Muscle protein breakdown during endotoxemia in rats and after treatment with interleukin-1 receptor antagonist (IL-1ra).

The purpose of this study was to examine the effect of endotoxemia on muscle protein degradation and to test the hypothesis that muscle proteolysis during endotoxemia is regulated by interleukin-1 (IL-1). Both total and myofibrillar protein breakdown rates in incubated extensor digitorum longus muscles were increased after the subcutaneous injection of 0.1 or 1.0 mg/kg endotoxin in rats. The endotoxin-induced increase in muscle protein breakdown was blunted by IL-1 receptor antagonist, administered intraperitoneally at a total dose of 45 or 105 mg/kg. Results suggest that endotoxemia in rats gives rise to sepsislike changes in muscle protein breakdown. Increased muscle protein breakdown during endotoxemia may be regulated, at least in part, by IL-1.     

Muscle protein metabolism during hemodialysis.

Despite improvement in many aspects of the care of maintenance hemodialysis (HD) patients, protein-calorie malnutrition, which is characterized by an insidious loss of somatic protein, is common and is a major risk factor for increased morbidity and mortality. We present here an overview of the current knowledge on protein metabolism in uremic patients with the expectation of providing insights into the mechanisms involved in HD-associated catabolism and outlining the rationale underlying intradialytic nutrition. We concentrate on the discussion of muscle protein metabolism because muscle is the predominant site of protein storage, and its integrity is mandatory for the maintenance of a good quality of life.     

Decreased myofibrillar protein breakdown following treatment with clenbuterol.

Daily treatment of Fischer-344 rats for 14 days with the beta 2-adrenergic agonist, clenbuterol, increased gastrocnemius muscle mass and protein content. Coadministration with the beta-adrenergic antagonist, nadolol, significantly reduced these anabolic effects of clenbuterol. Although clenbuterol treatment reduced food intake during the first 4 days, clenbuterol-treated rats were hyperphagic during the second week of drug administration. Nadolol treatment also blocked these effects of clenbuterol on feeding. In a second experiment, in vitro incubation of extensor digitorum longus muscles taken from post weaning food-deprived rats demonstrated decreased release of 3-methylhistidine by clenbuterol-treated rats, suggesting decreased breakdown of myofibrillar protein. Protein synthesis was not increased in vitro in the soleus muscles taken from these rats. These experiments demonstrate that the anabolic effect of clenbuterol is due in part to beta-adrenergic activity and may involve reduced myofibrillar protein degradation. These results appear to have direct application to nutrition and protein repletion in various catabolic diseases.     

Intravenous insulin decreases protein breakdown in infants on extracorporeal membrane oxygenation

Background/Purpose

Infants requiring extracorporeal membrane oxygenation (ECMO) have the highest rates of protein catabolism ever reported. Recent investigations have found that such extreme protein breakdown is refractory to conventional nutritional management. In this pilot study, the authors sought to use the anabolic hormone insulin to reduce the profound protein degradation in this cohort.

Methods

Four parenterally fed infants on ECMO were enrolled in a prospective, randomized, crossover trial. Subjects were administered an insulin infusion using a 4-hour hyperinsulinemic euglycemic clamp followed by a control saline infusion on consecutive days in random order. Whole-body protein flux and breakdown were quantified using a primed continuous infusion of the stable isotope l-[1-¹³C]leucine. Statistical analyses were performed using paired t tests.

Results

Serum insulin levels were increased 15-fold during the insulin clamp compared with the saline control (407 ± 103 v 26 ± 12 μU/mL; P < .05). During the insulin infusion, infants had decreased rates of total leucine flux (214 ± 25 v 298 ± 38 μmol/kg/h; P < .05) and leucine flux derived from protein breakdown (156 ± 40 v 227 ± 54 μmol/kg/h; P < .05) when compared with saline control. Overall, insulin administration produced a 32% reduction in protein breakdown (P < .05).

Conclusions

In this pilot study, the anabolic hormone insulin markedly reduced protein breakdown in critically ill infants on ECMO. Because elevated protein breakdown correlates with mortality and morbidity, the administration of intravenous insulin may ultimately have broad applicability to the metabolic management of critically ill infants.     

Evidence that cathepsin B contributes to skeletal muscle protein breakdown during sepsis

The mechanisms of accelerated skeletal muscle protein degradation during sepsis have not been fully elucidated. Activity of the lysosomal protease cathepsin B is increased in skeletal muscle during various catabolic states other than sepsis. In the present study the protein degradation rate and cathepsin B activity were determined in extensor digitorum longus and soleus muscles from nonseptic and septic rats. The protein degradation rate during incubation in vitro with or without the cathepsin B inhibitor leupeptin was also determined. Both protein degradation and cathepsin B activity were increased in muscles from septic rats. Incubation with leupeptin reduced, but did not normalize, the protein degradation rate in both extensor digitorum longus and soleus muscles from septic animals. These studies suggest that increased cathepsin B activity contributes to the accelerated muscle proteolysis seen during sepsis and that proteases other than cathepsin B are also involved.     

