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The mammalian target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that exquisitely regulates protein metabolism in skeletal muscle. mTOR integrates input from amino acids, growth factors, and intracellular cues to make or break muscle protein. mTOR accomplishes this task by stimulating the phosphorylation of substrates that control protein translation while simultaneously inhibiting proteasomal and autophagic protein degradation. In a metabolic twist of fate, sepsis induces muscle atrophy in part by the aberrant regulation of mTOR. In this review, we track the steps of normal mTOR signaling in muscle and examine where they go astray in sepsis and inflammation.

Loss of lean body mass is a characteristic feature of the septic response, and the mechanisms responsible for this decrease and means of prevention have not been fully elucidated. The present study tested the hypothesis that in vitro treatment of skeletal muscle with lithium chloride (LiCl), a glycogen synthase kinase (GSK) 3 inhibitor, would reverse both the sepsis-induced increase in muscle protein degradation and inhibition of protein synthesis. Sepsis decreased GSK-3[beta] phosphorylation and increased GSK-3[beta] activity, under basal conditions. Sepsis increased muscle protein degradation, with a concomitant increase in atrogin 1 and MuRF1 mRNA and 26S proteosome activity. Incubation of septic muscle with LiCl completely reversed the increased GSK-3[beta] activity and decreased proteolysis to basal nonseptic values, but only partially reduced proteosome activity and did not diminish atrogene expression. Lithium chloride also did not ameliorate the sepsis-induced increase in LC3-II, a marker for activated autophagy. In contrast, LiCl increased protein synthesis only in nonseptic control muscle. The inability of septic muscle to respond to LiCl was independent of its ability to reverse the sepsis-induced increase in eukaryotic initiation factor (eIF) 2B[varepsilon] phosphorylation, decreased eIF2B activity, or the reduced phosphorylation of FOXO3, but instead was more closely associated with the continued suppression of mTOR (mammalian target of rapamycin) kinase activity (e.g., reduced phosphorylation of 4E-BP1 and S6). These data suggest that in vitro lithium treatment, which inhibited GSK-3[beta] activity, (a) effectively reversed the sepsis-induced increase in proteolysis, but only in part by a reduction in the ubiquitin-proteosome pathway and not by a reduction in autophagy; and (b) was ineffective at reversing the sepsis-induced decrease in muscle protein synthesis. This lithium-resistant state seems mediated at the level of mTOR and not eIF2/eIF2B. Hence, use of GSK-3[beta] inhibitors in the treatment of sepsis may not be expected to fully correct the imbalance in muscle protein turnover.

Sepsis-induced muscle atrophy is produced in part by decreased protein synthesis mediated by inhibition of mTOR (mammalian target of rapamycin). The present study tests the hypothesis that alteration of specific protein-protein interactions within the mTORC1 (mTOR complex 1) contributes to the decreased mTOR activity observed after cecal ligation and puncture in rats. Sepsis decreased in vivo translational efficiency in gastrocnemius and reduced the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein (BP) 1, S6 kinase (S6K) 1, and mTOR, compared with time-matched pair-fed controls. Sepsis decreased T246-phosphorylated PRAS40 (proline-rich Akt substrate 40) and reciprocally increased S792-phosphorylated raptor (regulatory associated protein of mTOR). Despite these phosphorylation changes, sepsis did not alter PRAS40 binding to raptor. The amount of the mTOR-raptor complex did not differ between groups. In contrast, the binding and retention of both 4E-BP1 and S6K1 to raptor were increased, and, conversely, the binding of raptor with eIF3 was decreased in sepsis. These changes in mTORC1 in the basal state were associated with enhanced 5'-AMP activated kinase activity. Acute in vivo leucine stimulation increased muscle protein synthesis in control, but not septic rats. This muscle leucine resistance was associated with coordinated changes in raptor-eIF3 binding and 4E-BP1 phosphorylation. Overall, our data suggest the sepsis-induced decrease in muscle protein synthesis may be mediated by the inability of 4E-BP1 and S6K1 to be phosphorylated and released from mTORC1 as well as the decreased recruitment of eIF3 necessary for a functional 48S complex. These data provide additional mechanistic insight into the molecular mechanisms by which sepsis impairs both basal protein synthesis and the anabolic response to the nutrient signal leucine in skeletal muscle.

Background & Aims: Chronic ethanol consumption in the Long-Evans (LE) rat has been associated with hepatic p53 activation, and inhibition of the insulin/PI3K/AKT signal transduction cascade due to increased expression of PTEN. We hypothesize that p53 activation and altered insulin signaling may influence the susceptibility of rats to ethanol-induced liver damage. Furthermore, p53 not only activates programmed cell death pathways and suppresses hepatocellular survival signals, but also promotes gluconeogenesis to increase systemic insulin resistance due to a novel metabolic function., Methods: Fischer (F), Sprague-Dawley (SD) and LE rats were fed ethanol-containing or control liquid diet for 8 weeks. Histopathological and biochemical changes were assessed., Results: Here, we demonstrate that chronic ethanol feeding in rats promotes p53 activation, hepatic steatosis, oxidative stress, PUMA, and PTEN expression, which contribute to hepatocellular death and diminished insulin signaling in the liver. Such changes are pronounced in the LE, less prominent in SD, and virtually absent in the F rat strain. More importantly, there is activation of Tp53-induced glycolysis and apoptosis regulator (TIGAR) in the ethanol-fed LE rat. This event generates low hepatic fructose-2,6-bisphosphate (Fru-2,6-P₂) levels, reduced lactate/pyruvate ratio and may contribute to increased basal glucose turnover and high residual hepatic glucose production during euglycemic hyperinsulinemic clamp., Conclusions: p53 activation correlates with the susceptibility to ethanol-induced liver damage in different rat strains. p53 not only orchestrates apoptosis and suppresses cell survival, but by activating TIGAR and decreasing hepatic Fru-2,6-P₂) levels it promotes insulin resistance and therefore, contributes to the metabolic abnormalities associated with hepatic steatosis., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Objective: Porcine circovirus type 2 (PCV-2) and torque-teno-sus- viruses (TTSuV) are ubiquitous in all pig-producing countries. Both pathogens are transmitted horizontally as well as vertically, and semen could be an important means of transmission. Aim of this study was the investigation of the prevalence of these two viruses in boar semen., Material and Methods: Single semen samples from 100 boars from an artificial insemination centre were analysed by quantitative real time PCR for PCV2 and by conventional PCR for TTSuV-1 and TTSuV-2., Results: Only one Piétrain boar of age of 47months showed a measurable PCV-2 quantity of 7.56x10³/ml semen. Four samples were PCR-positive for TTSuV-1, and five for TTVSu-2, while in a single co-infection both TTSuVs were detected.All TTSuV-positive samples originated from Piétrain boars., Conclusion and Clinical Relevance: This study represents the first description of TTSuV presence in semen in Austria. The prevalence of TTSuV as well as PCV-2 was low. The clinical relevance of a simultaneous contamination of boar semen with both pathogens remains unclear.

Bacterial infection decreases skeletal muscle protein synthesis via inhibition of the mammalian target of rapamycin (mTOR), a key regulator of translation initiation. To better define the mechanism by which muscle mTOR activity is decreased, we used an in vitro model of C2C12 myotubes treated with endotoxin [lipopolysaccharide (LPS)]and interferon (IFN)-γ to determine whether stable lipophilic pyruvate derivatives restore mTOR signaling. Myotubes treated with a combination of LPS and IFNγ down-regulated the phosphorylation of the mTOR substrates S6 kinase-1 and 4E binding protein-1. The phosphorylation of ribosomal protein S6 was decreased, whereas phosphorylation of elongation factor-2 was enhanced; all results consistent with defects in both translation initiation and elongation. LPS/IFNγ decreased protein synthesis 60% in myotubes. Treatment with methyl or ethyl pyruvate partially protected against the LPS/IFNγ-induced fall in mTOR signaling. The protective effect of ethyl and methyl pyruvate could not be replicated by an equimolar amount of sodium pyruvate. Although LPS/IFNγ treated myotubes were initially IGF-I responsive, prolonged exposure (≥ 17 h) resulted in IGF-I resistance at the level of mTOR despite normal IGF-I receptor phosphorylation. Ethyl pyruvate treatment restored IGF-I sensitivity as evidenced by the left shift in the IGF-I dose-response curve and maintained IGF-I responsiveness for a prolonged period of time. Ethyl pyruvate also restored IGF-I-stimulated protein synthesis in LPS/IFNγ-treated myotubes. Cotreatment with N-acetyl cysteine or ascorbic acid also preserved IGF-I sensitivity and mTOR activity. The data suggest that the combination of LPS and IFNγ inhibits mTOR activity and that prolonged exposure induces IGF-I resistance in myotubes. Lipophilic pyruvate derivatives and antioxidants show promise at rescuing mTOR activity and muscle protein synthesis by maintaining IGF-I sensitivity in this model.

