Skeletal Muscle Protein Catabolism Research Articles

Conditioned Media from Head and Neck Cancer Cell Lines and Serum Samples from Head and Neck Cancer Patients Drive Catabolic Pathways in Cultured Muscle Cells

The role of secreted factors from the tumor cells in driving cancer cachexia and especially muscle loss is unknown. We wanted to study both the action of secreted factors from head and neck cancer (HNC) cell lines and circulating factors in HNC patients on skeletal muscle protein catabolism. Conditioned media (CM) made from head and neck cancer cell lines and mix of sera from head and neck cancer (HNC) patients were incubated for 48 h with human myotubes. The atrophy and the catabolic pathway were monitored in myotubes. The patients were classified regarding their skeletal muscle loss observed at the outset of management. Tumor CM (TCM) was able to produce atrophy on myotubes as compared with control CM (CCM). However, a mix of sera from HNC patients was not able to produce atrophy in myotubes. Despite this discrepancy on atrophy, we observed a similar regulation of the catabolic pathways by the tumor-conditioned media and mix of sera from cancer patients. The catabolic response after incubation with the mix of sera seemed to depend on the muscle loss seen in patients. This study found evidence that the atrophy observed in HNC patients cannot be solely explained by a deficit in food intake.

Особенности белкового метаболизма в скелетных мышцах костистых рыб

The review summarizes the literature and the data of own research on the traits of the structure, functions, and protein metabolism in the skeletal muscle of teleost fish (Teleostei). Despite the conservative fundamental mechanisms of muscle growth (myogenesis) and degradation in vertebrates, fish are characterized by unique features related to their poikilothermy, indeterminate growth, and a unique role of the muscle as a depot of plastic and energetic substrates. The skeletal muscle of fish reveals high plasticity defined as their ability to substantial anabolic or catabolic changes in response to environmental variables, such as temperature, photoperiod, and food availability. Under optimal (anabolic) conditions, the muscle tissue of fish grows at an extremely high rate due to both hypertrophy and hyperplasia ways, while at the periods of high energy demand, including migration, starvation, and gonad maturation, the skeletal muscle protein catabolism temporarily prevails. However, the muscle degradation could be deep enough to exceed their regenerative capacity; according to this scenario, both genetically determined programs and responses to exogenous signals of excessive strength and duration can be realized. An extreme and illustrative example of muscle protein reserve mobilization and resulting amino acid consumption in the processes of energy production and the synthesis of stage-specific gonad proteins are pacific salmonids which exhaustion at the spawning is so great to result in the death of individuals. The myopathies in fish and the potential of fish objects for modeling human diseases are also considered.

Skeletal muscle protein catabolism, protein anabolism, and myogenesis after various types of insults

Skeletal muscle protein catabolism, protein anabolism, and myogenesis after various types of insults

OR-017 Supplementation of Ala-Gln inhibits protein breakdown of skeletal muscle in rats with altitude training through TNF-α/NF-κB/MuRF1 pathway

