Total Protein Breakdown Research Articles

Phosphodiesterase 4B knockout prevents skeletal muscle atrophy in rats with burn injury.

The phosphodiesterase 4 (PDE4)-cAMP pathway plays a predominant role in mediating skeletal muscle proteolysis in burn injury. The present investigations to determine the PDE4 isoform(s) involved in this action revealed that burn injury increased the expression of rat skeletal muscle PDE4B mRNA by sixfold but had little or no effect on expression of other PDE4 isoforms. These observations led us to study the effects of burn in PDE4B knockout (KO) rats. As reported by us previously, burn injury significantly increased extensor digitorum longus (EDL) muscle total and myofibrillar proteolysis in wild-type (WT) rats, but there were no significant effects on either total or myofibrillar protein breakdown in EDL muscle of PDE4B KO rats with burn injury. Moreover, burn injury increased PDE4 activity in the skeletal muscle of WT rats, but this was reduced by >80% in PDE4B KO rats. Also, burn injury decreased skeletal muscle cAMP concentration in WT rats but had no significant effects in the muscles of PDE4B KO rats. Incubation of the EDL muscle of burn-PDE4B KO rats with an inhibitor of the exchange factor directly activated by cAMP, but not with a protein kinase A inhibitor, eliminated the protective effects of PDE4B KO on EDL muscle proteolysis and increased muscle proteolysis to the same extent as in the EDL of burn-WT rats. These novel findings confirm a major role for PDE4B in skeletal muscle proteolysis in burn injury and suggest that an innovative therapy based on PDE4B-selective inhibitors could be developed to treat skeletal muscle cachexia in burn injury without the fear of causing emesis, which is associated with PDE4D inhibition.

Systematic evaluation on effectiveness and safety of recombinant human growth hormone in treating adult patients with severe burn

Objective: To systcmatically evaluate the effectiveness and safety of recombinant human growth hormone (rhGH) in treating adults with severe burn. Methods: Databases including PubMed, Cochrane Library, and Embase were searched using key words " burns, thermal, human growth hormone, growth hormone, hGH, and somatropin (human)" , and China Biology Medicine disc, Chinese Journals Full-text Database, VIP Database, and Wanfang Database were searched using key words in Chinese version "," to obtain the randomized controlled trials about rhGH in the treatment of adults with severe burn from the establishment of each database to December 2016. The measurement indexes included hemoglobin (Hb) and plasma total protein, inflammatory factors [including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)], incidence rate of sepsis, incidence rate of hyperglycemia, wound healing time, length of stay, and mortality rate. Meta-analysis was conducted by RevMan 5.3 statistical software. Results: A total of 8 trials involving 534 patients were included; 276 patients in rhGH group were treated with rhGH and 258 patients in placebo control group were treated with placebo. One trial had low risk of bias, while the other 7 trials had unclear risk of bias. The levels of Hb and plasma total protein of patients in rhGH group were higher than those in placebo control group, with standardized mean differences (SMDs) respectively 2.00 and 2.23 [with 95% confidence intervals (CIs) respectively 0.19-3.82 and 1.21-3.26, P<0.05 or P<0.01]. The levels of IL-6 and TNF-α of patients in rhGH group were lower than those in placebo control group, with SMDs respectively -1.46 and -1.13 (with 95% CIs respectively -2.40--0.53 and -1.75--0.51, P values below 0.05). Incidence rate of sepsis and mortality rate of patients in rhGH group were lower than those in placebo control group, with relative risks (RRs) respectively 0.60 and 0.35 (with 95% CIs respectively 0.42-0.85 and 0.15-0.83, P values below 0.05). Incidence rate of hyperglycemia of patients in rhGH group was higher than that in placebo control group, with RR of 2.39 (with 95% CI 1.79-3.18, P<0.001). The wound healing time and length of stay of patients in rhGH group were lower than those in control group, with SMDs respectively -1.54 and -2.00 (with 95% CIs respectively -2.22--0.86 and -3.51--0.49, P<0.05 or P<0.01). Hb, plasma total protein, inflammatory factors, incidence rate of sepsis, wound healing time, length of stay, and mortality rate showed no significant publication bias (P values above 0.05), while there may be publication bias in incidence rate of hyperglycemia (P=0.026). Conclusions: rhGH can inhibit the breakdown of Hb and plasma total protein, reduce the level of inflammatory factors and incidence rate of sepsis, thus shorten the wound healing time and length of stay, thereby reduce mortality rate of adult patients with severe burn. However rhGH may cause hyperglycemia.