The present study determined whether the muscle atrophy produced by colitis is associated with altered rates of muscle protein synthesis or degradation, as well as the potential role of the local (eg, muscle) insulin-like growth factor (IGF) system and muscle-specific ubiquitin E3 ligases atrogin-1 and MuRF1 in mediating altered muscle protein balance. Colitis was induced in C57BL/6 mice by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and blood and tissues were collected on day 10. Mice with inflammatory bowel disease demonstrated reduced skeletal muscle mass and protein content, whereas colonic segment weight and gross damage score were both increased in mice with colitis, compared with time-matched control values. There was no change in muscle protein synthesis in mice with inflammatory bowel disease; but there was an increased protein breakdown (45%), proteasome activity (85%), and messenger RNA (mRNA) expression for atrogin-1 and MuRF1 (200%-300%) in muscle. These changes were associated with a reduction in liver (but not muscle) IGF-I mRNA as well as a reduction in both total and free IGF-I in the blood. Colitis decreased the hepatic content of IGF binding protein (IGFBP)-3 mRNA by 40% and increased IGFBP-1 mRNA by 100%. In contrast, colitis did alter IGFBP mRNAs in muscle. The tumor necrosis factor-α, interleukin-6, and nitric oxide synthase 2 mRNA content of both liver and skeletal muscle was increased in TNBS-treated mice; and plasma tumor necrosis factor-α and interleukin-6 concentrations were also elevated. These data suggest that TNBS-induced colitis is independent of a change in muscle protein synthesis but dependent on stimulation of protein degradation via increased expression of muscle-specific atrogenes, which may be mediated in part by the reduction in circulating concentration of IGF-I and the concomitant increase in inflammatory mediators observed in the blood and muscle per se., (Copyright © 2010 Elsevier Inc. All rights reserved.)

PRAS40 is an mTOR binding protein that has complex effects on cell metabolism. Our study tests the hypothesis that PRAS40 knockdown (KD) in C2C12 myocytes will increase protein synthesis via upregulation of the mTOR-S6K1 pathway. PRAS40 KD was achieved using lentiviruses to deliver short hairpin (sh)-RNA targeting PRAS40 or a scrambled control. C2C12 cells were used as either myoblasts or differentiated to myotubes. Knockdown reduced PRAS40 mRNA and protein content by >80% of time-matched control values but did not alter the phosphorylation of mTOR substrates, 4E-BP1 or S6K1, in neither myoblasts nor myotubes. No change in protein synthesis in myotubes was detected, as measured by the incorporation of (35)S-methionine. In contrast, protein synthesis was reduced 25% in myoblasts. PRAS40 KD in myoblasts also decreased proliferation rate with an increased percent of cells retained in the G1 phase. PRAS40 KD myoblasts were larger in diameter and had a decreased rate of myotube formation as assessed by myosin heavy chain content. Immunoblotting revealed a 25-30% decrease in total p21 and S807/811 phosphorylated Rb protein considered critical for G1 to S phase progression. Reduction in protein synthesis was not due to increased apoptosis, since cleaved caspase-3 and DNA laddering did not differ between groups. In contrast, the protein content of LC3B-II was decreased by 30% in the PRAS40 KD myoblasts, suggesting a decreased rate of autophagy. Our results suggest that a reduction in PRAS40 specifically impairs myoblast protein synthesis, cell cycle, proliferation and differentiation to myotubes.

The hindgut hypothesis posits improvements in Type 2 diabetes after gastric bypass surgery are due to enhanced delivery of undigested nutrients to the ileum, which increase incretin production and insulin sensitivity. The present study investigates the effect of ileal interposition (IT), surgically relocating a segment of distal ileum to the proximal jejunum, on glucose tolerance, insulin sensitivity, and glucose transport in the obese Zucker rat. Two groups of obese Zucker rats were studied: IT and sham surgery ad libitum fed (controls). Changes in food intake, body weight and composition, glucose tolerance, insulin sensitivity and tissue glucose uptake, and insulin signaling as well as plasma concentrations of glucagon-like peptide-1 and glucose-dependent insulinotropic peptide were measured. The IT procedure did not significantly alter food intake, body weight, or composition. Obese Zucker rats demonstrated improved glucose tolerance 3 wk after IT compared with the control group (P < 0.05). Euglycemic, hyperinsulinemic clamp and 1-[(14)C]-2-deoxyglucose tracer studies indicate that IT improves whole body glucose disposal, insulin-stimulated glucose uptake, and the ratio of phospho- to total Akt (P < 0.01 vs. control) in striated muscle. After oral glucose, the plasma concentration of glucagon-like peptide-1 was increased, whereas GIP was decreased following IT. Enhanced nutrient delivery to the ileum after IT improves glucose tolerance, insulin sensitivity and muscle glucose uptake without altering food intake, body weight, or composition. These findings support the concept that anatomic and endocrine alterations in gut function play a role in the improvements in glucose homeostasis after the IT procedure.

Endotoxin (LPS) and sepsis decrease mammalian target of rapamycin (mTOR) activity in skeletal muscle, thereby reducing protein synthesis. Our study tests the hypothesis that inhibition of branched-chain amino acid (BCAA) catabolism, which elevates circulating BCAA and stimulates mTOR, will blunt the LPS-induced decrease in muscle protein synthesis. Wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout mice were studied 4 h after Escherichia coli LPS or saline. Basal skeletal muscle protein synthesis was increased in knockout mice compared with WT, and this change was associated with increased eukaryotic initiation factor (eIF)-4E binding protein-1 (4E-BP1) phosphorylation, eIF4E.eIF4G binding, 4E-BP1.raptor binding, and eIF3.raptor binding without a change in the mTOR.raptor complex in muscle. LPS decreased muscle protein synthesis in WT mice, a change associated with decreased 4E-BP1 phosphorylation as well as decreased formation of eIF4E.eIF4G, 4E-BP1.raptor, and eIF3.raptor complexes. In BCATm knockout mice given LPS, muscle protein synthesis only decreased to values found in vehicle-treated WT control mice, and this ameliorated LPS effect was associated with a coordinate increase in 4E-BP1.raptor, eIF3.raptor, and 4E-BP1 phosphorylation. Additionally, the LPS-induced increase in muscle cytokines was blunted in BCATm knockout mice, compared with WT animals. In a separate study, 7-day survival and muscle mass were increased in BCATm knockout vs. WT mice after polymicrobial peritonitis. These data suggest that elevating blood BCAA is sufficient to ameliorate the catabolic effect of LPS on skeletal muscle protein synthesis via alterations in protein-protein interactions within mTOR complex-1, and this may provide a survival advantage in response to bacterial infection.

Sepsis and lipopolysaccharide (LPS) may decrease skeletal muscle protein synthesis by impairing mTOR (mammalian target of rapamycin) activity. The role of mTOR in regulating muscle protein synthesis was assessed in wild-type (WT) and mTOR heterozygous (+/-) mice under basal conditions and in response to LPS and/or leucine stimulation. No difference in body weight of mTOR(+/-) mice was observed compared with WT mice; whereas whole body lean body mass was reduced. Gastrocnemius weight was decreased in mTOR(+/-) mice, which was attributable in part to a reduced rate of basal protein synthesis. LPS decreased muscle protein synthesis in WT and mTOR(+/-) mice to the same extent. Reduced muscle protein synthesis in mTOR(+/-) mice under basal and LPS-stimulated conditions was associated with lower 4E-BP1 and S6K1 phosphorylation. LPS also decreased PRAS40 phosphorylation and increased phosphorylation of raptor and IRS-1 (Ser(307)) to the same extent in WT and mTOR(+/-) mice. Muscle atrogin-1 and MuRF1 mRNA content was elevated in mTOR(+/-) mice under basal conditions, implying increased ubiquitin-proteasome-mediated proteolysis, but the LPS-induced increase in these atrogenes was comparable between groups. Plasma insulin and IGF-I as well as tissue expression of TNFalpha, IL-6, or NOS2 did not differ between WT and mTOR(+/-) mice. Finally, whereas LPS impaired the ability of leucine to stimulate muscle protein synthesis and 4E-BP1 phosphorylation in WT mice, this inflammatory state rendered mTOR(+/-) mice leucine unresponsive. These data support the idea that the LPS-induced reduction in mTOR activity is relatively more important in regulating skeletal muscle mass in response to nutrient stimulation than under basal conditions.