Objective Objective: To explore the effects of alanyl-glutamine（Ala-Gln）or glutamine（Gln） supplementation on protein metabolism in rat skeletal muscle during simulated altitude training，and compare the intervention of Gln or Ala-Gln to provide the necessary experimental basis for finding nutritional interventions to inhibit skeletal muscle protein degradation during altitude training.
 Methods Methods: Forty SD rats aged 6 weeks were randomly divided into normoxic control group（NC group，n=10），hypoxic exercise group（HE group， n=10），hypoxic exercise + glutamine + alanine group（HEG group，n=10）， hypoxic exercise + alanyl glutamine group（HEAG group，n=10）. Rats were subjected to 6 weeks of 13.6% hypoxic exposure and 90% lactic acid threshold intensity weight-bearing swimming（load weight of 2.1% of body weight）exercise training，30 minutes after the end of each training，the mixed solution of Ala and Gln was administered according to the dose of 0.75g/Kg body weight in HEG group，and the solution of Ala-Gln was administered in the HEAG group at a dose of 1.5 g/kg body weight. After 6 weeks，the contents of rat skeletal muscle total protein（Pro），myosin heavy chain（Myo），tumor necrosis factor-α（TNF-α），nuclear transcription factor-κB （NF-κB），NF-κB inhibitory protein α（IkBα），and mRNA expression of muscle atrophy box F gene（MAFbx），muscle ring finger gene 1（MuRF1），and inhibitor of kappa B kinase complex-beta（IKKβ）were measured.
 Results Results:（1）Compared with NC group，the content of Pro and Myo in skeletal muscle in HE group was significantly decreased（P<0.05，P<0.01），and the mRNA expression of MAFbx and MuRF1 in skeletal muscle was significantly increased（P<0.05，P< 0.01），the levels of TNF-α and NF-κB were significantly increased（P<0.05），the content of IkBα was significantly decreased（P<0.05），and the expression of IKKβ mRNA was significantly increased（P<0.01）. （2）Compared with HE group，the content of Pro and Myo in skeletal muscle in HEG group increased，but there was no significant difference（P>0.05）. The expression of MuRF1 mRNA and the content of TNF-α and NF-κB in skeletal muscle decreased，IkBα content increased，there were no significant difference，but mRNA expression of MAFbx and IKKβ was significantly decreased（P<0.05， P<0.01）. （3）Compared with HE group，the content of Pro and Myo in skeletal muscle in HEAG group increased significantly（P<0.05），mRNA expression of IKKβ，MuRF1 and MAFb（P<0.01）and TNF-α，NF-κB content（P<0.05）in skeletal muscle was significantly decreased，and the IkBα content was significantly increased（P<0.05）.
 Conclusions Conclusion:（1）Simulated altitude training can activate TNF-α/NF-κB/MuRF1 pathway and enhance the catabolism of skeletal muscle protein，which is one of the important mechanisms for the reduction of skeletal muscle protein content caused by altitude training. （2）Supplementation of Ala-Gln during altitude training can significantly reduce the activation of TNF-α/NF-κB/MuRF1 pathway in skeletal muscle，and reduce the catabolism of skeletal muscle protein during altitude training，which plays a very important role in preventing the loss of skeletal muscle protein caused by altitude training. supplementation of Gln monomer during altitude training has little inhibitory effect on the activation of TNF-α/NF-κB/MuRF1 pathway in skeletal muscle.

Systemic inflammation is associated with exaggerated skeletal muscle protein catabolism in maintenance hemodialysis patients.

Systemic inflammation and muscle wasting are highly prevalent and coexist in patients on maintenance hemodialysis (MHD). We aimed to determine the effects of systemic inflammation on skeletal muscle protein metabolism in MHD patients. Whole body and skeletal muscle protein turnover were assessed by stable isotope kinetic studies. We incorporated expressions of E1, E214K, E3αI, E3αII, MuRF-1, and atrogin-1 in skeletal muscle tissue from integrin β1 gene KO CKD mice models. Among 129 patients with mean (± SD) age 47 ± 12 years, 74% were African American, 73% were male, and 22% had diabetes mellitus. Median high-sensitivity C-reactive protein (hs-CRP) concentration was 13 (interquartile range 0.8, 33) mg/l. There were statistically significant associations between hs-CRP and forearm skeletal muscle protein synthesis, degradation, and net forearm skeletal muscle protein balance (P < 0.001 for all). The associations remained statistically significant after adjustment for clinical and demographic confounders, as well as in sensitivity analysis, excluding patients with diabetes mellitus. In attempting to identify potential mechanisms involved in this correlation, we show increased expressions of E1, E214K, E3αI, E3αII, MuRF-1, and atrogin-1 in skeletal muscle tissue obtained from an animal model of chronic kidney disease. These data suggest that systemic inflammation is a strong and independent determinant of skeletal muscle protein homeostasis in MHD patients, providing rationale for further studies using anticytokine therapies in patients with underlying systemic inflammation. This study was in part supported by NIH grants R01 DK45604 and 1K24 DK62849, the Clinical Translational Science Award UL1-TR000445 from the National Center for Advancing Translational Sciences, the Veterans Administration Merit Award I01 CX000414, the SatelliteHealth Normon Coplon Extramural Grant Program, and the FDA grant 000943.

