Mammalian target of rapamycin is a serine/threonine kinase that directly regulates protein synthesis upon activation through an extensive network of upstream signalling. mTOR is involved in a vast array of cellular activities including transcription, translation, cell size, mRNA turnover, protein stability, cytoskeletal organization and autophagy. Activity of this mTOR signalling network is regulated largely by metabolic stimulants comprised of growth factors, nutrient availability such as amino acids and fatty acids, insulin, and cellular energy. It is known that mTOR is involved in the transcription and translation of all proteins, however, the specific influences and requirements of mTOR activation for skeletal muscle protein synthesis have been of recent interest. The immediate importance of this research area is highlighted by the growing occurrence of metabolic disorders such as diabetes mellitus, non-alcoholic fatty liver disease, hyperlipidemia and obesity and its downstream effect on the mTOR signalling network and subsequent protein synthesis. Metabolic disorders alter nutrient availability and growth factors which interrupt the classic mTOR complex, whereby the action and effect of specific dietary nutrients on this pathway is of large consequence. As a result, due to the intricacy of this network and the wealth of stimuli responsible for the activation of this protein, mTOR may emerge as a key contributor in obesity-related effects on skeletal muscle protein synthesis.
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Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that is at the crux of a complex network of upstream and downstream signalling involved in a multitude of cellular activities ranging from protein transcription and translation to cytoskeletal organization. Rapamycin is a compound originally isolated from soil samples on the South Pacific island of Rapa Nui, where it was identified to possess immunosuppressive and anti-tumor properties and later classified as a drug. This drug targets the protein suitably named TOR (target of rapamycin), which was originally identified in Saccharomyces cerevisiae, a unicellular budding yeast, that along with all other prokaryotes encompass two homologous TOR genes, TOR1 and TOR2, of which the cellular activities are shared. Conversely, eukaryotes only possess one TOR gene but seem to have two similar complexes where one complex, mTORC1, is responsible for translation, cell size and growth, transcription, autophagy, ribosome biogenesis and protein stability, whereas the second complex, mTORC2, controls the cytoskeleton organization. mTORC1 is of specific interest as it is the complex that is rapamycin sensitive, regulates protein translation and is found to be activated by nutrients such as specific amino acids, hormones, growth factors including insulin and cellular energy. mTORC1 is known to regulate mRNA translation by activating two downstream proteins, ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4EBP1) bound complex. The eIF4E·4E-BP1 complex remains inactive until dephosphorylated by mTORC1 where the bound proteins are released and eIF4E is free to form a complex with eIF4G, which then stimulates the initiation stage of mRNA translation.
At the cellular membrane level, the entire mTOR pathway is stimulated by the presence of amino acids, insulin and energy sufficiency, however the effect of varied levels on the regulation and activation on downstream signalling proteins and final protein synthesis is not well known. Given that nutrients and hormones play a large role in the activation of this pathway, irregularities in any of these stimulants due to metabolic disorders may create a negative reactive effect on the signalling network and resultant protein synthesis. It is has now been observed that insulin normally regulates downstream proteins and that amino acids are positive regulators in this network where impaired mTOR signalling occurs with inadequate intracellular amino acids. In theory, an extracellular influx of insulin and glucose in a hyperinsulinemic and hyperglycaemic state in the case of diabetes and insulin resistance should create dysregulation of Akt and mTOR due to debilitated insulin sensitivity leading to decreased phosphorylation of downstream proteins, hampered translation initiation and impaired protein synthesis. As a result, amino acids would be used as the main source for mTOR activation. Normally, insulin and amino acids are equal activators of the mTOR pathway, whereby insulin stimulates mTOR through a myriad of protein phosphorylation steps and amino acid mTOR activation is thought to be regulated through phosphoinositide 3 kinase (PI-3K) both facilitating mRNA translation. This review will provide an in depth examination of the stimulating factors and mechanisms regulating the mTOR signalling network and discuss amino acids and their function and influence on this complex system.
