REGULATORY ROLE OF REDUCING-EQUIVALENT TRANSFER FROM SUBSTRATE TO OXYGEN IN HEPATIC METABOLISM OF GLYCEROL AND SORBITOL
REGULATORY ROLE OF REDUCING-EQUIVALENT TRANSFER FROM SUBSTRATE TO OXYGEN IN HEPATIC METABOLISM OF GLYCEROL AND SORBITOL
复制标题
DOI：
10.1111/j.1432-1033.1973.tb02697.x
复制
发表时间：
1973-01-01
期刊：
影响因子：
--
通讯作者：
WERNER, HV
中科院分区：
文献类型：
--
作者：
BERRY, MN;KUN, E;WERNER, HV
形态学完整的分离大鼠肝细胞悬浮液与特定抑制剂一起用于鉴定和定量参与山梨醇或甘油还原等效物向O2转移的肝氢反式定位系统。氢的易位速率可以通过测量底物代谢的主要产物或底物利用率来确定。结果发现，在饱和底物浓度下，山梨醇或甘油3 -磷酸的氧化速率非常相似（约1.8 μmol × g湿重- 1× min - 1）。山梨醇的吸收速率与甘油的吸收速率呈反比关系，因此底物混合物中氢通量到o2的速率不大于单独添加任何一种底物的速率。丙酮酸作为细胞质的氢受体，增加了山梨醇和甘油的消耗速率。从这些观察中可以得出结论，在高底物水平和缺乏细胞质氢受体的情况下，氢易位是山梨醇和甘油肝脏代谢的限速过程，并且这两种底物还原成o2的通量涉及共享的氢易位系统。在底物含量较低的情况下，山梨醇或甘油的代谢速率也取决于底物浓度，但即使在这种情况下，研究发现，只要存在底物混合物，氢转运系统的能力就会控制整个代谢速率。这些系统的性质是通过使用特定抑制剂来评估的。大约15%的o2还原当量通量涉及抗霉素不敏感途径，可能是微粒体。另外40%通过鱼藤酮不敏感途径传递给o2，最有可能涉及黄素相关的线粒体甘油磷酸脱氢酶。其余的通量对鱼藤酮敏感，但其中不到一半利用苹果酸-草酰乙酸或苹果酸-天冬氨酸穿梭体。残留鱼藤酮敏感部分（约占总通量的20-30%）的途径仍有待澄清。这些数据表明，在正常大鼠肝脏实质细胞中，甘油- 3 -磷酸穿梭对于将还原性等价物从细胞质转移到线粒体可能比之前认识到的更重要。解偶联剂可抑制细胞对山梨糖醇的摄取达70%。这种抑制可以通过添加丙酮酸作为细胞质氢受体或人工电子受体来克服。这表明解偶联剂通过干扰细胞质和线粒体之间正常的氢穿梭体的运作来阻止细胞质NADH的氧化，而这些穿梭体是能量依赖性的。这些研究揭示了氢转运系统的速率限制和能量依赖性质，确定它们是代谢调节的潜在位点和激素作用的可能靶点。
Suspensions of morphologically intact isolated rat‐liver cells were used in conjunction with specific inhibitors to identify and quantitate the hepatic hy drogen‐trans ocating systems involved in the transfer of reducing‐equivalents from sorbitol or glycerol to O2. Rates of hydrogen translocation were derived either from measurement of the major products of substrate metabolism or from rates of substrate utilization. It was found that at saturating substrate concentrations, rates of sorbitol or glycerol 3‐phosphate oxidation were closely similar (about 1.8 μmol × g wet weight−1× min−1). There was an inverse relationship between rates of sorbitol and glycerol uptake so that the rate of hydrogen flux to O2from substrate mixtures was no greater than that from either substrate added separately. Rates of sorbitol and glycerol consumption were increased by pyruvate acting as a cytoplasmic hydrogen acceptor.It is concluded from these observations that at high substrate levels and in the absence of a cytoplasmic hydrogen acceptor, hydrogen translocation is the rate‐limiting process in the hepatic metabolism of sorbitol and glycerol and that the flux of reducing equivalents to O2from these two substrates involves shared hydrogen‐translocating systems. At low levels of substrate, more likely to be encounteredin vivo, the rate of sorbitol or glycerol metabolism is dependent also on substrate concentration, but even under these circumstances it was found that the capacity of the hydrogen‐translocating systems governs the over‐all rate of metabolism whenever substrate mixtures were present.The nature of these systems was assessed by the use of specific inhibitors. About 15% of the flux of reducing equivalents to O2involved antimycin‐insensitive pathways, presumably microsomal. A further 40% passed to O2by a rotenone‐insensitive path, most likely involving flavin‐linked mitochondrial glycerolphosphate dehydrogenase. The remainder of the flux was rotenone‐sensitive, but less than half of this utilized malate‐oxaloacetate or malate‐aspartate shuttles. The pathway for this residual rotenone‐sensitive fraction (about 20–30% of the total flux) remains to be clarified. These data suggest that in parenchymal cells from normal rat liver the glycerol 3‐phosphate shuttle may be more important for the transfer of reducing equivalents from cytoplasm to mitochondria than has been previously recognized.Sorbitol uptake by the cells was inhibited up to 70% by uncoupling agents. This inhibition could be overcome by addition of pyruvate as a cytoplasmic hydrogen acceptor or by artificial electron acceptors. This implies that uncoupling agents prevent the oxidation of cytoplasmic NADH by interfering with the operation of the normal hydrogen shuttles between cytoplasm and mitochondria and that these shuttles are energy‐dependent.The rate‐limiting and energy‐dependent nature of the hydrogen translocating systems as revealed by these studies identify them as potential sites for metabolic regulation and as possible targets for hormonal action.