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.     

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.     

Impaired translation initiation activation and reduced protein synthesis in weaned piglets fed a low-protein diet

Weanling mammals (including infants) often experience intestinal dysfunction when fed a high-protein diet. Recent work with the piglet (an animal model for studying human infant nutrition) shows that reducing protein intake can improve gut function during weaning but compromises the provision of essential amino acids (EAA) for muscle growth. The present study was conducted with weaned pigs to test the hypothesis that supplementing deficient EAA (Lys, Met, Thr, Trp, Leu, Ile and Val) to a low-protein diet may maintain the activation of translation initiation factors and adequate protein synthesis in tissues. Pigs were weaned at 21 days of age and fed diets containing 20.7, 16.7 or 12.7% crude protein (CP), with the low-CP diets supplemented with EAA to achieve the levels in the high-CP diet. On Day 14 of the trial, tissue protein synthesis was determined using the phenylalanine flooding dose method. Reducing dietary CP levels decreased protein synthesis in pancreas, liver, kidney and longissimus muscle. A low-CP diet reduced the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) in skeletal muscle and liver while increasing the formation of an inactive eIF4E.4E-BP1 complex in muscle. Dietary protein deficiency also decreased the phosphorylation of mammalian target of rapamycin (mTOR) and the formation of an active eIF4E.eIF4G complex in liver. These results demonstrate for the first time that chronic feeding of a low-CP diet suppresses protein synthesis in animals partly by inhibiting mTOR signaling. Additionally, our findings indicate that supplementing deficient EAA to low-protein diets is not highly effective in restoring protein synthesis or whole-body growth in piglets. We suggest that conditionally essential amino acids (e.g., glutamine and arginine) may be required to maintain the activation of translation initiation factors and optimal protein synthesis in neonates.     

Leucine reduces the duration of insulin-induced PI 3-kinase activity in rat skeletal muscle

Leucine (Leu) is known to stimulate translation initiation of protein synthesis at mammalian target of rapamycin (mTOR) in the insulin signaling pathway. However, potential feedback from mTOR to upstream aspects of the insulin signaling pathway remains controversial. This study evaluates the impact of a physiological oral dose of Leu and/or carbohydrate (CHO) on upstream elements of the insulin signaling pathway using phosphatidylinositol 3-kinase (PI 3-kinase) activity and glucose uptake as markers for insulin sensitivity and glucose homeostasis. Rats (approximately 200 g) were fasted 12 h and administered oral doses of CHO (1.31 g glucose, 1.31 g sucrose), Leu (270 mg), or CHO plus Leu. Animals were killed at 15, 30, 60, and 90 min after treatment. Plasma and gastrocnemius muscles were collected for analyses. Treatments were designed to produce elevated blood glucose and insulin with basal levels of Leu (CHO); elevated Leu with basal levels of glucose and insulin (Leu); or a combined increase of glucose, insulin, and Leu (CHO + Leu). The CHO treatment stimulated PI 3-kinase activity and glucose uptake with no effect on the downstream translation initiation factor eIF4E. Leu alone stimulated the release of the translation initiation factor eIF4E from 4E-BP1 with no effects on PI 3-kinase activity or glucose uptake. The CHO + Leu treatment reduced the magnitude and duration of the PI 3-kinase response but maintained glucose uptake similar to the CHO treatment and eIF4E levels similar to the Leu treatment. These findings demonstrate that Leu reduces insulin-stimulated PI 3-kinase activity while increasing downstream translation initiation and with no effect on net glucose transport in skeletal muscle.     

Rag GTPases and AMPK/TSC2/Rheb mediate the differential regulation of mTORC1 signaling in response to alcohol and leucine

