Proteasome proteolytic activity in skeletal muscle is increased in patients with sepsis

Patients with sepsis in the ICU (intensive care unit) are characterized by skeletal muscle wasting. This leads to muscle dysfunction that also influences the respiratory capacity, resulting in prolonged mechanical ventilation. Catabolic conditions are associated with a general activation of the ubiquitin-proteasome pathway in skeletal muscle. The aim of the present study was to measure the proteasome proteolytic activity in both respiratory and leg muscles from ICU patients with sepsis and, in addition, to assess the variation of proteasome activity between individuals and between duplicate leg muscle biopsy specimens. When compared with a control group (n=10), patients with sepsis (n=10) had a 30% (P<0.05) and 45% (P<0.05) higher proteasome activity in the respiratory and leg muscles respectively. In a second experiment, ICU patients with sepsis (n=17) had a 55% (P<0.01) higher proteasome activity in the leg muscle compared with a control group (n=10). The inter-individual scatter of proteasome activity was larger between the patients with sepsis than the controls. We also observed a substantial intra-individual difference in activity between duplicate biopsies in several of the subjects. In conclusion, the proteolytic activity of the proteasome was higher in skeletal muscle from patients with sepsis and multiple organ failure compared with healthy controls. It was shown for the first time that respiratory and leg muscles were affected similarly. Furthermore, the variation in proteasome activity between individuals was more pronounced in the ICU patients for both muscle types, whereas the intra-individual variation between biopsies was similar for ICU patients and controls.     

Role of ubiquitin-proteasome pathway in skeletal muscle wasting in rats with endotoxemia

OBJECTIVE: To investigate the mechanism of muscle protein breakdown under endotoxemia condition. DESIGN: Randomized, controlled, animal experiment in a hospital institute. SETTING: Experimental laboratory. INTERVENTION: Either saline or endotoxin (Escherichia coli O(55)B(5), 10 mg/kg) were administered into the peritoneal cavity in rats. MEASUREMENTS AND MAIN RESULTS: The rate of total protein breakdown was increased by 29% and 61% in extensor digitorum longus muscle at 2 hrs and 6 hrs, whereas the myofibrillar proteolytic rate was increased by 155%, 222%, and 40% at 2 hrs, 6 hrs, and 12 hrs, respectively, in the endotoxin treatment group compared with that of the pair-fed normal control group. Meanwhile, compared with the normal control group, the level of 2.4-kilobase (kb) messenger RNA (mRNA) for ubiquitin in extensor digitorum longus muscle in rats was increased by 153% and 470% at 2 hrs and 6 hrs. There were 87% and 117% increases in 1.2-kb mRNA for E2-14K, and 89% and 168% increase in RC2 mRNA expression in extensor digitorum longus muscle in endotoxemic rats than normal control rats at 2 hrs and 6 hrs after injection of endotoxin peritoneally. The tumor necrosis factor-alpha and interleukin-6 concentrations in rat plasma progressively increased after endotoxin treatment, but tumor necrosis factor-alpha peaked at the 2-hr time point, whereas interleukin-6 peaked at 12 hrs. Endotoxin administration resulted in a marked increase in endotoxin level at 2 hrs and 6 hrs. No significant change was observed in soleus muscle after endotoxin injection. A significantly positive correlation was found between the net release of 3-methylhistidine and respective values of endotoxin, intensity of mRNA expression of 2.-kb ubiquitin, 1.2-kb E2-14K, and subunit RC2 in extensor digitorum longus muscle (r =.9882, .9731, .9653, .9814, p <.05). However, no significant correlation was seen between tumor necrosis factor-alpha or interleukin-6 and respective values of 3-methylhistidine, mRNA expression of 2.4-kb ubiquitin, 1.2-kb E2-14K, and subunit RC2 (r =.3580, .4521, .5277, .4931, p >.05; r =.3950, .1767, .2136, .2519, p >.05, respectively.) in soleus muscle. CONCLUSIONS: Endotoxemia can induce enhancement of skeletal muscle protein breakdown, mainly involving myofibrillar protein and white, fast-twitch extensor digitorum longus muscle. Ubiquitin-proteasome proteolytic pathway plays an important and major role in skeletal muscle proteolysis. Endotoxin, tumor necrosis factor-alpha, and interleukin-6 can directly or indirectly regulate muscle protein breakdown.     

Sensitivity and protein turnover response to glucocorticoids are different in skeletal muscle from adult and old rats. Lack of regulation of the ubiquitin-proteasome proteolytic pathway in aging.