Addition of Calcium and Phosphorus to Preterm Donor Human Milk and the Impact on Protein, Fat, and Calcium Digestibility In Vitro

Infants born prematurely are at risk for metabolic bone disease and may need increased minerals for normal bone mineralization. In these situations, supplementation of human milk with calcium and phosphorus is common in the United States. The interaction of these nutrients and their carriers with other nutrition components of human milk has not been systematically investigated. The primary goal was to study the effect of calcium and phosphorus supplementation on the digestibility of protein, free fatty acids, and calcium in preterm donor human milk (DHM). An in vitro model of the premature infant’s gastrointestinal tract was used to simulate digestion. Protein, free fatty acids, and ionized calcium were measured before and after in vitro digestion in milk with and without added calcium glubionate and sodium potassium phosphate. Calcium and phosphorus supplementation did not negatively affect total protein, protein breakdown, protein digestibility, or fat breakdown when compared with unfortified DHM. Supplemental calcium increased ionized calcium, which may replace ionized calcium lost during milk expression, storage, and processing. Supplemental calcium glubionate and sodium potassium phosphate do not affect protein or fat breakdown in vitro. DHM contains less ionized calcium than fresh human milk because it loses CO2during expression and processing. Adding supplemental calcium increases ionized calcium in DHM.

Ghrelin receptor agonist, GHRP-2, attenuates burn injury-induced MuRF-1 and MAFbx expression and muscle proteolysis in rats

Thermal injury results in hypermetabolism, loss of body weight, and skeletal muscle wasting in mice and rats. Our earlier studies have demonstrated that ghrelin injection stimulates food intake and growth hormone release and inhibits skeletal muscle proteolysis in rats with thermal injury. We sought to develop a lower molecular weight, stable and longer acting peptide to combat the catabolic responses caused by thermal injury. Towards this goal, we examined the role of the hexapeptide mimetic of ghrelin, growth hormone-releasing peptide-2 (GHRP-2), on expression of E3 ubiquitin ligases and breakdown of muscle protein in rats with thermal injury. Slow in vivo release of GHRP-2 through minipump for 24 h attenuated the thermal injury-induced increase in mRNA expression of IL-6 and of the E3 ubiquitin ligases, MuRF-1 and MAFbx, in rat skeletal muscle. Furthermore, burn-induced increases in total and myofibrillar protein breakdown from rat EDL muscle were attenuated by GHRP-2. These findings suggest that catabolic responses resulting from thermal injury can be attenuated by GHRP-2.

Protein turnover during pregnancy

The response of whole body protein turnover in sows to increasing dietary threonine (THR) during pregnancy was determined using continuous oral delivery of L[1‐13C]phenylalanine (PHE) in six multiparous sows. Pregnancy (115 d) was divided into early (35 to 53 d) and late (93 to 110 d) gestation. Sows received diets with 4.9 ‐ 16.1 gd−1 THR. Blood was sampled every 30 min for 4 h. Data was analyzed with a nonlinear Mixed model.In early pregnancy, THR intake did not affect protein turnover. However, when expressed as a percent of flux, protein synthesis increased (R2=0.84) and PHE oxidation decreased (R2=0.79) at THR intakes below the dietary requirement. In late pregnancy, PHE oxidation decreased (R2=0.40) and protein synthesis increased (R2=0.40) linearly. The THR requirement based on protein synthesis in early pregnancy was 2‐fold lower (6.0 gd−1) than in late pregnancy (&gt;14.0 gd−1). There was no difference between early and late pregnancy in total protein breakdown (p=0.27, 44.1 ± 2.2 vs 46.2 ± 2.3 gd−1, respectively) and PHE flux (p=0.12, 57.0 ± 2.5 vs 60.0 ± 2.6 gd−1, respectively). However, PHE flux/kg BW was higher in early pregnancy compared to late pregnancy (82.2 ± 2.1 vs 71.2 ± 2.2 gd−1, respectively, p=0.0001).Funding: AB Pork, ON Pork, AB Livestock Industry Development Fund, ACAAF, Ajinomoto.

