Glucose stimulates protein synthesis in skeletal muscle of neonatal pigs through an AMPK- and mTOR-independent process

Skeletal muscle protein synthesis is elevated in neonates in part due to an enhanced response to the rise in insulin and amino acids after eating. In vitro studies suggest that glucose plays a role in protein synthesis regulation. To determine whether glucose, independently of insulin and amino acids, is involved in the postprandial rise in skeletal muscle protein synthesis, pancreatic-substrate clamps were performed in neonatal pigs. Insulin secretion was inhibited with somatostatin and insulin was infused to reproduce fasting or fed levels, while glucose and amino acids were clamped at fasting or fed levels. Fractional protein synthesis rates and translational control mechanisms were examined. Raising glucose alone increased protein synthesis in fast-twitch glycolytic muscles but not in other tissues. The response in muscle was associated with increased phosphorylation of protein kinase B (PKB) and enhanced formation of the active eIF4E.eIF4G complex but no change in phosphorylation of AMP-activated protein kinase (AMPK), tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mTOR), 4E-binding protein-1 (4E-BP1), ribosomal protein S6 kinase (S6K1), or eukaryotic elongation factor 2 (eEF2). Raising glucose, insulin, and amino acids increased protein synthesis in most tissues. The response in muscle was associated with phosphorylation of PKB, mTOR, S6K1, and 4E-BP1 and enhanced eIF4E.eIF4G formation. The results suggest that the postprandial rise in glucose, independently of insulin and amino acids, stimulates protein synthesis in neonates, and this response is specific to fast-twitch glycolytic muscle and occurs by AMPK- and mTOR-independent pathways.     

Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA 5' cap binding protein, which plays an important role in the control of translation. The activity of eIF4E is regulated by a family of repressor proteins, the 4E-binding proteins (4E-BPs), whose binding to eIF4E is determined by their phosphorylation state. When hyperphosphorylated, 4E-BPs do not bind to eIF4E. Phosphorylation of the 4E-BPs is effected by the phosphatidylinositol (PI) 3-kinase signal transduction pathway and is inhibited by rapamycin through its binding to FRAP/mTOR (FK506 binding protein-rapamycin-associated protein or mammalian target of rapamycin). Phosphorylation of 4E-BPs can also be induced by protein synthesis inhibitors. These observations led to the proposal that FRAP/mTOR functions as a "sensor" of the translational apparatus (E. J. Brown and S. L. Schreiber, Cell 86:517-520, 1996). To test this model, we have employed the tetracycline-inducible system to increase eIF4E expression. Removal of tetracycline induced eIF4E expression up to fivefold over endogenous levels. Strikingly, upon induction of eIF4E, 4E-BP1 became dephosphorylated and the extent of dephosphorylation was proportional to the expression level of eIF4E. Dephosphorylation of p70(S6k) also occurred upon eIF4E induction. In contrast, the phosphorylation of Akt, an upstream effector of both p70(S6k) and 4E-BP phosphorylation, was not affected by eIF4E induction. We conclude that eIF4E engenders a negative feedback loop that targets a component of the PI 3-kinase signalling pathway which lies downstream of PI 3-kinase.     

AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling

AMP-activated protein kinase (AMPK) is viewed as an energy sensor that acts to modulate glucose uptake and fatty acid oxidation in skeletal muscle. Given that protein synthesis is a high energy-consuming process, it may be transiently depressed during cellular energy stress. Thus, the intent of this investigation was to examine whether AMPK activation modulates the translational control of protein synthesis in skeletal muscle. Injections of 5-aminoimidazole-4-carboxamide 1-beta-d-ribonucleoside (AICAR) were used to activate AMPK in male rats. The activity of alpha1 AMPK remained unchanged in gastrocnemius muscle from AICAR-treated animals compared with controls, whereas alpha2 AMPK activity was significantly increased (51%). AICAR treatment resulted in a reduction in protein synthesis to 45% of the control value. This depression was associated with decreased activation of protein kinases in the mammalian target of rapamycin (mTOR) signal transduction pathway as evidenced by reduced phosphorylation of protein kinase B on Ser(473), mTOR on Ser(2448), ribosomal protein S6 kinase on Thr(389), and eukaryotic initiation factor eIF4E-binding protein on Thr(37). A reduction in eIF4E associated with eIF4G to 10% of the control value was also noted. In contrast, eIF2B activity remained unchanged in response to AICAR treatment and therefore would not appear to contribute to the depression in protein synthesis. This is the first investigation to demonstrate changes in translation initiation and skeletal muscle protein synthesis in response to AMPK activation.     