Acute alcohol intoxication decreases skeletal muscle protein synthesis by impairing mammalian target of rapamycin (mTOR). In 2 studies, we determined whether inhibition of branched-chain amino acid (BCAA) catabolism ameliorates the inhibitory effect of alcohol on muscle protein synthesis by raising the plasma BCAA concentrations and/or by improving the anabolic response to insulin-like growth factor (IGF)-I. In the first study, 4 groups of mice were used: wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout (KO) mice orally administered saline or alcohol (5 g/kg, 1 h). Protein synthesis was greater in KO mice compared with WT controls and was associated with greater phosphorylation of eukaryotic initiation factor (eIF)-4E binding protein-1 (4EBP1), eIF4E-eIF4G binding, and 4EBP1-regulatory associated protein of mTOR (raptor) binding, but not mTOR-raptor binding. Alcohol decreased protein synthesis in WT mice, a change associated with less 4EBP1 phosphorylation, eIF4E-eIF4G binding, and raptor-4EBP1 binding, but greater mTOR-raptor complex formation. Comparable alcohol effects on protein synthesis and signal transduction were detected in BCATm KO mice. The second study used the same 4 groups, but all mice were injected with IGF-I (25 microg/mouse, 30 min). Alcohol impaired the ability of IGF-I to increase muscle protein synthesis, 4EBP1 and 70-kilodalton ribosomal protein S6 kinase-1 phosphorylation, eIF4E-eIF4G binding, and 4EBP1-raptor binding in WT mice. However, in alcohol-treated BCATm KO mice, this IGF-I resistance was not manifested. These data suggest that whereas the sustained elevation in plasma BCAA is not sufficient to ameliorate the catabolic effect of acute alcohol intoxication on muscle protein synthesis, it does improve the anabolic effect of IGF-I.

The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GbetaL and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (EtOH). However, the mechanisms by which EtOH regulates mTORC1 activity have not been established. Here, we investigated the effect of EtOH on the phosphorylation and interaction of components of mTORC1 in C2C12 myocytes. We also examined the specific role that PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS-1. EtOH also caused changes in mTORC1 protein-protein interactions. EtOH enhanced the binding of raptor and PRAS40 with mTOR. These alterations occurred in concert with increased binding of 14-3-3 to raptor, while the PRAS40 and 14-3-3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to EtOH. PRAS40 KD increased raptor phosphorylation and its association with 14-3-3, whereas decreased GbetaL-mTOR binding. The effects of EtOH and PRAS40 KD were mediated by AMPK. Both factors increased in vitro AMPK activity towards the substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2. Collectively, our results indicate that EtOH stabilizes the association of raptor, PRAS40, and GbetaL with mTOR, while likewise increasing the interaction of raptor with 14-3-3. These data suggest a possible mechanism for the inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in myocytes.

Previous studies in monocytes and other cell types have provided evidence of a role for the NF-κB pathway in IL-6 induction. The purpose of the present study was to examine the involvement of NF-κB in the induction of the IL-6 promoter in skeletal muscle cells by endotoxin (LPS), TNFα or IL-1α. Transfection of C2C12 mouse myocytes with a luciferase reporter under the control of the IL-6 promoter indicated each immunomodulator enhanced IL-6 promoter activity. Mutation and inhibitor studies indicate this response was dependent on the IL-6 NF-κB binding site, but independent of NF-IL6, AP-1, CREB or C/EBP. Cotransfection with an expression vector which constitutively activates the RelA pathway increased IL-6 promoter activity, and activity could not be further enhanced by cytokines or LPS. However, cotransfecting various dominant negative upstream NF-κB kinase expression vectors which inhibited RelA or RelB pathways either individually or in combination had no effect on LPS-induced activation of the IL-6 promoter, but abolished induction from a NF-κB-based promoter. This lack of effect was not due to a lack of NF-κB pathway activation in C2C12 myocytes because both Western analysis and EMSA supershifting showed an LPS-induced increase in nuclear RelA and RelA phosphorylation. However, another protein was observed bound to the IL-6 NF-κB site that does not bind to a consensus NF-κB site. The present findings provide novel insights regarding inflammation-induced stimulation of IL-6 promoter activity in skeletal muscle which is an important but non-traditional component of the innate immune system.

Insulin-like growth factor-I (IGF-I) is a major anabolic hormone for skeletal muscle and a potent stimulus for protein synthesis and translation initiation. Recent studies suggest that translation can be inhibited by over expression of the mammalian target of rapamycin (mTOR) repressor REDD1. The purpose of the present study was to determine whether IGF-I alters the expression of REDD1 and whether this is associated with a concomitant change in protein synthesis in vitro. Subcutaneous injection of IGF-I or intravenous delivery of insulin for 3-4 h increased REDD1 mRNA in skeletal muscle 7-10-fold. A threefold increase in REDD1 was observed when C2C12 myotubes were treated with IGF-I. REDD1 protein continued to be expressed for up to 24 h after addition of IGF-I to cells. Withdrawal of IGF-I from myotubes lead to a rapid loss of REDD1 protein content. IGF-I-induced REDD1 mRNA and protein expression were prevented by inhibitors of transcription and translation. IGF-I had an additive effect with dexamethasone (Dex) on REDD1 protein content in myotubes. The PI3K inhibitor LY294002 blocked IGF-I but not Dex induced REDD1. IGF-I also stimulated REDD1 promoter activity. Although REDD1 protein was elevated 5-6 h after addition of IGF-I to myotubes, protein synthesis measured during this 1 h window was paradoxically greater in myotubes expressing more REDD1. In contrast to the IGF-I induced increase in REDD1 mRNA, REDD2 mRNA was decreased by IGF-I. We conclude that IGF-I stimulates REDD1 expression in skeletal muscle and myotubes but under these conditions the REDD1 response is not sufficient to repress protein synthesis.

Reduced testosterone as a result of catabolic illness or aging is associated with loss of muscle and increased adiposity. We hypothesized that these changes in body composition occur because of altered rates of protein synthesis under basal and nutrient-stimulated conditions that are tissue specific. The present study investigated such mechanisms in castrated male rats (75% reduction in testosterone) with demonstrated glucose intolerance. Over 9 wk, castration impaired body weight gain, which resulted from a reduced lean body mass and preferential sparing of adipose tissue. Castration decreased gastrocnemius weight, but this atrophy was not associated with reduced basal muscle protein synthesis or differences in plasma IGF-I, insulin, or individual amino acids. However, oral leucine failed to normally stimulate muscle protein synthesis in castrated rats. In addition, castration-induced atrophy was associated with increased 3-methylhistidine excretion and in vitro-determined ubiquitin proteasome activity in skeletal muscle, changes that were associated with decreased atrogin-1 or MuRF1 mRNA expression. Castration decreased heart and kidney weight without reducing protein synthesis and did not alter either cardiac output or glomerular filtration. In contradistinction, the weight of the retroperitoneal fat depot was increased in castrated rats. This increase was associated with an elevated rate of basal protein synthesis, which was unresponsive to leucine stimulation. Castration also decreased whole body fat oxidation. Castration increased TNFα, IL-1α, IL-6, and NOS2 mRNA in fat but not muscle. In summary, the castration-induced muscle wasting results from an increased muscle protein breakdown and the inability of leucine to stimulate protein synthesis, whereas the expansion of the retroperitoneal fat depot appears mediated in part by an increased basal rate of protein synthesis-associated increased inflammatory cytokine expression.

The purpose of the present study was to test the hypothesis that endogenous NO negatively affects translation in skeletal muscle cells after exposure to a combination of endotoxin (LPS) and interferon-gamma (IFN-gamma). Individually, LPS and IFN-gamma did not alter protein synthesis, but in combination, they inhibited protein synthesis by 80% in C2C12 myotubes. The combination of LPS and IFN-gamma dramatically downregulated the autophosphorylation of the mammalian target of rapamycin and its substrates S6K1 and 4EBP-1. The phosphorylation of ribosomal protein S6 was decreased, whereas phosphorylation of elongation factor 2 and raptor was enhanced, consistent with defects in both translation initiation and elongation. Reduced S6 phosphorylation occurred 8 to 18 h after LPS/IFN-gamma and coincided with a prolonged upregulation of NOS2 messenger RNA and protein. NOS2 protein expression and the LPS/IFN-gamma-induced fall in phosphorylated S6 were prevented by the proteasome inhibitor MG-132. The general NOS inhibitor, L-NAME, and the specific NOS2 inhibitor, 1400W, also prevented the LPS/IFN-gamma-induced decrease in protein synthesis and restored translational signaling. LPS/IFN-gamma downregulated the phosphorylation of multiple Akt substrates, including the proline-rich Akt substrate 40, while enhancing the phosphorylation of raptor on a 5'-AMP-activated kinase (AMPK)-regulated site. The negative effects of LPS/IFN-gamma were blunted by the AMPK inhibitor compound C. The data suggest that, in combination, LPS and IFN-gamma induce a prolonged expression of NOS2 and excessive production of NO that reciprocally alter Akt and AMPK activity and consequently downregulate translation via reduced mammalian target of rapamycin signaling.

