IGF-I stimulates protein synthesis in skeletal muscle through multiple signaling pathways during sepsis

Chronic septic abscess formation causes an inhibition of protein synthesis in gastrocnemius not observed in rats with a sterile abscess. Inhibition is associated with an impaired mRNA translation initiation that can be ameliorated by elevating IGF-I but not insulin. The present study investigated the ability of IGF-I signaling to stimulate protein synthesis in gastrocnemius by accelerating mRNA translation initiation. Experiments were performed in perfused hindlimb preparations from rats 5 days after induction of a septic abscess. Protein synthesis in gastrocnemius from septic rats was accelerated twofold by the addition of IGF-I (10 nM) to perfusate. IGF-I increased the phosphorylation of translation repressor 4E-binding protein-1 (4E-BP1). Hyperphosphorylation of 4E-BP1 in response to IGF-I resulted in its dissociation from the inactive eukaryotic initiation factor (eIF) 4E.4E-BP1 complex. Assembly of the active eIF4F complex (as assessed by the association eIF4G with eIF4E) was increased twofold by IGF-I in the perfusate. In addition, phosphorylation of eIF4G and ribosomal protein S6 kinase-1 (S6K1) was also enhanced by IGF-I. Activation of mammalian target of rapamycin, an upstream kinase implicated in phosphorylating both 4E-BP1 and S6K1, was also observed. Thus the ability of IGF-I to accelerate protein synthesis during sepsis may be related to a stimulation of signaling to multiple steps in translation initiation with an ensuing increased phosphorylation of eIF4G, eIF4E availability, and S6K1 phosphorylation.     

IGF-I/IGFBP-3 ameliorates alterations in protein synthesis, eIF4E availability, and myostatin in alcohol-fed rats

Chronic alcohol consumption decreases the concentration of the anabolic hormone IGF-I, and this change is associated with impaired muscle protein synthesis. The present study evaluated the ability of IGF-I complexed with IGF-binding protein (IGFBP)-3 to modulate the alcohol-induced inhibition of muscle protein synthesis in gastrocnemius. After 16 wk on an alcohol-containing diet, either the IGF-I/IGFBP-3 binary complex (BC) or saline was injected two times daily for three consecutive days. After the final injection of BC (3 h), plasma IGF-I concentrations were elevated in alcohol-fed rats to values not different from those of similarly treated control animals. Alcohol feeding decreased the basal rate of muscle protein synthesis by limiting translational efficiency. BC treatment of alcohol-fed rats increased protein synthesis back to basal control values, but the rate remained lower than that of BC-injected control rats. The BC partially reversed the alcohol-induced decrease in the binding of eukaryotic initiation factor (eIF)4E with eIF4G. This change was associated with reversal of the alcohol-induced dephosphorylation of eIF4G but was independent of changes in the phosphorylation of either 4E-BP1 or eIF4E. However, BC reversed the alcohol-induced increase in IGFBP-1 and muscle myostatin, known negative regulators of IGF-I action and muscle mass. Hence, exogenous IGF-I, administered as part of a BC to increase its circulating half-life, can in part reverse the decreased protein synthesis observed in muscle from chronic alcohol-fed rats by stimulating selected components of translation initiation. The data support the role of IGF-I as a mediator of chronic alcohol myopathy in rats.     

Insulin fails to stimulate muscle protein synthesis in sepsis despite unimpaired signaling to 4E-BP1 and S6K1