DNA microarray analysis identified molecular pathways mediating the effects of supplementation of branched‐chain amino acids on CCl4‐induced cirrhosis in rats

This study addresses the effects of branched-chain amino acids (BCAA) on global gene expression in liver and skeletal muscle and the molecular mechanisms underlying the improvement in liver cirrhosis using DNA microarray analysis combined with RNase protection assay. Male Wistar rats administered carbon tetrachloride (CCl(4) ) repeatedly for 19 weeks as a decompensated cirrhosis model were thereafter given BCAA-enriched diet (AL) or normal diet (LC) for 5 weeks. The control-diet rats without CCl(4) administration were used as a normal control group. Gene expression in AL was reversed by twofold greater than in LC in the microarray were selected to elucidate the improvements in nutritional and metabolic disorders. Downregulation of fatty acid translocase (FAT)/Cd36, glutamine synthetase, and pyruvate dehydrogenase kinase isoenzyme 4 is believed to promote lower uptake of fatty acids, lower ammonia incorporation, and higher uptake of glucose, and thus to provide an energy source without using BCAA. Ultimately, the catabolism of BCAA and skeletal muscle protein would be slowed, maintaining BCAA concentrations in blood. We established, for the first time, the regulatory gene pathways of processes involved in hepatic fibrosis and energy metabolism (hypoalbuminemia, hyperammonemia, and carbohydrate catabolism, and their relationships) under BCAA supplementation.

P055 Evaluation of cytokines in C2C12 myotubes treated with leucine and exposed to walker factor proteolysis-inducing factor-like

Introduction The proteolysis-inducing factor (PIF), produced by tumours, has been widely studied for cellular signalling processes, especially, for understanding the molecular mechanism of skeletal muscle protein catabolism induced by cachexia. Several studies also focus on muscle protein synthesis inhibition and its process during cachexia. In our studies, we have demonstrated that muscular protein breakdown increased, and the protein synthesis decreased under the effect of proteolysis-inducing factor (PIF) and also under treatment with Walker Factor, a molecule similar to PIF, in myotubes and myoblasts. Leucine plays an important role on muscle metabolism, increasing protein synthesis and decreasing muscle proteolysis. Methods The aim of this work was to investigate the cytokine profile in C2C12 myotubes culture under WF exposure and leucine modulation. C2C12 myoblast cells were maintained in DMEM supplemented with 10% foetal calf serum at 37 °C, and differentiated in myotubes after replaced medium (DMEM supplemented with 2% horse serum) for 4 days prior to the experimental procedures. C2C12 myotubes previously treated with 25 μM or 50 μM leucine for 2 h were exposed to different concentrations of WF (5 and 25 μg WF/well) for 24, 48 and 72 h. After 24 h, the culture medium and cells were collected to measure the amount of cytokines interferon-γ, interleukin-4, interleukin-6, interleukin-10 and TNF-α. Results The cytokine analysis of the DMEM medium, and also media supplemented with Factor Walker and/or leucine were measured and stabilised as control standard for each cytokine (pg/mL culture medium). Results showed that the culture of C2C12 myotube changed the cytokine concentration presented in standard medium; the control cells medium had an increase in INFγ and IL-6, reduction in IL-10 and IL-4, and similar TNF-α content. The results of experimental cells showed that the C2C12 myotubes exposed to WF had different cytokine pattern in medium culture compared to control cells. The C2C12 myotubes under high WF concentration (25 μg/mL) had a decrease in INFγ, IL-10, IL-4 and IL-6 content in the culture medium when compared to medium from control cells. These cytokine patterns for IL-10, IL-4 and IL-6 content had been reverted in these myotubes after previous 25 and 50 μM leucine exposition. Evaluating the C2C12 myotube cells, we suggest the higher IL-6 levels in the culture medium from these cells could be related probably to the normal cell activity. However, the WF exposition added to decrease IL-6 content changed cell activity and also leaded to protein wasting in these cells (increased proteasome enzymes activity and low myosin content). In C2C12 myotubes exposed to WF, the leucine treatment minimized those effects restoring the total protein net (inhibited the protein degradation and enhanced the protein synthesis). Conclusion In tumour-bearing animals, the IL-6 was higher and could be related among other pro-inflammatory cytokines with host wasting. This in vitro experiment suggested that this IL-6 cytokine can be related to hypertrophy and myogenesis during control state, but its lower release from C2C12 cells could be also leading to pro-cachectic process as an effect of Walker Factor, which could be modulated by leucine treatment. Statistical support Dr. J Marcondes; Financial support Fapesp #2006/6007-6, #2010/11328-9.