Mechanisms through which sulfur amino acids control protein metabolism and oxidative status

Amino acids regulate protein synthesis and breakdown (i.e., protein turnover) and consequently protein deposition, which corresponds to the balance between the two processes. Elucidating the mechanisms involved in such regulation is important from fundamental and applied points of view since it can provide a basis to optimize amino acid requirements and to control protein mass, body composition and so forth. Amino acids, which have long been considered simply as precursors of protein synthesis, are now recognized to exert other significant influences; that is, they are precursors of essential molecules, act as mediators or signal molecules and affect numerous functions. For example, amino acids act as mediators of metabolic pathways in the same manner as certain hormones. Thus, they modulate the activity of intracellular protein kinases involved in the regulation of metabolic pathways such as mRNA translation. We provide here an overview of the roles of amino acids as regulators of protein metabolism, by focusing particularly on sulfur amino acids. The potential importance of methionine as a "nutrient signal" is discussed in the light of recent findings. Emphasis is also placed on mechanisms controlling oxidative status since sulfur amino acids are involved in the synthesis of intracellular antioxidants (glutathione, taurine etc.) and in the methionine sulfoxide reductase antioxidant system.     

Manipulation of the host translation initiation complex eIF4F by DNA viruses

In the absence of their own translational machinery, all viruses must gain access to host cell ribosomes to synthesize viral proteins and replicate. Ribosome recruitment and scanning of capped host mRNAs is facilitated by the multisubunit eIF (eukaryotic initiation factor) 4F, which consists of a cap-binding protein, eIF4E and an RNA helicase, eIF4A, assembled on a large scaffolding protein, eIF4G. Although inactivated by many viruses to inhibit host translation, a growing number of DNA viruses are being found to employ diverse strategies to stimulate eIF4F activity in infected cells and maximize viral protein synthesis. These strategies include stimulation of cellular mTOR (mammalian target of rapamycin) signalling to inactivate 4E-BPs (eIF4E-binding proteins), a family of translational repressors that limit eIF4E availability and eIF4F complex formation, together with modulating the activity of the eIF4E kinase Mnk (mitogen-activated protein kinase signal-integrating kinase) in a variety of manners to regulate both host and viral mRNA translation. In some cases, specific viral proteins that mediate these signalling events have been identified, whereas others have been shown to interact with host translation initiation factors or complexes and modify their activity and/or subcellular localization. The present review outlines current understanding of the role of eIF4F in the life cycle of various DNA viruses and discusses its potential as a therapeutic target to suppress viral infection.     

eIF4A inactivates TORC1 in response to amino acid starvation

Amino acids regulate TOR complex 1 (TORC1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC1 recruitment to lysosomes where TORC1 is activated by binding Rheb. How the absence of amino acids inactivates TORC1 is less well understood. Amino acid starvation recruits the TSC1/TSC2 complex to the vicinity of TORC1 to inhibit Rheb; however, the upstream mechanisms regulating TSC2 are not known. We identify here the eIF4A‐containing eIF4F translation initiation complex as an upstream regulator of TSC2 in response to amino acid withdrawal in Drosophila. We find that TORC1 and translation preinitiation complexes bind each other. Cells lacking eIF4F components retain elevated TORC1 activity upon amino acid removal. This effect is specific for eIF4F and not a general consequence of blocked translation. This study identifies specific components of the translation machinery as important mediators of TORC1 inactivation upon amino acid removal.     

Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs

Previous studies have shown that intravenous infusion of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates and that insulin and amino acids act independently to produce this effect. The goal of the present study was to delineate the regulatory roles of insulin and amino acids on muscle protein synthesis in neonates by examining translational control mechanisms, specifically the eukaryotic translation initiation factors (eIFs), which enable coupling of initiator methionyl-tRNAi and mRNA to the 40S ribosomal subunit. Insulin secretion was blocked by somatostatin in fasted 7-day-old pigs (n = 8-12/group), insulin was infused to achieve plasma levels of approximately 0, 2, 6, and 30 microU/ml, and amino acids were clamped at fasting or fed levels or, at the high insulin dose, below fasting. Both insulin and amino acids increased the phosphorylation of ribosomal protein S6 kinase (S6K1) and the eIF4E-binding protein (4E-BP1), decreased the binding of 4E-BP1 to eIF4E, increased eIF4E binding to eIF4G, and increased fractional protein synthesis rates but did not affect eIF2B activity. In the absence of insulin, amino acids had no effect on these translation initiation factors but increased the protein synthesis rates. Raising insulin from below fasting to fasting levels generally did not alter translation initiation factor activity but raised protein synthesis rates. The phosphorylation of S6K1 and 4E-BP1 and the amount of 4E-BP1 bound to eIF4E and eIF4E bound to eIF4G were correlated with insulin level, amino acid level, and protein synthesis rate. Thus insulin and amino acids regulate muscle protein synthesis in skeletal muscle of neonates by modulating the availability of eIF4E for 48S ribosomal complex assembly, although other processes also must be involved.     