Mammalian target of rapamycin (mTOR)
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Rapamycin and its target protein, mammalian target of rapamycin, are relatively new discoveries in the field of cellular research albeit rapamycin has clinically been used as an anti-tumor and immunosuppressant agent for some time and originated as a traditional medicine in the South Pacific islands for its remedial properties. Recently this protein has become of great interest metabolically for its pivotal position in the highly integrated signalling network that regulates many cellular processes and is constantly maintained by a flow of hormones, nutrients and energy. Given that the efficiency and organization of this system is directly influenced by the working status of these stimulants the pathway of activation of all downstream and upstream signals of mTOR must be identified to recognize the interrelationship between metabolic irregularities and dysregulation of mTOR.
The most complex and understood downstream activator is insulin and begins with an influx of the hormone through the basal membrane into the intracellular space where the insulin-receptor substrate 1 (IRS1) is phosphorylated via tyrosine and activates phosphoinositide-3-kinase (PI-3K). The activation of PI-3K launches the phosphorylation of the membrane lipid phosphatidylinositol-4, 5-biphosphate (PIP2) creating the second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3). The presence of PIP3 stimulates phosphoinositide-dependent kinase (PDK1) to combine with and phosphorylate Akt/PKB at Thr308 (protein kinase B) to bind as a complex to PIP3. The activation of Akt/PKB phosphorylates the tuberous sclerosis protein (TSC) at Ser939 and Thr1462 inactivating the complex and moving downstream to increase Rheb (Ras homolog enriched in brain), which interrupts the action of FKBP38, an endogenous inhibitor of mTOR.
With the activation of mTOR, the same downstream effectors are in place regardless of cellular stimulant whereby two main proteins, p70 S6 kinase (S6K1) and 4e-binding protein (4EBP1) are activated leading to mRNA translation. Initially, under basal conditions S6K and 4EBP1 are bound together to a control protein, eukaryotic initiation factor 3 (eIF3) complex, where they are rendered inactive until dissociated from eIF3 through mTOR-mediated phosphorylation, Thr389 of S6K and Thr37 and Thr41 of 4EBP1. Once dissociated and active, S6K continues to phosphorylate ribosomal protein S6 where it contributes to direct regulation of mRNA containing a 5'- terminal oligopyrimidine tract (TOP) and eventual protein synthesis. Alternately, eIF4E, a cap-binding translation factor which binds to other translation factors such as eIF4G, together enable the assembly of initiation factors required in cap-dependent translation, is repressed by a 4EBP1·eIF4E bound complex. Once 4EBP1 is phosphorylated at Thr37 and Thr41 by mTOR, its affinity for eIF4E is reduced leading to the dissociation of the bound complex and allowing eIF4E to bind with eIF4G necessary for cap-dependent translation. Therefore, through mTOR-mediated phosphorylation of S6K and 4EBP1, protein synthesis is regulated via mRNA translation and cap-dependent translation through this cellular process.
It is well known that with sufficient supply of amino acids protein synthesis is triggered and maintained making amino acids the most potent activator of the mTOR pathway. Unlike insulin, amino acids and glucose are nutrient regulators of the mTOR signalling network and its mechanism of activation are much less complex but less understood. There are several competing hypotheses proposed for the amino acid-mediated mTOR activation however the most accepted model suggests that amino acids operate through a class 3 phosphoinositide-3 kinase (PI-3K) located upstream of mTOR and independent of all insulin signalling mechanisms. Comparatively, glucose is thought to activate mTOR by increasing ATP levels which subsequently inhibit AMPK leading to an inactivation of TSC where it then follows the insulin-mediated downstream pathway to activate mTOR. Therefore, in a hyperglycaemic event, an immediate influx of glucose would create an overstimulation of mTOR leading to upregulation in mRNA translation and protein synthesis.
Since this complex signalling pathway is entirely regulated by factors originating from or within the diet, the levels of these stimulants are constantly shifting leading to highly variable effects on mTOR activation. Due to the immense importance of an efficient mTOR signalling network on the cellular processes it is responsible for, a look into how these nutrients and hormones can affect the signalling pathway by downregulating or stimulating its activation is required.