The dose-dependent effects of endotoxin on protein metabolism in two types of rat skeletal muscle

Endotoxin administration is frequently used as a model of systemic inflammatory response which is considered the important pathogenetic factor in muscle wasting development in severe illness, such as sepsis, cancer, injury, AIDS and others. The main purpose of this study was determining the effect of various doses of endotoxin on protein and amino acid metabolism in two types of rat skeletal muscle. Sepsis was induced by intraperitoneal administration of endotoxin in a dose of 1, 3 and 5 mg/kg body weight (bw); control animals received a corresponding volume of the saline solution. After 24 h, extensor digitorum longus (EDL) and soleus (SOL) muscles were isolated and used for determination of total and myofibrillar proteolysis, protein synthesis, activity of cathepsins B and L, chymotrypsin-like activity of proteasome and amino acid release. The endotoxemia induced the body weight loss, the rise of total cholesterol and triglyceride plasma concentration and the protein catabolic state in skeletal muscle, which was caused by a higher increase in protein breakdown (due to activation of the proteasome system) than protein synthesis. The more significant effect of endotoxin was seen in EDL than SOL. The dose of 5 mg of endotoxin/kg bw induced the most significant changes in parameters of the protein and amino acid metabolism measured and could be therefore considered appropriate for studies of protein catabolism in young rat skeletal muscle at 24 h after endotoxin treatment.

The inhibition of phosphodiesterase 4 reduces skeletal muscle protein catabolism by suppressing autophagy/lysosomal and proteasomal pathways and atrophy‐specific gene transcription

This work investigated the effect of cAMP through PDE4 inhibition, the major PDE expressed in skeletal muscle, in modulating muscle protein degradation.The protein catabolism was monitored indirectly by measurement of interstitial tyrosine concentration in tibialis anterior muscle from Wistar rats (~250g) by microdialysis. Muscles were perfused with selective PDE 4 inhibitor, rolipram (1mM). Rates of proteolysis were determined by measuring the tyrosine release from extensor digital longus (EDL) and soleus from rats (~70g) incubated with different concentrations of PDE inhibitor. Gene expression was measured by real time‐PCR and cAMP level was estimated by ELISA. In situ muscle perfusion with rolipram reduced interstitial‐arterial tyrosine concentration difference, indicating a reduction in protein catabolism. Rolipram in vitro reduced overall proteolysis in a dose‐dependent manner and increased the muscle level of cAMP. This effect was accompanied by a decrease in the ubiquitin‐proteasome system (UPS) activity and suppression of autophagic and Ub‐ligases (atrogin‐1 and MuRF‐1) gene expression induced by fasting.These findings identify mechanisms by which PDE inhibitors reduces protein degradation, suggesting that inhibition of PDE 4 activates cAMP‐dependent pathway, which leads to the suppression of autophagic and UPS pathways and atrophy‐specific gene transcription.

