生长因子、能量状态、氨基酸和机械刺激对 mTORC1的调节作用

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Regulation of mTORC1 by growth factors, energy status, amino acids and mechanical stimuli at a glance

Abstract

摘要

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) plays a pivotal role in the regulation of skeletal muscle protein synthesis. Activation of the complex leads to phosphorylation of two important sets of substrates, namely eIF4E binding proteins and ribosomal S6 kinases. Phosphorylation of these substrates then leads to an increase in protein synthesis, mainly by enhancing translation initiation. mTORC1 activity is regulated by several inputs, such as growth factors, energy status, amino acids and mechanical stimuli. Research in this field is rapidly evolving and unraveling how these inputs regulate the complex. Therefore this review attempts to provide a brief and up-to-date narrative on the regulation of this marvelous protein complex. Additionally, some sports supplements which have been shown to regulate mTORC1 activity are discussed.

机械/哺乳动物靶雷帕霉素复合物1(mTORC1)在骨骼肌蛋白质合成的调控中起着关键作用。复合体的激活导致两组重要底物的磷酸化,即 eIF4E 结合蛋白和核糖体 S6激酶。这些底物的磷酸化会导致蛋白质合成的增加,主要是通过加强翻译起始。mTORC1活性受生长因子、能量状态、氨基酸和机械刺激等多种输入信号的调控。在这个领域的研究正在迅速发展和阐明这些输入如何调节复合体。因此,本综述试图提供一个简短的和最新的叙述调节这一奇妙的蛋白质复合体。此外,还讨论了一些已被证明能调节 mTORC1活性的运动补充剂。

Background

背景

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) has emerged as a key factor in regulation of skeletal muscle protein synthesis (MPS) [1]. mTORC1 is a protein complex comprised of the three core subunits mTOR, Raptor and mLST8 [2] and is regulated by several inputs, such as growth factors, energy status, amino acids and mechanical stimuli. mTOR forms the catalytic center of the two signaling complexes mTORC1 and mTORC2 [3], of which the first is primarily involved in regulation of MPS. Activation of the complex leads to phosphorylation of its two important sets of substrates which are involved in the translation of mRNA to protein. One comprising the eukaryotic initiation factor 4E (eIF4E)-binding proteins 4E-BP1 and 2. 4E-BPs inhibit the formation of the eIF4F complex which facilitates recruitment of the small (40S) ribosomal subunit to the 5’ end of mRNA [4]. Therefore, 4E-BPs inhibit mRNA translation initiation and phosphorylation by mTORC1 relieves this inhibition. The other important set of substrates of mTORC1 comprise the ribosomal S6 kinases S6K1 and 2. Phosphorylation of the S6Ks by mTORC1 activates them and resultingly modulates functions of translation initiation factors [5]. Additionally, S6Ks are thought to promote ribosome biogenesis and thereby increasing the translational capacity of the cell [6].

机械性/哺乳动物靶雷帕霉素复合物1(mTORC1)已成为调节骨骼肌蛋白质合成(MPS)的关键因子[1]。mTORC1是由 mTOR、 Raptor 和 mLST8[2]三个核心亚基组成的蛋白质复合体,它受到生长因子、能量状态、氨基酸和机械刺激等多种输入信号的调控。mTOR 是 mTORC1和 mTORC2[3]两个信号复合物的催化中心,其中 mTORC1和 mTORC2[3]主要参与 MPS 的调节。复合体的激活导致其两组重要底物的磷酸化,这两组底物参与了 mRNA 到蛋白质的翻译。其中一个包含真核起始因子结合蛋白4E-bp1和2。4E-BPs 抑制 eIF4F 复合物的形成,该复合物促进小的(40S)核糖体亚单位的补充到 mRNA [4]的5’端。因此,4E-BPs 抑制 mTORC1的 mRNA 翻译起始和磷酸化可以解除这种抑制作用。mTORC1的另一组重要底物包括核糖体 S6激酶 S6K1和2。被 mTORC1磷酸化的 S6Ks 激活它们并且结果调节翻译起始因子的功能[5]。此外,S6Ks 被认为促进核糖体的生物发生,从而提高细胞的翻译能力[6]。

This manuscript attempts to provide a brief and up-to-date narrative of some important factors which regulate mTORC1 activity at the cellular level. Additionally, some sports supplements which have been shown to regulate mTORC1 activity are discussed.

这份手稿试图提供一个简短的和最新的叙述一些重要因素调节 mTORC1活动在细胞水平。此外,还讨论了一些已被证明能调节 mTORC1活性的运动补充剂。

Regulation by growth factors

增长因素的调控

Research examining the regulation of mTORC1 by growth factors has mainly focused on the effect of insulin and insulin-like growth factor-1. The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) both belong to the class of tyrosine kinase receptors. Activation of either receptor leads to phosphorylation of the insulin receptor substrates (IRS) proteins. This, in turn, exposes binding sites on these proteins which enable interaction with other proteins which contain a Src Homology 2 (SH2) domain. Among the SH2 domain-containing proteins is phosphatidylinositol-3-kinase (PI3K). IRS activates PI3K by associating with the SH2 domain of the kinase [7]. Activated PI3K then phosphorylates inositol phospholipids embedded in the plasma membrane on a hydroxyl group located at carbon 3. This gives rise to phosphoinositides, such as phosphatidylinositol (3,4,5)-triphosphate (PIP3). PIP3 interacts with Pleckstrin homology (PH) domain-containing proteins, thereby recruiting these to the plasma membrane. Two important PH domain-containing proteins are 3-phosphoinositide dependent protein kinase (PDK1) and Akt. The interaction of PIP3 with Akt enhances phosphorylation (and thereby activation) of the latter. Additionally, interaction of PIP3 with PDK1 leads to phosphorylation of Akt by PDK1 (Fig. 1).

生长因子调节 mTORC1的研究主要集中在胰岛素和胰岛素样生长因子 -1的作用上。胰岛素受体和胰岛素样生长因子1受体都属于酪氨酸激酶受体。激活任一受体导致磷酸化的胰岛素受体底物(IRS)蛋白质。这反过来暴露了这些蛋白质的结合位点,使其能够与包含 Src 同源2(SH2)结构域的其他蛋白质相互作用。其中包含 SH2结构域的蛋白质是磷脂酰肌醇 -3- 激酶(PI3K)。IRS 通过与激酶的 SH2结构域相联系来激活 PI3K [7]。激活 PI3K,然后磷酸化肌醇磷脂嵌入在质膜上的位于碳3的羟基。这会产生磷脂酰肌醇,例如磷脂酰肌醇(3,4,5)-三磷酸(PIP3)。PIP3与 Pleckstrin 同源(PH)结构域含有的蛋白质相互作用,从而使这些蛋白质进入质膜。两种重要的 PH 结构域蛋白是3- 磷酸肌醇依赖性蛋白激酶(PDK1)和 Akt。PIP3与 Akt 的相互作用增强了后者的磷酸化(从而激活)。此外,PIP3与 PDK1的相互作用导致 PDK1磷酸化 Akt (图1)。

figure1
Fig. 1 图一

Akt is considered an important upstream regulator of mTORC1 [8]. The Akt family of proteins comprises the three isoforms Akt1, Akt2 and Akt3. Akt1 and Akt2 are expressed in skeletal muscle, while Akt3 is not [9]. PDK1 phosphorylates Akt1 and Akt2 at residues Thr308 and Thr309, respectively. However, full Akt kinase activity also requires phosphorylation at a serine residue [1011], Ser473 and Ser473 on Akt1 and Akt2, respectively. The Rictor-containing mTOR complex mTORC2 is possibly the kinase responsible for phosphorylation of the serine residue [12]. Mechanistic studies commonly measure the phosphorylation status of Akt1 at residues Thr308 and Ser473 in order to assess Akt activity.