Induction of sepsis in rats causes an inhibition of protein synthesis in skeletal muscle that is resistant to the stimulatory actions of insulin. To gain a better understanding of the underlying reason for this lack of response, the present study was undertaken to investigate sepsis-induced alterations in insulin signaling to regulatory components of mRNA translation. Experiments were performed in perfused hindlimb preparations from rats 5 days after induction of a septic abscess. Sepsis resulted in a 50% reduction in protein synthesis in the gastrocnemius. Protein synthesis in muscles from septic rats, but not controls, was unresponsive to stimulation by insulin. The insulin-induced hyperphosphorylation response of the translation repressor protein 4E-binding protein 1 (4E-BP1) and of the 70-kDa S6 kinase (S6K1) (1), two targets of insulin action on mRNA translation, was unimpaired in gastrocnemius of septic rats. Hyperphosphorylation of 4E-BP1 in response to insulin resulted in its dissociation from the inactive eukaryotic initiation factor (eIF)4E. 4E-BP1 complex in both control and septic rats. However, assembly of the active eIF4F complex as assessed by the association of eIF4E with eIF4G did not follow the pattern predicted by the increased availability of eIF4E resulting from changes in the phosphorylation of 4E-BP1. Indeed, sepsis caused a dramatic reduction in the amount of eIF4G associated with eIF4E in the presence or absence of insulin. Thus the inability of insulin to stimulate protein synthesis during sepsis may be related to a defect in signaling to a step in translation initiation involved in assembly of an active eIF4F complex.     

Endotoxin-induced decrease in muscle protein synthesis is associated with changes in eIF2B, eIF4E, and IGF-I

The present study examined potential mechanisms contributing to the inhibition of protein synthesis in skeletal muscle after administration of endotoxin (LPS). Rats implanted with vascular catheters were injected intravenously with a nonlethal dose of Escherichia coli LPS, and samples were collected at 4 and 24 h thereafter; pair-fed control animals were also included. The rate of muscle (gastrocnemius) protein synthesis in vivo was reduced at both time points after LPS administration. LPS did not alter tissue RNA content, but the translational efficiency was consistently reduced at both time points. To identify mechanisms responsible for regulating translation, we examined several eukaryotic initiation factors (eIFs). The content of eIF2alpha or the amount of eIF2alpha in the phosphorylated form did not change in response to LPS. eIF2B activity was decreased in muscle 4 h post-LPS but activity returned to control values by 24 h. A decrease in the relative amount of eIF2Balpha protein was not responsible for the LPS-induced reduction in eIF2B activity. LPS also markedly altered the distribution of eIF4E in muscle. Compared with control values, LPS-treated rats demonstrated 1) a transient increase in binding of the translation repressor 4E-binding protein-1 (4E-BP1) with eIF4E, 2) a transient decrease in the phosphorylated gamma-form of 4E-BP1, and 3) a sustained decrease in the amount of eIF4G associated with eIF4E. LPS also decreased insulin-like growth factor (IGF) I protein and mRNA expression in muscle at both times. A significant linear relationship existed between muscle IGF-I and the rate of protein synthesis or the amount of eIF4E bound to eIF4G. In summary, these data suggest that LPS impairs muscle protein synthesis, at least in part, by decreasing translational efficiency, resulting from an impairment in translation initiation associated with alterations in both eIF2B activity and eIF4E availability.     

Glucocorticoids and TNFalpha interact cooperatively to mediate sepsis-induced leucine resistance in skeletal muscle

Sepsis blunts the ability of nutrient signaling by leucine to stimulate skeletal muscle protein synthesis by impairing translation initiation. The present study tested the hypothesis that overproduction of either tumor necrosis factor (TNF)-alpha or glucocorticoids mediate the sepsis-induced leucine resistance. Prior to producing peritonitis, rats received either vehicle, TNF binding protein (TNF(BP)) to inhibit endogenous TNFalpha action, and/or the glucocorticoid receptor antagonist RU486. Leucine was orally administered to all rats 24 h thereafter and the gastrocnemius removed 20 min later to assess protein synthesis and signaling components important in controlling peptide-chain initiation. Muscle protein synthesis was 65% lower in septic rats administered leucine than in leucine-treated control animals. This reduction was not prevented by either TNF(BP) or RU486 alone, but was completely reversed by the combination. This sepsis-induced leucine resistance was associated with an 80% reduction in the amount of active eIF4E.eIF4G complex, a 5-fold increase in the formation of the inactive eIF4E.4E-BP1 complex as well as markedly reduced (at least 70%) phosphorylation of 4E-BP1, eIF4G, S6K1, S6, and mTOR. Pretreatment of septic rats with either TNF(BP) or RU486 individually only nominally improved the leucine action as assessed by the above-mentioned endpoints. In contrast, when TNF(BP) and RU486 were co-administered, the ability of sepsis to impair the leucine-stimulated phosphorylation of 4E-BP1, eIF4G, S6K1, and S6 as well as the redistribution of eIF4E was essentially prevented. No differences in the total amount or phosphorylation of eIF2alpha and eIF2Bepsilon were detected between the different groups, and changes could not be attributed to differences in the prevailing plasma concentration of insulin or leucine. Our data demonstrate the sepsis-induced leucine resistance in skeletal muscle results from the cooperative interaction of both TNFalpha and glucocorticoids.     

