Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR
Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR
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DOI：
10.1152/ajpendo.00153.2003
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发表时间：
2003-10-01
影响因子：
5.1
通讯作者：
Hutson, SM
中科院分区：
文献类型：
--
作者：
Lynch, CJ;Halle, B;Hutson, SM
亮氨酸已被证明可以刺激体内脂肪组织蛋白质合成，以及在使用新鲜分离的脂肪细胞的体外实验中刺激瘦素分泌、蛋白质合成、增生性生长和组织形态发生。最近，其他人提出，线粒体中的亮氨酸氧化可能需要激活哺乳动物雷帕霉素靶蛋白（mTOR），细胞溶质Ser/Thr蛋白激酶似乎介导这些效应中的一些。亮氨酸氧化的第一个不可逆和限速步骤是由支链α-酮酸脱氢酶（BCKD）复合物催化的。该复合物的活性受丝氨酸293（S293）处E1 α亚基磷酸化的剧烈调节，这使复合物失活。因为亮氨酸的α-酮酸调节BCKD激酶的活性，所以它被认为是亮氨酸调节mTOR的潜在靶标。为了研究BCKD磷酸化的调节及其与mTOR活化的潜在联系，开发并表征了识别该位点的磷酸肽特异性抗体。肝脏中磷酸化S293（pS293）的免疫反应性与饮食诱导的BCKD活性状态的变化密切相关。在通过药物诱导型启动子诱导BCKD激酶后，TREMK-4细胞中的免疫反应性也增加。脂肪组织和腓肠肌（在体内大部分是无活性的）中的BCKD S293磷酸化是相似的。这表明，来自食物剥夺大鼠的附睾脂肪组织中的BCKD复合物大多是无活性的（不能氧化亮氨酸），就像肌肉中的情况一样。为了开始检验mTOR活化的亮氨酸氧化假说，使用pS293抗体比较口服施用的亮氨酸对S6 K1（mTOR底物）和BCKD的急性活化的剂量依赖性作用。亮氨酸剂量的增加与血浆亮氨酸浓度的增加直接相关。脂肪组织中S6 K1的磷酸化（Thr（389），导致活化的磷酸化位点）在使血浆亮氨酸增加约三倍的亮氨酸剂量下最大。BCKD磷酸化状态的改变需要更高的血浆亮氨酸浓度。结果似乎与BCKD和BCKD激酶在亮氨酸代谢/氧化的活化中的作用比在亮氨酸信号至mTOR的活化中的作用更一致。
Leucine has been shown to stimulate adipose tissue protein synthesis in vivo as well as leptin secretion, protein synthesis, hyperplastic growth, and tissue morphogenesis in in vitro experiments using freshly isolated adipocytes. Recently, others have proposed that leucine oxidation in the mitochondria may be required to activate the mammalian target of rapamycin (mTOR), the cytosolic Ser/Thr protein kinase that appears to mediate some of these effects. The first irreversible and rate-limiting step in leucine oxidation is catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1alpha-subunit at Ser293 (S293), which inactivates the complex. Because the alpha-keto acid of leucine regulates the activity of BCKD kinase, it has been suggested as a potential target for leucine regulation of mTOR. To study the regulation of BCKD phosphorylation and its potential link to mTOR activation, a phosphopeptide-specific antibody recognizing this site was developed and characterized. Phospho-S293 (pS293) immunoreactivity in liver corresponded closely to diet-induced changes in BCKD activity state. Immunoreactivity was also increased in TREMK-4 cells after the induction of BCKD kinase by a drug-inducible promoter. BCKD S293 phosphorylations in adipose tissue and gastrocnemius (which is mostly inactive in vivo) were similar. This suggests that BCKD complex in epididymal adipose tissue from food-deprived rats is mostly inactive (unable to oxidize leucine), as is the case in muscle. To begin to test the leucine oxidation hypothesis of mTOR activation, the dose-dependent effects of orally administered leucine on acute activation of S6K1 (an mTOR substrate) and BCKD were compared using the pS293 antibodies. Increasing doses of leucine directly correlated with increases in plasma leucine concentration. Phosphorylation of S6K1 (Thr(389), the phosphorylation site leading to activation) in adipose tissue was maximal at a dose of leucine that increased plasma leucine approximately threefold. Changes in BCKD phosphorylation state required higher plasma leucine concentrations. The results seem more consistent with a role for BCKD and BCKD kinase in the activation of leucine metabolism/oxidation than in the activation of the leucine signal to mTOR.