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1.
Hepatic cirrhosis is associated with negative nitrogen balance and loss of lean body mass. This study aimed to identify the specific proteolytic pathways activated in skeletal muscles of cirrhotic rats. TNF-alpha can stimulate muscle proteolysis; therefore, a potential relationship between TNF-alpha and muscle wasting in liver cirrhosis was also evaluated. Cirrhosis was induced by bile duct ligation (BDL) in male adult Sprague-Dawley rats. mRNA and protein levels of various targets were determined by RT-PCR and Western blotting, respectively. The proteolytic rate was measured ex vivo using isolated muscles. Compared with sham-operated controls, BDL rats had an increased degradation rate of muscle proteins and enhanced gene expression of ubiquitin, 14-kDa ubiquitin carrier protein E2, and the proteasome subunits C2 and C8 (P < 0.01). The muscle protein levels of free ubiquitin and conjugated ubiquitin levels were also elevated (P < 0.01). However, there was no difference between the two groups with regard to cathepsin and calpain mRNA levels. Cirrhotic muscle TNF-alpha levels were increased and correlated positively with free and conjugated ubiquitin (P < 0.01). We conclude that the ubiquitin-proteasome system is involved in muscle wasting of rats with BDL-induced cirrhosis. TNF-alpha might play a role in mediating activation of this proteolytic pathway, probably through a local mechanism.  相似文献   

2.
Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.  相似文献   

3.
The ubiquitin-proteasome system is thought to play a major role in normal muscle protein turnover and to contribute to diabetes-induced protein wasting in skeletal muscle. However, its importance in cardiac muscle is not clear. We measured heart muscle mRNA for ubiquitin and for the C2 and C8 proteasomal subunits, the amount of free ubiquitin and the proteasome chymotrypsin-like proteolytic activity in control and diabetic rats. Results were compared to those in skeletal muscle (rectus). Heart ubiquitin, C2 and C8 subunit mRNA and proteolytic activity were significantly greater than in skeletal muscle (P 相似文献   

4.
The purpose of the present study was to determine whether catabolic stimuli that induce muscle atrophy alter the muscle mRNA abundance of insulin-like growth factor binding protein (IGFBP)-4 and -5, and if so determine the physiological mechanism for such a change. Catabolic insults produced by endotoxin (LPS) and sepsis decreased IGFBP-5 mRNA time- and dose-dependently in gastrocnemius muscle. This reduction did not result from muscle disuse because hindlimb immobilization increased IGFBP-5. Continuous infusion of a nonlethal dose of tumor necrosis factor-alpha (TNF-alpha) decreased IGFBP-5 mRNA 70%, whereas pretreatment of septic rats with a neutralizing TNF binding protein completely prevented the reduction in muscle IGFBP-5. The addition of LPS or TNF-alpha to cultured C(2)C(12) myoblasts also decreased IGFBP-5 expression. Although exogenously administered growth hormone (GH) increased IGFBP-5 mRNA 2-fold in muscle from control rats, muscle from septic animals was GH resistant and no such elevation was detected. In contrast, exogenous administration of IGF-I as part of a binary complex composed of IGF-I/IGFBP-3 produced comparable increases in IGFBP-5 mRNA in both control and septic muscle. Concomitant determinations of IGF-I mRNA content revealed a positive linear relationship between IGF-I and IGFBP-5 mRNA in the same muscle in response to LPS, sepsis, TNF-alpha, and GH treatment. Although dexamethasone decreased muscle IGFBP-5, pretreatment of rats with the glucocorticoid receptor antagonist RU486 did not prevent the sepsis-induced decrease in IGFBP-5 mRNA. In contrast, muscle IGFBP-4 mRNA abundance was not significantly altered by LPS, sepsis, or hindlimb immobilization. In summary, these data demonstrate that various inflammatory insults decrease muscle IGFBP-5 mRNA, without altering IGFBP-4, by a TNF-dependent glucocorticoid-independent mechanism. Finally, IGF-I appears to be a dominant positive regulator of IGFBP-5 gene expression in muscle under both normal and catabolic conditions.  相似文献   

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Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle‐specific ubiquitin ligases atrogin‐1 and MuRF1 but it is not known if atrogin‐1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin–proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 µg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin‐1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain‐, and caspase‐3‐dependent protein breakdown in addition to proteasome‐dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin‐1 and MuRF1 mRNA levels. The same treatment increased proteasome‐, cathepsin L‐, and calpain‐dependent proteolytic rates by approximately 40% but did not influence caspase‐3‐dependent proteolysis. The expression of atrogin‐1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin–proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. J. Cell. Biochem. 108: 963–973, 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