Signalling to translation: how signal transduction pathways control the protein synthetic machinery

Recent advances in our understanding of both the regulation of components of the translational machinery and the upstream signalling pathways that modulate them have provided important new insights into the mechanisms by which hormones, growth factors, nutrients and cellular energy status control protein synthesis in mammalian cells. The importance of proper control of mRNA translation is strikingly illustrated by the fact that defects in this process or its control are implicated in a number of disease states, such as cancer, tissue hypertrophy and neurodegeneration. Signalling pathways such as those involving mTOR (mammalian target of rapamycin) and mitogen-activated protein kinases modulate the phosphorylation of translation factors, the activities of the protein kinases that act upon them and the association of RNA-binding proteins with specific mRNAs. These effects contribute both to the overall control of protein synthesis (which is linked to cell growth) and to the modulation of the translation or stability of specific mRNAs. However, important questions remain about both the contributions of individual regulatory events to the control of general protein synthesis and the mechanisms by which the translation of specific mRNAs is controlled.     

Repressor binding to a dorsal regulatory site traps human eIF4E in a high cap-affinity state.

Eukaryotic translation initiation involves recognition of the 5' end of cellular mRNA by the cap-binding complex known as eukaryotic initiation factor 4F (eIF4F). Initiation is a key point of regulation in gene expression in response to mechanisms mediated by signal transduction pathways. We have investigated the molecular interactions underlying inhibition of human eIF4E function by regulatable repressors called 4E-binding proteins (4E-BPs). Two essential components of eIF4F are the cap-binding protein eIF4E, and eIF4G, a multi-functional protein that binds both eIF4E and other essential eIFs. We show that the 4E-BPs 1 and 2 block the interaction between eIF4G and eIF4E by competing for binding to a dorsal site on eIF4E. Remarkably, binding of the 4E-BPs at this dorsal site enhances cap-binding via the ventral cap-binding slot, thus trapping eIF4E in inactive complexes with high affinity for capped mRNA. The binding contacts and affinities for the interactions between 4E-BP1/2 and eIF4E are distinct (estimated K(d) values of 10(-8) and 3x10(-9) for 4E-BP1 and 2, respectively), and the differences in these properties are determined by three amino acids within an otherwise conserved motif. These data provide a quantitative framework for a new molecular model of translational regulation.     

Tissue-specific regulation of protein synthesis by insulin and free fatty acids

The purpose of the study described herein was to investigate how the mammalian target of rapamycin (mTOR)-signaling pathway and eukaryotic initiation factor 2B (eIF2B) activity, both having key roles in the translational control of protein synthesis in skeletal muscle, are regulated in cardiac muscle of rats in response to two different models of altered free fatty acid (FFA) and insulin availability. Protein synthetic rates were reduced in both gastrocnemius and heart of 3-day diabetic rats. The reduction was associated with diminished mTOR-mediated signaling and eIF2B activity in the gastrocnemius but only with diminished mTOR signaling in the heart. In response to the combination of acute hypoinsulinemia and hypolipidemia induced by administration of niacin, protein synthetic rates were also diminished in both gastrocnemius and heart. The niacin-induced changes were associated with diminished mTOR signaling and eIF2B activity in the heart but only with decreased mTOR signaling in the gastrocnemius. In the heart, mTOR signaling and eIF2B activity correlated with cellular energy status and/or redox potential. Thus FFAs may contribute to the translational control of protein synthesis in the heart but not in the gastrocnemius. In contrast, insulin, but not FFAs, is required for the maintenance of protein synthesis in the gastrocnemius.     

PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2alpha

Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.     

mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events

In response to nutrients, energy sufficiency, hormones, and mitogenic agents, S6K1 phosphorylates several targets linked to translation. However, the molecular mechanisms whereby S6K1 is activated, encounters substrate, and contributes to translation initiation are poorly understood. We show that mTOR and S6K1 maneuver on and off the eukaryotic initiation factor 3 (eIF3) translation initiation complex in a signal-dependent, choreographed fashion. When inactive, S6K1 associates with the eIF3 complex, while the S6K1 activator mTOR/raptor does not. Cell stimulation promotes mTOR/raptor binding to the eIF3 complex and phosphorylation of S6K1 at its hydrophobic motif. Phosphorylation results in S6K1 dissociation, activation, and subsequent phosphorylation of its translational targets, including eIF4B, which is then recruited into the complex in a phosphorylation-dependent manner. Thus, the eIF3 preinitiation complex acts as a scaffold to coordinate a dynamic sequence of events in response to stimuli that promote efficient protein synthesis.     