The present study tests the hypotheses that local bioavailability of insulin-like growth factor I (IGF-I) is capable of regulating muscle protein balance and that muscle-directed IGF-I can selectively maintain muscle mass during bacterial infection. Initial studies in C57BL/6 mice demonstrated that increasing or decreasing bioavailable IGF-I within muscle by local administration of either Leu(24) Ala(31) IGF-I or IGF binding protein 1, respectively, produced proportional changes in surrogate markers (eg, phosphorylation of 4E-BP1 and S6K1) of protein synthesis. We next examined the ability of a sustained local administration of IGF-I to prevent sepsis-induced muscle atrophy over a 5-day period. At the time of cecal ligation and puncture or sham surgery, mice had a time-release pellet containing IGF-I implanted next to the gastrocnemius and a placebo pellet placed in the contralateral limb. Data indicated that IGF-I released locally only affected the adjacent muscle and was not released into the circulation. Gastrocnemius from septic mice containing the placebo pellet was atrophied and had a reduced IGF-I protein content. In contrast, locally directed IGF-I increased IGF-I protein within adjacent muscle to basal control levels. This change was associated with a proportional increase in muscle weight and protein, as well as increased phosphorylation of 4E-BP1 and the redistribution of eIF4E from the inactive eIF4E4EBP1 complex to the active eIF4EeIF4G complex. Local IGF-I also prevented the sepsis-induced increase in atrogin-1 messenger RNA in the exposed muscle. Finally, local IGF-I prevented the sepsis-induced increase in muscle interleukin-6 messenger RNA. Thus, muscle-directed IGF-I attenuates the sepsis-induced atrophic response apparently by increasing muscle protein synthesis and potentially decreasing proteolysis. Collectively, our data suggest that agents that increase the bioavailability of IGF-I within muscle per se might be effective in ameliorating the sepsis-induced loss of muscle mass without having undesirable effects on metabolic processes in distant organs.

Objective: Obesity-related diabetes is caused by insulin resistance and beta-cell dysfunction. The current study examines changes in food intake, weight loss, body fat depots, oxygen consumption, insulin sensitivity, and incretin levels as potential mechanisms for improved glucose tolerance after Roux-en-Y gastric bypass (RYGB)., Methods: Three groups of genetically obese Zucker rats were studied: RYGB, sham surgery pair-fed (PF), and sham surgery ad libitum (AL) fed rats. Changes in body weight, visceral and subcutaneous fat depots, oral glucose tolerance, insulin sensitivity, and the plasma concentrations of insulin, glucagon, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide, and peptide YY (PYY) were measured., Results: Body weight and subcutaneous fat were decreased after RYGB, compared with the PF and AL groups. The reduction in visceral fat after RYGB appeared largely because of food restriction. Glucose tolerance and insulin sensitivity were significantly improved in only the RYGB group (P < 0.05 vs. AL, PF). Euglycemic, hyperinsulinemic clamp studies indicated RYGB improved the ability of insulin to stimulate peripheral (eg, skeletal muscle) glucose uptake. Fasting total GLP-1, glucose-dependent insulinotropic peptide, and PYY levels were similar between the groups, whereas postprandial plasma levels of intact GLP-1 (7-36) amide, total GLP-1, and PYY were increased in the RYGB group compared with PF and AL controls., Conclusions: Glucose homeostasis after RYGB is associated with decreased subcutaneous fat, increased postprandial PYY, GLP-1, and insulin, as well as improved insulin sensitivity/action. Changes in food intake and visceral fat do not seem to explain improvements in insulin action after RYGB in the Zucker rat model.

Background: Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation. However, these studies have been performed in relatively young rapidly growing rats in which muscle protein accretion is more sensitive to growth factor and nutrient stimulation. Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner. The hypothesis tested in the present study was that young rats will show a more pronounced decrement in muscle protein synthesis than older mature rats in response to acute EtOH intoxication., Methods: Male F344 rats were studied at approximately 3 (young) or 12 (mature) months of age. Young rats were injected intraperitoneally with 75 mmol/kg of EtOH, and mature rats injected with either 75 or 90 mmol/kg EtOH. Time-matched saline-injected control rats were included for both age groups. Gastrocnemius protein synthesis and the activity of the mTOR pathway were assessed 2.5 h after EtOH using [³H]-labeled phenylalanine and the phosphorylation of various protein factors known to regulate peptide-chain initiation., Results: Blood alcohol levels (BALs) were lower in mature rats compared to young rats after administration of 75 mmol/kg EtOH (154 ± 23 vs 265 ± 24 mg/dL). However, injection of 90 mmol/kg EtOH in mature rats produced BALs comparable to that of young rats (281 ± 33 mg/dL). EtOH decreased muscle protein synthesis similarly in both young and high-dose EtOH-treated mature rats. The EtOH-induced changes in both groups were associated with a concomitant reduction in 4E-BP1 phosphorylation, and redistribution of eIF4E between the active eIF4E.eIF4G and inactive eIF4E.4EBP1 complex. Moreover, EtOH increased the binding of mTOR with raptor in a manner which appeared to be AMPK- and TSC-independent. In contrast, although muscle protein synthesis was unchanged in mature rats given low-dose EtOH, compared to control values, the phosphorylation of rpS6 and eIF4G was decreased., Conclusion: These data indicate that muscle protein synthesis is equally sensitive to the inhibitory effects of EtOH in young rapidly growing rats and older mature rats which are growing more slowly, but that mature rats must be given a relatively larger dose of EtOH to achieve the same BAL. Based on the differential response in mature rats to low- and high-dose EtOH, the decreased protein synthesis was associated with a reduction in mTOR activity which was selectively mediated via a reduction in 4E-BP1 phosphorylation and an increase in mTOR.raptor formation.

TNF inhibits serine protease inhibitor 2.1 (Spi 2.1) and IGF-I gene expression by GH in CWSV-1 hepatocytes. The current study describes construction of a GH-inducible IGF-I promoter construct and investigates mechanisms by which TNF and nuclear factor-kappaB (NFkappaB) inhibit GH-inducible gene expression. CWSV-1 cells were transfected with GH-inducible Spi 2.1 or IGF-I promoter luciferase constructs, incubated with TNF signaling inhibitors (fumonisin B1 for sphingomyelinase and SP600125 for c-Jun N-terminal kinase), treated with or without TNF, and then stimulated with recombinant human GH. The 5- to 6-fold induction of Spi 2.1 and IGF-I promoter activity by GH was inhibited by TNF. Neither fumonisin B1 nor SP600125 prevented the inhibitory effects of TNF on GH-inducible promoter activity. Dominant-negative inhibitor-kappaBalpha (IkappaBalpha) expression vectors (IkappaBalphaS/A or IkappaBalphaTrunc), p65 and p50 expression vectors, and p65 deletion constructs were used to investigate the NFkappaB pathway. IkappaBalphaS/A and IkappaBalphaTrunc ameliorated the inhibitory effects of TNF on GH-inducible Spi 2.1 and IGF-I promoter activity. Cotransfection of CWSV-1 cells with expression vectors for p65 alone or p50 and p65 together inhibited GH-inducible Spi 2.1 and IGF-I promoter activity. Cotransfection with a C-terminal p65 deletion (1-450) enhanced GH-inducible promoter activity, whereas the N-terminal deletion (31-551) was inhibitory for IGF-I but not Spi 2.1. Cycloheximide did not antagonize the inhibitory effects of TNF on GH-inducible IGF-I expression. We conclude the inhibitory effects of TNF on GH-inducible promoter activity are mediated by NFkappaB, especially p65, by a mechanism that does not require protein synthesis.

HIV anti-retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor-2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose-dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP-activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK-eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K., ((c) 2008 Wiley-Liss, Inc.)

The mechanism by which human immunodeficiency virus (HIV)-1 infection in humans leads to the erosion of lean body mass is poorly defined. Therefore, the purpose of the present study was to determine whether transgenic (Tg) rats that constitutively overexpress HIV-1 viral proteins exhibit muscle wasting and to elucidate putative mechanisms. Over 7 mo, Tg rats gained less body weight than pair-fed controls exclusively as a result of a proportional reduction in lean, not fat, mass. Fast- and slow-twitch muscle atrophy in Tg rats did not result from a reduction in the in vivo-determined rate of protein synthesis. In contrast, urinary excretion of 3-methylhistidine, as well as the content of atrogin-1 and the 14-kDa actin fragment, was elevated in gastrocnemius of Tg rats, suggesting increased muscle proteolysis. Similarly, Tg rats had reduced cardiac mass, which was independent of a change in protein synthesis. This decreased cardiac mass was associated with a reduction in stroke volume, but cardiac output was maintained by a compensatory increase in heart rate. The HIV-induced muscle atrophy was associated with increased whole body energy expenditure, which was not due to an elevated body temperature or secondary bacterial infection. Furthermore, the atrophic response could not be attributed to the development of insulin resistance, decreased levels of circulating amino acids, or increased tissue cytokines. However, skeletal muscle and, to a lesser extent, circulating insulin-like growth factor I was reduced in Tg rats. Although hepatic injury was implicated by increased plasma levels of aspartate and alanine aminotransferases, hepatic protein synthesis was not different between control and Tg rats. Hence, HIV-1 Tg rats develop atrophy of cardiac and skeletal muscle, the latter of which results primarily from an increased protein degradation and may be related to the marked reduction in muscle insulin-like growth factor I.