Leucine (Leu) and insulin both stimulate muscle protein synthesis, albeit at least in part via separate signaling pathways. While alcohol (EtOH) suppresses insulin-stimulated protein synthesis in cultured myocytes, its ability to disrupt Leu signaling and Rag GTPase activity has not been determined. Likewise, little is known regarding the interaction of EtOH and Leu on the AMPK/TSC2/Rheb pathway. Treatment of myocytes with EtOH (100 mM) decreased protein synthesis, whereas Leu (2 mM) increased synthesis. In combination, EtOH suppressed the anabolic effect of Leu. The effects of EtOH and Leu were associated with coordinate changes in the phosphorylation state of mTOR, raptor, and their downstream targets 4EBP1 and S6K1. As such, EtOH suppressed the ability of Leu to activate these signaling components. The Rag signaling pathway was activated by Leu but suppressed by EtOH, as evidenced by changes in the interaction of Rag proteins with mTOR and raptor. Overexpression of constitutively active (ca)RagA and caRagC increased mTORC1 activity, as determined by increased S6K1 phosphorylation. Furthermore, the caRagA-caRagC heterodimer blocked the inhibitory effect of EtOH. EtOH and Leu produced differential effects on AMPK signaling. EtOH enhanced AMPK activity, resulting in increased TSC2 (S1387) and eEF2 phosphorylation, whereas Leu had the opposite effect. EtOH also decreased the interaction of Rheb with mTOR, and this was prevented by Leu. Collectively, our results indicate that EtOH inhibits the anabolic effects that Leu has on protein synthesis and mTORC1 activity by modulating both Rag GTPase function and AMPK/TSC2/Rheb signaling.     

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.     

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.     

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.     

Influence of benzoic acid and dietary protein level on performance, nitrogen metabolism and urinary pH in growing-finishing pigs

An experiment was conducted to examine the effect of benzoic acid and two dietary protein levels on pig performance, nitrogen balance and urinary pH. A total of 24 crossbred barrows (26 kg to 106 kg BW) received one of four diets: low protein level with and without 1% benzoic acid (LP- and LP+, respectively) and high protein level with and without 1% benzoic acid (HP- and HP+, respectively). The animals were fed restrictively grower and finisher diets and were kept in metabolic cages in weeks 3, 6, 9, and 12 of the experiment. The addition of benzoic acid did not improve weight gain and feed conversion ratio. N-intake and digested N were only influenced by dietary protein level (p< 0.01), while N-balance was similar in all four diets. Dietary benzoic acid improved N-digestibility in the grower period (p<0.01) but not in the finisher period. The addition of benzoic acid reduced urinary pH by about one pH-unit in both feeding periods independent of the protein level of the diet (p< 0.01) and increased the concentration of urinary hippuric acid markedly (p<0.01). The results of this study indicate a positive influence of dietary benzoic acid on pigs especially in case of feeding a low protein diet in the grower period.     

Lovastatin effect in rat neuroblasts of the CNS: inhibition of cap-dependent translation

Mevalonate biosynthesis pathway is important in cell growth and survival and its blockade by 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, statins, arrest brain neuroblasts growth and induce apoptosis. Translation is among the main biochemical mechanisms that controls gene expression and therefore cell growth or apoptosis. In the CNS, translation regulates synaptic plasticity. Thus, our aim was to investigate the effect of lovastatin in protein translation in rat neuroblasts of the CNS and the biochemical pathways involved. Lovastatin treatment in rat brain neuroblasts causes a significant time- and concentration-inhibition of protein synthesis, which is partially mediated by phosphatydilinositol 3-kinase/mammalian target of rapamycin (mTOR) pathway inhibition. Lovastatin treatment decreases the phosphorylation state of mTOR substrates, p70S6K and eukaryotic translation initiation factor (eIF) 4E-binding protein 1 and simultaneously increases eIF4E-binding protein 1 in a time-dependent manner. Concomitantly, lovastatin causes a decrease in eIF4G cellular amount, which is partially mediated by caspase(s) activity excluding caspase 3. These biochemical pathways affected by lovastatin might explain the protein translation inhibition observed in neuroblasts. Cycloheximide treatment, which blocked protein synthesis, does not induce neuroblasts apoptosis. Therefore, we suggest that lovastatin-induced protein synthesis inhibition might not contribute to the concomitant neuroblasts apoptosis previously observed.     

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.     

Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-dependent process

Protein synthesis is repressed in both skeletal muscle and liver after a short-term fast and is rapidly stimulated in response to feeding. Previous studies in rats and pigs have shown that the feeding-induced stimulation of protein synthesis is associated with activation of the 70-kDa ribosomal protein S6 kinase (S6K1) as well as enhanced binding of eukaryotic initiation factor eIF4E to eIF4G to form the active eIF4F complex. In cells in culture, hormones and nutrients regulate both of these events through a protein kinase termed the mammalian target of rapamycin (mTOR). In the present study, the involvement of mTOR in the feeding-induced stimulation of protein synthesis in skeletal muscle and liver was examined. Pigs at 7 days of age were fasted for 18 h, and then one-half of the animals were fed. In addition, one-half of the animals in each group were administered rapamycin (0.75 mg/kg) 2 h before feeding. The results reveal that treating 18-h fasted pigs with rapamycin, a specific inhibitor of mTOR, before feeding prevented the activation of S6K1 and the changes in eIF4F complex formation observed in skeletal muscle and liver after feeding. Rapamycin also ablated the feeding-induced stimulation of protein synthesis in liver. In contrast, in skeletal muscle, rapamycin attenuated, but did not prevent, the stimulation of protein synthesis in response to feeding. The results suggest that feeding stimulates hepatic protein synthesis through an mTOR-dependent process involving enhanced eIF4F complex formation and activation of S6K1. However, in skeletal muscle, these two processes may account for only part of the stimulation of protein synthesis, and thus additional steps may be involved in the response.     

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.     

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.     

Effects of in ovo feeding of l-arginine on breast muscle growth and protein deposition in post-hatch broilers

In ovo feeding (IOF) of l-arginine (Arg) can affect growth performance of broilers, but the response of IOF of Arg on breast muscle growth is unclear, and the mechanism involved in protein deposition remains unknown. Hense, this experiment was conducted to evaluate the effects of IOF of Arg on breast muscle growth and protein-deposited signalling in post-hatch broilers. A total of 720 fertile eggs were collected from 34-week-old Arbor Acres breeder hens and distributed to three treatments: (1) non-injected control group; (2) 7.5 g/l (w/v) NaCl diluent-injected control group; (3) 0.6 mg Arg/egg solution-injected group. At 17.5 days of incubation, fertile eggs were injected 0.6 ml solutions into the amnion of the injected groups. Upon hatching, 80 male chicks were randomly assigned to eight replicates of 10 birds each and fed ad libitum for 21 days. The results indicated that IOF of Arg increased relative breast muscle weight compared with those of control groups at hatch, 3-, 7- and 21-day post-hatch (P<0.05). In the Arg-injected group, the plasma total protein and albumen concentrations were higher at 7- and 21-day post-hatch than those of control groups (P<0.05). The alanine aminotransferase activity in Arg group was higher at hatch than that of control groups (P<0.05). The levels of triiodothyronine at four time points and thyroxine hormones at hatch, 7- and 21-day post-hatch in Arg group were higher than those of control groups (P<0.05). In addition, IOF of Arg increased the amino acid concentrations of breast muscle at hatch, 7- and 21-day post-hatch (P<0.05). In ovo feeding of Arg also enhanced mammalian target of rapamycin, ribosomal protein S6 kinase-1 and eIF4E-bindingprotein-1 messenger RNA expression levels at hatch compared with those of control groups (P<0.05). It was concluded that IOF of Arg treatment improved breast muscle growth, which might be associated with the enhancement of protein deposition.     

Endotoxin induces differential regulation of mTOR-dependent signaling in skeletal muscle and liver of neonatal pigs

In the present study, differential responses of regulatory proteins involved in translation initiation in skeletal muscle and liver during sepsis were studied in neonatal pigs treated with lipopolysaccharide (LPS). LPS did not alter eukaryotic initiation factor (eIF) 2B activity in either tissue. In contrast, binding of eIF4G to eIF4E to form the active mRNA-binding complex was repressed in muscle and enhanced in liver. Phosphorylation of eIF4E-binding protein, 4E-BP1, and ribosomal protein S6 kinase, S6K1, was reduced in muscle during sepsis but increased in liver. Finally, changes in 4E-BP1 and S6K1 phosphorylation were associated with altered phosphorylation of the protein kinase mammalian target of rapamycin (mTOR). Overall, the results suggest that translation initiation in both skeletal muscle and liver is altered during neonatal sepsis by modulation of the mRNA-binding step through changes in mTOR activation. Moreover, the LPS-induced changes in factors that regulate translation initiation are more profound than previously reported changes in global rates of protein synthesis in the neonate. This finding suggests that the initiator methionyl-tRNA-rather than the mRNA-binding step in translation initiation may play a more critical role in maintaining protein synthesis rates in the neonate during sepsis.     

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.     

S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover

A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR. signal transduction; atrophy; autophagy