Elevated plasma free fatty acids decrease basal protein synthesis, but not the anabolic effect of leucine, in skeletal muscle

Elevations in free fatty acids (FFAs) impair glucose uptake in skeletal muscle. However, there is no information pertaining to the effect of elevated circulating lipids on either basal protein synthesis or the anabolic effects of leucine and insulin-like growth factor I (IGF-I). In chronically catheterized conscious rats, the short-term elevation of plasma FFAs by the 5-h infusion of heparin plus Intralipid decreased muscle protein synthesis by approximately 25% under basal conditions. Lipid infusion was associated with a redistribution of eukaryotic initiation factor (eIF)4E from the active eIF4E.eIF4G complex to the inactive eIF4E.4E-BP1 complex. This shift was associated with a decreased phosphorylation of eIF4G but not 4E-BP1. Lipid infusion did not significantly alter either the total amount or phosphorylation state of mTOR, TSC2, S6K1, or the ribosomal protein S6 under basal conditions. In control rats, oral leucine increased muscle protein synthesis. This anabolic response was not impaired by lipid infusion, and no defects in signal transduction pathways regulating translation initiation were detected. In separate rats that received a bolus injection of IGF-I, lipid infusion attenuated the normal redistribution of eIF4E from the active to inactive complex and largely prevented the increased phosphorylation of 4E-BP1, eIF4G, S6K1, and S6. This IGF-I resistance was associated with enhanced Ser(307) phosphorylation of insulin receptor substrate-1 (IRS-1). These data indicate that the short-term elevation of plasma FFAs impairs basal protein synthesis in muscle by altering eIF4E availability, and this defect may be related to impaired phosphorylation of eIF4G, not 4E-BP1. Moreover, hyperlipidemia impairs IGF-I action but does not produce leucine resistance in skeletal muscle.     

TNFalpha mediates sepsis-induced impairment of basal and leucine-stimulated signaling via S6K1 and eIF4E in cardiac muscle

Decreased translation initiation adversely impacts protein synthesis and contributes to the myocardial dysfunction produced by sepsis. Therefore, the purpose of the present study was to identify sepsis-induced changes in signal transduction pathways known to regulate translation initiation in cardiac muscle and to determine whether the stimulatory effects of leucine can reverse the observed defects. To address this aim, sepsis was produced by cecal ligation and puncture (CLP) in anesthetized rats and the animals studied in the fasted condition 24 h later. Separate groups of septic and time-matched control rats also received an oral gavage of leucine. To identify potential mechanisms responsible for regulating cap-dependent mRNA translation in cardiac muscle, several eukaryotic initiation factors (eIFs) were examined. Under basal conditions, hearts from septic rats demonstrated a redistribution of the rate-limiting factor eIF4E due to increased binding of the translational repressor 4E-BP1 with eIF4E. However, this change was independent of an alteration in the phosphorylation state of 4E-BP1. The phosphorylation of mTOR, S6K1, the ribosomal protein (rp) S6, and eIF4G was not altered in hearts from septic rats under basal conditions. In control rats, leucine failed to alter eIF4E distribution but increased the phosphorylation of S6K1 and S6. In contrast, in hearts from septic rats leucine acutely reversed the alterations in eIF4E distribution. However, the ability of leucine to increase S6K1 and rpS6 phosphorylation in septic hearts was blunted. Sepsis increased the content of tumor necrosis factor (TNF)-alpha in heart and pre-treatment of rats with a TNF antagonist prevented the above-mentioned sepsis-induced changes. These data indicate that oral administration of leucine acutely reverses sepsis-induced alterations eIF4E distribution observed under basal conditions but the anabolic actions of this amino acid on S6K1 and rpS6 phosphorylation remain blunted, providing evidence for a leucine resistance. Finally, TNFalpha, either directly or indirectly, appears to mediate the sepsis-induced defects in myocardial translation initiation.     