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.     

Role of eIF4E in stimulation of protein synthesis by IGF-I in perfused rat skeletal muscle

Insulin-like growth factor I (IGF-I) promotes anabolism by stimulating protein synthesis in skeletal muscle. In the present study, we have examined mechanisms by which IGF-I stimulates protein synthesis in skeletal muscle with a perfused rat hindlimb preparation. IGF-I (10 nM) stimulated protein synthesis over 2.7-fold. Total RNA content was unaffected, but translational efficiency was increased by IGF-I. We next examined the effect of IGF-I on eukaryotic initiation factor (eIF) 4E as a mechanism regulating translation initiation. IGF-I did not alter either the amount of eIF4E associated with the eIF4E binding protein 4E-BP1 or the phosphorylation state of 4E-BP1. Likewise, the phosphorylation state of eIF4E was unaltered by IGF-I. In contrast, the amount of eIF4E bound to eIF4G was increased threefold by IGF-I. We conclude that IGF-I regulates protein synthesis in skeletal muscle by enhancing formation of the active eIF4E x eIF4G complex.     

Effect of sepsis on eIE4E availability in skeletal muscle

Chronic septic abscess formation causes an inhibition of protein synthesis in gastrocnemius that is not observed in rats with a sterile abscess. The inhibition is associated with an impaired translation initiation. The present study was designed to investigate the effects of sepsis on phosphorylation and availability of eukaryotic initiation factor (eIF)4E in gastrocnemius 5 days after induction of a sterile or septic abscess. Neither sepsis nor sterile inflammation altered the extent of eIF4E phosphorylation. Moreover, no changes in the amount of the binding protein 4E-BP1 associated with eIF4E or in the phosphorylation of 4E-BP1 were observed during sepsis or sterile inflammation. In contrast, sepsis and sterile inflammation caused a reduction in the relative amount of eIF4G bound to eIF4E compared with controls. The diminished amount of eIF4G bound to eIF4E was not the result of a reduced abundance of eIF4E. Sepsis, but not sterile inflammation, caused an increase in the cellular abundance of eIF4E. The results provide evidence that alterations in the eIF4E system are probably not rate controlling for the synthesis of total, mixed proteins in gastrocnemius during sepsis. Instead, on the basis of our previous studies, changes in eIF2B appear to be responsible for limiting protein synthesis in skeletal muscle during sepsis.     

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.     

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.     

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.     

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.     

Impaired myocardial protein synthesis induced by acute alcohol intoxication is associated with changes in eIF4F