Akt 被认为是 mTORC1的重要上游调节因子。Akt 家族的蛋白质包括三种亚型 Akt1、 Akt2和 Akt3。Akt1和 Akt2在骨骼肌中表达,而 Akt3不表达[9]。PDK1分别在 Thr308和 Thr309的残基上磷酸化 Akt1和 Akt2。然而,完整的 Akt 激酶活性也需要丝氨酸残基[10,11]、丝氨酸473和丝氨酸473分别在 Akt1和 Akt2上磷酸化。含有蓖麻毒素的 mTOR 复合物 mTORC2可能是丝氨酸残基磷酸化的激酶[12]。机械研究通常测量 Akt1在 Thr308和 Ser473残基处的磷酸化状态,以评估 Akt 活性。

Myostatin, a potent negative regulator of skeletal muscle growth [13], has also been found to regulate Akt phosphorylation [14]. Myostatin is a member of the transforming growth factor- β superfamily and a ligand for activin type II receptors (ActRIIA and ActRIIB). After binding to its receptor, it phosphorylates and activates activin type I receptors [15]. These receptors then phosphorylate and activate the transcription factors Smad2 and Smad3 which then form a heterotrimeric complex by joining with Smad4. After formation, the complex can translocate to the nucleus where it regulates several key genes involved in skeletal muscle growth. Knockout of myostatin in animal models has been found to dramatically increase skeletal muscle fiber size and number [1618]. In postnatal skeletal muscle, inhibition of myostatin signaling mainly affects fiber size rather than number [1920]. Importantly, incubation of human myoblasts with myostatin has been found to reduce Akt phosphorylation at residue Ser473 by 50 % [14]. The reduction of Akt phosphorylation by myostatin might underlie its inhibiting effect on muscle hypertrophy. Recently, researchers discovered that this effect is mediated via the microRNA miR-486 [21]. miR-486 increases Akt phosphorylation, likely by inhibiting phosphatase and tensin homolog (PTEN), a protein which opposes the action of PI3K by dephosphorylating PIP3 to PIP2 [22]. Myostatin negatively regulates the expression of miR-486 at the transcriptional level and therefore inhibits Akt phosphorylation mediated by PI3K.

肌肉生长抑制素,一个强有力的骨骼肌生长负调节剂[13] ,也被发现调节 Akt 磷酸化[14]。肌肉生长抑制素是转化生长因子-β 超家族的成员,是激活素 II 型受体(ActRIIA 和 ActRIIB)的配体。在与其受体结合之后,它磷酸化并激活 i 型激活素受体[15]。这些受体然后磷酸化并激活转录因子 Smad2和 Smad3,通过与 Smad4结合形成异三聚体复合体。形成后,复合体可以转移到细胞核,在那里它调节骨骼肌生长的几个关键基因。在动物模型中,肌肉生长抑制素基因敲除已被发现显著增加骨骼肌纤维的大小和数量[16-18]。在出生后的骨骼肌,抑制肌肉生长抑制素信号主要影响纤维大小而不是数量[19,20]。重要的是,人类成肌细胞与肌肉生长抑制素的孵育作用已被发现可以减少 Akt 在残基 Ser473磷酸化50% [14]。肌肉生长抑制素对 Akt 磷酸化的减少可能是其抑制肌肉肥大的作用的基础。最近,研究人员发现这种效应是通过 microRNA miR-486介导的。miR-486增加 Akt 磷酸化,可能是通过抑制同源性磷酸酶-张力蛋白(PTEN) ,一种通过去磷酸化 PIP3到 PIP2而反对 PI3K 作用的蛋白质。肌肉生长抑制素负性调节 miR-486的表达在转录水平,因此抑制 Akt 磷酸化介导的 PI3K。

After Akt is activated it phosphorylates several other proteins. The best researched substrates of Akt are glycogen synthase kinase 3 β (GSK3 β) [23], proline-rich Akt substrate of 40 KDa (PRAS40) [24], tuberous sclerosis complex 2 (TSC2) [25] and forkhead box class O (FoxO) proteins [26]. Both TSC2 and PRAS40 act as negative regulators of mTORC1. TSC2 forms a protein complex with TSC1 and the recently discovered protein TBC1D7 [27]. When the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is formed, it inhibits mTORC1 activity by means of its GTPase-activating protein (GAP) domain [2728]. GTP-bound Rheb proteins (Rheb-GTP) activate mTORC1 at the lysosomal membrane [29]. The mechanism for this activation is currently unknown although interaction with the mTOR kinase domain appears to be involved [29]. By virtue of its GAP domain, the TSC-TBC complex can thus regulate the amount of Rheb-GTP and therefore mTORC1 activity. Akt phosphorylates TSC2 at multiple sites (Ser939, Ser981, Ser1130, Ser1132 and Thr1462) in order to inhibit the GAP activity of the TSC-TBC complex towards Rheb-GTP, possibly by dissociating the complex from the lysosome [30]. Moreover, it should be noted that the TSC-TBC complex has the highest affinity for Rheb-GDP rather than Rheb-GTP [30]. This might suggest a mechanism in which the complex acts to prevent the exchange of GDP for GTP in order to keep the Rheb proteins from reloading GTP. Akt further acts by relieving mTORC1 of the inhibition imposed by PRAS40. PRAS40 binds to the mTORC1 subunit Raptor, thereby inhibiting its association with substrates. PRAS40 is phosphorylated at one threonine residue (Thr246) and two serine residues (Ser181 and Ser221) [31]. The threonine residue is phosphorylated by Akt, whereas the serine residues appear to be phosphorylated by mTORC1.

Akt 被激活后,它可以磷酸化其他几种蛋白质。Akt 的最佳底物是糖原合成酶激酶3 β (GSK3 β)[23] ,富含脯氨酸的 Akt 底物40 KDa (PRAS40)[24] ,结节性硬化症2(TSC2)[25]和叉头盒级 o (FoxO)蛋白[26]。TSC2和 PRAS40都是 mTORC1的负调节剂。TSC2与 TSC1和最近发现的蛋白 TBC1D7形成蛋白复合物[27]。TSC1-TSC2-TBC1D7(TSC-TBC)复合物形成后,通过其 GTPase-activating protein (GAP)结构域抑制 mTORC1活性[27,28]。结合谷胱甘肽的 Rheb 蛋白(Rheb-GTP)激活溶酶体膜上的 mTORC1[29]。这种激活的机制目前还不清楚,虽然与 mTOR 激酶结构域的相互作用似乎涉及[29]。TSC-TBC 复合物凭借其 GAP 结构域,可以调节 Rheb-GTP 的量,从而调节 mTORC1的活性。Akt 在多个位点(Ser939,Ser981,Ser1130,Ser1132和 Thr1462)磷酸化 TSC2,以抑制 TSC-TBC 复合物对 Rheb-GTP 的 GAP 活性,可能是通过从溶酶体[30]中解离该复合物来实现的。此外,应该注意的是 TSC-TBC 复合物对 Rheb-GDP 的亲和力最高,而不是 Rheb-GTP [30]。这可能暗示了一个机制,在这个机制中,复杂的行为阻止 GTP 的 GDP 交换,以防止 Rheb 蛋白重新载入 GTP。Akt 通过减轻 mTORC1对 PRAS40的抑制作用而进一步发挥作用。PRAS40与 mTORC1亚基 Raptor 结合,从而抑制其与底物的结合。在一个苏氨酸残基(th246)和两个丝氨酸残基(Ser181和 Ser221)上磷酸化 PRAS40。苏氨酸残基被 Akt 磷酸化,而丝氨酸残基似乎被 mTORC1磷酸化。

GSK3 β is a negative regulator of the Wnt/ β-catenin signaling pathway as it forms a complex with other proteins and phosphorylates β-catenin leading to degradation of the molecule [32]. Akt phosphorylates GSK3 β, which inactivates the enzyme and thereby stimulates Wnt/ β-catenin signaling through removal of its inhibiton. β-catenin seems to play an important role in skeletal muscle hypertrophy by functioning as a transcription factor [33] and inhibition of GSK3 β stimulates hypertrophy in C2C12 myotubes [34]. The kinase has also been found to inhibit mRNA translation by blocking the GDP-GTP exhange of eIF2B [35] which is required to form a functional ternary complex for translation initiation [36].

