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.     

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.     

Leucine stimulates HGF production by hepatic stellate cells through mTOR pathway

Branched chain amino acids modulate various cellular functions in addition to providing substrates for the production of proteins. We examined the mechanism underlying the stimulation by leucine of hepatocyte growth factor (HGF) production by hepatic stellate cells. Both p70 S6 kinase activity and phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were up-regulated rapidly after leucine treatment of a rat hepatic stellate cell clone. No such activation was observed following treatment with valine or isoleucine. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), suppressed leucine-induced activation of p70 S6 kinase and 4E-BP1 and negated the stimulatory effect of leucine on HGF production. An mTOR-dependent signaling pathway mediates the stimulatory effect of leucine on the production of HGF by hepatic stellate cells.     

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.     

Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs

Leucine is unique among the amino acids in its ability to promote protein synthesis by activating translation initiation via the mammalian target of rapamycin (mTOR) pathway. Previously, we showed that leucine infusion acutely stimulates protein synthesis in fast-twitch glycolytic muscle of neonatal pigs but this response cannot be maintained unless the leucine-induced fall in amino acids is prevented. To determine whether leucine can stimulate protein synthesis in muscles of different fiber types and in visceral tissues of the neonate in the long-term if baseline amino acid concentrations are maintained, overnight fasted neonatal pigs were infused for 24 h with saline, leucine (400 μmol kg⁻¹ h⁻¹), or leucine with replacement amino acids to prevent the leucine-induced hypoaminoacidemia. Changes in the fractional rate of protein synthesis and activation of mTOR, as determined by eukaryotic initiation factor 4E binding protein (4E-BP1) and S6 kinase 1 (S6K1) phosphorylation, in the gastrocnemius and masseter muscles, heart, liver, jejunum, kidney, and pancreas were measured. Leucine increased mTOR activation in the gastrocnemius and masseter muscles, liver, and pancreas, in both the absence and presence of amino acid replacement. However, protein synthesis in these tissues was increased only when amino acids were infused to maintain baseline levels. There were no changes in mTOR signaling or protein synthesis in the other tissues we examined. Thus, long-term infusion of leucine stimulates mTOR signaling in skeletal muscle and some visceral tissues but the leucine-induced stimulation of protein synthesis in these tissues requires sustained amino acid availability.     

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.     

Leucine-stimulated mTOR signaling is partly attenuated in skeletal muscle of chronically uremic rats

The branched-chain amino acid leucine stimulates muscle protein synthesis in part by directly activating the mTOR signaling pathway. Furthermore, leucine, if given in conjunction with resistance exercise, enhances the exercise-induced mTOR signaling and protein synthesis. Here we tested whether leucine can activate the mTOR anabolic signaling pathway in uremia and whether it can enhance work overload (WO)-induced signaling through this pathway. Chronic kidney disease (CKD) and control rats were studied after 7 days of surgically induced unilateral plantaris muscle WO and a single leucine or saline load. In the basal state, 4E-BP1 phosphorylation was modestly depressed in non-WO muscle of CKD rats, whereas rpS6 phosphorylation was nearly completely suppressed. After oral leucine mTOR, S6K1 and rpS6 phosphorylation increased similarly in both groups, whereas the phospho-4E-BP1 response was modestly attenuated in CKD. WO alone activated the mTOR signaling pathway in control and CKD rats. In WO CKD, muscle leucine augmented mTOR and 4E-BP1 phosphorylation, but its effect on S6K1 phosphorylation was attenuated. Taken together, this study has established that the chronic uremic state impairs basal signaling through the mTOR anabolic pathway, an abnormality that may contribute to muscle wasting. However, despite this abnormality, leucine can stimulate this signaling pathway in CKD, although its effectiveness is partially attenuated, including in skeletal muscle undergoing sustained WO. Thus, although there is some resistance to leucine in CKD, the data suggest a potential role for leucine-rich supplements in the management of uremic muscle wasting.     

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.     

Effects of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists on leucine-induced phosphorylation of translational targets in C2C12 cells

Effect of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.     