Several stress conditions are characterized by activation of 5'-AMP-activated protein kinase (AMPK) and the development of leucine resistance in skeletal muscle. In the present study, we determined whether direct activation of the AMPK by 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) prevents the characteristic leucine-induced increase in protein synthesis by altering mammalian target of rapamycin (mTOR) signal transduction. Rats were injected with AICAR or saline (Sal) and 1 h thereafter received an oral gavage of leucine (or Sal). Efficacy of AICAR was verified by increased AMPK phosphorylation. AICAR decreased basal in vivo muscle (gastrocnemius) protein synthesis and completely prevented the leucine-induced increase, independent of a change in muscle adenine nucleotide concentration. AICAR also prevented the hyperphosphorylation of eukaryotic initiation factor (eIF) 4E binding protein (4E-BP1), ribosomal protein S6 kinase (S6K1), S6, and eIF4G in response to leucine, suggesting a decrease in mTOR activity. Moreover, AICAR prevented the leucine-induced redistribution of eIF4E from the inactive eIF4E.4E-BP1 to the active eIF4E.eIF4G complex. This ability of AICAR to produce muscle leucine resistance could not be attributed to a change in phosphorylation of tuberous sclerosis complex (TSC)2, the formation of a TSC1.TSC2 complex, the binding of raptor with mTOR, or the phosphorylation of eukaryotic elongation factor-2. However, the inhibitory actions of AICAR were associated with reduced phosphorylation of proline-rich Akt substrate-40 and increased phosphorylation of raptor, which represent potential mechanisms by which AICAR might be expected to inhibit leucine-induced increases in mTOR activity and protein synthesis under in vivo conditions.

Acute alcohol intoxication decreases muscle protein synthesis, but there is a paucity of data on the ability of alcohol to regulate muscle protein degradation. Furthermore, various types of atrophic stimuli appear to regulate ubiquitin-proteasome-dependent proteolysis by increasing the muscle-specific E3 ligases atrogin-1 and MuRF1 (i.e., "atrogenes"). Therefore, the present study was designed to test the hypothesis that acute alcohol intoxication increases atrogene expression leading to an elevated rate of muscle protein breakdown. In male rats, the intraperitoneal injection of alcohol dose- and time-dependently increased atrogin-1 and MuRF1 mRNA in gastrocnemius, the latter of which was most pronounced. A comparable change was absent in the soleus and heart. The ability of in vivo-administered ethanol to increase atrogene expression was independent of the route of alcohol administration (intraperitoneal vs. oral), as well as of nutritional status (fed vs. fasted) and gender (male vs. female). The increase in atrogin-1 and MuRF1 was independent of alcohol metabolism, and the overproduction of endogenous glucocorticoids and could not be prevented by maintaining the circulating concentration of insulin-like growth factor-I. Despite marked changes in atrogene expression, acute alcohol in vivo did not alter the release of either 3-methylhistidine (MH) or tyrosine from the isolated perfused hindlimb, suggesting that the rate of muscle proteolysis remains unchanged. Moreover, alcohol did not increase the directly determined rate of protein degradation in isolated epitrochlearis muscles or cultured myocytes. Finally, no increase in atrogene expression or 3-MH release was detected in muscle from rats fed an alcohol-containing diet. Our results indicate that although acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA preferentially in fast-twitch skeletal muscle, this change was not associated with increased rates of muscle proteolysis. Therefore, the loss of muscle mass/protein in response to chronic alcohol abuse appears to result primarily from a decrement in muscle protein synthesis, not an increase in degradation.

Background: The mechanism by which acute alcohol (EtOH) intoxication decreases basal muscle protein synthesis via inhibition of the Ser/Thr kinase mammalian target of rapamycin (mTOR) is poorly defined. In this regard, mTOR activity is impaired after over expression of the regulatory protein REDD1. Hence, the present study assessed the ability of REDD1 as a potential mediator of the EtOH-induced decrease in muscle protein synthesis., Methods: The effect of acute EtOH intoxication on REDD1 mRNA and protein was determined in striated muscle of rats and mouse myocytes using an RNase protection assay and Western blotting, respectively. Other components of the mTOR signaling pathway were also assessed by immunoblotting. For comparison, REDD1 mRNA/protein was also determined in the muscle of rats chronically fed an alcohol-containing diet for 14 weeks., Results: Intraperitoneal (IP) injection of EtOH increased gastrocnemius REDD1 mRNA in a dose- and time-dependent manner, and these changes were associated with reciprocal decreases in the phosphorylation of 4E-BP1, which is a surrogate marker for mTOR activity and protein synthesis. No change in REDD1 mRNA was detected in the slow-twitch soleus muscle or heart. Acute EtOH produced comparable increases in muscle REDD1 protein. The EtOH-induced increase in gastrocnemius REDD1 was independent of the route of EtOH administration (oral vs. IP), the nutritional state (fed vs. fasted), gender, and age of the rat. The nonmetabolizable alcohol tert-butanol increased REDD1 and the EtOH-induced increase in REDD1 was not prevented by pretreatment with the alcohol dehydrogenase inhibitor 4-methylpyrazole. In contrast, REDD1 mRNA and protein were not increased in the isolated hindlimb perfused with EtOH or in C2C12 myocytes incubated with EtOH, under conditions previously reported to decrease protein synthesis. Pretreatment with the glucocorticoid receptor antagonist RU486 failed to prevent the EtOH-induced increase in REDD1. Finally, the EtOH-induced increase in REDD1 was not associated with altered formation of the TSC1*TSC2 complex or the phosphorylation of TSC2 which is down stream in the REDD1 stress response pathway. In contradistinction to the changes observed with acute EtOH intoxication, REDD1 mRNA/protein was not changed in gastrocnemius from chronic alcohol-fed rats despite the reduction in 4E-BP1 phosphorylation., Conclusions: These data indicate that in fast-twitch skeletal muscle (i) REDD1 mRNA/protein is increased in vivo by acute EtOH intoxication but not in response to chronic alcohol feeding, (ii) elevated REDD1 in response to acute EtOH appears due to the production of an unknown secondary mediator which is not corticosterone, and (iii) the EtOH-induced decrease in protein synthesis can be dissociated from a change in REDD1 suggesting that the induction of this protein is not responsible for the rapid decrease in protein synthesis after acute EtOH administration or for the development of alcoholic myopathy in rats fed an alcohol-containing diet.

In mice fed trypsin inhibitor (camostat) to elevate endogenous CCK, pancreatic growth plateaus by 7 days. It is unknown whether this represents the maximum growth capacity of the pancreas. To test the ability of CCK to drive further growth, mice were fed chow containing camostat (0.1%) for 1 wk, then fed standard chow for 1 wk, and finally returned to the camostat diet for a week. Pancreatic mass increased to 245% of initial value (iv) following 1 wk of camostat feeding, decreased to 147% iv following a 1 wk return to normal chow, and increased to 257% iv with subsequent camostat feeding. Camostat feeding was associated with significant increases in circulating CCK and changes in pancreatic mass were paralleled by changes in protein and DNA content. Moreover, regression of the pancreas following camostat feeding was associated with changes in the expression of the autophagosome marker LC3. Pancreatic protein synthetic rates were 130% of control after 2 days on camostat but were equivalent to control after 7 days. Changes in the phosphorylation of 4E-BP1 and S6, downstream effectors of mammalian target of rapamycin (mTOR), paralleled changes in protein synthetic rates. Cellular content of Akt, an upstream activating kinase of mTOR, decreased after 7 days of camostat feeding whereas expression of the E3 ubiquitin-ligases and the cell cycle inhibitor p21 increased after 2 days. These results indicate that CCK-stimulated growth of the pancreas is not limited by acinar cell mitogenic capacity but is due, at least in part, to inhibition of promitogenic Akt signaling.