The purpose of the present study was to examine potential mechanisms for the known inhibitory effect of acute alcohol exposure on myocardial protein synthesis. Rats were injected intraperitoneally with either ethanol (75 mmol/kg) or saline, and protein synthesis was measured in vivo 2.5 h thereafter by use of the flooding-dose L-[(3)H]phenylalanine technique. Rates of myocardial protein synthesis and translational efficiency in alcohol-treated rats were decreased compared with control values. Free (nonpolysome bound) 40S and 60S ribosomal subunits were increased 50% after alcohol treatment, indicating an impaired peptide-chain initiation. To identify mechanisms responsible for this impairment, several eukaryotic initiation factors (eIF) were analyzed. Acute alcohol intoxication did not significantly alter the myocardial content of eIF2 alpha or eIF2B epsilon, the extent of eIF2 alpha phosphorylation, or the activity of eIF2B. Acute alcohol exposure increased the binding of 4E-binding protein 1 (4E-BP1) to eIF4E (55%), diminished the amount of eIF4E bound to eIF4G (70%), reduced the amount of 4E-BP1 in the phosphorylated gamma-form (40%), and decreased the phosphorylation of p70S6 kinase and the ribosomal protein S6. There was no significant difference in either the plasma insulin-like growth factor (IGF) I concentration (total or free) or expression of IGF-I or IGF-II mRNA in heart between the two groups. These data suggest that the acute alcohol-induced impairment in myocardial protein synthesis results, in part, from an inhibition in peptide-chain initiation, which is associated with marked changes in eIF4E availability and p70S6 kinase phosphorylation but is independent of changes in the eIF2/2B system and IGFs.     

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.     

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.     

Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4E-BP1).

BACKGROUND INFORMATION: The translational inhibitor protein 4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5' cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E-BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. RESULTS: We now report that activation of p53 also results in modification of 4E-BP1 to a truncated form. Unlike full-length 4E-BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full-length 4E-BP1. Inhibitor studies indicate that the p53-induced cleavage of 4E-BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full-length 4E-BP1. Measurements of the turnover of 4E-BP1 indicate that the truncated form is much more stable than the full-length protein. CONCLUSIONS: The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E-BP1, which may exert a long-term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth-inhibitory and pro-apoptotic effects of p53.     

Alcohol impairs insulin and IGF-I stimulation of S6K1 but not 4E-BP1 in skeletal muscle

The present study determined whether acute alcohol (ethanol; EtOH) intoxication in rats impaired components of the insulin- and IGF-I-signaling pathway in skeletal muscle. Rats were administered EtOH, and 2.5 h thereafter either insulin, IGF-I, or saline was injected and the gastrocnemius removed. EtOH did not alter the total amount or tyrosine phosphorylation of the insulin receptor, IGF-I receptor, insulin receptor substrate (IRS)-1, or protein kinase B (PKB)/Akt under basal or hormone-stimulated conditions. In contrast, the ability of insulin or IGF-I to phosphorylate T389 and T421/S424 on S6K-1 was markedly diminished by EtOH, and these changes were associated with a reduction in the phosphorylation of the ribosomal protein S6. Under basal conditions, EtOH altered the distribution of eukaryotic initiation factor (eIF)4E, as evidenced by a decreased amount of active eIF4E. eIF4G complex, an increased amount of inactive eIF4E. 4E-binding protein (BP)1 complex, and decreased 4E-BP1 phosphorylation. In contrast, EtOH did not impair the ability of either hormone to reverse the changes in eIF4E distribution or 4E-BP1 phosphorylation. Pretreatment with a glucocorticoid receptor antagonist was unable to attenuate either the basal EtOH-induced changes in eIF4E distribution or the impaired ability of IGF-I to stimulate S6K1 and S6 phosphorylation. Hence, acute alcohol intoxication alters selected aspects of translational control under both basal and anabolic hormone-stimulated conditions in skeletal muscle in a glucocorticoid-independent manner.     

Availability of eIF4E regulates skeletal muscle protein synthesis during recovery from exercise

