Human Nutrition

The age-related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein-7 (Pax7) or laminin-2a, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the agerelated rise in apoptotic nuclei was essentially due to stromal cells. Myofibre-associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7 + satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age-dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing.

Multiple lines of evidence suggest that the ubiquitin-proteasome-dependent proteolytic pathway is the major degradative process responsible for the loss of muscle proteins seen in various pathological states and following food deprivation. The first step in this pathway is the covalent attachment of polyubiquitin chains to protein substrates. This signal targets the substrates for subsequent hydrolysis into peptides by the 26S proteasome. Several metabolic abnormalities (reduced food intake, impaired mobility, and perturbations in the production or responsiveness of catabolic and anabolic hormones, cytokines and/or proteolysis inducing factors) act in concert to contribute to muscle wasting in disease states. We cite recent evidence that insulin, glucocorticoids, thyroid hormones, and nutrients regulate the rates of ubiquitinylation of protein substrates and of proteasome-dependent proteolysis in skeletal muscle.

Ubiquitin-dependent proteolysis is activated in skeletal muscle atrophying in response to various catabolic stimuli. Previous studies have demonstrated activation of ubiquitin conjugation. Because ubiquitination can also be regulated by deubiquitinating enzymes, we used degenerate oligonucleotides derived from conserved sequences in the ubiquitin-specific protease (UBP) family of deubiquitinating enzymes in RT-PCR with skeletal muscle RNA to amplify putative deubiquitinating enzymes. We identified USP19, a 150-kDa deubiquitinating enzyme that is widely expressed in various tissues including skeletal muscle. Expression of USP19 mRNA increased by ∼30–200% in rat skeletal muscle atrophying in response to fasting, streptozotocin-induced diabetes, dexamethasone treatment, and cancer. Increased mRNA levels during fasting returned to normal with refeeding, but 1 day later than the normalization of rates of proteolysis and coincided instead with recovery of muscle mass. Indeed, in all catab...

Immobilization produces morphological, physiological, and biochemical alterations in skeletal muscle leading to muscle atrophy and long periods of recovery. Muscle atrophy during disuse results from an imbalance between protein synthesis and proteolysis but also between apoptosis and regeneration processes. This work aimed to characterize the mechanisms underlying muscle atrophy and recovery following immobilization by studying the regulation of the mitochondria-associated apoptotic and the ubiquitin-proteasome-dependent proteolytic pathways. Animals were subjected to hindlimb immobilization for 4–8 days (I4 to I8) and allowed to recover after cast removal for 10–40 days (R10 to R40). Soleus and gastrocnemius muscles atrophied from I4 to I8 to a greater extent than extensor digitorum longus and tibialis anterior muscles. Gastrocnemius muscle atrophy was first stabilized at R10 before being progressively reduced until R40. Polyubiquitinated proteins accumulated from I4, whereas the i...

Sustained muscle wasting due to immobilization leads to weakening and severe metabolic consequences. The mechanisms responsible for muscle recovery after immobilization are poorly defined. Muscle atrophy induced by immobilization worsened in the lengthened tibialis anterior (TA) muscle but not in the shortened gastrocnemius muscle. Here, we investigated some mechanisms responsible for this differential response. Adult rats were subjected to unilateral hindlimb casting for 8 days (I8). Casts were removed at I8, and animals were allowed to recover for 10 days (R1 to R10). The worsening of TA atrophy following immobilization occurred immediately after cast removal at R1 and was sustained until R10. This atrophy correlated with a decrease in type IIb myosin heavy chain (MyHC) isoform and an increase in type IIx, IIa, and I isoforms, with muscle connective tissue thickening, and with increased collagen (Col) I mRNA levels. Increased Col XII, Col IV, and Col XVIII mRNA levels during TA im...

Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C2C12 myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts....