Treating hyperglycemia improves skeletal muscle protein metabolism in cancer patients after major surgery

Cancer and surgical stress interact to aggravate insulin resistance, protein catabolism, and glutamine depletion in skeletal muscle. We compared the effects of insulin-mediated euglycemia and moderate hyperglycemia on kinetics of protein and selected amino acids in skeletal muscle of female cancer patients after major surgery. In each patient, a 24-hr period of insulin-mediated tight euglycemia (mean blood glucose, 5.8 +/- 0.4 mmol/L) preceded or followed a 24-hr control period of moderate hyperglycemia (mean blood glucose, 9.6 +/- 0.6 mmol/L) on the first and second day after surgery, in randomized order, according to a crossover experimental design. Intensive care unit, cancer hospital. Cancer patients after abdominal radical surgery combined with intraoperative radiation therapy. Intensive (57 +/- 11 units/24 hrs) and conventional (25 +/- 5 units/24 hrs) insulin treatment during total parenteral nutrition. Muscle metabolism was assessed at the end of each 24-hr period of euglycemia and of hyperglycemia by leg arteriovenous catheterization with stable isotopic tracers. We found that euglycemia as compared with hyperglycemia was associated with higher (p < .05) fractional glucose uptake (16% +/- 4% vs. 9% +/- 3%); higher (p < .05) muscle protein synthesis and neutral net protein balance (-3 +/- 3 vs. -11 +/- 3 nmol phenylalanine x 100 mL(-1) x min(-1), respectively); lower (-52% +/- 12%, p < .01) muscle nonprotein leucine disposal (an index of leucine oxidation) and higher (p < .05) plasma leucine concentrations; and higher (3.6 +/- 1.7 times, p < .01) net de novo muscle glutamine synthesis and plasma glutamine concentrations (p < .05). Euglycemia was associated with higher (23% +/- 7%, p < .05) plasma concentrations of arginine but did not affect either arginine release from muscle or plasma concentration and muscle flux of asymmetrical dimethylarginine. Rate of muscle proteolysis correlated (p < .05) with muscle release of asymmetrical dimethylarginine. Treating hyperglycemia improves skeletal muscle protein and amino acid metabolism in cancer patients after major surgery.

Treatment of hyperglycemia for skeletal muscle metabolism in cancer patients after major surgery

20578 Background: Cancer and surgical stress interact to aggravate insulin resistance, protein catabolism and glutamine depletion in skeletal muscle. In the clinical setting, hyperglycemia and glutamine depletion were associated with poor outcome in critically ill patients. The present study was designed to explore the effects of insulin mediated euglycemia and moderate hyperglycemia on kinetics of protein and selected amino acids in skeletal muscle of cancer patients after major surgery. Methods: Adult cancer patients were studied after abdominal radical surgery. In each patient a 24-h period of insulin-mediated tight euglycemia (mean blood glucose: 5.8± 0.4 mmol/l) preceded or followed a 24-h period of moderate hyperglycemia (mean blood glucose: 9.6± 0.6 mmol/l) on the first and the second day after surgery (cross-over design with randomized sequence of treatments). Intensive (57±11 U/24-h) or conventional (25±5 U/24-h) insulin treatments were used to obtain euglycemia or moderate hyperglycemia during total parenteral nutrition. Muscle metabolism was assessed at the end of each 24-h period of tight glycemic control and of hyperglycemia by leg arteriovenous catheterization with stable isotopic tracers. Results in the two different experimental conditions were compared using the Wilcoxon Matched-Pairs Signed-Ranks Test. P values < 0.05 were taken as significantly different. Results: Plasma insulin levels were not significantly different at the end of intensive or conventional insulin treatment period. Strict glycemic control as compared to hyperglycemia was associated with higher (p<0.05) fractional glucose uptake (16±4 vs 9±3 percent); higher (p<0.05) muscle protein synthesis and neutral net protein balance (-3±3 vs - 11±3 mmol PHE · 100cc-1 · min-1, respectively); higher (3.6±1.7 times, p<0.01) net de novo muscle glutamine production. Conclusions: This study shows that, in cancer patients after major surgery, hyperglycemia with relative insulin deficiency promotes muscle wasting and blunted the ability of muscle to provide glutamine to extra-muscular tissues. This latter effect could contribute to increased morbidity and mortality in surgical cancer patients with poor metabolic control. No significant financial relationships to disclose.