GSK3 β 是 Wnt/β- 连环蛋白信号通路的负调节因子,与其他蛋白形成复合物,磷酸化 β- 连环蛋白导致分子降解[32]。Akt 磷酸化 GSK3 β,使酶失活,通过去除其抑制因子刺激 Wnt/β-catenin 信号转导。β 连环蛋白似乎在骨骼肌肉肥大中扮演重要角色,其功能是作为一个转录因子蛋白[33] ,抑制 GSK3 β 刺激 C2C12肌管肥大[34]。此外,还发现该激酶通过阻断 eIF2B [35]的 GDP-GTP 外显子而抑制 mRNA 的翻译,该外显子是形成翻译起始功能性三元复合物[36]所必需的。

Besides the regulation of anabolic processes through inhibition of GSK3 β, PRAS40 and TSC2 activity, Akt is also closely involved in inhibiting protein breakdown by modulating the activity of the FoxO family of proteins. FoxO proteins are key regulators of protein breakdown modulating ubiquitin-proteasome, as well as autophagy-lysosomal proteolytic pathways [37]. Especially the first seems important in muscle protein breakdown and two E3 ubiquitin ligases, muscle atrophy F-box (MAFbx/atrogin-1) and muscle ring finger 1 (MuRF1) [3839], appear to be the two main downstream effectors of FoxO signaling affecting protein breakdown. FoxO proteins are phosphorylated, and thereby inhibited, by Akt [40].

Akt 除了通过抑制 GSK3 β、 PRAS40和 TSC2活性来调节蛋白合成过程外,还通过调节 FoxO 家族蛋白的活性来抑制蛋白质分解。FoxO 蛋白是蛋白质分解的关键调节因子,调节泛素-蛋白酶体,以及自噬-溶酶体蛋白水解途径[37]。尤其是在肌肉蛋白质分解和两种 E3泛素连接酶中,肌肉萎缩的 F-box (MAFbx/atrogin-1)和肌肉环指1(MuRF1)[38,39]似乎是影响蛋白质分解的两个主要的下游效应因子。FoxO 蛋白被 Akt [40]磷酸化,从而被抑制。

Aside from the regulation of Akt by insulin and IGF-I, some studies [4146], but not all [4750], suggest androgens also increase Akt phosphorylation. The large heterogeneity across these studies, such as differences in experimental animal models, differences in the type of androgen used as well as its dosage, timepoint of measurement, among others, might explain why some studies did not find an increase in Akt phosphorylation. Interestingly, one study examining the rapid effects of testosterone in cultured rat myotubes directly implicates the PI3K/Akt/mTORC1 pathway as a mediator of androgens’ effect on contractile protein synthesis [46]. Basualto-Alarcón et al., incubated the myotubes with testosterone (100 nM) and performed measurements of total Akt and phosphorylated Akt (at Ser473) 1, 5, 15, 30 and 60 m after incubation. Measurements of α-actin mRNA and protein were taken 6 and 12 h after incubation with testosterone and both were significantly increased, thus indicating an increase in contractile protein synthesis. Indeed, the cross-sectional area (CSA) was significantly increased after 12 h. Moreover, Akt phosphorylation was increased 15 m after incubation. When the authors inhibited PI3K, Akt or mTOR the effect on α-actin was blocked. As such, it appears likely that androgens exert rapid effects by activation of the PI3K/Akt/mTOR pathway. Given that PI3K operates at the cell membrane and that the effect on Akt phosphorylation occured rapidly (after 15 m), it appears highly likely that a cell membrane-localized receptor is involved. Indeed, multiple lines of evidence implicate a cell membrane-localized receptor in the rapid effects of androgens [51]. The G-protein coupled receptor (GPCR) GPRC6A has been shown to mediate a rapid signaling response, including involvement of PI3K, to testosterone [52]. In the experiment by Basualto-Alarcón et al., the addition of the androgen receptor (AR) antagonist bicalutamide blocked the increase in CSA, despite an increase in α-actin protein level. This indicates crosstalk between the intracellular AR and the PI3K pathway activated by testosterone. Strikingly, the intracellular AR has been shown to interact with the p85 α regulatory subunit of PI3K in androgen-sensitive epithelial cells, enhancing its activity [53]. However, the addition of bicalutamide to these androgen-sensitive epithelial cells blocked the androgen-induced Akt phosphorylation. This is in contrast with the experiment by Basualto-Alarcón et al., which showed that inhibition of Akt phosphorylation blocked the increase in α-actin protein level, whereas bicalutamide did not affect α-actin protein level, thus suggesting that bicalutamide did not inhibit Akt phosphorylation in this experiment. If bicalutamide also affected Akt phosphorylation in the experiment by Basualto-Alarcón et al., this should therefore be observed in α-actin protein level, but it remained unaltered by addition of bicalutamide. This difference between both studies might be due to the differences in cell lines and AR ligands used. Nevertheless, AR-PI3K crosstalk might, partly, underlie the absence of an increase in CSA with the addition of bicalutamide in the experiment by Basualto-Alarcón et al., despite an increase in α-actin. Additionally, activation of the PI3K/Akt pathway can, in turn, regulate AR activity, since Akt has been shown to post-translationally modify the AR by phosphorylation [54]. Further research might further elucidate the mechanisms through which the cell membrane-localized and intracellular AR regulate mTORC1 activity.

除了通过胰岛素和胰岛素样生长因子 i 调节 Akt 外,一些研究[41-46] ,但不是全部[47-50] ,提示雄激素也增加 Akt 磷酸化。这些研究的巨大差异性,比如实验动物模型的差异,雄激素的使用类型以及剂量的差异,测量的时间点,等等,可能解释了为什么一些研究没有发现 Akt 磷酸化水平的增加。有趣的是,一项研究检查了培养大鼠肌管中睾酮的快速作用,直接提示 PI3K/Akt/mTORC1途径作为雄激素影响收缩蛋白合成的介质[46]。Basualto-Alarcón 等人,用睾酮(100nm)孵化肌管,并在孵化后进行总 Akt 和磷酸化 Akt (在 Ser473)1,5,15,30和60m 的测量。与睾酮孵育6小时和12小时后,肌动蛋白 α mRNA 和蛋白含量均显著升高,表明肌肉收缩蛋白合成增加。实际上,孵育12小时后,细胞的横截面积(CSA)明显增加,Akt 磷酸化水平在孵育15小时后增加。当作者抑制 PI3K、 Akt 或 mTOR 时,对 α- 肌动蛋白的影响被阻断。因此,雄激素可能通过激活 PI3K/Akt/mTOR 途径而迅速发挥作用。考虑到 PI3K 作用于细胞膜,并且对 Akt 磷酸化的影响发生得很快(15米后) ,似乎很有可能涉及细胞膜定位受体。事实上,许多证据表明,细胞膜上的受体与雄激素的快速作用有关。G 蛋白偶联受体(GPCR) GPRC6A 已被证明能够介导快速的信号反应,包括 PI3K 对睾酮的参与[52]。在巴萨阿尔托-阿拉尔孔等人的实验中,尽管 α- 肌动蛋白水平增加,但是加入雄激素受体(AR)拮抗剂比卡鲁胺可以阻断 CSA 的增加。这表明细胞内 AR 与睾酮激活的 PI3K 通路之间存在交叉。引人注目的是,在雄激素敏感的上皮细胞中,细胞内 AR 与 PI3K 的 p85α 调节亚基相互作用,提高其活性[53]。然而,在这些雄激素敏感的上皮细胞中加入比卡鲁胺阻断了雄激素诱导的 Akt 磷酸化。这与 Basualto-Alarcón 等人的实验相反,实验表明抑制 Akt 磷酸化阻碍了 α- 肌动蛋白水平的增加,而重卡鲁胺并不影响 α- 肌动蛋白水平,因此表明重卡鲁胺在这个实验中并不抑制 Akt 磷酸化。如果在巴苏阿托-阿拉尔孔等人的实验中,比卡鲁胺也影响 Akt 磷酸化,那么这应该在 α- 肌动蛋白水平上观察到,但是加入比卡鲁胺后仍然没有改变。这两项研究之间的差异可能是由于细胞系和 AR 配体使用的不同。然而,AR-PI3K 的串扰可能部分地解释了在巴苏阿尔托-阿拉尔孔等人的实验中加入比卡鲁胺后 CSA 没有增加的原因,尽管 α-actin 有所增加。此外,PI3K/Akt 通路的激活反过来可以调节 AR 活性,因为 Akt 已被证明通过磷酸化在翻译后修饰 AR [54]。进一步的研究可能进一步阐明细胞膜和细胞内 AR 调节 mTORC1活性的机制。