The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-α (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of highmolecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expres

Alteration of skeletal muscle protein breakdown is a hallmark of a set of pathologies, including sepsis, with negative consequences for recovery. The aim of the present study was to search for muscle markers associated with protein loss, which could help in predicting and understanding pathological wasting. With the use of differential display reverse transcription-PCR, we screened differentially expressed genes in muscle from septic rats in a longlasting catabolic state. One clone was isolated, confirmed as being overexpressed in septic skeletal muscle and identified as encoding the lysosomal cysteine endopeptidase cathepsin L. Northern-and Western-blot analysis of cathepsin L in gastrocnemius or tibialis anterior muscles of septic rats confirmed an elevation (up to 3-fold) of both mRNA and protein levels as early as 2 days post-infection, and a further increase 6 days postinfection (up to 13-fold). At the same time, the increase in mRNAs encoding other lysosomal endopeptidases or com

Recent studies highlight other, nonhomeostatic controls of feeding related to food reward and learning. The question is whether the regions highlighted in these studies should be considered as functionally apart from or interacting with homeostatic controls.

Unintentional loss of over 10% of body weight occurred in 11% of patients in the 6 months prior to admission and in 37% of those undernourished on admission. At discharge, 62% of cases assessed had lost weight in hospital and only 40% of the 62 undernourished patients were referred for nutritional support. The patients found to be malnourished on admission in this study may represent those most difficult to manage successfully, as their mortality rate in hospital (6.5%) was almost four times that of the adequately nourished group (1.8%).

Purpose" Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the catabolism of the pyrimidine bases. 5-Fluorouracil (5FU), a pyrimidine analogue widely used in chemotherapy for digestive tract cancers, is also catabolized by DPD. In this study, we examined the effect of parenteral nutrition on 5FU metabolism in rats.

Background: Glutathione serves as an important intracellular scavenger against reactive oxygen metabolites and its concentration in skeletal muscle decreases following elective surgery. However, it is not known if this is related to a lower rate of synthesis or other mechanisms. Aims: To determine the activities of the two enzymes involved in glutathione synthesis, ~'-glutamyl-cysteine synthetase and glutathione synthetase, in skeletal muscle of patients after surgical trauma. Methods: Patients (n = 8) undergoing elective abdominal surgery were studied. Percutaneous muscle biopsies were taken preoperatively, 24 and 72 h postoperatively. The activities of the two enzymes and the concentrations of reduced and oxidized glutathione were assayed. Results: Reduced glutathione decreased by 40% at 24 h postoperatively and remained low 72 h after the operation. The oxidized glutathione was 0.064 _+ 0.014 (mmol/kg wet weight) preoperatively and did not change significantly postoperatively. The activity of ~'-glutamylcysteine synthetase was 52.72 _+ 8.47 (mmol/kg protein/min) before surgery and it remained unaltered, whilst the activity of glutathione synthetase decreased after the operation. A statistical correlation between glutathione synthetase and reduced glutathione was seen (t 2 = 0.4, P< 0.05). Pre-operative 24-h postop, 72-h postop. Reduced glutathione 1.55-+ 0.10 0.92 _+ 0.12"* 1.10-+ 0.06* (mmoi/kg wet weight) Glutathione synthetase 63.51 _+ 8.29 35.17 _+ 4.63** 37.51 _+ 3.56* (mmol/kg protein/rnin) Data are expressed as mean _+ SEM, *P < 0.05, **P < 0.01. Conclusions: Surgical trauma depletes cellular glutathione level in skeletal muscle by decreasing its synthetic capacity. The alteration of glutathione metabolism influences the redox status of the tissue.

