5mM glucose + 30 mM galactose, respectively). RESULTS: Lens galactitol levels in 2, 4, 6, 8, 10, and 14-day galactose-fed rats were 48 +/- 8, 58 +/- 9, 68 +/- 8, 73 +/- 5, 81 +/- 20, and 75 +/- 11 mmol/g wet weight (mean +/- SD), respectively. NAD+/NADH ratios were indistinguishable from controls after 2-6 days on the galactose diet, but fell dramatically between 8 and 10 days, and did not correlate with polyol accumulation per se. The pattern of glycolytic intermediates (no change in G6P, F6P, and 3-PG, increase in GA3P, decrease in FDP, PEP, pyruvate, and lactate), as well as reduced in vitro lactate and pyruvate production, suggest inhibition of glycolysis at the sites of phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, enolase, and pyruvate kinase. ATP levels as well as total ATP/ADP, ATP/ADP x Pi, adenylate charge, and cytosolic phosphate potential were decreased in galactose-fed rats, while galactose 1-phosphate and a-glycerophosphate levels as well as NADP+/NADPH ratio were increased. Lens galactitol levels were reduced approximately 57% in 10-day galactose-fed rats treated with the ARI (tolrestat, 100 mg/kg bwt/day, 6-day pretreatment); the changes in the lower segment of glycolysis, alpha-glycerophosphate levels, redox state of NAD-couples, and energy metabolism were partially prevented while NADP+/ NADPH ratios were unchanged and galactose 1-phosphate levels were further increased. CONCLUSIONS: Depressed glycolysis in lens in galactose-fed rats is consistent with decreased NAD+/NADPH and adenine nucleotide phosphorylation. Early changes in lens glucose utilization, redox state of NAD-couples, and energy metabolism in this model of galactosemia are similar to those in diabetes, are at least in part mediated by aldose reductase involved mechanisms, and can be partially prevented by an aldose reductase inhibitor.
PURPOSE: The present study was aimed at evaluating early changes in glycolysis, the redox state of free cytosolic NAD(P)-couples, and the adenine nucleotide system in lens in both control and 50% galactose-fed rats, with the possibility of preventing these with an aldose reductase inhibitor (ARI). METHODS: Experiments were performed on male Sprague-Dawley rats fed the galactose diet for 2-14 days. The levels of glucose, galactose, glycolytic intermediates, alpha-glycerophosphate, malate, NAD, ATP, ADP, AMP were assayed spectrofluorometrically in individual lenses by enzymatic procedures, while galactitol and myo-inositol were quantified by GC-MS. Free cytosolic NAD+/NADH, NADP+/NADPH, and ATP/ADP x P(i) (phosphate potential) were estimated from lactate dehydrogenase, malic enzyme, and triose phosphate isomerase-glyceraldehyde 3-phosphate dehydrogenase-3-phosphoglycerate kinase systems. Lactate and pyruvate production by lenses of both control and galactose-fed rats was measured in a set of in vitro incubation studies (2 hr, 37 degrees C, Krebs bicarbonate-Hepes buffer, pH 7.45, with 5mM glucose or 5mM glucose + 30 mM galactose, respectively). RESULTS: Lens galactitol levels in 2, 4, 6, 8, 10, and 14-day galactose-fed rats were 48 +/- 8, 58 +/- 9, 68 +/- 8, 73 +/- 5, 81 +/- 20, and 75 +/- 11 mmol/g wet weight (mean +/- SD), respectively. NAD+/NADH ratios were indistinguishable from controls after 2-6 days on the galactose diet, but fell dramatically between 8 and 10 days, and did not correlate with polyol accumulation per se. The pattern of glycolytic intermediates (no change in G6P, F6P, and 3-PG, increase in GA3P, decrease in FDP, PEP, pyruvate, and lactate), as well as reduced in vitro lactate and pyruvate production, suggest inhibition of glycolysis at the sites of phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, enolase, and pyruvate kinase. ATP levels as well as total ATP/ADP, ATP/ADP x Pi, adenylate charge, and cytosolic phosphate potential were decreased in galactose-fed rats, while galactose 1-phosphate and a-glycerophosphate levels as well as NADP+/NADPH ratio were increased. Lens galactitol levels were reduced approximately 57% in 10-day galactose-fed rats treated with the ARI (tolrestat, 100 mg/kg bwt/day, 6-day pretreatment); the changes in the lower segment of glycolysis, alpha-glycerophosphate levels, redox state of NAD-couples, and energy metabolism were partially prevented while NADP+/ NADPH ratios were unchanged and galactose 1-phosphate levels were further increased. CONCLUSIONS: Depressed glycolysis in lens in galactose-fed rats is consistent with decreased NAD+/NADPH and adenine nucleotide phosphorylation. Early changes in lens glucose utilization, redox state of NAD-couples, and energy metabolism in this model of galactosemia are similar to those in diabetes, are at least in part mediated by aldose reductase involved mechanisms, and can be partially prevented by an aldose reductase inhibitor.
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Authors: Subir K Roy Chowdhury; Elena Zherebitskaya; Darrell R Smith; Eli Akude; Sharmila Chattopadhyay; Corinne G Jolivalt; Nigel A Calcutt; Paul Fernyhough Journal: Diabetes Date: 2010-01-26 Impact factor: 9.461