Identification of a conserved motif required for mTOR signaling

BACKGROUND: The mammalian target of rapamycin (mTOR) controls the translation machinery via activation of S6 kinases 1 and 2 (S6K1/2) and inhibition of the eukaryotic initiation factor 4E (eIF4E) binding proteins 1, 2, and 3 (4E-BP1/2/3). S6K1 and 4E-BP1 are regulated by nutrient-sensing and mitogen-activated pathways. The molecular basis of mTOR regulation of S6K1 and 4E-BP1 remains controversial. RESULTS: We have identified a conserved TOR signaling (TOS) motif in the N terminus of all known S6 kinases and in the C terminus of the 4E-BPs that is crucial for phosphorylation and regulation S6K1 and 4E-BP1 activities. Deletion or mutations within the TOS motif significantly inhibit S6K1 activation and the phosphorylation of its hydrophobic motif, Thr389. In addition, this sequence is required to suppress an inhibitory activity mediated by the S6K1 C terminus. The TOS motif is essential for S6K1 activation by mTOR, as mutations in this motif mimic the effect of rapamycin on S6K1 phosphorylation, and render S6K1 insensitive to changes in amino acids. Furthermore, only overexpression of S6K1 with an intact TOS motif prevents 4E-BP1 phosphorylation by a common mTOR-regulated modulator of S6K1 and 4E-BP1. CONCLUSIONS: S6K1 and 4E-BP1 contain a conserved five amino acid sequence (TOS motif) that is crucial for their regulation by the mTOR pathway. mTOR seems to regulate S6K1 by two distinct mechanisms. The TOS motif appears to function as a docking site for either mTOR itself or a common upstream activator of S6K1 and 4E-BP1.     

Requirement for the eIF4E Binding Proteins for the Synergistic Down-Regulation of Protein Synthesis by Hypertonic Conditions and mTOR Inhibition

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.     

Glucocorticoids modulate amino acid-induced translation initiation in human skeletal muscle

Amino acids are unique anabolic agents in that they nutritively signal to mRNA translation initiation and serve as substrates for protein synthesis in skeletal muscle. Glucocorticoid excess antagonizes the anabolic action of amino acids on protein synthesis in laboratory animals. To examine whether excessive glucocorticoids modulate mixed amino acid-signaled translation initiation in human skeletal muscle, we infused an amino acid mixture (10% Travasol) systemically to 16 young healthy male volunteers for 6 h in the absence (n = 8) or presence (n = 8) of glucocorticoid excess (dexamethasone 2 mg orally every 6 h for 3 days). Vastus lateralis muscles were biopsied before and after amino acid infusion, and the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (p70(S6K)), and eIF2alpha and the guanine nucleotide exchange activity of eIF2B were measured. Systemic infusion of mixed amino acids significantly stimulated the phosphorylation of 4E-BP1 (P < 0.04) and p70(S6K) (P < 0.001) and the dephosphorylation of eIF2alpha (P < 0.003) in the control group. Dexamethasone treatment did not alter the basal phosphorylation state of 4E-BP1, p70(S6K), or eIF2alpha; however, it abrogated the stimulatory effect of amino acid infusion on the phosphorylation of 4E-BP1 (P = 0.31) without affecting amino acid-induced phosphorylation of p70(S6K) (P = 0.002) or dephosphorylation of eIF2alpha (P = 0.003). Neither amino acid nor dexamethasone treatment altered the guanine nucleotide exchange activity of eIF2B. We conclude that changes of amino acid concentrations within the physiological range stimulate mRNA translation by enhancing the binding of mRNA to the 43S preinitiation complex, and the activity of p70(S6K) and glucocorticoid excess blocks the former action in vivo in human skeletal muscle.     

Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3′-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.     

Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis

Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.     

mTOR的研究进展

mTOR(mammaliantargetofrapamycin)是丝氨酸/苏氨酸蛋白激酶,在感受营养信号、调节细胞生长与增殖中起着关键性的作用。mTOR可磷酸化p70S6K和4E-BP1,促进蛋白质合成。mTOR的活性受氨基酸尤其是亮氨酸浓度的调节,生长因子及能量水平也能通过AMPK调节mTOR活性。PI3K/Akt和Akt/TSC1-TSC2两条信号通路都可调控mTOR活性,进而调节细胞的生长与增殖。mTOR信号通路的异常会导致肿瘤的发生,可以针对mTOR研制出治疗肿瘤的靶向药物。