Endotoxin disrupts the leucine-signaling pathway involving phosphorylation of mTOR, 4E-BP1, and S6K1 in skeletal muscle

Endotoxin (i.e., lipopolysaccharide, LPS) impairs skeletal muscle protein synthesis. Although this impairment is not acutely associated with a decreased plasma concentration of total amino acids, LPS may blunt the anabolic response to amino acids. To examine this hypothesis, rats were injected intraperitoneally with LPS or saline (Sal) and 4 h thereafter were orally administered either leucine (Leu) or Sal. The gastrocnemius was removed 20 min later to assess signaling components important in the translational control of protein synthesis. In the Sal-Leu group phosphorylation of 4E-BP1 in muscle was markedly increased, compared to values from time-matched saline-treated control rats. This change was associated with a redistribution of eukaryotic initiation factor (eIF) 4E from the inactive eIF4E x 4E-BP1 complex to the active eIF4E x eIF4G complex. In LPS-treated rats, the Leu-induced phosphorylation of 4E-BP1 and changes in eIF4E distribution were partially or completely abrogated. LPS also antagonized the Leu-induced increase in phosphorylation of S6K1, ribosomal protein S6 and mTOR. Neither LPS nor leu altered the total amount or phosphorylation of TSC2 in muscle. The ability of LPS to blunt the anabolic effects of Leu could not be attributed to differences in the plasma concentrations of insulin or Leu between groups. Furthermore, the replacement of plasma insulin-like growth factor (IGF)-I in LPS-treated rats to basal levels also did not ameliorate the defect in leucine-induced phosphorylation of S6K1 or S6, although it did reverse the LPS-induced decrease in the constitutive phosphorylation of mTOR, S6 and 4E-BP1. Pretreatment with the glucocorticoid receptor antagonist RU486 was unable to prevent the LPS-induced leucine resistance. In contrast, to the abovementioned results with leucine, LPS did not prevent the ability of pharmacological levels of IGF-I to phosphorylate 4E-BP1, S6K1, mTOR or alter the availability of eIF4E. Hence, LPS working via a glucocorticoid-independent mechanism produces a leucine resistance in skeletal muscle that might be expected to impair the ability of this amino acid to stimulate translation initiation and protein synthesis.     

Clofibrate treatment promotes branched-chain amino acid catabolism and decreases the phosphorylation state of mTOR, eIF4E-BP1, and S6K1 in rat liver

Leucine stimulates protein synthesis by modulating the mammalian target of rapamycin (mTOR) signaling pathway. We hypothesized that promotion of the branched-chain amino acid (BCAA) catabolism might influence the leucine-induced protein synthesis. Clofibric acid (an active metabolite of clofibrate) is known to promote the BCAA catabolism by activation of branched-chain alpha-keto acid dehydrogenase complex (BCKDC), the rate-limiting enzyme of the BCAA catabolism. In the present study, we examined the phosphorylation state of mTOR, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), and ribosomal protein S6 kinase 1 (S6K1) in liver of rats with or without activation of the BCKDC by clofibrate treatment. Clofibrate-treated rats were prepared by oral administration of clofibrate 5 h before sacrifice. In order to stimulate phosphorylation of components in the mTOR signaling pathway, rats were orally administered with leucine 1 h before sacrifice. Clofibrate treatment almost fully activated hepatic BCKDC and significantly decreased the plasma leucine concentration in rats without leucine administration, resulting in decreased mTOR and 4E-BP1 phosphorylation. Similarly, in rats administered with leucine, clofibrate treatment attenuated the predicted increase in plasma leucine concentration as well as the phosphorylation of mTOR, 4E-BP1, and S6K1. These results suggest that BCAA catabolism enhanced by clofibrate treatment has significant influences on the leucine-induced activation of translation initiation processes.     

Adding protein to a carbohydrate supplement provided after endurance exercise enhances 4E-BP1 and RPS6 signaling in skeletal muscle.

To examine the role of both endurance exercise and nutrient supplementation on the activation of mRNA translation signaling pathways postexercise, rats were subjected to a 3-h swimming protocol. Immediately following exercise, the rats were provided with a solution containing either 23.7% wt/vol carbohydrates (CHO), 7.9% wt/vol protein (Pro), 31.6% wt/vol (23.7% wt/vol CHO + 7.9% wt/vol Pro) carbohydrates and Pro (CP), or a placebo (EX). The rats were then killed at 0, 30, and 90 min postexercise, and phosphorylation states of mammalian target of rapamycin (mTOR), ribosomal S6 kinase (p70(S6K)), ribosomal protein S6 (rpS6), and 4E-binding protein 1 (4E-BP1), were analyzed by immunoblot analysis in the red and white quadriceps muscle. Results demonstrated that rat groups provided with any of the three nutritional supplements (CHO, Pro, CP) transiently increased the phosphorylation states of mTOR, 4E-BP1, rpS6, and p70(S6K) compared with EX rats. Although CHO, Pro, and CP supplements phosphorylated mTOR and p70(S6K) after exercise, only CP elevated the phosphorylation of rpS6 above all other supplements 30 min postexercise and 4E-BP1 30 and 90 min postexercise. Furthermore, the phosphorylation states of 4E-BP1 (r(2) = 0.7942) and rpS6 (r(2) = 0.760) were highly correlated to insulin concentrations in each group. These results suggest that CP supplementation may be most effective in activating the mTOR-dependent signaling pathway in the postprandial state postexercise, and that there is a strong relationship between the insulin concentration and the activation of enzymes critical for mRNA translation.     