7.
Smoking causes multiple organ dysfunction. The effect of smoking on skeletal muscle protein metabolism is unknown. We hypothesized that the rate of skeletal muscle protein synthesis is depressed in smokers compared with non-smokers. We studied eight smokers (> or =20 cigarettes/day for > or =20 years) and eight non-smokers matched for sex (4 men and 4 women per group), age (65 +/- 3 and 63 +/- 3 yr, respectively; means +/- SEM) and body mass index (25.9 +/- 0.9 and 25.1 +/- 1.2 kg/m(2), respectively). Each subject underwent an intravenous infusion of stable isotope-labeled leucine in conjunction with blood and muscle tissue sampling to measure the mixed muscle protein fractional synthesis rate (FSR) and whole body leucine rate of appearance (Ra) in plasma (an index of whole body proteolysis), the expression of genes involved in the regulation of muscle mass (myostatin, a muscle growth inhibitor, and MAFBx and MuRF-1, which encode E3 ubiquitin ligases in the proteasome proteolytic pathway) and that for the inflammatory cytokine TNF-alpha in muscle, and the concentration of inflammatory markers in plasma (C-reactive protein, TNF-alpha, interleukin-6) which are associated with muscle wasting in other conditions. There were no differences between nonsmokers and smokers in plasma leucine concentration, leucine rate of appearance, and plasma concentrations of inflammatory markers, or TNF-alpha mRNA in muscle, but muscle protein FSR was much less (0.037 +/- 0.005 vs. 0.059 +/- 0.005%/h, respectively, P = 0.004), and myostatin and MAFBx (but not MuRF-1) expression were much greater (by approximately 33 and 45%, respectivley, P < 0.05) in the muscle of smokers than of nonsmokers. We conclude that smoking impairs the muscle protein synthesis process and increases the expression of genes associated with impaired muscle maintenance; smoking therefore likely increases the risk of sarcopenia.  相似文献   

8.
There is little information on the mechanisms responsible for muscle recovery following a catabolic condition. To address this point, we reloaded unweighted animals and investigated protein turnover during recovery from this highly catabolic state and the role of proteolysis in the reorganization of the soleus muscle. During early recovery (18 h of reloading) both muscle protein synthesis and breakdown were elevated (+65%, P<0.001 and +22%, P<0.05, respectively). However, only the activation of non-lysosomal and Ca(2+)-independent proteolysis was responsible for increased protein breakdown. Accordingly, mRNA levels for ubiquitin and 20S proteasome subunits C8 and C9 were markedly elevated (from +89 to +325%, P<0.03) and actively transcribed as shown by the analysis of polyribosomal profiles. In contrast, both cathepsin D and 14-kDa-ubiquitin conjugating enzyme E2 mRNA levels decreased, suggesting that the expression of such genes is an early marker of reversed muscle wasting. Following 7 days of reloading, protein synthesis was still elevated and there was no detectable change in protein breakdown rates. Accordingly, mRNA levels for all the proteolytic components tested were back to control values even though an accumulation of high molecular weight ubiquitin conjugates was still detectable. This suggests that soleus muscle remodeling was still going on. Taken together, our observations suggest that enhanced protein synthesis and breakdown are both necessary to recover from muscle atrophy and result in catch-up growth. The observed non-coordinate regulation of proteolytic systems is presumably required to target specific classes of substrates (atrophy-specific protein isoforms, damaged proteins) for replacement and/or elimination.  相似文献   

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Intra-amniotic (IA) lipopolysaccharide (LPS) induces intrauterine and fetal lung inflammation and increases lung surfactant and compliance in preterm sheep; however, the mechanisms are unknown. Prostaglandins (PGs) are inflammatory mediators, and PGE(2) has established roles in fetal lung surfactant production. The aim of our first study was to determine PGE(2) concentrations in response to IA LPS and pulmonary gene expression for PG synthetic [prostaglandin H synthase-2 (PGHS-2) and PGE synthase (PGES)] and PG-metabolizing [prostaglandin dehydrogenase (PGDH)] enzymes and PGE(2) receptors. Our second study aimed to block LPS-induced increases in PGE(2) with a PGHS-2 inhibitor (nimesulide) and determine lung inflammation and surfactant protein mRNA expression. Pregnant ewes received an IA saline or LPS injection at 118 days of gestation. In study 1, fetal plasma and amniotic fluid were sampled before and at 2, 4, 6, 12, and 24 h after injection and then daily, and fetuses were delivered 2 or 7 days later. Amniotic fluid PGE(2) concentrations increased (P < 0.05) 12 h and 3-6 days after LPS. Fetal lung PGHS-2 mRNA and PGES mRNA increased 2 (P = 0.0084) and 7 (P = 0.014) days after LPS, respectively. In study 2, maternal intravenous nimesulide or vehicle infusion began immediately before LPS or saline injection and continued until delivery 2 days later. Nimesulide inhibited LPS-induced increases in PGE(2) and decreased fetal lung IL-1β and IL-8 mRNA (P ≤ 0.002) without altering lung inflammatory cell infiltration. Nimesulide decreased surfactant protein (SP)-A (P = 0.05), -B (P = 0.05), and -D (P = 0.0015) but increased SP-C mRNA (P = 0.023). Thus PGHS-2 mediates, at least in part, fetal pulmonary responses to inflammation.  相似文献   