Aims: Pentoxifylline (PTX) is a very inexpensive haemorheological agent, which is widely used in humans, and also inhibits tumour necrosis factor-c~ (TNF-c 0 transcription. TNF-c~ has been reported to activate skeletal muscle proteolysis in various catabolic conditions. We aimed to determine whether PTX may reduce muscle wasting in cachectic cancer rats. Methods: Young Wistra rats were divided into control (CT), tumour-bearing (TB) and PTX-treated tumour-bearing (TB-PTX) rats, n = 6-10 per group). TB animals received an injection of a Yoshida sarcoma homogenate. TB-PTX rats received a daily IP injection of PTX (100 mg/kg bodt wt.), and the CT and TB groups a daily IP injection of PTX vehicle. Rats were killed 9 days after tumour implantation. Protein synthesis and breakdown were measured in incubated extensor digitorum Iongus muscles, by following the incorporation of [UJ4C]L-phenylalanine into proteins, and the release of tyrosine into incubation media containing 0.5 mM cycloheximide, respectively. Methylamine and E-64c were added to Ca2+-free media to estimate non-lysosomal and Ca2%independent proteolysis. The expression of proteolytic genes was measured by Northern blots. Data were compared using the Student's t-test. Results: The mass of the EDL, tibialis anterior, and gastrocnemius muscles was reduced by 24, 19 and 28%, respectively (P < 0.05) in TB vs CT. PTX significantly improved the mass of these muscles in TB-PTX vs TB (P < 0.05), without affecting tumour growth. The atrophy of EDL in TB rats was only due to increased proteolysis (+27%, P < 0.05), as there was no change in protein synthesis. PTX abolished the enhanced proteolysis seen in TB rats by inhibiting a nonlysosomal Ca2+-independent proteolytic process, and eliminated the enhanced expression (P < 0.03) of ubiquitin (Ub) (+4480%), 14-kDa Ub-conjugating enzyme E2 (+159%), and the 20S proteasome subunit C2 (+380%). The 19S complex and 11S regulator associate with and regulate the 20S proteasome peptidase activities. The mRNA levels for the ATPase subunit MSS1 of the 19S complex increased by 105% (P<0.02) in TB rats, in contrast with mRNAs of other subunits. This adaptation was also suppressed by PTX, suggesting that the drug inhibited the activation of the 26S proteasome. Conclusion: The data suggest that PTX may prevent muscle wasting when TNF-~ production rises (e.g. cancer, sepsis, AIDS and trauma) by inhibiting the activation of the Ub-proteasome proteolytic pathway.

Background & aims: Ornithine a-ketoglutarate (OKG) is recognized to improve nutritional status in various catabolic states, such as burn injury, trauma, and sepsis. However, in wasting diseases, such as induced by cancer, the data are scarce and the precise mechanisms by which OKG acts on protein metabolism are still unclear. The aim of this study was to evaluate the ability of OKG to affect protein metabolism in an aggressive model of cancer and to modulate the ubiquitin-proteasome-dependent pathway, which in skeletal muscle is the critical degradative pathway implicated in many catabolic states, including cancer-associated cachexia. Methods: Experiments were carried out in Yoshida sarcoma-bearing and healthy pair-fed rats. Three groups of 16 young male rats were studied during 9 days following tumor implantation: two groups of tumor-bearing rats fed a balanced regimen enriched with either OKG (5 g/kg body weight/day, OKG-K) or an isonitrogenous mixture of non-essential amino acids (C-K), and one group of healthy pair-fed rats (PF). Results: As expected, Yoshida sarcoma induced muscle atrophy, decreased nitrogen balance, enhanced 3-methylhistidine (3-MH) excretion and increased mRNA levels for ubiquitin and 14-kDa ubiquitin-conjugating enzyme E2. OKG supplementation did not improve muscle mass or protein balance and did not reduce enhanced 3-MH excretion in

In order to characterize the poorly defined mechanisms that account for the anti-proteolytic effects of insulin in skeletal muscle, we investigated in rats the effects of a 3 h systemic euglycaemic hyperinsulinaemic clamp on lysosomal, Ca 2 +-dependent proteolysis, and on ubiquitin/proteasome-dependent proteolysis. Proteolysis was measured in incubated fast-twitch mixed-fibre extensor digitorum longus (EDL) and slow-twitch red-fibre soleus muscles harvested at the end of insulin infusion. Insulin inhibited proteolysis (P 0n05) in both muscles. This anti-proteolytic effect disappeared in the presence of inhibitors of the lysosomal/Ca 2 +-dependent proteolytic pathways in the soleus, but not in the EDL, where only the proteasome inhibitor MG 132 (benzyloxycarbonyl-leucyl-leucyl-leucinal) was effective. Furthermore, insulin depressed ubiquitin mRNA levels in the mixed-fibre tibialis anterior, but not in the red-fibre diaphragm muscle, suggesting that insulin inhibits ubiquitin/proteasome-dependent proteolysis in mixedfibre muscles only. However, depressed ubiquitin mRNA levels in such muscles were not associated with significant decreases in the amount of ubiquitin conjugates, or in mRNA levels or protein content for the 14 kDa ubiquitin-conjugating enzyme E2 and 20 S proteasome subunits. Thus alternative, as yet unidentified, mechanisms are likely to contribute to inhibit the ubiquitin/proteasome system in mixed-fibre muscles.