Skeletal muscle demonstrates great plasticity in response to environmental and hormonal factors including pathogen-associated molecules, inflammatory cytokines, and growth factors. These signals impinge on muscle by forcing individual muscle fibers to either grow or atrophy. We recently demonstrated that skeletal muscle cells express multiple Toll-like receptors (TLR) that recognize bacterial cell wall components, such as lipopolysaccharide (LPS). Exposure of myocytes to LPS and other TLR ligands initiates an inflammatory response culminating in the autocrine production of cytokines and NO by NO synthase (NOS)2. The TLR signal through protein kinases that phosphorylate and promote the degradation of an inhibitory protein that normally retains the transcription factor, nuclear factor kappaB (NFkappaB), in the cytoplasm. Phosphorylation and degradation of the inhibitor of NFkappaB allows for translocation of NFkappaB to the nucleus and activation of inflammatory genes. Overexpression of a constitutively active inhibitor of NFkappaB kinase in skeletal muscle causes severe wasting, and we found that inhibitors of either the phosphorylation of IkappaB or its proteolytic degradation prevent TLR ligand-induced expression of cytokines and NOS2. The combination of LPS and interferon gamma dramatically enhances the magnitude and duration of LPS-stimulated NOS2 expression and reduces protein translation. Lipopolysaccharide and interferon gamma also downregulates signaling from the mammalian target of rapamycin, a kinase that directs changes in cell size. Inhibitors of NOS block the fall in muscle cell protein synthesis and restore translational signaling, indicating that activation of the NOS2-NO pathway is responsible for the observed decrease in muscle protein synthesis. Our work provides a molecular explanation for reduced muscle growth during infection. Muscle is largely self-sufficient because it expresses receptors, signaling pathways, and effectors to regulate its own size. Prolonged activation of NFkappaB and NOS2 have emerged as detrimental facets of the immune response in muscle. The interplay between inflammatory components and growth factor signaling clearly places muscle at the interface between growth and immunity.

Acute alcohol intoxication leads to an inhibition of protein synthesis in heart that results in part through altered phosphorylation of protein factors controlling mRNA translation initiation. The purpose of the present set of experiments was designed to examine the effects of inhibitors of ethanol metabolism on the phosphorylation of 4E-binding protein (4EBP1) and S6k1(Thr(389)), two factors regulating mRNA translation initiation. Phosphorylation of 4E-BP1, S6k1(Thr(389)), and Erk 1/2 was reduced 2 h following IP injection of alcohol. Pretreatment with 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase (ADH), did not attenuate the ethanol-induced decrease in phosphorylation of 4EBP1 and S6k1(Thr(389)). In contrast, 4-MP prevented the decrease in Erk 1/2 phosphorylation observed with acute ethanol intoxication. Pretreatment with cyanamide, an inhibitor of aldehyde dehydrogenase, did not attenuate the ethanol-induced decrease in phosphorylation S6k1(Thr(389)), but partially prevented the ethanol-induced lowering of 4EBP1 phosphorylation. The studies indicate that modulation of ethanol metabolism through inhibition of ADH or aldehyde dehydrogenase leads to preferential modulation of the phosphorylation of distinct myocardial signaling systems involved in regulating protein synthesis.

Background: Survival following human immunodeficiency virus (HIV) infection has improved significantly following the advent of highly active antiretroviral therapy. A large percentage of HIV-infected patients consume and abuse alcohol. Erosion of lean body mass is an important contributing factor to patient morbidity and mortality, and is a common feature of both chronic alcohol (ALC) consumption and acquired immunodeficiency syndrome (AIDS). We hypothesized that alcohol-induced loss in lean body mass is likely to exacerbate the AIDS wasting syndrome, particularly at the terminal stage of AIDS (SAIDS)., Methods: This study examined the impact of chronic, intra-gastric ALC (5 h/d x 4 d/wk; blood alcohol levels = 55 mM to 60 mM) administration on body composition and muscle mass in simian immunodeficiency virus (SIV)-infected male Rhesus macaques in contrast to SIV-infected isocaloric (22 kcal/kg/d) sucrose (SUC)-infused control animals at the terminal stage of SIV infection., Results: At terminal stage, ALC/SIV+ animals had significantly lower body weight, body mass index, and limb muscle area than SUC/SIV+ animals. Both ALC/SIV+ and SUC/SIV+ animals had suppressed expression of insulin-like growth factor-I and increased expression of the ubiquitin ligase muscle-specific RING finger-1 mRNA. ALC increased mRNA expression of atrogin-1 (pre-SIV and at SAIDS) and tumor necrosis factor (TNF)-alpha (SAIDS). These changes were not associated with significant differences in fractional rates of muscle protein synthesis or in overall survival rate. These data show that chronic ALC exacerbated the loss of muscle mass at terminal SAIDS., Conclusion: Our findings suggest the involvement of TNF-alpha and increased muscle proteolysis via atrogin-1 for the greater erosion of lean body mass at terminal SAIDS in ALC-treated Rhesus macaques.

Although much is known regarding the posttranslational regulation of the FoxO transcription factors, there is little appreciation of how stressors which regulate cellular energy status effect the various FoxO family members at the mRNA level. The hypothesis of the present study was that exposure of differentiated muscle cells to agonists of AMP-activated protein kinase (AMPK) would increase the mRNA content of various FoxO mRNA transcripts. Stimulation of AMPK in vivo by the injection of AICAR into mice increased FoxO1 and FoxO3 (but not FoxO4) mRNA in skeletal muscle. A comparable increase in these FoxO mRNAs was seen in skeletal muscle in response to sepsis which also increased AMPK phosphorylation. In contrast to the in vivo data, FoxO1, 3 and 4 mRNA content was decreased dose-dependently, with the decrement in FoxO1 being the largest, in C(2)C1(2) myotubes incubated with the AMPK agonists AICAR or metformin. Treatment of myotubes with 2-deoxyglucose or reducing the media glucose concentration also decreased mRNA content for FoxO1 and FoxO4. All stressors increased AMPK phosphorylation under in vitro conditions. Incubation of myotubes with AICAR decreased the rate of protein synthesis and increased protein degradation. Finally, treatment with the AMPK inhibitor compound C prevented both the AICAR-induced changes in FoxO mRNA and changes in protein metabolism. Our data indicate FoxO mRNA expression is down-regulated by AMPK activation and energy depletion in cultured myotubes, but that a contrasting increase in FoxO1 and FoxO3 mRNA is observed in vivo with the agent (and in response to sepsis) suggesting the expression of these FoxOs may be controlled by other hormonal or energy sensing cues under in vivo conditions.

The development of alcoholic muscle disease, which affects both cardiac and skeletal muscle, leads to increased morbidity and mortality in patients who abuse alcohol. The disease pathology includes myocyte degeneration, loss of striations, and myofilament dissolution, which is consistent with alterations in structural and myofibrillar proteins. One explanation for the changes in myofibrillar architecture is that the expression of cellular proteins may be compromised by ethanol consumption. The dynamic balance of proteins in striated muscle is dependent upon rates of protein synthesis and protein degradation. We have shown that protein synthesis is depressed in striated muscle after either acute alcohol intoxication or chronic alcohol ingestion. The loss of myofibrillar proteins occurs prior to any detection of abnormal muscle function in vivo. It is therefore of major importance to evaluate the regulation of protein turnover after ethanol consumption. This review describes protocols to study protein synthesis either in vivo or under in vitro conditions. The methods can be modified for studies involving transgenic mice allowing mechanisms responsible for the defects in protein synthesis to be dissected.

Background: Muscle disuse atrophy is observed in patients recovering from trauma and there is an increased risk and severity of injury in patients abusing alcohol (EtOH). However, the interaction of EtOH and disuse on muscle protein balance has not been examined. Therefore, the present study addressed the hypothesis that EtOH accelerates the disuse atrophy and/or impairs the accretion of muscle protein during muscle recovery., Methods: To address this aim, disuse atrophy was induced in rats by 3 days of unilateral hindlimb immobilization (casting), using the contralateral leg as control, with EtOH or saline being orally gavaged twice, each day during this period. In a separate study, EtOH-treated rats received Velcade to inhibit proteasomal degradation. Finally, in the last study, rats had 1 limb casted for 5 days, the cast removed, and EtOH or saline gavaged twice daily during a 5-day recovery period. Muscle protein metabolism was assessed using surrogate markers of protein synthesis [i.e., phosphorylation of 4E-binding protein 1 (BP1) and S6 kinase 1 (S6K1)] and protein degradation (i.e., mRNA content of the ubiquitin E3 ligases atrogin-1 and MuRF1)., Results: Ethanol alone did not decrease muscle weight in the uncasted muscle. However, the loss of mass of immobilized muscle from EtOH-gavaged rats was 80% greater than in the animals not receiving EtOH. This atrophic response was not associated with a change in Akt, 4E-BP1 or S6K1 phosphorylation among groups. In contrast, immobilization alone increased both atrogin-1 and MuRF1 mRNA, and EtOH further increased their expression in immobilized muscle. The proteasome inhibitor Velcade attenuated atrophy produced by EtOH + disuse. When administered during the recovery period, EtOH prevented the normal accretion of muscle mass. This EtOH effect was associated with increased atrogin-1 mRNA, a reduction in 4E-BP1 and S6 phosphorylation, and an increased AMP-activated kinase phosphorylation., Conclusions: Based on the changes in these surrogate markers, our data suggest that EtOH accelerates disuse atrophy by stimulating ubiquitin-mediated proteolysis, and blunts repletion of muscle protein during recovery from disuse by increasing proteolysis and decreasing protein synthesis.