Ghrelin inhibits skeletal muscle protein breakdown in rats with thermal injury through normalizing elevated expression of E3 ubiquitin ligases MuRF1 and MAFbx

We previously determined that ghrelin synthesis was downregulated after burn injury and that exogenous ghrelin retained its ability both to stimulate food intake and to restore plasma growth hormone levels in burned rats. These observations and the finding that anabolic hormones can attenuate skeletal muscle catabolism led us to investigate whether ghrelin could attenuate burn-induced skeletal muscle protein breakdown in rats. These studies were performed in young rats (50-60 g) 24 h after approximately 30% total body surface area burn injury. Burn injury increased total and myofibrillar protein breakdown in extensor digitorum longus (EDL) muscles assessed by in vitro tyrosine and 3-methyl-histidine release, respectively. Continuous 24-h administration of ghrelin (0.2 mg.kg(-1).h(-1)) significantly inhibited both total and myofibrillar protein breakdown in burned rats. Ghrelin significantly attenuated burn-induced changes in mRNA expression of IGFBP-1 and IGFBP-3 in liver. In EDL, ghrelin attenuated the increases in mRNA expression of the binding proteins, but had no significant effect on reduced expression of IGF-I. Ghrelin markedly reduced the elevated mRNA expression of TNF-alpha and IL-6 in EDL muscle that occurred after burn. Moreover, ghrelin normalized plasma glucocorticoid levels, which were elevated after burn. Expression of the muscle-specific ubiquitin-ligating enzyme (E3) ubiquitin ligases MuRF1 and MAFbx were markedly elevated in both EDL and gastrocnemius and were normalized by ghrelin. These results suggest that ghrelin is a powerful anticatabolic compound that reduces skeletal muscle protein breakdown through attenuating multiple burn-induced abnormalities.

Latent and Active Polyphenol Oxidase (PPO) in Red Clover (Trifolium pratense) and Use of a Low PPO Mutant To Study the Role of PPO in Proteolysis Reduction

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzyme's own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.

Protein breakdown on whole-body and organ level in non-cachectic tumour-bearing mice undergoing surgery

Since both cancer and surgery are known to alter protein turnover, we investigated how the presence of tumour affects post-operative protein breakdown. Controls and tumour-bearing non-cachectic mice were studied, both with and without laparotomy (n=8 per group). One day after laparotomy, stable isotopes of phenylalanine and 3-methylhistidine were used in a steady-state protocol to assess total protein breakdown on whole-body level and in muscle, intestines, liver and kidney, in addition to whole-body myofibrillar protein breakdown and body composition. Proteasomal chymotrypsin-like activity was measured to determine activity of the ATP-dependent ubiquitin pathway. On whole-body level, the presence of tumour increased total protein breakdown from 47+/-6 to 58+/-4 nmol/10 g/min (p<0.05) and myofibrillar protein breakdown from 0.70+/-0.04 to 1.22+/-0.14 nmol/10 g/min (p<0.05) without affecting protein breakdown in organs, body composition or proteasomal activity. Laparotomy increased myofibrillar protein breakdown in controls (from 0.70+/-0.04 to 0.98+/-0.12 nmol/10 g/min, p<0.05) and tumour-bearing mice (from 1.22+/-0.14 to 1.54+/-0.22 nmol/10 g/min, p=0.15) to a similar extent. Whole-body total protein breakdown, total protein breakdown across organs, body composition or proteasomal activity were not affected by laparotomy. Tumour-bearing mice had increased total and myofibrillar protein breakdown on whole-body level even before weight loss was obvious. However, this did not affect the post-operative response in protein breakdown or body composition.