Effects of peroxisome proliferator-activated receptor α (PPARα) agonists on leucine-induced phosphorylation of translational targets in C2C12 cells

Effect of peroxisome proliferator-activated receptor α (PPARα) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.     

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.     

Deficiency of dietary EAA preferentially inhibits mRNA translation of ribosomal proteins in liver of meal-fed rats

The goal of these studies was to investigate the mechanisms by which amino acid supply regulates global rates of protein synthesis as well as the translation of ribosomal protein (rp) mRNAs in liver. In the experiments conducted, male weanling rats were trained over a 2-wk period to consume their daily food intake within 3 h. On day 14, rats were fed the control diet or an isocaloric, isonitrogenous diet lacking glycine, tryptophan, leucine, or the branched-chain amino acids (BCAA) for 1 h. Feeding Trp-, Leu-, or BCAA-deficient diets resulted in significant reductions in serum insulin, hepatic protein synthesis, eukaryotic initiation factor 2B (eIF2B) activity, and phosphorylation of eIF4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K1). Phosphorylation of eIF2alpha was inversely related to eIF2B activity under all conditions. Alterations in the hepatic synthesis of rp were assessed by changes in the distribution of rp (S4, S8, L26) mRNAs across sucrose density gradients and compared with non-rp (beta-actin, albumin) mRNAs. In all dietary treatments, non-rp mRNAs were mostly polysome associated. Conversely, the proportion of rp mRNAs residing in polysomes was two- to fivefold less in rats fed diets lacking tryptophan, leucine, or BCAA compared with rats fed the control diet. Total hepatic abundance of all mRNAs examined did not differ among treatment groups. For all parameters examined, there were no differences between rats fed the glycine-deficient diet and rats fed the control diet. The data suggest that essential amino acid (EAA) deficiency inhibits global rates of liver protein synthesis via a block in translation initiation. Additionally, the translation of rp mRNAs is preferentially repressed in association with decreased S6K1 phosphorylation.     

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.     

Amino acid availability and age affect the leucine stimulation of protein synthesis and eIF4F formation in muscle

We have previously shown that a physiological increase in plasma leucine for 60 and 120 min increases translation initiation factor activation in muscle of neonatal pigs. Although muscle protein synthesis is increased by leucine at 60 min, it is not maintained at 120 min, perhaps because of the decrease in plasma amino acids (AA). In the present study, 7- and 26-day-old pigs were fasted overnight and infused with leucine (0 or 400 micromol.kg(-1).h(-1)) for 120 min to raise leucine within the postprandial range. The leucine was infused in the presence or absence of a replacement AA mixture (without leucine) to maintain baseline plasma AA levels. AA administration prevented the leucine-induced reduction in plasma AA in both age groups. At 7 days, leucine infusion alone increased eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) phosphorylation, decreased inactive 4E-BP1.eIF4E complex abundance, and increased active eIF4G.eIF4E complex formation in skeletal muscle; leucine infusion with replacement AA also stimulated these, as well as 70-kDa ribosomal protein S6 kinase, ribosomal protein S6, and eIF4G phosphorylation. At 26 days, leucine infusion alone increased 4E-BP1 phosphorylation and decreased the inactive 4E-BP1.eIF4E complex only; leucine with AA also stimulated these, as well as 70-kDa ribosomal protein S6 kinase and ribosomal protein S6 phosphorylation. Muscle protein synthesis was increased in 7-day-old (+60%) and 26-day-old (+40%) pigs infused with leucine and replacement AA but not with leucine alone. Thus the ability of leucine to stimulate eIF4F formation and protein synthesis in skeletal muscle is dependent on AA availability and age.     

Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine

Enhanced protein synthesis in skeletal muscle after ingestion of a balanced meal in postabsorptive rats is mimicked by oral leucine administration. To assess the contribution of insulin to the protein synthetic response to leucine, food-deprived (18 h) male rats (approximately 200 g) were intravenously administered a primed-constant infusion of somatostatin (60 microg + 3 microg.kg(-1).h(-1)) or vehicle beginning 1 h before administration of leucine (1.35 g L-leucine/kg) or saline (control). Rats were killed 15, 30, 45, 60, or 120 min after leucine administration. Compared with controls, serum insulin concentrations were elevated between 15 and 45 min after leucine administration but returned to basal values by 60 min. Somatostatin maintained insulin concentrations at basal levels throughout the time course. Protein synthesis was increased between 30 and 60 min, and this effect was blocked by somatostatin. Enhanced assembly of the mRNA cap-binding complex (composed of eukaryotic initiation factors eIF4E and eIF4G) and hyperphosphorylation of the eIF4E-binding protein 1 (4E-BP1), the 70-kDa ribosomal protein S6 kinase (S6K1), and the ribosomal protein S6 (rp S6) were observed as early as 15 min and persisted for at least 60 min. Somatostatin attenuated the leucine-induced changes in 4E-BP1 and S6K1 phosphorylation and completely blocked the change in rp S6 phosphorylation but had no effect on eIF4G small middle dot eIF4E assembly. Overall, the results suggest that the leucine-induced enhancement of protein synthesis and the phosphorylation states of 4E-BP1 and S6K1 are facilitated by the transient increase in serum insulin. In contrast, assembly of the mRNA cap-binding complex occurs independently of increases in insulin and, by itself, is insufficient to stimulate rates of protein synthesis in skeletal muscle after leucine administration.     

Effect of branched-chain amino acids on muscle atrophy in cancer cachexia

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.     

Leucine regulates translation of specific mRNAs in L6 myoblasts through mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6

Regulation of translation of mRNAs coding for specific proteins plays an important role in controlling cell growth, differentiation, and transformation. Two proteins have been implicated in the regulation of specific mRNA translation: eukaryotic initiation factor eIF4E and ribosomal protein S6. Increased phosphorylation of eIF4E as well as its overexpression are associated with stimulation of translation of mRNAs with highly structured 5'-untranslated regions. Similarly, phosphorylation of S6 results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5'-end of the message. In the present study, leucine stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6 myoblasts, resulting in dissociation of eIF4E from the inactive eIF4E.4E-BP1 complex. The increased availability of eIF4E was associated with a 1.6-fold elevation in ornithine decarboxylase relative to global protein synthesis. Leucine also stimulated phosphorylation of the ribosomal protein S6 kinase, p70(S6k), resulting in increased phosphorylation of S6. Hyperphosphorylation of S6 was associated with a 4-fold increase in synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the protein kinase mTOR, prevented all of the leucine-induced effects. Thus, leucine acting through an mTOR-dependent pathway stimulates the translation of specific mRNAs both by increasing the availability of eIF4E and by stimulating phosphorylation of S6.     

Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways

Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5′-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5′-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.     

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.     

Reactive oxygen species (ROS) mediate the effects of leucine on translation regulation and type I collagen production in hepatic stellate cells

The amino acid leucine causes an increase of collagen alpha1(I) synthesis in hepatic stellate cells through the activation of translational regulatory mechanisms and PI3K/Akt/mTOR and ERK signaling pathways. The aim of the present study was to evaluate the role played by reactive oxygen species on these effects. Intracellular reactive oxygen species levels were increased in hepatic stellate cells incubated with leucine 5 mM at early time points, and this effect was abolished by pretreatment with the antioxidant glutathione. Preincubation with glutathione also prevented 4E-BP1, eIF4E and Mnk-1 phosphorylation induced by leucine, as well as enhancement of procollagen alpha1(I) protein levels. Inhibitors for MEK-1 (PD98059), PI3K (wortmannin) or mTOR (rapamycin) did not affect leucine-induced reactive oxygen species production. However, preincubation with glutathione prevented ERK, Akt and mTOR phosphorylation caused by treatment with leucine. The mitochondrial electron chain inhibitor rotenone and the NADPH oxidase inhibitor apocynin prevented reactive oxygen species production caused by leucine. Leucine also induced an increased phosphorylation of IR/IGF-R that was abolished by pretreatment with either rotenone or apocynin. Therefore, leucine exerts on hepatic stellate cells a prooxidant action through NADPH oxidase and mitochondrial Reactive oxygen species production and these effects mediate the activation of IR/IGF-IR and signaling pathways, finally leading to changes in translational regulation of collagen synthesis.