Introduction Controlled mechanical ventilation (CMV) induces profound modifications of diaphragm protein metabolism, including muscle atrophy and severe ventilator-induced diaphragmatic dysfunction. Diaphragmatic modifications could be decreased by spontaneous breathing. We hypothesized that mechanical ventilation in pressure support ventilation (PSV), which preserves diaphragm muscle activity, would limit diaphragmatic protein catabolism. Methods Forty-two adult Sprague-Dawley rats were included in this prospective randomized animal study. After intraperitoneal anesthesia, animals were randomly assigned to the control group or to receive 6 or 18 hours of CMV or PSV. After sacrifice and incubation with 14 C-phenylalanine, in vitro proteolysis and protein synthesis were measured on the costal region of the diaphragm. We also measured myofibrillar protein carbonyl levels and the activity of 20S proteasome and tripeptidylpeptidase II. Results Compared with control animals, diaphragmatic protein catabolism was significantly increased after 18 hours of CMV (33%, P = 0.0001) but not after 6 hours. CMV also decreased protein synthesis by 50% (P = 0.0012) after 6 hours and by 65% (P < 0.0001) after 18 hours of mechanical ventilation. Both 20S proteasome activity levels were increased by CMV. Compared with CMV, 6 and 18 hours of PSV showed no significant increase in proteolysis. PSV did not significantly increase protein synthesis versus controls. Both CMV and PSV increased protein carbonyl levels after 18 hours of mechanical ventilation from +63% (P < 0.001) and +82% (P < 0.0005), respectively. Conclusions PSV is efficient at reducing mechanical ventilation-induced proteolysis and inhibition of protein synthesis without modifications in the level of oxidative injury compared with continuous mechanical ventilation. PSV could be an interesting alternative to limit ventilator-induced diaphragmatic dysfunction.

Glucocorticoids mediate muscle atrophy in many catabolic states. Myostatin expression, a negative regulator of muscle growth, is increased by glucocorticoids and myostatin overexpression is associated with lower muscle mass. This suggests that myostatin is required for the catabolic effects of glucocorticoids. We therefore investigated whether myostatin gene disruption could prevent muscle atrophy caused by glucocorticoids. Male myostatin knockout (KO) and wild-type mice were subjected to dexamethasone treatment (1 mg/kg⅐d for 10 d or 5 mg/kg⅐d for 4 d). In wild-type mice, daily administration of lowdose dexamethasone for 10 d resulted in muscle atrophy (tibialis anterior: ؊15%; gastrocnemius: ؊13%; P < 0.01) due to 15% decrease in the muscle fiber cross-sectional area (1621 ؎ 31 vs. 1918 ؎ 64 m 2 , P < 0.01). In KO mice, there was no reduction of muscle mass nor fiber cross-sectional area after dexamethasone treatment. Muscle atrophy after 4 d of high-dose dexamethasone was associated with increased mRNA of enzymes involved in proteolytic pathways (atrogin-1, muscle ring finger 1, and cathepsin L) and increased chymotrypsin-like proteasomal activity. In contrast, the mRNA of these enzymes and the proteasomal activity were not significantly affected by dexamethasone in KO mice. Muscle IGF-I mRNA was paradoxically decreased in KO mice (؊35%, P < 0.05); this was associated with a potentially compensatory increase of IGF-II expression in both saline and dexamethasone-treated KO mice (2-fold, P < 0.01). In conclusion, our results show that myostatin deletion prevents muscle atrophy in glucocorticoid-treated mice, by blunting the glucocorticoid-induced enhanced proteolysis, and suggest an important role of myostatin in muscle atrophy caused by glucocorticoids. (Endocrinology 148: 452-460, 2007) Materials and Methods Animals Eight-week-old male myostatin knockout (KO) FVB mice harboring a constitutive deletion of the third myostatin exon (n ϭ 28 KO mice) (9)

The ubiquitin-proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin-protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.