Dynamics of albumin synthesis after major rectal operation

Major abdominal operations were found to be associated with long-lasting metabolic changes, such as accelerated release of stress hormones and carbohydrate turnover. It is unknown currently whether acute changes of hepatic protein metabolism persist in a similar way. We wanted to determine the long-term dynamics of albumin synthesis and its relationship to whole body protein breakdown and albumin concentration after major rectal operations. We used stable isotope tracer techniques to determine albumin synthesis and whole body protein breakdown (rate of appearance of leucine, Ra) in postoperative patients about 1 week after low anterior rectal resection and also during convalescence (about 4 months after operation), and in healthy controls. Consecutive blood sampling was carried out during continuous isotope infusion (1-[(13)C]-leucine, 0.16 micromol/kg min). Serum albumin concentrations were close to the lower normal limit in patients early after operation but were comparable to controls in convalescent patients. Simultaneously, albumin synthesis was increased in the early postoperative phase (0.53 +/- 0.0.5%/h) compared with convalescent patients (0.32 +/- 0.04) and controls (0.28 +/- 0.04) (P < .01 each). A significant inverse correlation could be found between plasma albumin concentration and corresponding rates of albumin synthesis. Early after operation patients showed an increased leucine Ra (3.25 +/- 0.23 micromol/kg min) that was greater than that of convalescent patients (2.37 +/- 0.06 micromol/kg min, P < .05). Leucine Ra in both patient groups were greater than the rates in controls (2.01 +/- 0.07 micromol/kg min, P < .01) Albumin synthesis correlated weakly with whole body protein breakdown rate. Albumin synthesis and total body protein breakdown are increased after major abdominal operation, but albumin synthesis returns to control values only during convalescence. Hypoalbuminemia after rectal operations may be associated with high rates of albumin synthesis and is, therefore, not necessarily an indicator of insufficient hepatic function or poor nutritional status in that particular situation.

Demonstration of a reduction in postoperative body protein breakdown using the Clinitron fluidized bed with an ambient temperature of 32°C

Patients managed at an elevated ambient temperature after major surgery have a less pronounced rise in postoperative urinary nitrogen excretion. To investigate the mechanism involved, body protein breakdown was assessed, using a tracer dose of labelled amino acid, in patients following aorto-bifemoral bypass surgery nursed on either a Clinitron fluidized bed at 32 degrees C or a hospital bed at 22 degrees C and correlated with urinary total nitrogen excretion. Results showed a small reduction in measured body protein breakdown on the second postoperative day in patients managed on the Clinitron fluidized bed at 32 degrees C (2.92 +/- 0.91 versus 3.23 +/- 0.84 g protein kg-1 day-1; mean +/- s.d.), which was equivalent to the mean protein sparing (0.29g protein kg-1 day-1) demonstrated by the significant improvement in urinary total nitrogen excretion (9.20 +/- 2.0 versus 12.48 +/- 3.9 g N day-1; mean +/- s.d.: P less than 0.05). Urinary total nitrogen excretion (N) and body protein breakdown (B) showed a weak though significant positive correlation (B = 1.25 + 13.13N; r = +0.55: P = 0.05), whereas no correlation existed between urinary total nitrogen excretion and the derived rate of body protein synthesis. There was also a significant decrease in postoperative stress, measured during the isotope infusion, in patients managed on the Clinitron fluidized bed at 32 degrees C (12.3 +/- 2.2 versus 16.1 +/- 3.2 per cent activity incorporated into plasma proteins; mean +/- s.d.: P less than 0.05). These results show the beneficial effect of managing postoperative patients on a Clinitron fluidized bed at 32 degrees C in conserving body nitrogen through a reduction in body protein breakdown, probably as a consequence of decreased postoperative stress.

Signalling pathways in the induction of proteasome expression by proteolysis-inducing factor in murine myotubes

The mechanism by which the tumour product proteolysis-inducing factor (PIF) induced increased expression of the ubiquitin–proteasome proteolytic pathway was studied in C 2C 12 murine myotubes. PIF directly increased total protein breakdown at concentrations between 4 and 16 nM, and the effect was attenuated by eicosapentaenoic acid (EPA) and the 12/15-lipoxygenase inhibitor 2,3,5-trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504). PIF induced an increased expression of mRNA for proteasome α (C2) and β (C5) subunits over the same concentration range as that inducing protein degradation and with a maximal effect 4 h after PIF addition. The effect was attenuated by both EPA and CV-6504, suggesting the role of a lipoxygenase metabolite in the increased gene transcription. 15( S)-Hydroxyeicosatetraenoic acid [15( S)-HETE], an intermediate in intracellular signalling by PIF was shown to activate protein kinase C α(PKC) over the same concentration range as that inducing proteasome expression and both effects were attenuated by calphostin C, a highly specific inhibitor of PKC. 15( S)-HETE induced phosphorylation and degradation of IκBα at the same concentrations as those inducing 20S proteasome expression, and this effect was attenuated by calphostin C, suggesting the mediation of PKC. These results suggest potential control points in proteasome activation that could be useful for therapeutic intervention.