Alcohol consumption induces a dose-dependent noxious effect on skeletal muscle, leading to progressive functional and structural damage of myocytes, with concomitant reductions in lean body mass. Nearly half of high-dose chronic alcohol consumers develop alcoholic skeletal myopathy. The pathogenic mechanisms that lie between alcohol intake and loss of muscle tissue involve multiple pathways, making the elucidation of the disease somewhat difficult. This review discusses the recent advances in basic and clinical research on the molecular and cellular events involved in the development of alcohol-induced muscle disease. The main areas of recent research interest on this field are as follows: (i) molecular mechanisms in alcohol exposed muscle in the rat model; (ii) gene expression changes in alcohol exposed muscle; (iii) the role of trace elements and oxidative stress in alcoholic myopathy; and (iv) the role of apoptosis and preapoptotic pathways in alcoholic myopathy. These aforementioned areas are crucial in understanding the pathogenesis of this disease. For example, there is overwhelming evidence that both chronic alcohol ingestion and acute alcohol intoxication impair the rate of protein synthesis of myofibrillar proteins, in particular, under both postabsorptive and postprandial conditions. Perturbations in gene expression are contributory factors to the development of alcoholic myopathy, as ethanol-induced alterations are detected in over 400 genes and the protein profile (i.e., the proteome) of muscle is also affected. There is supportive evidence that oxidative damage is involved in the pathogenesis of alcoholic myopathy. Increased lipid peroxidation is related to muscle fibre atrophy, and reduced serum levels of some antioxidants may be related to loss of muscle mass and muscle strength. Finally, ethanol induces skeletal muscle apoptosis and increases both pro- and antiapoptotic regulatory mechanisms.

We report on the advancement of apertureless terahertz microscopy by active shear force control of the scanning probe. Extreme subwavelength spatial resolution and a maximized image contrast are achieved by maintaining a tip-surface distance of about 20 nm. The constant distance between scanning tip and surface results in terahertz images that mirror the dielectric permittivity of the surface.

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.

This article contains the proceedings of a symposium at the 2006 ISBRA meeting in Sydney Australia, organized and cochaired by Martin J. Ronis and Thomas M. Badger. The presentations were (1) Effect of long-term ethanol consumption on liver injury and repair, by Jack R. Wands; (2) Alcohol-induced insulin resistance in liver: potential roles in regulation of ADH expression, ethanol clearance, and alcoholic liver disease, by Thomas M. Badger; (3) Chronic gestational exposure to ethanol causes brain insulin and insulin-like growth factor resistance, by Suzanne M de la Monte; (4) Disruption of IGF-1 signaling in muscle: a mechanism underlying alcoholic myopathy, by Charles H. Lang; (5) The role of reduced plasma estradiol and impaired estrogen signaling in alcohol-induced bone loss, by Martin J. Ronis; and (6) Short-term influence of alcohol on appetite-regulating hormones in man, by Jan Calissendorff.

Although the boundaries of skeletal muscle size are fundamentally determined by genetics, this dynamic tissue also demonstrates great plasticity in response to environmental and hormonal factors. Recent work indicates that contractile activity, nutrients, growth factors, and cytokines all contribute to determining muscle mass. Muscle responds not only to endocrine hormones but also to the autocrine production of growth factors and cytokines. Skeletal muscle synthesizes anabolic growth factors such as insulin-like growth factor (IGF)-I and potentially inhibitory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and myostatin. These self-regulating inputs in turn influence muscle metabolism, including the use of nutrients such as glucose and amino acids. These changes are principally achieved by altering the activity of the protein kinase known as protein kinase B or Akt. Akt plays a central role in integrating anabolic and catabolic responses by transducing growth factor and cytokine signals via changes in the phosphorylation of its numerous substrates. Activation of Akt stimulates muscle hypertrophy and antagonizes the loss of muscle protein. Here we review the many signals that funnel through Akt to alter muscle mass.

During systemic inflammation, the liver becomes unresponsive to growth hormone (GH), resulting in decreased plasma insulin-like growth factor-I (IGF-I) with concomitant reductions in lean body mass. Transgenic mice that overexpress IL-6 also demonstrate impaired growth and decreased IGF-I. To determine whether IL-6 directly inhibits GH-inducible gene expression, CWSV-1 hepatocytes were incubated with IL-6 (10 ng/ml), then stimulated with recombinant human GH (500 ng/ml, 18 h). The increase in IGF-I and serine protease inhibitor 2.1 (Spi 2.1) mRNA in GH-treated cells was inhibited by treatment with IL-6 for 24 h. To investigate potential mechanisms, we examined the effects of IL-6 on GH receptor (GHR) expression and GH signaling via the JAK/signal transducer and activator of transcription (STAT) and MAP kinase pathways. Incubation of cells with IL-6 (10 ng/ml, 24 h) had no effect on GHR abundance or signaling proteins JAK2, STAT5b, and ERK1/2. Although GH transiently increased (2- to 5-fold) the tyrosine phosphorylation of GHR, JAK2, STAT5b, and ERK1/2, IL-6 did not alter these phosphorylation events. However, nuclear protein from IL-6-treated cells demonstrated reduced STAT5 DNA binding (by EMSA) at 15 min (-20%) and 60 min (-43%) after GH stimulation. To determine whether IL-6 inhibits GH-inducible promoter activity, CWSV-1 cells were transfected with Spi 2.1 or prolactin receptor promoter luciferase vectors, incubated with or without IL-6, then stimulated with GH. The induction of both Spi 2.1 (7.5-fold) and prolactin receptor (4-fold) promoter activity by GH was inhibited by IL-6. In summary, IL-6 mediates hepatic GH resistance by a time-dependent inhibition of GH-inducible promoter activity that is associated with reductions in STAT5 DNA binding.

The hypothesis of the present study was that exposure of differentiated muscle cells to agonists of the AMP-activated protein kinase (AMPK) would increase the mRNA content of the muscle-specific ubiquitin ligases muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). C(2)C(12) cells were incubated with incremental doses of 5-aminoimidazol-4-carboximide ribonucleoside (AICAR) or metformin for 24 h. Both MAFbx and MuRF1 mRNA increased dose dependently in response to these AMPK activators. AICAR, metformin, and 2-deoxy-d-glucose produced time-dependent alterations in ubiquitin ligase expression, typified by a biphasic pattern of expression marked by an acute repression followed by a sustained induction. AMPK-activating treatments in conjunction with dexamethasone produced a pronounced synergistic effect on ligase mRNA expression at later time points. This cooperative response occurred in the absence of a dexamethasone-dependent increase in AMPK expression or activity, as determined by immunoblotting for phosphorylation and expression of AMPKalpha and its downstream target acetyl-CoA carboxylase (ACC). These responses elicited by AMPK activation singly or in combination with dexamethasone did not extend to the mRNA expression of the UBR box family E3s UBR1/E3alphaI and UBR2/E3alphaII. Treatment with the AMPK inhibitor compound C prevented increases in MAFbx and MuRF1 mRNA in response to serum deprivation, as well as AICAR and dexamethasone treatment individually or jointly. Stimulation of AMPK activity in vivo via AICAR injection increased both MAFbx and MuRF1 mRNA in murine skeletal muscle. These data suggest that activation of AMPK in skeletal muscle results in a specific upregulation of MAFbx and MuRF1, responses that are reminiscent of the proposed atrophic transcriptional program executed under various conditions of skeletal muscle wasting. Therefore, AMPK may be a critical component of the intercalated network of signaling pathways governing skeletal muscle atrophy, where its input acts to modify anti- and proatrophic signals to influence gene expression in reaction to catabolic perturbations.

Epidemiological evidence suggests alcoholic myopathy is more severe in females than males, but comparable animal studies are lacking that make elucidating the biochemical locus for this defect problematic. The present study determined whether skeletal muscle protein synthesis and markers of degradation exhibit a sexual dimorphic response to either chronic alcohol consumption or acute intoxication. Male and female rats were fed an alcohol-containing diet, pair-fed for 26 wk (chronic), or received an intraperitoneal injection of alcohol (acute). In males, chronic alcohol decreased gastrocnemius protein synthesis by 20%. This reduction was associated with a twofold increase in the inactive eukaryotic initiation factor (eIF) 4E.4E-binding protein 1 (4E-BP1) complex and a 60% reduction in the active eIF4E.eIF4G complex. This redistribution of eIF4E was associated with decreased phosphorylation of both 4E-BP1 and eIF4G (50-55%). The phosphorylation of ribosomal protein S6 was also reduced 60% in alcohol-consuming male rats. In contrast, neither rates of protein synthesis nor indexes of translation initiation in muscle were altered in alcohol-fed female rats despite blood alcohol levels comparable to males. Chronic alcohol ingestion did not alter atrogin-1 or muscle RING finger-1 mRNA content (biomarkers of muscle proteolysis) in males but increased their expression in females 50-100%. Acute alcohol intoxication produced a comparable decrease in muscle protein synthesis and translation initiation in both male and female rats. Our data demonstrate a sexual dimorphism for muscle protein synthesis, translation initiation, and proteolysis in response to chronic, but not acute, alcohol intoxication; however, they do not support evidence indicating females are more sensitive toward the development of alcoholic skeletal muscle myopathy.

Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mm EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.

Various atrophic stimuli increase two muscle-specific E3 ligases, muscle RING finger 1 (MuRF1) and atrogin-1, and knockout mice for these "atrogenes" display resistance to denervation-induced atrophy. The present study determined whether increased atrogin-1 and MuRF1 mRNA are mediated by overproduction of endogenous glucocorticoids or inflammatory cytokines in adult rats and whether atrogene expression can be downregulated by anabolic agents such as insulin-like growth factor (IGF)-I and the nutrient-signaling amino acid leucine. Both atrogin-1 and MuRF1 mRNA in gastrocnemius was upregulated dose and time dependently by endotoxin. Additionally, peritonitis produced by cecal ligation and puncture increased atrogin-1 and MuRF1 mRNA in gastrocnemius (but not soleus or heart) by 8 h, which was sustained for 72 and 24 h, respectively. Whereas the sepsis-induced increase in atrogin-1 expression was completely prevented by IGF-I, the increased MuRF1 was not altered. In contrast to the IGF-I effect, the sepsis-induced increased mRNA of both atrogenes was unresponsive to either acute or repetitive administration of leucine. Whereas exogenous infusion of TNF-alpha increased atrogin-1 and MuRF1 in gastrocnemius, pretreatment of septic rats with the TNF antagonist TNF-binding protein did not prevent increased expression of either atrogene. Similarly, whereas dexamethasone increased atrogene expression, pretreatment with the glucocorticoid receptor antagonist RU-486 failed to ameliorate the sepsis-induced increase in atrogin-1 and MuRF1. Thus, under in vivo conditions in mature adult rats, the sepsis-induced increase in muscle atrogin-1 and MuRF1 mRNA appears both glucocorticoid and TNF independent and is unresponsive to leucine.

The present study determined whether thermal injury increases the expression of the ubiquitin (Ub) E3 ligases referred to as muscle ring finger (MuRF)-1 and muscle atrophy F-box (MAFbx; aka atrogin-1), which are muscle specific and responsible for the increased protein breakdown observed in other catabolic conditions. After 48 h of burn injury (40% total body surface area full-thickness scald burn) gastrocnemius weight was reduced, and this change was associated with an increased mRNA abundance for atrogin-1 and MuRF-1 (3.1- to 8-fold, respectively). Similarly, burn increased polyUb mRNA content in the gastrocnemius twofold. In contrast, there was no burn-induced atrophy of the soleus and no significant change in atrogin-1, MuRF-1, or polyUb mRNA. Burns also did not alter E3 ligase expression in heart. Four hours after administration of the anabolic agent insulin-like growth factor (IGF)-I to burned rats, the mRNA content of atrogin-1 and polyUb in gastrocnemius had returned to control values and the elevation in MuRF-1 was reduced 50%. In contrast, leucine did not alter E3 ligase expression. In a separate study, in vivo administration of the proteasome inhibitor Velcade prevented burn-induced loss of muscle mass determined at 48 h. Finally, administration of the glucocorticoid receptor antagonist RU-486 did not prevent burn-induced atrophy of the gastrocnemius or the associated elevation in atrogin-1, MuRF-1, or polyUb. In summary, the acute muscle wasting accompanying thermal injury is associated with a glucocorticoid-independent increase in the expression of several Ub E3 ligases that can be downregulated by IGF-I.

Inhibition of translational efficiency is responsible at least in part for the sepsis-induced decrease in protein synthesis observed in skeletal muscle. Moreover, infusion of the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) into naive rats produces a comparable decrement. Therefore, the purpose of the present study was to determine whether inhibition of TNF action under in vivo conditions could prevent the sepsis-induced decrease in translation initiation observed in the postabsorptive state. To address this aim, sepsis was produced by cecal ligation and puncture (CLP) and rats were studied in the fasted condition 20 to 24 hours thereafter. Both septic and time-matched nonseptic control rats were pretreated with TNF-binding protein (TNF(BP)) before CLP or sham surgery to neutralize endogenously produced TNF. Sepsis altered the distribution of eukaryotic initiation factor 4E (eIF4E) in the gastrocnemius by increasing the amount associated with 4E-BP1 (inactive complex) and decreasing the amount bound to eIF4G (active complex). This change in eIF4E availability was associated with a decreased phosphorylation of 4E-BP1. Furthermore, the phosphorylation of ribosomal protein S6 and mammalian target of rapamycin (mTOR) was also decreased in the gastrocnemius from septic rats. Pretreatment of septic rats with TNF(BP) largely ameliorated the altered distribution of eIF4E as well as the reduced phosphorylation of 4E-BP1, S6, and mTOR. In contrast, sepsis did not change either the total amount or the phosphorylation state of eIF2alpha or eIF2Bepsilon. Furthermore, no sepsis-induced change in eIFs was detected in the slow-twitch soleus muscle. The ability of TNF(BP) to prevent the sepsis-induced alterations in translation initiation was independent of change in plasma insulin and proportional to the insulinlike growth factor I content in blood and muscle but was associated with a reduction in plasma corticosterone. Hence, the decreased constitutive protein synthesis observed in fast-twitch skeletal muscle in response to peritonitis is mediated by a TNF-dependent mechanism affecting mTOR regulation of translation initiation.

Background: Alcohol abuse has been reported to have a high prevalence in the human immunodeficiency virus (HIV)-infected population. However, its impact on disease progression is unknown. Studies dissecting the drug-induced or alcohol-induced metabolic derangements that are likely to alter the course of disease progression are lacking. This is particularly important because of the substantial reduction in morbidity and mortality of patients on highly active antiretroviral therapy (HAART). HIV infection has become a more chronic disease during which alcohol-induced metabolic alterations may become more prevalent and pronounced., Methods: The present study used a model of chronic intragastric alcohol administration initiated 3 months before intravenous simian immunodeficiency (SIV) inoculation and continued thereafter throughout the course of SIV infection, to investigate the impact of chronic alcohol binge-like consumption during the initial 10-month asymptomatic phase of SIV infection in nonhuman primate rhesus macaques. Anthropometric, metabolic, biochemical, nutritional, and immune state indicators were examined before infection and at 3-month intervals in asymptomatic chronic alcohol-treated SIV-infected macaques and time-matched isocaloric and uninfected controls., Results: Intravenous SIV(DeltaB670) infection resulted in increased viral load, decreased circulating CD4(+)/CD8(+) lymphocyte ratio, and increased lymphocyte proliferation (Ki67/CD3(+)). Chronic alcohol/SIV(+) animals showed a higher viral load at 3 months post-SIV infection as well as a significant and early decrease in caloric intake and nitrogen balance associated with a change in food choice. Rates of skeletal muscle protein synthesis and breakdown, mRNA expression of IGF-I, myostatin, or the ubiquitin ligase muscle atrophy F-box protein (MAFbx) did not differ from basal during the 10-month asymptomatic period of infection. However, muscle TNF-alpha mRNA expression was markedly increased at 10 months post-SIV infection in alcohol/SIV(+) animals., Discussion: These findings suggest that chronic alcohol accelerates nutritional and metabolic dysregulation during SIV infection and may favor a skeletal muscle proinflammatory state, possibly conducive to subsequent muscle wasting.

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 the level of phosphorylated eukaryotic initiation factor (eIF) 4G in gastrocnemius after induction of a chronic intra-abdominal sterile or septic abscess as a possible mechanism to account for the impairment of translation initiation during sepsis. The extent of phosphorylated eIF4G was reduced by more than 50% (P< 0.05) and 68% (P < 0.01) in gastrocnemius after 3 and 5 days, respectively, and returned to control values after 14 days of abscess formation in septic rats compared with sterile inflammatory animals. To examine the mediators of the septic process contributing to the decreased levels of phosphorylated eIF4G, the cytokine response to sepsis was pharmacologically modulated. First, treatment of septic rats with tumor necrosis factor (TNF) binding protein or interleukin (IL) 1 receptor antagonist increased the level of phosphorylated eIF4G. Second, infusion of TNF-alpha for 24 h in control rats resulted in a 70% decrease in phosphorylated eIF4G. Third, infusion of IL-1ra led to an increase in the level of phosphorylation of eIF4G in rats infused with TNF-alpha. Taken together, the data indicate that a cytokine-dependent decrease in the steady state phosphorylation of eIF4G is a possible mechanism accounting for the inhibition of skeletal muscle protein synthesis during sepsis. Furthermore, the findings support a role of IL-1 as the proinflammatory mediator responsible for the reduced level of phosphorylated eIF4G.

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.