Regulation by energy status

能源状况调节

The regulation of mTORC1 by energy status of the cell is less well described than that of growth factors and appears primarily mediated through the AMP-activated kinase (AMPK). AMPK is a heterotrimeric protein comprising a combination of αβ and γ subunits. Currently there are two isoforms known of both the α (α1 and α2) and β (β1, β2) subunits. There are three isoforms known of the γ subunit (γ1, γ2, γ3). The α-subunit functions as the catalytic subunit of the complex, whereas the other two subunits ’sense’ the energy status of the cell. The β-subunit can interact with glycogen [55] and the γ-subunit with the nucleotides adenosinetriphosphate (ATP), adenosinediphosphate (ADP) and adenosinemonophosphate (AMP) [56]. The interaction between glycogen and the β-subunit leads to allosteric inhibition of AMPK activity, a decrease in glycogen will therefore lead to relieve of this inhibition and thus activation of the complex. In sum, the β and γ subunits allow the kinase to measure the energy status of the cell as reflected by its glycogen content and ATP to ADP or AMP ratio. A decrease in glycogen or the ATP to ADP or AMP ratio signals a decrease in available energy to the kinase and activates it. In general, activation of AMPK promotes catabolic pathways in order to recover cellular energy homeostasis and attenuates anabolic pathways to preserve energy (Fig. 2) [5657].

细胞能量状态对 mTORC1的调节作用不如生长因子的调节作用明显,主要通过 AMPK 介导。AMPK 是一种由 α、 β 和 γ 三个亚基组成的异三聚体蛋白。目前已知 α (α1和 α2)亚基和 β (β1,β2)亚基有两种亚型。已知 γ 亚基有三种亚型(γ1,γ2,γ3)。α 亚基是复合物的催化亚基,而另外两个亚基则是细胞的能量状态。该 β- 亚基能与糖原[55]和 γ- 亚基相互作用,与腺苷三磷酸(ATP)、腺苷二磷酸(ADP)和腺苷酸(AMP)相互作用。糖原和 β 亚基之间的相互作用导致 AMPK 活性的变构抑制,糖原的减少将导致缓解这种抑制,从而激活复合物。总而言之,β 和 γ 亚基允许激酶测量细胞的能量状态,这反映在它的糖原含量和 ATP 对 ADP 或 AMP 的比例上。糖原或 ATP 与 ADP 或 AMP 比值的减少标志着对激酶的可利用能量的减少并激活它。一般来说,活化的 AMPK 促进分解代谢途径,以恢复细胞能量稳态和衰减代谢途径,以保存能量(图2)[56,57]。

figure2
Fig. 2 图二

Theoretically, the different isoforms of the subunits allows for twelve unique combinations. However, to date only three different combinations have been found in human skeletal muscle: α2/ β2/ γ1, α2/ β2/ γ3 and α1/ β2/ γ1 [58]. The quantitative distribution of these heterotrimeric proteins has been estimated at 15 % α1/ β2/ γ1, 65% α2/ β2/ γ1 and 20 % α2/ β2/ γ3. The three heterotrimers show differential regulation and effects [59]. The α2/ β2/ γ3 heterotrimer is rapidly activated following physical activity, whereas the other two take far longer to activate. Additionally, only the α1-containing heterotrimer appears to attenuate muscle growth, whereas the α2-containing heterotrimers do not appear to do so [60].

从理论上讲,不同的亚单位的同构形式允许十二种独特的组合。然而,迄今为止,在人类骨骼肌中只发现了三种不同的组合: α2/β2/γ1,α2/β2/γ3和 α1/β2/γ1[58]。这些异三聚体蛋白的定量分布估计为15% α1/β2/γ1,65% α2/β2/γ1和20% α2/β2/γ3。这三种异三聚体表现出差异调节和影响[59]。α2/β2/γ3异三聚体在体力活动后迅速激活,而另外两种异三聚体激活时间要长得多。此外,只有含 α1的异三聚体似乎减弱了肌肉生长,而含 α2的异三聚体似乎没有这样做[60]。

The antagonizing effect AMPK has on muscle growth is mediated, atleast in part, by inhibiting mTORC1 activity. AMPK phosphorylates two residues (Thr1227 and Ser1345) on TSC2 which are important for its activation [61]. TSC2 then acts to inhibit mTORC1 by formation of the TSC-TBC complex as described earlier. Moreover, Raptor, one of the proteins compromising mTORC1, has also been found to be a substrate of AMPK [62]. Phosphorylation of Raptor at residues Ser722 and Ser792 likewise inhibits mTORC1 activity.

AMPK 对肌肉生长的拮抗作用是通过抑制 mTORC1活性而介导的,至少部分是这样。AMPK 磷酸化 TSC2上的两个残基(Thr1227和 Ser1345) ,这两个残基对 TSC2的活化很重要[61]。如前所述,TSC2通过形成 TSC-TBC 复合物来抑制 mTORC1。此外,猛禽,其中一个蛋白质的损害 mTORC1,也被发现是 AMPK [62]的底物。在 Ser722和 Ser792位点磷酸化的 Raptor 同样抑制 mTORC1的活性。

In sum, the antagonistic effect of AMPK on mTOR is mediated through phosphorylation of TSC2 and Raptor.

总之,AMPK 对 mTOR 的拮抗作用是通过 TSC2和 Raptor 的磷酸化介导的。

Regulation by amino acids

氨基酸的调节

It should come as no surprise that the availability of the basic building blocks of protein control its synthesis. When a cell is deprived of amino acids, mTOR can be found throughout the cytoplasm, whereas addition of amino acids rapidly localizes mTOR to the peri-nuclear region of the cell, to large vesicular structures, or to both [63]. The amino acid-induced locatization is similar to that of Rab7, a late endosome-/lysosome-associated small GTPase. This suggests that amino acids might stimulate mTORC1 activity by localizing it to lysosomal surface where it can be activated by Rheb-GTP. The Ragulator-Rag complex was found responsible for targeting mTORC1 to the lysosomal surface [64]. At the lysosomal surface, mTORC1 associates with Ras-related GTPases (Rags). There are four different Rags: RagA, RagB, RagC and RagD. RagA and RagB (RagA/B) bind to RagC and RagD (RagC/D) to form heterodimeric pairs. Rags, in turn, associate with the protein complex Ragulator which is anchored in the lysosomal membrane. The interaction of Rags with mTORC1 is dependent on their guaninenucleotide binding state. In an amino acid-deprived cell, the RagA/B are bound to GDP, and the RagC/D are bound to GTP. The addition of amino acids induce a nucleotide exchange favoring the GTP bound state of RagA/B and the GDP bound state of RagC/D. The Ragulator, anchored in the lyosomal membrane, associates with Rags, therefore localizing them to the lysosomal membrane. Importantly, the Ragulator functions as a guanine nucleotide exchange factor (GEF) for RagA/B [65], thereby facilitating the exchange of GDP bound RagA/B for GTP bound RagA/B (the active form). The GEF activity of Ragulator is regulated by v-ATPase [65]. v-ATPase consumes ATP in order to pump hydrogens up their concentration gradient into the lysosome in order to maintain its acidic environment. Ragulator is associated with v-ATPase and amino acids induce a conformational change to the protein which then acts to activate Ragulator’s GEF activity. As of yet it is unclear how amino acids induce this conformational change, but the signal appears to originate from inside the lysosome due to accumulation of amino acids in its lumen (Fig. 3) [66].