Sepsis induces the transcription of the glucocorticoid receptor in skeletal muscle cells.

Evidence from a recent study indicates that glucocorticoids (GCs) mediate skeletal muscle proteolysis during sepsis via the GC receptor (GR) pathway. Attempts to identify the mechanisms regulating GR gene expression in skeletal muscle during sepsis have been hampered by the lack of an appropriate in vitro model system that can mimic in vivo septic conditions. In the present study, we report that GR gene transcription in L6 myocytes in vitro is up-regulated by treatment with sera from septic rats in a manner similar to that measured in septic rats in vivo. Sera from septic rats were collected from animals in which sepsis was induced by caecal ligation and puncture and from control rats that were sham-operated. Finally, by treating L6 myotubes with the GR antagonist RU 38486, thereby preventing sepsis-induced GR transcription, we confirmed that the possible septic effect on the GR was due to increased GCs. L6 myocytes treated with sera from septic rats might therefore be useful as an experimental model for identifying the molecular mechanisms by which the GR regulates muscle cachexia during sepsis. Furthermore, RU 38486 inhibited the sepsis-induced increase in total and myofibrillar energy-dependent protein breakdown rates in incubated extensor digitorum longus muscles from septic and sham-operated rats, as measured by release of tyrosine and 3-methylhistidine respectively. Our results demonstrate for the first time that sepsis induces GR transcription in skeletal muscle, and supports the hypothesis that the GC-induced proteolysis under sepsis is partially a consequence of GR activation.

Protein metabolism in liver cirrhosis: from albumin to muscle myofibrils.

Liver cirrhosis in the advanced state is characterized by protein wasting, as indicated by the loss of muscle mass, hypoalbuminemia, and an abnormal amino acid profile. The protein wasting condition cirrhosis is associated with a poor prognosis and reduced survival. Poor nutrition, metabolic and hormonal abnormalities, and other disease-associated alterations may all concur to protein wasting. An understanding of the causes and mechanisms leading to protein wasting in cirrhosis may help in the development of nutritional interventions and new therapies. Albumin and muscle protein turnover in cirrhotic patients have been studied in vivo with the aid of isotope dilution techniques or organ catheterization. Albumin synthesis appears to parallel liver function, i.e. the more compromised is the liver, the less is the albumin production rate. Meal-induced albumin synthesis is impaired even in compensated cirrhotic patients. Skeletal muscle protein synthesis is diminished in cirrhosis, and total muscle protein breakdown also appears to be increased, thus explaining the reduced muscle mass. Either hormone or substrate resistance, or newly involved substances (cytokines, insulin-like growth factor 1, leptin) may play a role in the reduced synthesis of both albumin and muscle proteins in liver cirrhosis. Abnormalities of both albumin and muscle protein turnover have been demonstrated in liver cirrhotic patients. The possible role of the multiple hormonal and metabolic abnormalities of this disease, as well that of cytokines and other recently discovered substances, need to be investigated further.

A Mechanism Regulating Proteolysis of Specific Proteins during Renal Tubular Cell Growth