Glucagon represses signaling through the mammalian target of rapamycin in rat liver by activating AMP-activated protein kinase

The opposing actions of glucagon and insulin on glucose metabolism within the liver are essential mechanisms for maintaining plasma glucose concentrations within narrow limits. Less well studied are the counterregulatory actions of glucagon on protein metabolism. In the present study, the effect of glucagon on amino acid-induced signaling through the mammalian target of rapamycin (mTOR), an important controller of the mRNA binding step in translation initiation, was examined using the perfused rat liver as an experimental model. The results show that amino acids enhance signaling through mTOR resulting in phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP)1, the 70-kDa ribosomal protein (rp)S6 kinase, S6K1, and rpS6. In contrast, glucagon repressed both basal and amino acid-induced signaling through mTOR, as assessed by changes in the phosphorylation of 4E-BP1 and S6K1. The repression was associated with the activation of protein kinase A and enhanced phosphorylation of LKB1 and the AMP-activated protein kinase (AMPK). Surprisingly, the phosphorylation of two S6K1 substrates, rpS6 and eukaryotic initiation factor 4B, was not repressed but instead was increased by glucagon treatment, regardless of the amino acid concentration. The latter finding could be explained by the glucagon-induced phosphorylation of the ERK1 and the 90-kDa rpS6 kinase p90(rsk). Thus, glucagon represses phosphorylation of 4E-BP1 and S6K1 through the activation of a protein kinase A-LKB-AMPK-mTOR signaling pathway, while simultaneously enhancing phosphorylation of other downstream effectors of mTOR through the activation of the extracellular signal-regulated protein kinase 1-p90(rsk) signaling pathway. Amino acids also enhance AMPK phosphorylation, although to a lesser extent than glucagon and amino acids combined.     

Alcohol impairs leucine-mediated phosphorylation of 4E-BP1, S6K1, eIF4G,and mTOR in skeletal muscle

Acute alcohol (EtOH) intoxication impairs skeletal muscle protein synthesis. Although this impairment is not associated with a decrease in the total plasma amino acid concentration, EtOH may blunt the anabolic response to amino acids. To examine this hypothesis, rats were administered EtOH or saline (Sal) and 2.5 h thereafter were orally administered either leucine (Leu) or Sal. The gastrocnemius was removed 20 min later to assess protein synthesis and signaling components important in translational control of protein synthesis. Oral Leu increased muscle protein synthesis by the same magnitude in Sal- and EtOH-treated rats. However, the increase in the latter group was insufficient to overcome the suppressive effect of EtOH, and the rate of synthesis remained lower than that observed in rats from the Sal-Sal group. Leu markedly increased phosphorylation of Thr residues 36, 47, and 70 on 4E-binding protein (BP)1 in muscle from rats not receiving EtOH, and this response was associated with a redistribution of eukaryotic initiation factor (eIF) 4E from the inactive eIF4E. 4E-BP1 to the active eIF4E. eIF4G complex. In EtOH-treated rats, the Leu-induced phosphorylation of 4E-BP1 and changes in eIF4E availability were partially abrogated. EtOH also prevented the Leu-induced increase in phosphorylation of eIF4G, the serine/threonine protein kinase S6K1, and the ribosomal protein S6. Moreover, EtOH attenuated the Leu-induced phosphorylation of the mammalian target of rapamycin (mTOR). The ability of EtOH to blunt the anabolic effects of Leu could not be attributed to differences in the plasma concentrations of insulin, insulin-like growth factor I, or Leu. Finally, although EtOH increased the plasma corticosterone concentration, inhibition of glucocorticoid action by RU-486 was unable to prevent EtOH-induced defects in the ability of Leu to stimulate 4E-BP1, S6K1, and mTOR phosphorylation. Hence, ethanol produces a leucine resistance in skeletal muscle, as evidenced by the impaired phosphorylation of 4E-BP1, eIF4G, S6K1, and mTOR, that is independent of elevations in endogenous glucocorticoids.     

