Inhibition of contraction-stimulated AMP-activated protein kinase inhibits contraction-stimulated increases in PAS-TBC1D1 and glucose transport without altering PAS-AS160 in rat skeletal muscle.
Inhibition of contraction-stimulated AMP-activated protein kinase inhibits contraction-stimulated increases in PAS-TBC1D1 and glucose transport without altering PAS-AS160 in rat skeletal muscle.
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作者：
Funai K;Cartee GD
TBC 1结构域蛋白家族的两个成员，160 kDa的Akt底物（AS 160，也称为TBC 1D 4）和TBC 1D 1的磷酸化与骨骼肌中葡萄糖转运的调节有关。胰岛素刺激的AS 160和TBC 1D 1磷酸化（使用磷酸化Akt底物[PAS]抗体测量）似乎以Akt依赖性方式发生，但负责收缩刺激的PAS-AS 160和PAS-TBC 1D 1的激酶仍不清楚。AMP活化蛋白激酶（AMPK）和Akt都是通过收缩激活的，它们可以在无细胞测定中分别磷酸化AS 160和TBC 1D 1。为了评价AMPK和Akt对胰岛素或收缩刺激的PAS-AS 160、PAS-TBC 1D 1和葡萄糖转运的作用，在胰岛素刺激或收缩之前和期间，将大鼠上滑车与化合物C（AMPK抑制剂）或Wortmannin（磷脂酰肌醇[PI] 3-激酶抑制剂，Akt上游）一起孵育。Wortmannin完全抑制胰岛素刺激的葡萄糖转运和AS 160和TBC 1D 1的磷酸化。Wortmannin消除了磷酸-Ser 21/9糖原合成酶激酶3α/β（pGSK 3; Akt底物）和PAS-AS 160的收缩刺激，但未显著改变收缩刺激肌肉中的pAMPK、磷酸-Ser 79乙酰CoA羧化酶（pACC; AMPK底物）、PAS-TBC 1D 1或葡萄糖转运。化合物C完全抑制收缩刺激的pACC和PAS-TBC 1D 1，并部分阻断葡萄糖转运，但它没有显著改变pAkt、pGSK 3或PAS-AS 160。这些数据表明，1）胰岛素以PI 3-激酶/Akt依赖性方式刺激葡萄糖转运和AS 160和TBC 1D 1的磷酸化，2）收缩以PI 3-激酶/Akt依赖性方式刺激PAS-AS 160（但不刺激PAS-TBC 1D 1或葡萄糖转运），3）收缩以AMPK依赖性方式刺激PAS-TBC 1D 1和葡萄糖转运（但不刺激PAS-AS 160）。
Phosphorylation of two members of the TBC1 domain family of proteins, Akt substrate of 160 kDa (AS160, also known as TBC1D4) and TBC1D1, has been implicated in the regulation of glucose transport in skeletal muscle. Insulin-stimulated phosphorylation (measured using the phospho-Akt substrate [PAS] antibody) of AS160 and TBC1D1 appears to occur in an Akt-dependent manner, but the kinases responsible for contraction-stimulated PAS-AS160 and PAS-TBC1D1 remain unclear. AMP-activated protein kinase (AMPK) and Akt, both activated by contraction, can each phosphorylate AS160 and TBC1D1 in cell-free assays. To evaluate the roles of AMPK and Akt on insulin- or contraction-stimulated PAS-AS160, PAS-TBC1D1, and glucose transport, rat epitrochlearis was incubated with and without compound C (inhibitor of AMPK) or Wortmannin (inhibitor of phosphatidylinositol [PI] 3-kinase, which is upstream of Akt) before and during insulin stimulation or contraction. Insulin-stimulated glucose transport and phosphorylation of both AS160 and TBC1D1 were completely inhibited by Wortmannin. Wortmannin eliminated contraction stimulation of phospho-Ser21/9glycogen synthase kinase 3α/β (pGSK3; Akt substrate) and PAS-AS160 but did not significantly alter pAMPK, phospho-Ser79acetyl CoA carboxylase (pACC; AMPK substrate), PAS-TBC1D1, or glucose transport in contraction-stimulated muscle. Compound C completely inhibited contraction-stimulated pACC and PAS-TBC1D1 and partially blocked glucose transport, but it did not significantly alter pAkt, pGSK3, or PAS-AS160. These data suggest that 1) insulin stimulates glucose transport and phosphorylation of AS160 and TBC1D1 in a PI 3-kinase/Akt–dependent manner, 2) contraction stimulates PAS-AS160 (but not PAS-TBC1D1 or glucose transport) in a PI 3-kinase/Akt–dependent manner, and 3) contraction stimulates PAS-TBC1D1 and glucose transport (but not PAS-AS160) in an AMPK-dependent manner.