We studied glucocorticoid-induced muscle wasting and subsequent recovery in adult (7-mo-old) and old (22-mo-old) rats, since the increased incidence of various disease states may result in glucocorticoids hypersecretion in aging. Adult and old rats received dexamethasone in their drinking water and were then allowed to recover. Muscle wasting occurred more rapidly in old rats and the recovery of muscle mass was impaired, suggesting that glucocorticoids may be involved in the emergence of muscle atrophy with advancing age. According to measurements in incubated epitrochlearis muscles, dexamethasone-induced muscle wasting mainly resulted from increased protein breakdown in the adult, but from depressed protein synthesis in the aged animal. Increased expression of cathepsin D, m-calpain, and ubiquitin was observed in the muscles from both dexamethasone-treated adult and old rats. By contrast, the disappearance of the stimulatory effect of glucocorticoids on protein break-down in aging occurred along with a loss of ability of steroids to enhance the expression of the 14-kD ubiquitin carrier protein E2, which is involved in protein substrates ubiquitinylation, and of subunits of the 20 S proteasome (the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates). Thus, if glucocorticoids play any role in the progressive muscle atrophy seen in aging, this is unlikely to result from an activation of the ubiquitin-proteasome proteolytic pathway.     

Exercise stimulates the mitogen-activated protein kinase pathway in human skeletal muscle.

Physical exercise can cause marked alterations in the structure and function of human skeletal muscle. However, little is known about the specific signaling molecules and pathways that enable exercise to modulate cellular processes in skeletal muscle. The mitogen-activated protein kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. We tested the hypothesis that a single bout of exercise activates the MAPK signaling pathway. Needle biopsies of vastus lateralis muscle were taken from nine subjects at rest and after 60 min of cycle ergometer exercise. In all subjects, exercise increased MAPK phosphorylation, and the activity of its downstream substrate, the p90 ribosomal S6 kinase 2. Furthermore, exercise increased the activities of the upstream regulators of MAPK, MAP kinase kinase, and Raf-1. When two additional subjects were studied using a one-legged exercise protocol, MAPK phosphorylation and p90 ribosomal S6 kinase 2, MAP kinase kinase 1, and Raf-1 activities were increased only in the exercising leg. These studies demonstrate that exercise activates the MAPK cascade in human skeletal muscle and that this stimulation is primarily a local, tissue-specific phenomenon, rather than a systemic response to exercise. These findings suggest that the MAPK pathway may modulate cellular processes that occur in skeletal muscle in response to exercise.     

Sepsis is frequent in initially non-critical hypotensive emergency department patients and is associated with increased mortality

ObjectiveHypotension, defined as a mean arterial pressure of maximum 70 mmHg, is associated with significant morbidity and mortality. The objective of this study was to determine in initially non-critical hypotensive adult patients the proportion of sepsis and if septic patients had different outcome and clinical factors than non-septic patients.MethodsThis retrospective observational study was conducted over a year on adult hypotensive emergency department patients initially considered by triage as non-critical. Patients were separated into three groups: hypotensive septic patients (HSP), hypotensive non-septic infected patients (HNSIP), and other hypotensive patients (OHP). Clinical scores, signs, length of stay (LOS), and mortality were compared using analysis of variance for continuous variables and chi-square analysis for categorical variables.ResultsThere were 136 (35.5%) septic patients, 37 (9.7%) with non-septic infection, and 210 (54.8%) with another cause of hypotension. Overall in-hospital mortality was 12.0% and total mortality was greater in HSP than in HNSIP (20.6% vs. 5.4%, p = 0.031) or OHP (20.6 vs. 7.6%, p < 0.001). LOS was greater for HSP when compared to HNSIP (median(IQR): 9(6–17) vs. 6(1?13), p = 0.004) and OHP (median(IQR): 9(6–17) vs. 3(1–8) days, p < 0.0001).ConclusionSepsis in a priori non-critical hypotensive adult patients, when compared with other causes of hypotension, is associated with significantly higher mortality and increased LOS. Patients that present to the emergency department and have a MAP of 70mmHg or less must be rigorously evaluated and have consistent follow-up.     

Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle.