Rapamycin blunts nutrient stimulation of eIF4G, but not PKCepsilon phosphorylation, in skeletal muscle

Phosphorylation of eukaryotic initiation factor 4G (eIF4G) is hypothesized to be an important contributor to the stimulation of protein synthesis in skeletal muscle following meal feeding. The experiments reported herein examined the potential role for a rapamycin-sensitive signaling pathway in mediating the meal feeding-induced elevations in phosphorylation of eIF4G. Gastrocnemius from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled prior to and following 3 h of having the meal provided in the presence or absence of treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Pretreatment with rapamycin prevented the feeding-induced phosphorylation of mTOR, eIF4G, and S6K1 but only partially attenuated the shift in 4E-BP1 into the gamma-form. In contrast, the feeding-induced increase in phosphorylation of PKCepsilon was not reduced by rapamycin. Rapamycin also prevented the augmented association of eIF4G with eIF4E and the decreased association of eIF4E with 4E-BP1. Similar findings were observed in gastrocnemius from animals after oral administration of leucine. Perfusion of gastrocnemius with medium containing rapamycin partially prevented the leucine-induced increase in phosphorylation of eIF4G. Thus, rapamycin attenuated a feeding- or leucine-induced phosphorylation of eIF4G in skeletal muscle both in vivo and in situ. The latter observation implies that the effects observed with rapamycin were the result of modulation of skeletal muscle signaling mechanisms responsible for eIF4G phosphorylation.     

Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway.

Ribosomal S6 kinase 1 (S6K1) is a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and plays a regulatory role in translation initiation, protein synthesis, and muscle hypertrophy. AMP-activated protein kinase (AMPK) is a cellular energy sensor, a negative regulator of mTOR, and an inhibitor of protein synthesis. The purpose of this study was to determine whether the hypertrophy/cell growth-associated mTOR pathway was downregulated during muscle atrophy associated with chronic paraplegia. Soleus muscle was collected from male Sprague-Dawley rats 10 wk following complete T(4)-T(5) spinal cord transection (paraplegic) and from sham-operated (control) rats. We utilized immunoprecipitation and Western blotting techniques to measure upstream [AMPK, Akt/protein kinase B (PKB)] and downstream components of the mTOR signaling pathway [mTOR, S6K1, SKAR, 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor (eIF) 4G and 2alpha]. Paraplegia was associated with significant soleus muscle atrophy (174 +/- 8 vs. 240 +/- 13 mg; P < 0.05). There was a reduction in phosphorylation of mTOR, S6K1, and eIF4G (P < 0.05) with no change in Akt/PKB or 4E-BP1 (P > 0.05). Total protein abundance of mTOR, S6K1, eIF2alpha, and Akt/PKB was decreased, and increased for SKAR (P < 0.05), whereas 4E-BP1 and eIF4G did not change (P > 0.05). S6K1 activity was significantly reduced in the paraplegic group (P < 0.05); however, AMPKalpha2 activity was not altered (3.5 +/- 0.4 vs. 3.7 +/- 0.5 pmol x mg(-1) x min(-1), control vs. paraplegic rats). We conclude that paraplegia-induced muscle atrophy in rats is associated with a general downregulation of the mTOR signaling pathway. Therefore, in addition to upregulation of atrophy signaling during muscle wasting, downregulation of muscle cell growth/hypertrophy-associated signaling appears to be an important component of long-term muscle loss.     