Growth factors suppress the degradation of cellular proteins in lysosomes in renal epithelial cells. Whether this process also involves specific classes of proteins that influence growth processes is unknown. We investigated chaperone-mediated autophagy, a lysosomal import pathway that depends on the 73-kDa heat shock cognate protein and allows the degradation of proteins containing a specific lysosomal import consensus sequence (KFERQ motif). Epidermal growth factor (EGF) or ammonia, but not transforming growth factor beta1, suppresses total protein breakdown in cultured NRK-52E renal epithelial cells. EGF or ammonia prolonged the half-life of glyceraldehyde-3-phosphate dehydrogenase, a classic substrate for chaperone-mediated autophagy, by more than 90%, whereas transforming growth factor beta1 did not. EGF caused a similar increase in the half-life of the KFERQ-containing paired box-related transcription factor, Pax2. The increase in half-life was accompanied by an increased accumulation of proteins with a KFERQ motif including glyceraldehyde-3-phosphate dehydrogenase and Pax2. Ammonia also increased the level of the Pax2 protein. Lysosomal import of KFERQ proteins depends on the abundance of the 96-kDa lysosomal glycoprotein protein (lgp96), and we found that EGF caused a significant decrease in lgp96 in cellular homogenates and associated with lysosomes. We conclude that EGF in cultured renal cells regulates the breakdown of proteins targeted for destruction by chaperone-mediated autophagy. Because suppression of this pathway results in an increase in Pax2, these results suggest a novel mechanism for the regulation of cell growth.

Dantrolene reduces serum TNFalpha and corticosterone levels and muscle calcium, calpain gene expression, and protein breakdown in septic rats.

The effects of dantrolene on serum TNFalpha and corticosterone levels and on muscle calcium, calpain gene expression, and protein breakdown were studied in rats with abdominal sepsis induced by cecal ligation and puncture. Treatment of rats with 10 mg/kg of dantrolene 2 h before and 8 h after induction of sepsis reduced serum TNFalpha and corticosterone, muscle calcium levels, mRNA levels for m- and mu-calpain, and the muscle specific calpain p94, as well as total and myofibrillar protein breakdown rates, determined as release of tyrosine and 3-methylhistidine, respectively, from incubated extensor digitorum longus muscles. The results support the concept that increased calcium concentrations may be an important mechanism of sepsis-induced muscle protein breakdown. The data also indicate that other mechanisms, in addition to reduced muscle calcium concentrations such as decreased levels of TNFalpha and glucocorticoids, may contribute to the anti-catabolic effects of dantrolene during sepsis. The observations are important from a clinical standpoint because they suggest that the catabolic response in skeletal muscle during sepsis may be prevented by treatment with a calcium antagonist.

The breakdown of autophagic vesicles inside the vacuole depends on Aut4p.

Autophagy is a degradative transport pathway that delivers cytosolic proteins to the lysosome (vacuole). Cytosolic proteins appear inside the vacuole enclosed in autophagic vesicles. These autophagic vesicles are broken down in the vacuole together with their cytosolic content. The breakdown of vesicular transport intermediates is a unique feature of autophagy. We here identify Aut4p, a component essential for the disintegration of autophagic vesicles, inside the vacuole of S. cerevisiae cells. Aut4p is a putative integral membrane protein with limited homologies to permeases. Chromosomal deletion of AUT4 has no obvious influence on growth, vacuolar acidification and the activities of vacuolar proteinases. Like proteinase B-deficient cells, aut4-deleted cells show a partial reduction in total protein breakdown during nitrogen starvation. A biologically active fusion protein of Aut4p and the green fluorescent protein is visualized at the vacuolar membrane and in punctate structures attached to the vacuole.

Insulin-Like Growth Factor I Reduces Ubiquitin and Ubiquitin-Conjugating Enzyme Gene Expression but Does Not Inhibit Muscle Proteolysis in Septic Rats

We examined the effect of insulin-like growth factor I (IGF-I), administered in vivo, on protein turnover rates and gene expression of the ubiquitin-proteasome proteolytic pathway in skeletal muscle of septic rats. Sepsis was induced by cecal ligation and puncture. Other rats were sham-operated. Miniosmotic pumps were implanted sc, and groups of rats received IGF-I (7 mg/kg·24 h) or saline. Protein synthesis and breakdown rates were determined in incubated extensor digitorum longus muscles. Messenger RNA levels for ubiquitin and the ubiquitin-conjugating enzyme E214k were determined by Northern blot analysis. Sepsis resulted in an approximately 30% reduction of muscle protein synthesis, and this effect of sepsis was blunted in rats treated with IGF-I. In contrast, IGF-I did not prevent the sepsis-induced increase in total and myofibrillar muscle protein breakdown. Ubiquitin and E214k messenger RNA levels were increased several fold in muscle from septic rats, and this effect of sepsis was abolished in IGF-I treated rats. The results suggest that administration of IGF-I may improve sepsis-induced muscle cachexia by stimulating protein synthesis. However, because muscles were resistant to IGF-I, with regard to regulation of protein breakdown, the use of IGF-I to treat muscle cachexia during sepsis remains unclear. An additional important implication of the present study is that changes in messenger RNA levels for ubiquitin and the ubiquitin-conjugating enzyme E214k do not always reflect changes in muscle protein breakdown rates.