Insulin-stimulated glycogen synthase activity in human skeletal muscle correlates with insulin-mediated glucose disposal rate (M) and is reduced in insulin-resistant subjects. We have previously reported reduced insulin-stimulated glycogen synthase activity associated with reduced fasting glycogen synthase phosphatase activity in skeletal muscle of insulin-resistant Pima Indians. In this study we investigated the time course for insulin stimulation of glycogen synthase and synthase phosphatase during a 2-h high-dose insulin infusion (600 mU/min per m2) in six insulin-sensitive caucasians (group S) and in five insulin-resistant Pima Indians (group R). Percutaneous muscle biopsies were obtained from the quadriceps femoris muscle after insulin infusion for 0, 10, 20, 40, and 120 min. In group S, insulin-stimulated glycogen synthase activity increased with time and was significantly higher than in group R. In group S, synthase phosphatase activity increased significantly by 25% at 10 min and then decreased gradually. No significant change in synthase phosphatase was seen in group R and activity was lower than group S at 0 to 20 min. These data suggest that a low basal synthase phosphatase activity and a defect in its response to insulin explain, at least in part, reduced insulin stimulation of skeletal muscle glycogen synthase associated with insulin resistance.     

Role of the ubiquitin-proteasome pathway in the diagnosis of human diseases

The ubiquitin–proteasome pathway constitutes the major system for nuclear and extralysosomal cytosolic protein degradation in eukaryotic cells. A plethora of cell proteins implicated in the maintenance and regulation of essential cellular processes undergoes processing and functional modification by proteolytic degradation via the ubiquitin–proteasome pathway. Deregulations of the pathway have been shown to contribute to the pathogenesis of several human diseases, such as cancer, neurodegenerative, autoimmune, genetic and metabolic disorders, most of them exhibiting abnormal accumulation and altered composition of components of the pathway that is suitable for diagnostic proceedings. While the ubiquitin–proteasome pathway is currently exploited to develop novel therapeutic strategies, it is less regarded as a diagnostic area. Future research should lead to an improved understanding of the pathophysiology of the ubiquitin–proteasome pathway with the aim of allowing the development of subtle diagnostic strategies.     

Skeletal muscle mRNA levels for cathepsin B, but not components of the ubiquitin-proteasome pathway, are increased in patients with lung cancer referred for thoracotomy

Muscle wasting is a common and prominent feature of advanced cancer, including lung cancer. Evidence from animal experiments suggests that accelerated proteolysis via the ubiquitin--proteasome pathway is the primary cause of cancer-related cachexia. However, there are few data on the role of this pathway in determining muscle wasting in human cancer. The present study was designed to measure whether skeletal muscle gene expression of components of the ubiquitin-proteasome pathway and/or the lysosomal proteolytic pathway was increased in patients with early lung cancer. A total of 36 patients with lung cancer referred for curative resection and 10 control subjects had biopsies of latissimus dorsi muscle taken at operation. mRNA levels of four components of the ubiquitin-proteasome pathway, i.e. polyubiquitin, C2 alpha proteasome subunit, 14 kDa ubiquitin-carrier protein and ubiquitin-activating protein, and of two lysosomal proteolytic enzymes, i.e. cathepsin B and cathepsin D, were measured using quantitative Northern blotting. mRNA levels for cathepsin B, but not for components of the ubiquitin--proteasome pathway, were higher in patients with cancer compared with controls (P=0.01). Among lung cancer patients, cathepsin B mRNA levels correlated with fat-free mass index (r = -0.57, P=0.003) and tumour stage (r(s)=0.45, P=0.03), and were higher in smokers (P=0.04). Thus gene expression of the lysosomal protease cathepsin B is increased in the skeletal muscle of patients with early lung cancer, and the strong inverse relationship with fat-free mass suggests that cathepsin B may have a role in inducing muscle wasting in the early stages of lung cancer.     

Decrease in erythrocyte glycophorin sialic acid content is associated with increased erythrocyte aggregation in human diabetes.

1. Sialic acid moieties of erythrocyte membrane glycoproteins are the principal determinants of the negative charge on the cell surface. The resultant electrostatic repulsion between the cells reduces erythrocyte aggregation and hence the low shear rate viscosity and yield stress of blood. 2. Using g.c.-m.s., a decrease in sialic acid content has been observed in the major erythrocyte membrane glycoprotein, glycophorin A, obtained from nine diabetic patients compared with that from seven normal control subjects [median (range): 3.30 (0.01-11.90) versus 18.60 (3.20-32.60) micrograms/100 micrograms of protein, P less than 0.02]. 3. Erythrocyte aggregation, measured by viscometry as the ratio of suspension viscosity to supernatant viscosity (LS/S) in fibrinogen solution, was increased in ten diabetic patients compared with ten normal control subjects (mean +/- SEM, 37.6 +/- 1.3 versus 33.8 +/- 0.6, P less than 0.02). 4. In the patients in whom both viscometry and carbohydrate analysis were performed, the decrease in erythrocyte glycophorin sialylation and the increase in erythrocyte aggregation in fibrinogen solution were related statistically (LS/S correlated negatively with glycophorin sialic acid content, r = 0.73, P less than 0.05). 5. Decreased glycophorin sialylation provides an explanation at the molecular level for increased erythrocyte aggregation and it may be important in the pathogenesis of vascular disease in diabetes.     