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A 48-h starvation period resulted in a great increase in muscle proteolysis-as measured following the release of tyrosine into the medium-in incubated isolated rat extensor digitorum longus (EDL) muscles. We have quantified the contribution of the different proteolytic systems to the increased protein degradation and observed a considerable activation in the ATP-dependent proteolytic (60%) and in the calcium-dependent (125%) systems, while no increases were observed in lysosomal proteolysis. The addition of 10 mM leucine to the incubation medium did not result in any changes in either total proteolytic rate or the activity rates of any of the different systems studied. In addition, the presence of the amino acid did not influence the levels of mRNA for the different genes studied-ubiquitin, C8 proteasome subunit, E2 conjugating enzyme, m-calpain, and cathepsin B. In a similar way, as observed during starvation, tumor growth resulted in increased protein degradation in incubated isolated EDL muscles from animals bearing the Yoshida AH-130 ascites hepatoma. The increased rate of protein degradation affected all the proteolytic systems studied: ATP- and calcium-dependent and lysosomal. Finally, leucine addition (10 mM), although not able to revert the increased proteolytic rate, resulted in a decrease in the gene expression for ubiquitin, C8 proteasome subunit and cathepsin B.  相似文献   

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To clarify the molecular changes of sublesional muscle in the acute phase of spinal cord injury (SCI), a moderately severe injury (40 g cm) was induced in the spinal cord (T10 vertebral level) of adult male Sprague–Dawley rats (injury) and compared with sham (laminectomy only). Rats were sacrificed at 48 h (acute) post injury, and gastrocnemius muscles were excised. Morphological examination revealed no significant changes in the muscle fiber diameter between the sham and injury rats. Western blot analyses performed on the visibly red, central portion of the gastrocnemius muscle showed significantly higher expression of muscle specific E3 ubiquitin ligases (muscle ring finger-1 and muscle atrophy f-box) and significantly lower expression of phosphorylated Akt-1/2/3 in the injury group compared to the sham group. Cyclooxygenase 2, tumor necrosis factor alpha (TNF-α), and caspase-1, also had a significantly higher expression in the injury group; although, the mRNA levels of TNF-α and IL-6 did not show any significant difference between the sham and injury groups. These results suggest activation of protein degradation, deactivation of protein synthesis, and development of inflammatory reaction occurring in the sublesional muscles in the acute phase of SCI before overt muscle atrophy is seen.  相似文献   

16.
The efficacy of structured lipid, a triacylglycerol of medium and long chain fatty acids, as an element of nutritional support therapies in cancer cachexia was investigated. Using the Yoshida sarcoma to induce cachexia, male Sprague Dawley rats (90 g) were injected subcutaneously with tumor cells (n = 17) or sterile saline (n = 16). Seven days later, rats were randomized to two intravenous diets for 3 days at 220 kcal/kg body weight/d, including 2 g nitrogen/kg body weight/d and 39% of total calories as either structured lipid or long chain triglyceride. Nitrogen balance, tumor growth rate, energy metabolism, and plasma albumin and free fatty acid levels were measured, and whole-body protein kinetics and liver, muscle, and tumor fractional protein synthetic rates were evaluated by adding (14)C-leucine to the diet during the last 4 hours of feeding. Nitrogen balance improved (P < .05) in both tumor and control rats receiving structured lipid-enriched total parenteral nutrition, and was also greater in tumor rats compared with controls. There were no differences in tumor growth or protein kinetics between diet groups. Albumin was lower (P < .05) in tumor rats, but significantly higher in both tumor and control rats given structured lipid-enriched total parenteral nutrition. Free fatty acid was significantly higher in tumor rats versus controls. Whole-body protein kinetics were similar among the four groups. Liver weight, liver weight to body weight ratio, and liver protein synthetic rate were higher in tumor rats. Also, liver weight to body weight ratio was lower in tumor and control animals given structured lipid-enriched total parenteral nutrition. Muscle protein synthetic rate was significantly lower in tumor rats, but higher in tumor and control rats given long chain triglyceride-enriched total parenteral nutrition. The nutritional benefits of structured lipid-enriched total parenteral nutrition favor support of host tissue without promoting tumor growth.  相似文献   