Differential effect of sepsis on ability of leucine and IGF-I to stimulate muscle translation initiation

Polymicrobial sepsis impairs skeletal muscle protein synthesis, which results from impairment in translation initiation under basal conditions. The purpose of the present study was to test the hypothesis that sepsis also impairs the anabolic response to amino acids, specifically leucine (Leu). Sepsis was induced by cecal ligation and puncture, and 24 h later, Leu or saline (Sal) was orally administered to septic and time-matched nonseptic rats. The gastrocnemius was removed 20 min later for assessment of protein synthesis and signaling components important in peptide-chain initiation. Oral Leu increased muscle protein synthesis in nonseptic rats. Leu was unable to increase protein synthesis in muscle from septic rats, and synthetic rates remained below those observed in nonseptic + Sal rats. In nonseptic + Leu rats, phosphorylation of eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1) in muscle was markedly increased compared with values from time-matched Sal-treated nonseptic rats. This change was associated with redistribution of eIF4E from the inactive eIF4E.4E-BP1 to the active eIF4E.eIF4G complex. In septic rats, Leu-induced phosphorylation of 4E-BP1 and changes in eIF4E distribution were completely abrogated. Sepsis also antagonized the Leu-induced increase in phosphorylation of S6 kinase 1 and ribosomal protein S6. Sepsis attenuated Leu-induced phosphorylation of mammalian target of rapamycin and eIF4G. The ability of sepsis to inhibit anabolic effects of Leu could not be attributed to differences in plasma concentrations of insulin, insulin-like growth factor I, or Leu between groups. In contrast, the ability of exogenous insulin-like growth factor I to stimulate the same signaling components pertaining to translation initiation was not impaired by sepsis. Hence, sepsis produces a relatively specific Leu resistance in skeletal muscle that impairs the ability of this amino acid to stimulate translation initiation and protein synthesis.     

Regulation of global and specific mRNA translation by oral administration of branched-chain amino acids

The importance of branched-chain amino acids as nutrient regulators of protein synthesis in skeletal muscle was recognized more than 20 years ago. Of the branched-chain amino acids, leucine in particular was shown to play a central role in promoting muscle protein synthesis. However, it was only recently that the mechanism(s) involved in the stimulation of protein synthesis by leucine has begun to be defined. Studies performed in our laboratory during the past few years have revealed that oral administration of leucine to fasted rats enhances protein synthesis in association with increased phosphorylation of two proteins downstream of the protein kinase referred to as the mammalian target of rapamycin (mTOR). These proteins, eukaryotic initiation factor eIF4E binding protein (4E-BP)1 and ribosomal protein S6 kinase S6K1, control in part the step in translation initiation involving the binding of mRNA to the 40S ribosomal subunit. In theory the translation of all mRNAs can be regulated through such mechanisms, however, some mRNAs are more sensitive to the changes than others, resulting in modulation of gene expression through altered patterns of translation of specific mRNAs. Moreover, although a basal amount of plasma insulin is required for leucine to enhance signaling downstream of mTOR, the concentration observed in plasma of fasted rats is sufficient to observe maximal changes in phosphorylation of 4E-BP1 and S6K1.     

Regulation of muscle protein synthesis during sepsis and inflammation

Prolonged sepsis and exposure to an inflammatory milieu decreases muscle protein synthesis and reduces muscle mass. As a result of its ability to integrate diverse signals, including hormones and nutrients, the mammalian target of rapamycin (mTOR) is a dominant regulator in the translational control of protein synthesis. Under postabsorptive conditions, sepsis decreases mTOR kinase activity in muscle, as evidenced by reduced phosphorylation of both eukaryotic initiation factor (eIF)4E-binding protein (BP)-1 and ribosomal S6 kinase (S6K)1. These sepsis-induced changes, along with the redistribution of eIF4E from the active eIF4E.eIF4G complex to the inactive eIF4E.4E-BP1 complex, are preventable by neutralization of tumor necrosis factor (TNF)-alpha but not by antagonizing glucocorticoid action. Although the ability of mTOR to respond to insulin-like growth factor (IGF)-I is not disrupted by sepsis, the ability of leucine to increase 4E-BP1 and S6K1 phosphorylation is greatly attenuated. This "leucine resistance" results from a cooperative interaction between both TNF-alpha and glucocorticoids. Finally, although septic animals are not IGF-I resistant, the anabolic actions of IGF-I are nonetheless reduced because of the development of growth hormone resistance, which decreases both circulating and muscle IGF-I. Herein, we highlight recent advances in the mTOR signaling network and emphasize their connection to the atrophic response observed in skeletal muscle during sepsis. Although many unanswered questions remain, understanding the cellular basis of the sepsis-induced decrease in translational activity will contribute to the rational development of therapeutic interventions and thereby minimize the debilitating affects of the atrophic response that impairs patient recovery.     

Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation

Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML) can be phenotypically dormant (quiescent), we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells) by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448) and its downstream targets ribosomal protein S6 (rpS6, S235/236) and 4E-BP1 (T36/45), we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML.     

Nutrient regulation of PKCepsilon is mediated by leucine, not insulin, in skeletal muscle

Nutrients enhance signaling pathways involved in skeletal muscle growth through an increased rate of protein synthesis. These studies have led to an understanding of the potential role of the mammalian target of rapamycin (mTOR) in this process. However, activation of mTOR cannot account for all the stimulatory effects of nutrients. The purpose of these experiments was to examine the effect of nutrients on the cellular distribution and activation state of novel PKC isoforms (PKCepsilon and PKCdelta) in the gastrocnemius of rats by use of modification state-dependent phosphopeptide-specific antibodies. The phosphorylation of PKCepsilon on the catalytic domain autophosphorylation site (Ser(729)) was elevated during feeding and then returned to basal levels when the feeding period ended. Meal feeding augmented the phosphorylation of the downstream effectors of mTOR, namely S6K1 and 4E-BP1. In contrast, the phosphorylation of PKCdelta on either the catalytic domain autophosphorylation site (Ser(643)) or activation loop site (Thr(505)) was unaffected. Similar results were obtained when animals were given leucine either acutely via gavage or chronically by dietary supplementations. The effect of leucine was not mimicked by injecting animals with insulin but could be induced by gavage with norleucine, a structural analog of leucine that does not increase plasma insulin concentration. Thus rises in insulin secondary to meal intake or leucine gavage are probably not responsible for increased phosphorylation of PKCepsilon in response to meal feeding. Elevating the leucine concentration stimulated the phosphorylation of PKCepsilon in gastrocnemius from perfused hindlimb and caused a shift in the distribution of PKCepsilon from the membrane fraction to the cytosolic fraction. The results indicate that leucine leads to an activation (autophosphorylation) and subcellular redistribution of PKCepsilon, but not PKCdelta, in gastrocnemius both in vivo and in vitro. Furthermore, activation of the mTOR signaling pathway above basal conditions does not appear to be necessary to induce phosphorylation or translocation of PKCepsilon, suggesting that multiple signaling pathways become activated with leucine.     

Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.     

Glutamine is a key regulator for amino acid-controlled cell growth through the mTOR signaling pathway in rat intestinal epithelial cells

Amino acids, especially branched-chain amino acids such as l-leucine, have been shown to regulate activation of p70 S6 kinase and phosphorylation of 4E-BP1 through the mTOR signaling pathway. In our recent study, l-arginine was also shown to activate the mTOR signaling pathway in rat intestinal epithelial cells. l-Glutamine is an amino acid that is required for culturing of numerous cell types, including rat intestinal epithelial cells. In this study, we showed that l-glutamine inhibited the activation of p70 S6 kinase and phosphorylation of 4E-BP1 induced by arginine or leucine in rat intestinal epithelial cells. Although the molecular mechanism of l-glutamine-induced inhibition of the mTOR signaling pathway is still unknown, the presence of this novel signal pathway may indicate that individual amino acids play specific roles for cellular proliferation and growth.     

Indinavir alters regulators of protein anabolism and catabolism in skeletal muscle

The HIV protease inhibitor indinavir adversely impairs carbohydrate and lipid metabolism, whereas its influence on protein metabolism under in vivo conditions remains unknown. The present study tested the hypothesis that indinavir also decreases basal protein synthesis and impairs the anabolic response to insulin in skeletal muscle. Indinavir was infused intravenously for 4 h into conscious rats, at which time the homeostasis model assessment of insulin resistance was increased. Indinavir decreased muscle protein synthesis by 30%, and this reduction was due to impaired translational efficiency. To identify potential mechanisms responsible for regulating mRNA translation, several eukaryotic initiation factors (eIFs) were examined. Under basal fasted conditions, there was a redistribution of eIF4E from the active eIF4E.eIF4G complex to the inactive eIF4E.4E-BP1 complex, and this change was associated with a marked decrease in the phosphorylation of 4E-BP1 in muscle. Likewise, indinavir decreased constitutive phosphorylation of eIF4G and mTOR in muscle, but not S6K1 or the ribosomal protein S6. In contrast, the ability of a maximally stimulating dose of insulin to increase the phosphorylation of PKB, 4E-BP1, S6K1, or mTOR was not altered 20 min after intravenous injection. Indinavir increased mRNA expression of the ubiquitin ligase MuRF1, but the plasma concentration of 3-methylhistidine remained unaltered. These indinavir-induced changes were associated with a marked reduction in the plasma testosterone concentration but were independent of changes in plasma levels of IGF-I, corticosterone, TNF-alpha, or IL-6. In conclusion, indinavir acutely impairs basal protein synthesis and translation initiation in skeletal muscle but, in contrast to muscle glucose uptake, does not impair insulin-stimulated signaling of protein synthetic pathways.     