Nutrient Regulation of mTOR
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It has been shown through many in vitro and in vivo studies that resultant protein synthesis through mRNA translation initiation can be largely influenced through the levels of amino acids available and the type of amino acid supplied through the diet. Although there have been recent advances in mTOR signalling research, the molecular mechanisms of amino acid regulation of mTOR are still poorly understood. However there are areas within the amino acid sensing module that are in the process of becoming well recognized, such as intracellular import, the roles of amino acid metabolites as activators, and current findings in specific amino acid activation among others. It is known that when sufficient amino acids are lacking, mTOR signalling is quickly inhibited even when adequate insulin and other growth factors are present, thus strengthening the requirement for a better understanding of this sensoring mechanism.
Intracellular Amino Acid Import
The mTOR signalling pathway responds very well to amino acids as an activation factor, especially branched chain amino acids, leucine in particular. These amino acids enter the cell through various amino acid transporters located at the cell membrane where the signalling for mTOR activations begins. The current opinion is that a system L amino acid transporter is responsible for all amino acid import since this transport system has low specificity for the type of amino acid or structure and size of the molecule. This transporter imports many amino acids such as isoleucine, methionine, phenylalanine, valine, histidine including leucine, however the question arose of whether an additional transport system is in place when leucine was still imported into the cell when treated with a system L amino acid transporter inhibitor. An alternate mode of intracellular amino acid transport accountable for this observation is a transport system based on intracellular osmolarity, where Na+ dependent transporters import amino acids into the cells based on changing osmolarity due to an accumulation in amino acid metabolites. This mode of intracellular import is based on amino acid concentration rather than a specific membrane-based amino acid transporter making amino acid-mediated mTOR activation efficient and not hampering mRNA translation in cases of amino acid deprivation. Ultimately, the most proficient intracellular transport for leucine in particular is the system L amino acid transporter, which is not dependent on amino acid concentration and is Na+ independent. Therefore, there are two main modes of intracellular amino acid import, one which is based on intracellular amino acid concentrations and the other which allows for intracellular import regardless of concentration, molecular structure, size and type of amino acid.
Amino Acid Metabolites as mTOR Activators
It has been shown in several reports that amino acid-mediated mTOR activation may be partly attributed to specific products of leucine metabolism and directly contributing to downstream of mTOR protein phosphorylation and mRNA translation. Leucine, a branched chain amino acid, is the most studied factor of amino acid activation and the metabolism of this particular amino acid has stimulated new interest in potential novel mTOR activators. Leucine is catalyzed by a reversible aminotransferase to α-ketoisocaproic acid and further metabolized by branched-chain α-ketoacid dehydrogenase (BCKDH) into numerous TCA cycle intermediates. In addition, one of the main metabolites resulting from leucine catalysis is the production of ATP and is suggested to be a factor in leucine-induced mTOR activation. It is proposed that mTOR is activated through an intracellular ATP sensor and that in ATP-starved situations the phosphorylation and activation of downstream-mTOR proteins, S6K and 4EBP1, is inhibited and mRNA translation is attenuated. Conversely, new evidence questions whether leucine itself or its metabolites contribute to mTOR activation since various studies inhibiting leucine catalysis and the production of metabolic intermediates halted S6K phosphorylation. Furthermore, upon treatment with a nonmetabolized leucine analogue as well as with α-ketoisocaproic acid, a direct leucine metabolite, subsequent downstream phosphorylation was inhibited. These findings strongly suggest that the mTOR signalling network is potently activated by leucine through its metabolites, yet the mechanisms for activations are still to be elucidated. Moreover, since leucine has a fairly low rate of transamination it is not efficiently metabolized yet it remains as the most effective amino acid for mTOR activation leading to suggest that leucine metabolites are not solely responsible for mTOR signalling. In fact, when α-ketoisocaproic acid is reversely metabolized back to leucine, leucine itself has a stronger phosphorylation effect on 4EBP1 than the metabolite α-ketoisocaproic acid demonstrating that the nonmetabolized leucine remains as a key contributor in amino acid-mediated mTOR activation.
Current Research in Leucine-Induced mTOR Signaling
It is known that leucine is one of the most potent amino acid stimulators of the mTOR signalling network, but the direct effect on resultant protein synthesis