Contribution Of Oxidative Stress To Protein Catabolism In Skeletal Muscle

Increase of oxidative stress causes inflammation, necrosis, and, catabolism in various tissues. In skeletal muscle, oxidative stress could also promote protein catabolism while the mechanism is unclear. PURPOSE: We studied the contribution of oxidative stress to protein catabolism in skeletal muscle associated with redox-sensitive signal cascade. METHODS: Lipid peroxides and oxidant-modified proteins were measured in gastrocnemius muscle obtained from young (3-mo) and old (24-mo) mice. Activity of redox signaling pathway in muscle was examined. Further, protein content and ubiquitination of myosin heavy chain (MHC) were measured after stimulation of oxidants in myotube cell. Effect of the inhibitor of redox-sensitive transcription factor was examined. RESULTS: The muscle weight in old mice was significantly decreased compared with young mice (7. 1±0.66 vs. 12.2±0.65 mg/g BW, p<0.05) with accumulation of peroxides. Markedly elevation of nuclear factor-kappa B (NF-KB) pathway was observed in the aged muscle. In myotube, oxidants decreased MHC content dose-dependently with ubiquitination although the NF-?B inhibitor prevented this decrease. CONCLUSIONS: The increase of oxidative stress contributes to protein catabolism in skeletal muscle via abnormal activation of redox-sensitive signaling cascade.

Low postabsorptive net protein degradation in male cancer patients: Lack of sensitivity to regulatory amino acids?

Autophagic (lysosomal) and proteasomic protein degradation are important regulatory mechanisms in the homeostasis of muscle mass, that may be profoundly disturbed in cancer and other wasting syndromes. Due to the inhibiting effect of amino acids and insulin, net proteolysis is restricted to the fasted state, and in autophagy certain amino acids have been identified as 'regulatory' in the rat, including leucine, tyrosine, phenylalanine, methionine, and histidine (i.e. LYFMH). The present cross-sectional study assessed postabsorptive net protein catabolism in male cancer patients as well as in healthy male volunteers, to analyse its relation to such 'regulatory amino acids'. Postabsorptive amino acid exchange rates across the leg were determined in patients with gastrointestinal cancer (GIC, n=47) or renal cell carcinoma (RCC, n=15), age-matched (n=33), and young male control subjects (n=42). Both groups of cancer patients revealed a significantly lower postabsorptive net protein catabolism than control subjects. Furthermore, in the control subjects, the postabsorptive net protein catabolism was found to be inversely and significantly correlated with the arterial concentrations of the 8 amino acids YSHMFGI and L which include 5 of the 'regulatory amino acids'. Cancer patients, in contrast, revealed no such significant correlations. These results may indicate i) that postabsorptive net protein catabolism in skeletal muscle of healthy subjects may be sensitive to amino acids which reportedly regulate autophagy and ii) that such amino acid-sensitive mechanism of protein catabolism may be disturbed in cancer patients.

Effects of long-term hypoxia on enzymes of carbohydrate metabolism in the Gulf killifish,Fundulus grandis

The goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l(-1) dissolved oxygen (approximately 3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis (in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy demand in this tissue during chronic hypoxia. In contrast, several enzyme specific activities were higher in liver tissue after hypoxic exposure, suggesting increased capacity for carbohydrate metabolism. Hypoxic exposure affected fewer enzymes in heart and brain than in skeletal muscle and liver, and the changes were smaller in magnitude, perhaps due to preferential perfusion of heart and brain during hypoxia. The specific activities of some gluconeogenic enzymes increased in liver during long-term hypoxic exposure, which may be coupled to increased protein catabolism in skeletal muscle. These results demonstrate that when intact fish are subjected to prolonged hypoxia, enzyme activities respond in a tissue-specific fashion reflecting the balance of energetic demands, metabolic role and oxygen supply of particular tissues. Furthermore, within glycolysis, the effects of hypoxia varied among enzymes, rather than being uniformly distributed among pathway enzymes.

