Wood John P M, Chidlow Glyn, Graham Mark, Osborne Neville N
Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom.
Invest Ophthalmol Vis Sci. 2004 Apr;45(4):1272-80. doi: 10.1167/iovs.03-0693.
To determine the metabolic conditions that provide maintenance of viability for cultured rat RPE cells and to determine whether monocarboxylates such as lactate or pyruvate, which are known to exist at high concentrations in the subretinal space, can provide an alternative energy source to maintain cells when other nutritive supplies are limited.
Cultured rat RPE cells (passage 2-4), in the absence of serum, were subjected to different metabolic challenges relating to glucose, amino acid, or oxygen deprivation. Lactate or pyruvate was added to some cells in each instance to determine whether cells could be maintained by using these substances as fuel sources for metabolic reactions. Cell viability was assessed after treatments, and in some cases proliferation rates and appearance of apoptosis-like DNA cleavage were also investigated by terminal deoxynucleotidyl-transferase UTP-linked nick-end labeling (TUNEL). Western blot analysis was used to determine the expression of transporters for glucose and monocarboxylates in these cells.
RPE cell viability was partially reduced in the absence of glucose or with glycolytic inhibition. Lactate or pyruvate did not prevent these reductions. Inhibition of transaminase reactions with aminooxyacetic acid (AOAA) in the absence of glucose caused a complete loss of viability that was reversed by pyruvate or lactate. MCT inhibition was detrimental to RPE cell viability only at high concentrations (500 microM) in the presence of glucose but blocked the protective effect of pyruvate-lactate in the presence of AOAA at 1 microM.
Rat RPE cells require glucose as their primary metabolic substrate in culture, but can metabolize glutamine in its absence. When glucose and glutamine are limiting, RPE cells can metabolize monocarboxylates such as lactate or pyruvate. These data provide evidence that such cells are able to withstand various types of insult brought about by nutrient deprivation, by altering their pathways of energy production.
确定能维持培养的大鼠视网膜色素上皮（RPE）细胞活力的代谢条件，并确定诸如乳酸或丙酮酸等单羧酸盐（已知在视网膜下间隙中高浓度存在）在其他营养供应有限时是否能提供替代能源以维持细胞存活。
在无血清条件下，对培养的大鼠RPE细胞（传代2 - 4代）进行与葡萄糖、氨基酸或氧剥夺相关的不同代谢挑战。在每种情况下，向一些细胞中添加乳酸或丙酮酸，以确定细胞是否可以利用这些物质作为代谢反应的燃料来源来维持存活。处理后评估细胞活力，在某些情况下，还通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）研究增殖率和凋亡样DNA裂解的出现情况。采用蛋白质免疫印迹分析来确定这些细胞中葡萄糖和单羧酸盐转运体的表达。
在无葡萄糖或糖酵解受抑制的情况下，RPE细胞活力部分降低。乳酸或丙酮酸不能阻止这些降低。在无葡萄糖时用氨基氧乙酸（AOAA）抑制转氨酶反应会导致细胞活力完全丧失，而丙酮酸或乳酸可使其恢复。单羧酸转运体（MCT）抑制仅在存在葡萄糖时高浓度（500微摩尔）下对RPE细胞活力有害，但在存在1微摩尔AOAA时会阻断丙酮酸 - 乳酸的保护作用。
大鼠RPE细胞在培养中需要葡萄糖作为其主要代谢底物，但在无葡萄糖时可以代谢谷氨酰胺。当葡萄糖和谷氨酰胺受限，RPE细胞可以代谢诸如乳酸或丙酮酸等单羧酸盐。这些数据提供了证据，表明此类细胞能够通过改变其能量产生途径来承受营养剥夺带来的各种类型的损伤。