First-trimester ultrasound diagnosis of skeletal dysplasia associated with increased nuchal translucency thickness.

A series of five cases of skeletal dysplasia is reported in which the diagnosis was reached at the 11-14-week routine ultrasound examination in our referral center. All five cases had increased nuchal translucency thickness (NT) associated with bone abnormalities. We review the current literature on skeletal dysplasia in the first trimester of pregnancy associated with increased NT.     

Activity of some proteolytic enzymes in normal and dystrophic human muscle.

1. The following proteolytic enzymes were measured in muscles of control subjects and patients with muscular dystrophies and related neuromuscular diseases: an elastase-like enzyme, carboxypeptidase A, carboxypeptidase B and pyroglutamyl peptidase. 2. Elastase-like enzyme and carboxypeptidase B did not show significant alterations in various disease conditions that were examined. 3. Carboxypeptidase A was moderately elevated in dystrophic as well as other diseased muscles. 4. Pyroglutamyl peptidase was not markedly altered in any disease condition except that is was slightly lower in dystrophic muscles.     

Insulin-like growth factor I-mediated skeletal muscle hypertrophy is characterized by increased mTOR-p70S6K signaling without increased Akt phosphorylation.

BACKGROUND: Insulin-like growth factor I (IGF-I) is an anabolic hormone that is known to induce skeletal muscle hypertrophy. However, the signaling pathways mediating IGF-I's hypertrophic effect in vivo are unknown. METHOD: The phosphorylation of 46 proteins was investigated by Kinetworks proteomic analysis in the gastrocnemius muscle of transgenic mice overexpressing IGF-I myosin light chain/muscle specific IGF-I (MLC/mlgf-I) and wild-type littermates. RESULTS: In the hypertrophic muscle of MLC/mlgf-I mice, we observed increased phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1; 53% increase), the mammalian target of rapamycin (mTOR; 112% increase), and p70 S6 kinase (p70S6K) (254% increase) but no significant change in Akt phosphorylation (4% decrease). Furthermore, we found reduced phosphorylation of MAP kinase kinase 1 and 2 (MEK1/2) (60% decrease) and of mitogen-activated protein kinase kinases 3 and 6 (MKK3/6) (50% decrease) in muscle from transgenic mice, suggesting that the hypertrophic and mitogenic effects of IGF-I are mediated via distinct signaling pathways in skeletal muscle and that inhibition of the mitogen-activated protein (MAP) kinase pathway may be required for the IGF-I-induced hypertrophic effect. Single-fiber analysis revealed a trend toward a higher percentage of the fast twitch fibers (IIb and IIx) in the transgenic mice. CONCLUSION: Persistent overexpression of IGF-I in mice skeletal muscle results in hypertrophy, which is likely mediated via the mTOR/p70S6K pathway, potentially via an Akt-independent signaling pathway.     

Sepsis stimulates nonlysosomal, energy-dependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle.

We tested the role of different intracellular proteolytic pathways in sepsis-induced muscle proteolysis. Sepsis was induced in rats by cecal ligation and puncture; controls were sham operated. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Lysosomal proteolysis was assessed by using the lysosomotropic agents NH4Cl, chloroquine, leupeptin, and methylamine. Ca(2+)-dependent proteolysis was determined in the absence or presence of Ca2+ or by blocking the Ca(2+)-dependent proteases calpain I and II. Energy-dependent proteolysis was determined in muscles depleted of ATP by 2-deoxyglucose and 2.4-dinitrophenol. Muscle ubiquitin mRNA and the concentrations of free and conjugated ubiquitin were determined by Northern and Western blots, respectively, to assess the role of the ATP-ubiquitin-dependent proteolytic pathway. Total and myofibrillar protein breakdown was increased during sepsis by 50 and 440%, respectively. Lysosomal and Ca(2+)-dependent proteolysis was similar in control and septic rats. In contrast, energy-dependent total and myofibrillar protein breakdown was increased by 172% and more than fourfold, respectively, in septic muscle. Ubiquitin mRNA was increased severalfold in septic muscle. The results suggest that the increase in muscle proteolysis during sepsis is due to an increase in nonlysosomal energy-dependent protein breakdown, which may involve the ubiquitin system.