蛋白质的基本组成部分控制着蛋白质的合成,这一点不足为奇。当一个细胞被剥夺了氨基酸时,mTOR 可以在整个细胞质中被发现,而氨基酸的加入使 mTOR 迅速定位于细胞核周围区域、大的泡状结构或两者[63]。氨基酸诱导的定位与 Rab7的定位相似,Rab7是一种晚期内生体/溶酶体相关的小 g 蛋白酶。这表明氨基酸可能通过将其定位于溶酶体表面而刺激 mTORC1的活性,在那里它可以被 Rheb-GTP 激活。Ragulator-Rag 复合物被发现负责将 mTORC1靶向溶酶体表面[64]。在溶酶体表面,mTORC1与 ras 相关的 GTPases (Rags)结合。有四种不同的 Rags: RagA,RagB,RagC 和 RagD。RagA 和 RagB (RagA/b)与 RagC 和 RagD (RagC/d)结合形成异二聚体对。破布依次与锚定在溶酶体膜上的蛋白复合物 Ragulator 结合。Rags 与 mTORC1的相互作用取决于它们的鸟嘌呤核苷酸结合状态。在氨基酸去除的细胞中,RagA/B 与 GDP 结合,RagC/D 与 GTP 结合。氨基酸的加入引起核苷酸交换有利于 RagA/B 的 GTP 结合态和 RagC/D 的 GDP 结合态。Ragulator 锚定在溶酶体膜上,与抹布结合,因此定位于溶酶体膜上。重要的是,Ragulator 的功能是作为 RagA/B [65]的鸟苷酸交换因子,从而促进了与 GDP 相关的 RagA/B 与与 GTP 相关的 RagA/B 的交换(活动形式)。Ragulator 的 GEF 活性受到 v-ATPase [65]的调控。ATP 酶消耗 ATP 是为了将其浓度梯度泵入溶酶体以维持其酸性环境。Ragulator 与 v-ATPase 相关,氨基酸诱导蛋白质产生构象改变,从而激活 Ragulator 的 GEF 活性。到目前为止,还不清楚氨基酸是如何引起这种构象改变的,但是这种信号似乎来自于溶酶体内部,因为它的腔内积累了大量的氨基酸。

figure3
Fig. 3 图3

Whereas Ragulator acts as a GEF for RagA/B, the GAP activity towards Rags (GATOR1) complex functions as a GAP towards RagA/B [67]. The GATOR1 complex thus exchanges the GTP for GDP of RagA/B, leading to deactivation of the Rags and subsequently inhibition of mTORC1. Another protein complex dubbed GATOR2 is responsible for inhibiting GATOR1 activity [67] and therefore relieves mTORC1 from its inhibition. The inhibiting effect of GATOR2 on GATOR1 is mediated by Sestrin proteins in response to amino acids [68]. However, it is unknown how GATOR2 mediates its inhibiting effect and how amino acids regulate the complex.

Ragulator 作为 RagA/B 的全球环境基金,GAP 对 Rags 复合体(GATOR1)的活动作为 GAP 对 RagA/B 的活动[67]。GATOR1复合物因此将 GTP 与 RagA/B 的 GDP 进行交换,导致 RagA/B 的失活和 mTORC1的抑制。另一种被称为 GATOR2的蛋白复合物负责抑制 GATOR1的活性[67] ,因此减轻了 mTORC1的抑制作用。GATOR2对 GATOR1的抑制作用是通过 Sestrin 蛋白对氨基酸的反应介导的[68]。然而,GATOR2是如何介导其抑制作用以及氨基酸是如何调节复合物的还不清楚。

Lastly, there is evidence that the guanine nucleotide binding state of RagC/D is regulated by leucyl tRNA-synthetase (LRS), the enzyme responsible for loading tRNA with leucine. The enzyme acts as a GAP for RagD GTPase, in a leucine depedent manner [69]. However, a later study found that purified LRS did not act as a GAP for any of the Rags [70]. Instead the authors propose that folliculin tumor suppressor (FLCN) and its binding partners act as Rag-interacting proteins with GAP activity for RagC/D, leading to mTORC1 activation. Moreover, leucine specifically appears to regulate mTORC1 through Sestrin2 [71].

最后,有证据表明,鸟嘌呤核苷酸结合状态的 RagC/D 是由亮氨酸 tRNA-synthetase (LRS) ,负责负载 tRNA 与亮氨酸。该酶作为一个 GAP 的 RagD GTPase,在一个亮氨酸的方式[69]。然而,后来的一项研究发现,纯化的 LRS 并没有作为任何破布的 GAP [70]。相反,作者提出,滤泡蛋白肿瘤抑制因子(FLCN)及其结合伙伴作为雷格相互作用蛋白的 RagC/D 活性,导致 mTORC1激活。此外,亮氨酸似乎通过 Sestrin2[71]特异性地调节 mTORC1。

Regulation by mechanical stimuli

机械刺激的调节

It is well known that physical activity, resistance exercise in particular, increases skeletal muscle mass in healthy persons under most conditions. Currently, two important mechanisms have been identified which regulate mTORC1 by mechanical stimuli. One of these mechanisms shows close resemblance with the PI3K/Akt-pathway in that it leads to dissociation of TSC2 from the lysosomal membrane [72]. Eccentric contractions lead to phosphorylation of TSC2 which leads to the dissociation observed. Since Rheb-GTP, the target of the TSC-TBC complex its GAP activity, is located at the lysosomal membrane, mechanical stimuli effectively prevents the GTP/GDP-exchange. Moreover, mechanical stimuli increase the levels of mTORC1 at the lysosomal membrane, further supporting its activation [72]. The mechanism for this remains uncertain (Fig. 4).

众所周知,身体活动,特别是抗阻力运动,增加骨骼肌质量的健康人在大多数情况下。目前,机械刺激对 mTORC1的调节机制主要有两个。其中一个机制与 pi3k/akt 途径非常相似,因为它导致 TSC2从溶酶体膜上解离[72]。偏心收缩导致 TSC2磷酸化,从而导致观察到的离解。由于 TSC-TBC 复合物的 GAP 活性靶点 Rheb-GTP 位于溶酶体膜上,机械刺激有效地阻止了 GTP/GDP-exchange。此外,机械性刺激增加了溶酶体膜上 mTORC1的水平,进一步支持其激活[72]。这种机制仍然不确定(图4)。

figure4
Fig. 4 图4

Secondly, mechanical stimuli regulate mTORC1 by regulating levels of phosphatidic acid (PA), a diacylglycerol phospholipid which has been found to directly activate mTORC1 [73]. A twofold effect mediates the stimulating effect of PA on mTORC1: i) displacing the endogeneous mTORC1 inhibitor FK506 binding protein 38 (FKBP38) through competitive inhibition, ii) allosteric activation of mTORC1.