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.

Plasma amino acids in critically ill patients

Stress states, sepsis and trauma markedly increase protein catabolism in skeletal muscle, gut, and connective tissue. Catabolism is potentiated by a sepsis-induced decrease in amino acid uptake in skeletal muscle. One-third of the total amino acid output from skeletal muscle is glutamine and one-third is alanine.

Omega-3 fatty acids in colorectal cancer prevention.

Omega-3 fatty acids in colorectal cancer prevention.

Signal transduction pathways involved in proteolysis-inducing factor induced proteasome expression in murine myotubes.

The proteolysis-inducing factor (PIF) is produced by cachexia-inducing tumours and initiates protein catabolism in skeletal muscle. The potential signalling pathways linking the release of arachidonic acid (AA) from membrane phospholipids with increased expression of the ubiquitin–proteasome proteolytic pathway by PIF has been studied using C2C12 murine myotubes as a surrogate model of skeletal muscle. The induction of proteasome activity and protein degradation by PIF was blocked by quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor and trifluroacetyl AA, an inhibitor of cytosolic PLA2. PIF was shown to increase the expression of calcium-independent cytosolic PLA2, determined by Western blotting, at the same concentrations as those inducing maximal expression of 20S proteasome α-subunits and protein degradation. In addition, both U-73122, which inhibits agonist-induced phospholipase C (PLC) activation and D609, a specific inhibitor of phosphatidylcholine-specific PLC also inhibited PIF-induced proteasome activity. This suggests that both PLA2 and PLC are involved in the release of AA in response to PIF, and that this is important in the induction of proteasome expression. The two tyrosine kinase inhibitors genistein and tryphostin A23 also attenuated PIF-induced proteasome expression, implicating tyrosine kinase in this process. PIF induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) at the same concentrations as that inducing proteasome expression, and the effect was blocked by PD98059, an inhibitor of MAPK kinase, as was also the induction of proteasome expression, suggesting a role for MAPK activation in PIF-induced proteasome expression.

Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes.

Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H2O2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C2C12 myotubes with 100 microM H2O2 increased the rate of 125I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H2O2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H2O2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H2O2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E214k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system.

Biochemical mechanisms of cellular catabolism.

To provide an in-depth analysis of current developments concerning biochemical mechanisms of cellular catabolism. There have been a number of important developments in this area over the past 12 months, particularly with respect to protein catabolism. Protein degradation in a range of catabolic conditions is mediated primarily through the ubiquitin-proteasome proteolytic pathway. Glucocorticoids have been suggested to activate this system in sepsis, while in cancer cachexia a tumour-produced sulphated glycoprotein, proteolysis-inducing factor, induces protein catabolism in skeletal muscle by increasing expression of proteasome subunits and the ubiquitin carrier protein, E2(14k). Apoptosis may also be important in the loss of muscle protein during the early stage of cachexia. Induction of proteasome expression by glucocorticoids appears to be a direct result of the downregulation of the activity of nuclear factor kappaB, while proteolysis-inducing factor acts through 15-hydroxyeicosatetraenoic acid as an intracellular transducer. Formation of 15-hydroxyeicosatetraenoic acid is inhibited by eicosapentaenoic acid, which has been shown to attenuate the development of weight loss in patients with pancreatic cancer. When eicosapentaenoic acid is combined with an energy dense nutritional supplement, there is an increase in body weight of cachectic cancer patients through an increase in lean body mass. Eicosapentaenoic acid also prevents protein catabolism and activation of the ubiquitin-proteasome proteolytic pathway during acute starvation in mice, suggesting a similar pathway is involved. Thus eicosapentaenoic acid may be effective in the treatment of protein catabolism in conditions other than cancer.