Insulin-like growth factor I reduces ubiquitin and ubiquitin-conjugating enzyme gene expression but does not inhibit muscle proteolysis in septic rats.

We examined the effect of insulin-like growth factor I (IGF-I), administered in vivo, on protein turnover rates and gene expression of the ubiquitin-proteasome proteolytic pathway in skeletal muscle of septic rats. Sepsis was induced by cecal ligation and puncture. Other rats were sham-operated. Miniosmotic pumps were implanted sc, and groups of rats received IGF-I (7 mg/kg x 24 h) or saline. Protein synthesis and breakdown rates were determined in incubated extensor digitorum longus muscles. Messenger RNA levels for ubiquitin and the ubiquitin-conjugating enzyme E2(14k) were determined by Northern blot analysis. Sepsis resulted in an approximately 30% reduction of muscle protein synthesis, and this effect of sepsis was blunted in rats treated with IGF-I. In contrast, IGF-I did not prevent the sepsis-induced increase in total and myofibrillar muscle protein breakdown. Ubiquitin and E2(14k) messenger RNA levels were increased several fold in muscle from septic rats, and this effect of sepsis was abolished in IGF-I treated rats. The results suggest that administration of IGF-I may improve sepsis-induced muscle cachexia by stimulating protein synthesis. However, because muscles were resistant to IGF-I, with regard to regulation of protein breakdown, the use of IGF-I to treat muscle cachexia during sepsis remains unclear. An additional important implication of the present study is that changes in messenger RNA levels for ubiquitin and the ubiquitin-conjugating enzyme E2(14k) do not always reflect changes in muscle protein breakdown rates.

Sepsis-Induced Muscle Proteolysis Is Prevented by a Proteasome Inhibitor in Vivo

Sepsis-induced muscle proteolysis mainly reflects ubiquitin-proteasome-dependent protein degradation. The effect of in vivo administration of a proteasome inhibitor on muscle protein breakdown during sepsis is not known. We treated rats with the proteasome inhibitor N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal (PSI) or corresponding volume of vehicle i.p. 2 h before sham-operation or induction of sepsis by cecal ligation and puncture. The sepsis-induced increase in total and myofibrillar muscle protein breakdown was inhibited in rats treated in vivo with PSI and a maximal effect was seen following 15 mg/kg of the proteasome inhibitor. Results from in vitro experiments in which incubated muscles were treated with 100 μM PSI suggest that the drug has a direct effect on muscle and that the effect is specific for the proteasome. The results are important because they suggest that it may be possible to prevent or improve the cachectic response in skeletal muscle during sepsis by treatment with a proteasome inhibitor.

Role of the ubiquitin-proteasome pathway in sepsis-induced muscle catabolism.

Several lines of evidence suggest that the ubiquitin-proteasome pathway is involved in sepsis-induced muscle catabolism. The gene expression of ubiquitin and several of the proteasome subunits was increased in muscle from both septic rats and patients. In other studies, the activity of isolated 20S proteasomes was stimulated in septic muscles. Sepsis-induced increase in muscle total and myofibrillar protein breakdown was inhibited with specific proteasome blockers. Although the ubiquitin-proteasome pathway is upregulated in septic muscle, it is still unclear how the myofibrillar proteins actin and myosin are ubiquitinated and become substrates for the 26S proteasome. Recent studies suggest that a calcium-dependent, calpain-mediated process releases myofilaments from the Z-disks during sepsis. It is possible that this process exposes destabilizing N-terminal residues on actin and myosin, making them suitable substrates for the N-end rule pathway involving the 14 kD ubiquitin-conjugating enzyme E214k and the ubiquitin-protein ligase E3alpha.