其次,机械刺激通过调节磷脂酸(PA)的水平来调节 mTORC1,这是一种二酰基甘油磷脂,已发现它能直接激活 mTORC1[73]。通过双重作用介导 PA 对 mTORC1的刺激作用: i)通过竞争性抑制剂取代内源性 mTORC1抑制剂 FK506结合蛋白38(FKBP38) ,ii) mTORC1的变构体激活。

The [PA] is regulated by five classes of enzymes [74]. Three are responsible for the synthesis of PA and two regulate its degradation. A delicate balance between the activities of these enzymes determines cellular PA levels. Glycerol-3-phosphate (G3P), phosphatidylcholine (PC) and diacylglycerol (DAG) are precursors for the biosynthesis of PA. G3P is acetylated twice in order to produce PA. First glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first acetylation reaction, after which lysophosphatidic acid acyltransferase (LPAAT) catalyzes the second. PC is hydrolyzed in order to produce PA. This reaction is catalyzed by phospholipase D (PLD). For long it had been assumed PLD was crucial in mediating the mechanical stimuli-induced increase in PA. This assumption was mainly based on experiments which applied the PLD inhibitor 1-butanol, which effectively inhibited mTORC1 activity in several experiments [75]. However, later it was found that not all biological activity induced by 1-butanol could be attributed to its PLD inhibiting effect. Moreover, earlier findings already reported that PLD activity induced by mechanical stimuli poorly correlated with the cellular increase of PA [76]. Recent evidence suggests that the mechanical stimuli-induced increase of PA might be attributed to an increased synthesis from DAG rather than PC. PA is produced from DAG by phosphorylation catalyzed by diacylglycerolkinases (DGK). Many DGKs have been identified and it appears the ζ-isoform is primarily responsible for the mechanical stimuli-induced increase of PA [77].

[ PA ]受五类酶的调节[74]。三种负责 PA 的合成,两种负责调节 PA 的降解。这些酶活性之间的人海万花筒(电影)决定了细胞 PA 的水平。甘油 -3- 磷酸酯(G3P)、磷脂酰胆碱(PC)和甘油二酯(DAG)是生物合成 pa 的前体。G3P 通过两次乙酰化反应生成 pa。第一个甘油 -3- 磷酸酰基转移酶(GPAT)催化第一个乙酰化反应,其次是溶血磷脂酸酰基转移酶(LPAAT)催化第二个。对 PC 进行水解制备 pa。这个反应是由磷脂酶 d (PLD)催化的。长期以来,它一直被认为是至关重要的介导机械刺激诱导增加 pa。这一假设主要是基于在多个实验中应用 PLD 抑制剂正丁醇,有效地抑制 mTORC1活性的实验[75]。然而,后来发现并非所有丁醇诱导的生物活性都与其 PLD 抑制作用有关。此外,早期的研究已经报道机械刺激诱导的 PLD 活性与细胞内 PA [76]的增加不相关。最近的证据表明,机械刺激引起的 PA 的增加可能是由于 DAG 而不是 PC 的合成增加。二酰基甘油激酶(DGK)催化 DAG 磷酸化生成 PA。许多 dgk 已经被确定,似乎 ζ 亚型是主要负责机械刺激诱导 PA [77]的增加。

The regulation of the enzymes responsible for degradation of PA are currently poorly understood.

目前对 PA 降解酶的调控知之甚少。

Sports supplements and mTORC1 signaling

运动补充剂与 mTORC1信号传导

In 2011, Kunkel et al. performed an elegant study to identify a compound which might help against skeletal muscle atrophy [78]. The authors screened for changes in mRNA expression in both human and rodent skeletal muscle during fasting and spinal cord injury. Both fasting and spinal cord injury involve dramatic muscle atrophy over time and this effect is driven by changes in muscle gene expression. The authors therefore hypothesized that pharmacologic compounds with opposite effects on gene expression might inhibit skeletal muscle atrophy. By querying the Connectivity Map [79] with the data they gathered, they identified ursolic acid as a potential pharmacologic compound which might inhibit skeletal muscle atrophy. After identification of the compound they continued to test its effects in mice and found it to reduce muscle atrophy and stimulate muscle hypertrophy. Interestingly, IGF-I mRNA was upregulated in skeletal muscle of the mice treated with ursolic acid. Moreover, Akt phosphorylation was also increased. The researchers also evaluated the effect of C2C12 myoblasts incubated with ursolic acid and found that, on its own, it did not increase Akt phosphorylation. However, in the presence of IGF-I ursolic acid did increase Akt phosphorylation. Similarly, ursolic acid alone did not upregulate S6K1 phosphorylation, but it did enhance IGF-I- and insulin-mediated S6K1 phosphorylation. Later research confirmed these findings and found that ursolic acid stimulates mTORC1 signaling in rat skeletal muscle [80]. This was evidenced by an increase in phosphorylation of Akt (at Thr308, but not Ser473), PRAS40 and S6K1 after resistance exercise. A recent clinical study also found an improvement in body composition and strength in sixteen Korean men with over 3 years resistance exercise experience who were supplemented ursolic acid compared to placebo [81].

在2011年,Kunkel 等人进行了一项优雅的研究,以确定一种化合物,可能有助于防止骨骼肌萎缩。作者筛选了禁食和嵴髓损伤期间人类和啮齿动物骨骼肌 mRNA 表达的变化。随着时间的推移,禁食和嵴髓损伤都会引起严重的肌肉萎缩,这种效应是由肌肉基因表达的变化所驱动的。因此,作者推测对基因表达具有相反作用的药物化合物可能抑制骨骼肌萎缩。通过查询连接图[79]和他们收集的数据,他们确定熊果酸作为一种可能抑制骨骼肌萎缩的潜在药理复合物。在确认了这种化合物之后,他们继续在小鼠身上测试它的效果,发现它可以减少肌肉萎缩并刺激肌肉肥大。有趣的是,熊果酸处理后小鼠骨骼肌中 IGF-I mRNA 表达上调。此外,Akt 磷酸化也增加。研究人员还评估了与熊果酸孵育的 C2C12成肌细胞的效果,发现它本身并不增加 Akt 的磷酸化。然而,在存在的 IGF-I 熊果酸确实增加 Akt 磷酸化。同样,熊果酸单独使 S6K1磷酸化不上调,但它确实增强胰岛素样生长因子 -i 和胰岛素介导的 S6K1磷酸化。后来的研究证实了这些发现,并发现熊果酸刺激大鼠骨骼肌中的 mTORC1信号[80]。抗阻训练后 Akt (Thr308,但 Ser473)、 PRAS40和 S6K1的磷酸化水平增加,可以证明这一点。最近的一项临床研究还发现,在十六名韩国男子身体成分和力量的改善,超过3年的抗阻力运动的经验,谁补充熊果酸相比,安慰剂[81]。

Some evidence suggests that the popular ergogenic aid creatine might also stimulate mTORC1 signaling. In a double-blind placebo-controlled study, participants received either placebo or creatine for 5 days [82]. Muscle biopsies were then taken at rest, immediately after exercise, 24 and 72 h later. The phosphorylation of Akt at Ser473 and Thr308 were determined, as well as the phosphorylation of 4E-BP1 and S6K1. Surprisingly, creatine supplementation decreased Akt phosphorylation at Thr308 in rest, whereas it was unaffected immediately, 24 and 72 h post-exercise. Akt phosphorylation at Ser473 was unaffected at all time points. Similar results were obtained for 4E-BP1 and S6K1 phosphorylation: 4E-BP1 phosphorylation showed a decrease 24 h after training, while it remained unaffected at all other time points and S6K1 phosphorylation remained unchanged at all time points. Nevertheless, MHCIIA mRNA expression showed an increase immediately after exercise and MHC1 mRNA expression showed an increase during rest after creatine supplementation compared to placebo. However, another study with a similar experimental design found an increase in phosphorylated 4E-BP1 24 h after exercise in the creatine group compared to placebo, but found no difference in phosphorylated S6K1 between both groups [83]. Again, no difference was found in phosphorylated 4E-BP1 and S6K1 3 h post-exercise. Notably, an increase in IGF-I mRNA expression was also observed 24 h post-exercise in the creatine group compared to placebo. These results suggest that creatine might activate mTORC1 by increasing IGF-I activity at rest, but does not further potentiate mTORC1 signaling in the hours after exercise. Interestingly, a clinical study also found that creatine supplementation amplified the resistance exercise-induced decrease in serum myostatin [84]. Although no markers of the mTORC1 pathway were measured in this study, it might be that a decrease in serum myostatin might enhance Akt phosphorylation and thus mTORC1 activity.