Nutritionally essential amino acids and metabolic signaling in aging

Aging is associated with a gradual decline in skeletal muscle mass and strength leading to increased risk for functional impairments. Although basal rates of protein synthesis and degradation are largely unaffected with age, the sensitivity of older muscle cells to the anabolic actions of essential amino acids appears to decline. The major pathway through which essential amino acids induce anabolic responses involves the mammalian target of rapamycin (mTOR) Complex 1, a signaling pathway that is especially sensitive to regulation by the branched chain amino acid leucine. Recent evidence suggests that muscle of older individuals require increasing concentrations of leucine to maintain robust anabolic responses through the mTOR pathway. While the exact mechanisms for the age-related alterations in nutritional signaling through the mTOR pathway remain elusive, there is increasing evidence that decreased sensitivity to insulin action, reductions in endothelial function, and increased oxidative stress may be underlying factors in this decrease in anabolic sensitivity. Ensuring adequate nutrition, including sources of high quality protein, and promoting regular physical activity will remain among the frontline defenses against the onset of sarcopenia in older individuals.     

The Translation Regulatory Subunit eIF3f Controls the Kinase-Dependent mTOR Signaling Required for Muscle Differentiation and Hypertrophy in Mouse

The mTORC1 pathway is required for both the terminal muscle differentiation and hypertrophy by controlling the mammalian translational machinery via phosphorylation of S6K1 and 4E-BP1. mTOR and S6K1 are connected by interacting with the eIF3 initiation complex. The regulatory subunit eIF3f plays a major role in muscle hypertrophy and is a key target that accounts for MAFbx function during atrophy. Here we present evidence that in MAFbx-induced atrophy the degradation of eIF3f suppresses S6K1 activation by mTOR, whereas an eIF3f mutant insensitive to MAFbx polyubiquitination maintained persistent phosphorylation of S6K1 and rpS6. During terminal muscle differentiation a conserved TOS motif in eIF3f connects mTOR/raptor complex, which phosphorylates S6K1 and regulates downstream effectors of mTOR and Cap-dependent translation initiation. Thus eIF3f plays a major role for proper activity of mTORC1 to regulate skeletal muscle size.     

Impaired overload-induced hypertrophy is associated with diminished mTOR signaling in insulin-resistant skeletal muscle of the obese Zucker rat

Recent data have suggested that insulin resistance may be associated with a diminished ability of skeletal muscle to undergo hypertrophy (Paturi S, Gutta AK, Kakarla SK, Katta A, Arnold EC, Wu M, Rice KM, Blough ER. J Appl Physiol 108: 7-13, 2010). Here we examine the effects of insulin resistance using the obese Zucker (OZ) rat with increased muscle loading on the regulation of the mammalian target of rapamycin (mTOR) and its downstream signaling intermediates 70-kDa ribosomal protein S6 kinase (p70S6k), ribosomal protein S6 (rpS6), eukaryotic elongation factor 2 (eEF2), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Compared with that observed in lean Zucker (LZ) rats, the degree of soleus muscle hypertrophy as assessed by changes in muscle wet weight (LZ: 35% vs. OZ: 16%) was significantly less in the OZ rats after 3 wk of muscle overload (P < 0.05). This diminished growth in the OZ rats was accompanied by significant impairments in the ability of the soleus to undergo phosphorylation of mTOR (Ser(2448)), p70S6k (Thr(389)), rpS6 (Ser(235/236)), and protein kinase B (Akt) (Ser(473) and Thr(308)) (P < 0.05). Taken together, these data suggest that impaired overload-induced hypertrophy in insulin-resistant skeletal muscle may be related to decreases in the ability of the muscle to undergo mTOR-related signaling.