Feasibility of a mitochondrial pyruvate malate shuttle in pancreatic islets. Further implication of cytosolic NADPH in insulin secretion.
Feasibility of a mitochondrial pyruvate malate shuttle in pancreatic islets. Further implication of cytosolic NADPH in insulin secretion.
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DOI：
10.1074/jbc.270.34.20051
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发表时间：
1995-08
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影响因子：
--
通讯作者：
M. MacDonald
中科院分区：
文献类型：
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作者：
M. MacDonald
以往的研究表明，在胰岛中，葡萄糖衍生的丙酮酸通过羧化进入线粒体代谢的量与通过脱羧化进入线粒体的量大致相同，并且羧化和脱羧化都与葡萄糖代谢的能力和胰岛素的释放有关。相对较高的羧化率与目前研究发现，丙酮酸羧化酶在胰岛中的含量与在肝脏和肾脏中一样丰富。由于胰岛不含磷酸烯醇式丙酮酸羧酸激酶，因此不能由丙酮酸进行糖异生作用，因此该羧基酶可能存在于胰岛中以参与新的无性反应。这个想法最初是通过将各种组织的线粒体与丙酮酸孵育而被探索出来的。线粒体来自组织，如胰岛、肝脏和肾脏，其中丙酮酸羧基酶丰富，输出大量的苹果酸，很少或没有柠檬酸、异柠檬酸和天冬氨酸到介质中。线粒体中的苹果酸含量是培养基中苹果酸含量的1%。当胰岛线粒体与[1-14C]丙酮酸孵育时，放射性碳主要出现在苹果酸中。氨基酸中的放射性很小，柠檬酸和异柠檬酸中几乎没有或几乎没有放射性。丙酮酸的碳1只能通过羧化作用结合到苹果酸和其他柠檬酸循环中间体中，因为当丙酮酸通过丙酮酸脱氢酶反应以乙酰辅酶A的形式进入柠檬酸循环时，这种碳会通过脱羧基丢失。苹果酸的生成量等于14CO2的生成量，从苹果酸中回收的丙酮酸C-1的放射性略超过14CO2的生成量，这与我们之前报道的完整胰岛中丙酮酸的高羧化率的研究是一致的。当完整的胰岛与[U-14C]琥珀酸甲酯作为四碳二元酸的线粒体源孵育时，丙酮酸和乳酸中出现放射性。结合前人的研究，目前的结果表明，在葡萄糖诱导的胰岛素分泌过程中，存在一个跨越线粒体膜的梭形结构，其中葡萄糖衍生的丙酮酸被线粒体摄取，并通过丙酮酸羧基酶被羧化为草酰乙酸酯。草酰乙酸酯被转化为苹果酸，苹果酸离开线粒体，在胞浆中，在苹果酸酶催化的反应中，草酰乙酸酯被脱羧为丙酮酸。这种丙酮酸重新进入线粒体池。这样的循环在细胞质中产生NADPH。由于它是一个周期，这种穿梭方式可以产生比磷酸戊糖途径多得多的NADPH，后者是已知的胰岛葡萄糖代谢的一条非常微小的途径。
Previous studies indicated that in pancreatic islets the amount of glucose-derived pyruvate that enters mitochondrial metabolism via carboxylation is approximately equal to that entering via decarboxylation and that both carboxylation and decarboxylation are correlated with capacitation of glucose metabolism and insulin release. The relatively high rate of carboxylation is consistent with the current study's finding that pyruvate carboxylase is as abundant in pancreatic islets as it is in liver and kidney. Since islets do not contain phosphoenolpyruvate carboxykinase and, therefore, cannot carry out glyconeogenesis from pyruvate, the carboxylase might be present in the islet to participate in novel anaplerotic reactions. This idea was first explored by incubating mitochondria from various tissues with pyruvate. Mitochondria from tissues, such as pancreatic islets, liver, and kidney, in which pyruvate carboxylase is abundant, exported a large amount of malate and little or no citrate, isocitrate, and aspartate to the medium. The amount of malate within the mitochondria was < 1% that in the medium. When pancreatic islet mitochondria were incubated with [1-14C]pyruvate, radioactive carbon appeared in the medium primarily in malate. Very little radioactivity appeared in amino acids, and little or no radioactivity appeared in citrate and isocitrate. Carbon 1 of pyruvate can be incorporated into malate and other citric acid cycle intermediates only via carboxylation, as this carbon would be lost via decarboxylation when pyruvate enters the citric acid cycle as acetyl-CoA via the pyruvate dehydrogenase reaction. The amount of malate formed equaled the 14CO2 formed and the radioactivity from C-1 of pyruvate recovered in malate slightly exceeded the formation of 14CO2 in agreement with our previous studies that reported a high rate of carboxylation of pyruvate in intact islets. When intact pancreatic islets were incubated with methyl [U-14C]succinate as a mitochondrial source of four-carbon dicarboxylic acids, radioactivity appeared in pyruvate and lactate. Taken together with previous studies, the current results suggest that during glucose-induced insulin secretion there is a shuttle operating across the mitochondrial membrane in which glucose-derived pyruvate is taken up by mitochondria and carboxylated to oxaloacetate by pyruvate carboxylase. The oxaloacetate is converted to malate which exits the mitochondrion, where, in the cytosol, it is decarboxylated to pyruvate in the reaction catalyzed by malic enzyme. This pyruvate re-enters mitochondrial pools. Such a cycle produces NADPH in the cytosol. Since it is a cycle, this shuttle can produce far more NADPH than the pentose phosphate pathway, which is known to be a very minor route of glucose metabolism in the islet.(ABSTRACT TRUNCATED AT 400 WORDS)