一些证据表明,普遍使用的促进人体生长的辅助性肌酸可能也会刺激 mTORC1信号。在一项双盲安慰剂对照研究中,参与者服用安慰剂或肌酸5天[82]。然后在休息时、运动后立即、24小时和72小时进行肌肉活检。测定了在 Ser473和 Thr308位点 Akt 的磷酸化水平,以及4E-BP1和 S6K1的磷酸化水平。令人惊讶的是,在运动后24和72小时,补充肌酸在静息状态下降低了 Akt 磷酸化,而在运动后24小时和72小时没有立即发生变化。Akt 磷酸化在 Ser473是未受影响的所有时间点。4E-BP1和 S6K1磷酸化的结果相似: 4E-BP1磷酸化在训练后24h 下降,但在其他时间点没有变化,S6K1磷酸化在各时间点保持不变。然而,与安慰剂相比,运动后 MHCIIA mRNA 表达立即增加,补充肌酸后 MHC1 mRNA 表达增加。然而,另一项类似实验设计的研究发现,肌酸组运动后24小时磷酸化的4E-BP1比安慰剂组有所增加,但两组之间磷酸化的 S6K1没有差异[83]。同样,运动后3小时磷酸化的4E-BP1和 S6K1也没有差异。值得注意的是,与安慰剂组相比,肌酸组在运动后24小时内胰岛素样生长因子 i 的 mRNA 表达也有所增加。这些结果表明,肌酸可能通过增加静息状态下的 IGF-I 活性来激活 mTORC1,但在运动后小时内并不进一步增强 mTORC1信号。有趣的是,一项临床研究也发现补充肌酸放大了阻力运动诱导的血清肌肉生长抑制素[84]。虽然在这项研究中没有检测到 mTORC1途径的标志物,但是血清肌肉生长抑制素的减少可能会增强 Akt 磷酸化,从而增强 mTORC1的活性。

The mTORC1 signaling pathway is also thought to be involved in the anabolic effects of the leucine metabolite β-hydroxy β-methylbutyrate (HMB) [8586]. In rats fed HMB, mTOR protein expression increased significantly compared to treatment with saline [87]. Moreover, phosphorylated S6K1 also increased significantly in the HMB treated rats compared to the control group. Similar results were obtained in an in vitro experiment [88]. C2C12 myoblasts were incubated with proteolysis-inducing factor (PIF, a protein which stimulates proteolysis and inhibits protein synthesis) and addition of HMB increased S6K1 phosphorylation. Notably, in the rat study no differences were found in Akt phosphorylation between both groups. However, another in vivo experiment did find an increase in phosphorylated Akt in differentiated C2C12 myoblasts 10 and 30 m after incubation with HMB, as well as an increase in phosphorylated mTOR 30 m after incubation [89]. These results might seem conflicting, but the measurements in the rat study were taken 15 h to 18 h after HMB supplementation. Thus it might be that the activation of Akt/mTORC1 signaling was short-lived and was therefore missed in the rat study. Interestingly, another leucine metabolite, α-hydroxy-isocaproic acid (HICA), has shown to increase whole lean body mass when compared to placebo in a small sample of soccer players [90]. Rats fed HICA and recovering from hindlimb immobilization also showed a sustained increase in protein synthesis and phosphorylation of S6K1 and 4E-BP1 after 14 days when compared to placebo and leucine [91]. Further research might further clarify the role of mTORC1 signaling in the anabolic effects of these leucine metabolites.

mTORC1信号通路也被认为参与了亮氨酸代谢物 β- 羟基 β- 甲基丁酸(HMB)[85,86]的合成作用。在喂食 HMB 的大鼠中,mTOR 蛋白表达明显高于盐水组[87]。此外,磷酸化 S6K1在 HMB 治疗大鼠也明显增加比对照组。在体外实验中也得到了类似的结果[88]。C2C12成肌细胞与蛋白水解诱导因子(PIF,一种刺激蛋白水解和抑制蛋白质合成的蛋白质)孵育,加入 HMB 增加 S6K1磷酸化。值得注意的是,在大鼠的研究中,两组之间在 Akt 磷酸化方面没有发现差异。然而,另一个体内实验确实发现,与 HMB 孵育10和30m 后,分化的 C2C12成肌细胞的 Akt 磷酸化水平增加,孵育30m 后的 mTOR 磷酸化水平增加[89]。这些结果可能看起来相互矛盾,但在大鼠研究中的测量是在补充 HMB 后15小时至18小时进行的。因此,可能 Akt/mTORC1信号转导的激活是短暂的,因此在大鼠研究中没有被发现。有趣的是,另一种亮氨酸代谢物—— α- 羟基异佐菌酸(HICA) ,在一小部分足球运动员样本中与安慰剂相比,显示出增加整个瘦体质量。与安慰剂和亮氨酸相比,喂食 HICA 和后肢制动恢复的大鼠在14天后也表现出 S6K1和4E-BP1蛋白质合成和磷酸化的持续增加[91]。进一步的研究可能会进一步阐明 mTORC1信号在这些亮氨酸代谢物的合成效应中的作用。

Trimethylglycine (TMG), a methyl derivate of the amino acid glycine and also known as betaine, was recently shown to improve body composition when supplemented to trained athletes [92]. TMG is hypothesized to work as an ergogenic aid by functioning as both an osmolyte as well as a methyl donor in cells [93]. In a small double-blinded crossover trial, participants underwent 2 weeks of supplementation with either TMG or placebo [94]. Before and after the 2-week period, participants performed an acute exercise session. Both before the supplementation period, as well as 10 m before and after exercise, muscle biopsies were taken from the vastus lateralis muscle. Total Akt protein content was significantly increased in the TMG group compared to placebo. There was no difference in phosphorylated Akt and S6K1 in rest, but there was a decrease in phosphorylated Akt and S6K1 after the acute exercise session in the placebo group which did not occur in the TMG group. AMPK phosphorylation at Thr172 was also measured, but there was no difference between both groups. Notably, an increase in circulating growth hormone (GH) and IGF-I concentrations was observed in the TMG group, but not in the placebo group. This makes it appealing to speculate that the increase in circulatory GH and IGF-I underlies the effect of TMG on Akt. However, it should be taken into account that local GH and IGF-I, rather than circulatory, appear to affect skeletal muscle hypertrophy [95]. Nevertheless, an in vitro experiment in C2C12 myoblasts showed an increase in IGF-1 receptor protein expression after incubation with TMG [96]. An increase in Akt and myosin heavy chain protein content was also observed. Taken together these observations suggest that TMG activates the IGF-I/Akt/mTORC1 pathway.