We examined the association of the mRNA cap binding proteineIF4E with the translational inhibitor 4E-BP1 in the acute modulation of skeletal muscle protein synthesis during recovery from exercise. Fasting male rats were run on a treadmill for 2 h at 26 m/min and wererealimented immediately after exercise with either saline, acarbohydrate-only meal, or a nutritionally complete meal (54.5% carbohydrate, 14% protein, and 31.5% fat). Exercised animals and nonexercised controls were studied 1 h postexercise. Muscle protein synthesis decreased 26% after exercise and was associated with afourfold increase in the amount of eIF4E present in the inactive eIF4E · 4E-BP1 complex and a concomitant 71%decrease in the association of eIF4E with eIF4G. Refeeding the completemeal, but not the carbohydrate meal, increased muscle protein synthesisequal to controls, despite similar plasma concentrations of insulin.Additionally, eIF4E · 4E-BP1 association wasinversely related and eIF4E · eIF4G association waspositively correlated to muscle protein synthesis. This studydemonstrates that recovery of muscle protein synthesis after exerciseis related to the availability of eIF4E for 48S ribosomal complexformation, and postexercise meal composition influences recovery viamodulation of translation initiation.

     

Amino acids and insulin are both required to regulate assembly of the eIF4E. eIF4G complex in rat skeletal muscle

The respective roles of insulin and amino acids in regulation of skeletal muscle protein synthesis and degradation after feeding were examined in rats fasted for 17 h and refed over 1 h with either a 25 or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained (control groups: C(25) and C(0)) or blocked with diazoxide injections (diazoxide groups: DZ(25) and DZ(0)). Muscle protein metabolism was examined in vitro in epitrochlearis muscles. Only feeding the 25% amino acid/protein meal in the presence of increased plasma insulin concentration (C(25) group) stimulated protein synthesis and inhibited proteolysis in skeletal muscle compared with the postabsorptive state. The stimulation of protein synthesis was associated with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1), reduced binding of eIF4E to 4E-BP1, and increased assembly of the active eIF4E. eIF4G complex. The p70 S6 kinase (p70(S6k)) was also hyperphosphorylated in response to the 25% amino acid/protein meal. Acute postprandial insulin deficiency induced by diazoxide injections totally abolished these effects. Feeding the 0% amino acid/protein meal with or without postprandial insulin deficiency did not stimulate muscle protein synthesis, reduce proteolysis, or regulate initiation factors and p70(S6k) compared with fasted rats. Taken together, our results suggest that both insulin and amino acids are required to stimulate protein synthesis, inhibit protein degradation, and regulate the interactions between eIF4E and 4E-BP1 or eIF4G in response to feeding.     

IGF-I and insulin regulate eIF4F formation by different mechanisms in muscle and liver in the ovine fetus

The mechanisms by which insulin-like growth factor I (IGF-I) and insulin regulate eukaryotic initiation factor (eIF)4F formation were examined in the ovine fetus. Insulin infusion increased phosphorylation of eIF4E-binding protein (4E-BP1) in muscle and liver. IGF-I infusion did not alter 4E-BP1 phosphorylation in liver. In muscle, IGF-I increased 4E-BP1 phosphorylation by 27%; the percentage in the gamma-form in the IGF-I group was significantly lower than that in the insulin group. In liver, only IGF-I increased eIF4G. Both IGF-I and insulin increased eIF4E. eIF4G binding in muscle, but only insulin decreased the amount of 4E-BP1 associated with eIF4E. In liver, only IGF-I increased eIF4E. eIF4G binding. Insulin increased the phosphorylation of p70 S6 kinase (p70(S6k)) in both muscle and liver and protein kinase B (PKB/Akt) in muscle, two indicative signal proteins in the phosphatidylinositol (PI) 3-kinase pathway. IGF-I increased PKB/Akt phosphorylation in muscle but had no effect on p70(S6k) phosphorylation in muscle or liver. We conclude that insulin and IGF-I modulate eIF4F formation; however, the two hormones have different regulatory mechanisms. Insulin increases phosphorylation of 4E-BP1 and eIF4E. eIF4G binding in muscle, whereas IGF-I regulates eIF4F formation by increasing total eIF4G. Insulin, but not IGF-I, decreased 4E-BP1 content associated with eIF4E. Insulin regulates translation initiation via the PI 3-kinase-p70(S6k) pathway, whereas IGF-I does so mainly via mechanisms independent of the PI 3-kinase-p70(S6k) pathway.