17.
Muscle atrophy is a prominent feature of catabolic conditions and in animal models of these conditions there is accelerated muscle proteolysis that is dependent on the ubiquitin-proteasome system. However, ubiquitin system cannot degrade actomyosin or myofibrils even though it rapidly degrades actin or myosin. We identified caspase-3 as the initial and potentially rate-limiting proteolytic step that cleaves actomyosin/myofibrils. In rodent models of catabolic conditions, we find that caspase-3 is activated to cleave muscle proteins and actomyosin to fragments that are rapidly degraded by the ubiquitin system. This initial proteolytic step in muscle can be recognized because it leaves a footprint of a characteristic 14-kDa actin band. Stimulation of caspase-3 activity depends on activation of phosphatidylinositol 3-kinase. When we suppressed this enzyme in muscle cells, protein breakdown increased as did the expression of caspase-3. In addition, there was increased expression of E3-ubiquitin-conjugating enzymes that are involved in muscle proteolysis, atrogin-1/MAFbx and MuRF1. Thus, when phosphatidylinositol 3-kinase activity is low in muscle cells or rat muscle, both caspase-3 and the ubiquitin-proteasome system are stimulated to degrade protein. Additional investigations will be needed to define the cell signaling processes that activate muscle proteolysis in uremia and catabolic conditions.  相似文献   

18.
Previous studies suggest that elevated temperature stimulates protein degradation in skeletal muscle, but the intracellular mechanisms are not fully understood. We tested the role of different proteolytic pathways in temperature-dependent degradation of long- and short-lived proteins in cultured L6 myotubes. When cells were cultured at different temperatures from 37 to 43 degrees C, the degradation of both classes of proteins increased, with a maximal effect noted at 41 degrees C. The effect of high temperature was more pronounced on long-lived than on short-lived protein degradation. By using blockers of individual proteolytic pathways, we found evidence that the increased degradation of both long-lived and short-lived proteins at high temperature was independent of lysosomal and calcium-mediated mechanisms but reflected energy-proteasome-dependent degradation. mRNA levels for enzymes and other components of different proteolytic pathways were not influenced by high temperature. The results suggest that hyperthermia stimulates the degradation of muscle proteins and that this effect of temperature is regulated by similar mechanisms for short- and long-lived proteins. Elevated temperature may contribute to the catabolic response in skeletal muscle typically seen in sepsis and severe infection.  相似文献   

19.
目的:探讨L-精氨酸(L-Arg)对脂多糖(LPS)诱导的急性肺损伤大鼠肺表面活性物质和肺泡巨噬细胞功能的影响。方法:舌下静脉注射脂多糖(LPS)复制肺损伤模型。健康雄性SD大鼠48只,随机分为对照组、模型组(LPS组)和L-Arg治疗组(L-Arg组)(n=16)。分别于给予LPS 3 h或6 h后给予生理盐水(对照组及LPS组,ip)和L-Arg(500 mg/kg ip)(L-Arg治疗组),治疗3 h。原位杂交法(ISH)检测肺组织中肺表面活性蛋白A(SP-A)mRNA的表达;测定肺泡灌洗液(BALF)中的总蛋白(TP)。体外分离培养大鼠肺泡巨噬细胞,以LPS(终浓度10 mg/L)处理巨噬细胞,观察L-Arg对肺泡巨噬细胞的影响。结果:与对照组比较,大鼠肺损伤后SP-A mRNA表达减弱,BALF中TP增多(P<0.01)。肺损伤3 h用L-Arg治疗3 h后,SP-A mRNA阳性细胞表达明显增强,BALF中TP较LPS组相同时间点明显降低(P<0.05,P<0.01),肺损伤减轻。体外实验中,与正常对照组相比,LPS组细胞培养上清中乳酸脱氢酶(LDH)、一氧化氮(NO)、肿瘤坏死因子-α(TNFα-)和白细胞介素-6(IL-6)浓度明显增高(P<0.01);L-Arg明显减少LPS所致的LDH的释放,降低TNFα-和IL-6浓度。结论:L-Arg可减轻内毒素性肺损伤,此机制可能与增强SP-AmRNA表达有关;LPS可刺激巨噬细胞分泌促炎因子和NO,L-Arg可抑制LPS对巨噬细胞的作用。  相似文献   

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