甜菜碱,一种氨基酸甘氨酸的甲基衍生物,又称甜菜碱,最近被证明补充训练有素的运动员时可以改善身体成分[92]。TMG 被假设作为一种促进作用,既作为渗透剂又作为细胞中的甲基供体[93]。在一个小型的双盲交叉试验中,参与者接受2周的 TMG 或安慰剂的补充。在为期两周的训练前后,参与者进行了一次急性运动训练。在补充期之前,以及运动前后10米,肌肉活组织检查都是从外侧广肌中心取得的。与安慰剂组相比,TMG 组 Akt 蛋白质总含量显著增加。安慰剂组在休息时磷酸化 Akt 和 S6K1没有差异,但在急性运动后,磷酸化 Akt 和 S6K1有所下降,而 TMG 组没有出现这种情况。还测定了 Thr172位点的 AMPK 磷酸化水平,但两组之间没有差异。值得注意的是,在 TMG 组中观察到循环生长激素(GH)和 IGF-I 浓度的增加,而在安慰剂组中没有。这使得推测 TMG 对 Akt 的影响可能是由于循环 GH 和 IGF-I 的增加。然而,应该考虑的是局部生长激素和胰岛素样生长因子 -i,而不是循环系统,似乎影响骨骼的肌肉肥大。然而,C2C12成肌细胞体外实验显示,与 TMG [96]孵育后 IGF-1受体蛋白表达增加。Akt 和肌球蛋白重链蛋白含量也有所增加。综合这些观察表明 TMG 激活了 IGF-I/Akt/mTORC1通路。

A recent study also showed that PA supplementation activated mTORC1 and improved responses in skeletal muscle hypertrophy, lean body mass, and maximal strength to resistance exercise [97]. A sample of 28 resistance trained men received either PA or placebo and took part in an 8 week periodized resistance training program. The PA group showed a larger increase in lean body mass than the placebo group and also the CSA of the rectus femoris muscle showed a larger increase in the PA group than the placebo group. The authors also performed an in vitro experiment assessing phosphorylation of S6K1 in C2C12 myoblasts after incubation with two different sources of PA (egg and soy). While both showed a large increase in phosphorylation of S6K1, the soy-derived PA showed the largest increase. As the authors note, the difference might be due to soy and egg derived PA having varying degrees of unsaturated and saturated fatty acid chains which influence its action. A later study carried out both an in vivo and in vitro experiment to examine the effects of PA on anabolic signaling [98]. In the in vivo experiment, male Wister rats received either tap water (CON), PA (PA), whey protein concentrate (WPC) or PA + WPC (PA+WPC) after an overnight fast. Samples were taken after 3 h. Ribosomal protein S6 (rpS6) phosphorylation was increased in the PA and PA+WPC groups compared to the CON group, whereas it was not increased in the WPC group. S6K1 phosphorylation was also only significantly increased compared to control in the PA+WPC group. However, while PA showed an increase in MPS compared to CON, the largest increase in MPS was observed in the WPC group. There was no synergistic effect of PA+WPC in MPS when compared to WPC alone. The authors therefore speculate that combined PA and WPC might alter mTOR pathway activation dynamics, thus shifting MPS levels to the left or right of the sampling point or that PA might interfere with WPC-induced increases in MPS. Future research might clarify this matter. Their in vitro experiment in C2C12 myoblasts confirmed that PA increased MPS and mTOR signaling.

最近的一项研究也表明,补充 PA 能够激活 mTORC1,改善骨骼和肌肉肥大的反应,瘦体重和最大力量抵抗运动[97]。28名接受抗药性训练的男性样本接受 PA 或安慰剂,并参加为期8周的抗药性训练计划。与安慰剂组相比,PA 组的瘦体重增加更多,而且股直肌的 CSA 组的瘦体重增加更多。作者还进行了体外实验,评估了两种不同来源的 PA (鸡蛋和大豆)孵育后 C2C12成肌细胞 S6K1的磷酸化。虽然两者都显示了 S6K1磷酸化的大幅度增加,但大豆衍生的 PA 显示了最大的增加。正如作者指出的,这种差异可能是由于大豆和鸡蛋衍生的 PA 具有不同程度的不饱和脂肪酸和饱和脂肪酸链影响其作用。后来的研究进行了体内和体外实验,以检查 PA 对合成代谢信号传导的影响[98]。在体内实验中,雄性 Wister 大鼠在隔夜禁食后分别接受自来水(CON)、 PA (PA)、乳清浓缩蛋白(WPC)或 PA + WPC (PA + WPC)。与 CON 组相比,PA 和 PA + WPC 组的 S6(rpS6)磷酸化水平在3小时后提高,而 WPC 组的核糖体蛋白质水平没有提高。与对照组相比,pa + wpc 组 S6K1磷酸化水平也仅显著升高。然而,尽管 PA 显示与 CON 相比 MPS 增加,WPC 组 MPS 增加最大。与单独使用 WPC 相比,多磺酸钠中 pa + WPC 没有协同作用。因此,作者推测,PA 和 WPC 的结合可能改变 mTOR 通路的激活动力学,从而将 MPS 水平移动到取样点的左侧或右侧,或者 PA 可能干扰了 WPC-诱导的 MPS 的增加。未来的研究可能会澄清这个问题。他们的 C2C12成肌细胞体外实验证实,PA 增加 MPS 和 mTOR 信号。

Conclusions

结论

In the past few years our knowledge of mTORC1 regulation in skeletal muscle has increased tremendously. This review therefore attempted to provide a brief and up-to-date narrative on its regulation. Energy intake, protein intake, mechanical stumuli, as well as growth factors, have been shown to regulate the mTORC1 complex. All these elements provide signals to muscle cells which are then sensed, transduced and integrated which leads to changes in cellular functions. Ultimately, these signals are sensed by proteins such as cell surface receptors or intracellular kinases. For example, the IR senses the concentration of insulin outside the cell and relays this signal through the PI3K/Akt/mTORC1 pathway, whereas energy availability is directly relayed through AMPK by the nucleotides ATP, ADP and AMP as well as stored glycogen. Finally, these signals are integrated by the cell in order to respond accordingly by changing cellular functions such as protein synthesis and protein breakdown. mTORC1 plays a pivotal role in integrating several of these signals such as growth factors, energy status, amino acids availability and mechanical stumuli. All these signals together affect the cellular response. Sports supplements might benefit the athlete in optimizing these signals, in addition to resistance exercise training, to maximize muscle hypertrophy. While ultimately clinical trials are required to properly evaluate their effects, they are expensive and sometimes difficult to carry out. For example, it can be challenging to find enough participants which conform the criteria of interest (e.g. young adults with several years of weightlifting experience) to yield enough statistical power. Additionally, strictly controlling all variables, such as dietary intake, can be hard. This is of special concern in studies of several weeks or months of duration. Insights in the mechanistic features of sports supplements might therefore aid clinical trials by providing hypothesizes under which conditions supplements might work best, as well as which combinations of supplements might provide additive effects. Additionally, it might aid in discovering new supplements of interest. The increasing knowledge of mTORC1 regulation therefore helps to refine these matters.

在过去的几年中,我们对骨骼肌中 mTORC1调节的知识有了极大的增加。因此,这次审查试图就其规章制度提供一个简短和最新的叙述。能量的摄入,蛋白质的摄入,机械性的三通,以及生长因子,已经被证明可以调节 mTORC1复合物。所有这些元件提供信号给肌肉细胞,然后被感知,传递和整合,导致细胞功能的变化。最终,这些信号被细胞表面受体或细胞内激酶等蛋白质感知。例如,IR 感知细胞外的胰岛素浓度,并通过 PI3K/Akt/mTORC1途径传递这一信号,而能量可用性则通过核苷酸 ATP、 ADP 和 AMP 以及储存的糖原直接传递 AMPK。最后,这些信号被细胞整合,以便通过改变细胞功能(如蛋白质合成和蛋白质分解)作出相应的反应。mTORC1在整合生长因子、能量状态、氨基酸利用率和机械通路等多种信号中起着关键作用。所有这些信号共同影响细胞的反应。运动补充剂可能有利于运动员优化这些信号,除了阻力训练,最大限度地提高肌肉肥大。虽然最终需要临床试验来正确评估它们的效果,但它们代价昂贵,有时难以实施。例如,要找到足够多的符合兴趣标准的参与者(例如有多年举重经验的年轻人)来产生足够的统计能力是一个挑战。此外,严格控制所有的变量,如饮食摄入量,可能是困难的。这在持续数周或数月的研究中特别值得关注。因此,了解运动补充剂的机械特性可能有助于临床试验,因为可以提供这样的假设: 在哪种条件下补充剂可能效果最好,以及哪种补充剂的组合可能提供附加效应。此外,它可能有助于发现新的兴趣补充剂。因此,对 mTORC1规则的日益了解有助于完善这些问题。

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