Glycolytic pathway, redox state of NAD(P)-couples and energy metabolism in lens in galactose-fed rats: Effect of an aldose reductase inhibitor
Glycolytic pathway, redox state of NAD(P)-couples and energy metabolism in lens in galactose-fed rats: Effect of an aldose reductase inhibitor
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DOI：
10.1076/ceyr.16.1.34.5113
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发表时间：
1997-01-01
影响因子：
2
通讯作者：
Williamson, JR
中科院分区：
文献类型：
--
作者：
Obrosova, I;Faller, A;Williamson, JR
目的.本研究旨在评估糖酵解的早期变化，游离胞质NAD（P）-对的氧化还原状态，以及透镜中的腺嘌呤核苷酸系统在对照组和50%半乳糖喂养的大鼠中，与预防这些与醛糖还原酶抑制剂（ARI）的可能性。实验通过酶促方法用荧光分光光度法测定各个晶状体中葡萄糖、半乳糖、糖酵解中间体、α-甘油磷酸盐、苹果酸盐、NAD、ATP、ADP、AMP的水平，同时通过GC-MS定量半乳糖醇和肌醇。游离胞质NAD+/NADH、NADP+/NADPH、从乳酸脱氢酶、苹果酸酶和磷酸丙糖异构酶-甘油醛3-磷酸脱氢酶-3-磷酸甘油酸激酶系统中测定ATP/ADP x P-1（磷酸电位）。在一组体外孵育研究中测量对照和半乳糖喂养的大鼠的晶状体的乳酸盐和丙酮酸盐产生（2小时，37 ℃，Krebs碳酸氢盐-Hepes缓冲液。pH7.45，分别用5 mM葡萄糖或5 mM葡萄糖+30mM半乳糖）。喂食半乳糖2、4、6、8、10和14天的大鼠的透镜半乳糖醇水平分别为48 +/- 8、58 +/- 9、68 +/- 8、73 +/- 5、81 +/- 20和75 +/- 11 mmol/g湿重（平均值+/- SD）。半乳糖饮食2-6天后，NAD+/NADH比值与对照组无区别，但在8 - 10天之间急剧下降，并且与多元醇积累本身无关。糖酵解中间产物的模式（G6 P、F6 P和3-PG无变化，GA 3 P增加，FDP减少。PEP、丙酮酸和乳酸），以及体外乳酸和丙酮酸产生减少，表明磷酸果糖激酶位点的糖酵解受到抑制。甘油醛3-磷酸脱氢酶、烯醇酶和丙酮酸激酶。在半乳糖喂养的大鼠中，ATP水平以及总ATP/ADP、ATP/ADP x P-1、腺苷酸电荷和胞质磷酸电位降低，而半乳糖1-磷酸和α-甘油磷酸水平以及NADP+/NADPH比值升高，透镜半乳糖醇水平降低，与ARI加热的半乳糖喂养的大鼠相似，降低了57（托司他，100 mg/kg bwt/天，预处理6天）：糖酵解下段的变化，α-甘油磷酸水平。NADP+/NADPH比值未改变，半乳糖1-磷酸水平进一步升高。半乳糖喂养大鼠透镜中糖酵解抑制与NAD+/NADH和腺嘌呤核苷酸磷酸化降低一致。半乳糖血症模型中透镜葡萄糖利用率、NAD-对的氧化还原状态和能量代谢的早期变化与糖尿病相似，至少部分由醛糖还原酶相关机制介导，并且可通过醛糖还原酶抑制剂部分预防。
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 P-i, 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 similar to 57% in 10-day galactose-fed rats heated with the ARI (tolrestat, 100 mg/kg bwt/day, 6-day pretreatment): the changes in the lower segment of glycolysis, alpha-glycerophosphate levels. redux stale 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+/NADH 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.