Intraoperative epidural blockade prevents the increase in protein breakdown after abdominal surgery

BACKGROUND: The aim of this study was to investigate the effect of epidural blockade with bupivacaine, restricted to the intraoperative period, on protein catabolism after major abdominal surgery. METHODS: Fourteen patients undergoing cystoprostatectomy were randomly assigned to receive either general anaesthesia with isoflurane (control group, n=7) or a combination of general anaesthesia and epidural blockade with bupivacaine from segment T4 to S5 (epidural group, n=7). Rates of urea (Ra urea) and glucose production (Ra glucose) were measured three days before and three days after the operation using stable isotope tracers ([15N2]urea, [6,6-2H2]glucose). Protein breakdown was calculated from the urea production rate. Plasma concentrations of metabolic substrates (urea, glucose, lactate, glycerol, amino acids) and hormones (insulin, glucagon, cortisol, adrenaline, noradrenaline) were also determined. RESULTS: Protein breakdown significantly increased after surgery in the control group (P<0.05), while it remained unaltered in the epidural group (control; 66 (54-76), epidural; 43 (29-58) mg x kg(-1) x h(-1), P<0.05, median (range)). Glucose plasma concentration and Ra glucose increased in both groups to a similar extent (P<0.05). Plasma concentration of branched chain amino acids decreased after epidural analgesia to a value significantly lower than in the control group (P<0.05). Glutamine plasma concentration decreased in the control group (P<0.05), but did not change in the epidural group. There were no differences in plasma concentrations of insulin, cortisol and catecholamines between the two groups. Glucagon plasma concentration in the epidural group was significantly lower than in the control group (P<0.05). CONCLUSION: Intraoperative epidural blockade inhibits the increase in protein breakdown after abdominal surgery.     

Cellular protein breakdown and systemic inflammation are unaffected by pulmonary rehabilitation in COPD

BACKGROUND: Pulmonary rehabilitation can improve the functional capacity, but has a variable effect on the low fat-free mass (FFM) in patients with chronic obstructive pulmonary disease. HYPOTHESIS: Pulmonary rehabilitation would not affect catabolic drives such as systemic inflammation and also protein breakdown. METHODS: Patients (n = 40) were studied at the start of an 8-week in-patient pulmonary rehabilitation programme, at the end of the programme and 4 weeks later. FFM and functional capacity (quadriceps strength, handgrip strength and peak workload) were assessed. Pseudouridine (PSU) urinary excretion (cellular protein breakdown) and inflammatory status were determined. Healthy participants had a single baseline assessment (n = 18). RESULTS: PSU, (IL)-6 and soluble tumour necrosis factor (sTNF)alpha R75 were increased in patients compared with healthy participants, whereas FFM and functional capacity were reduced (all p < 0.01). PSU was inversely related to both FFM and skeletal muscle function. FFM and functional parameters increased with rehabilitation, but PSU and inflammatory status were unaffected. The gain in FFM was lost 4 weeks after the completion of rehabilitation (p < 0.01). CONCLUSION: The anabolic effect of pulmonary rehabilitation improved FFM, but it did not reverse the increased protein breakdown or systemic inflammation. Thus, on cessation of pulmonary rehabilitation the FFM gains were lost owing to a loss of anabolic drive.     

Increased muscle protein breakdown in chronic hemodialysis patients with type 2 diabetes mellitus

BACKGROUND: The presence of diabetes mellitus (DM) in chronic hemodialysis (CHD) patients has potential to increase body protein losses and muscle wasting. METHODS: In this study, we examined whole-body and skeletal muscle protein metabolism in 6 CHD patients with type 2 (T2) DM (2 male, 44.4 +/- 6.1 years old, 2 white/4 African American HbA(1)C = 9.5 +/- 1.1%), and 6 non-DM CHD patients (2 male, 43.3 +/- 6.7 years old, 2 white/4 African American) in a fasting state, using a primed-constant infusion of L-(1-(13)C) leucine and L-(ring-(2)H(5)) phenylalanine. RESULTS: CHD patients with T2DM had significantly increased (83%) skeletal muscle protein breakdown (137 +/- 27 vs. 75 +/- 25 microg/100 mL/min). There was no significant difference in muscle protein synthesis between groups (78 +/- 27 vs. 66 +/- 21 microg/100 mL/min, for DM and non-DM respectively), resulting in significantly more negative net protein balance in the muscle compartment in the DM group (-59 +/- 4 vs. -9 +/- 6 microg/100 mL/min, P < 0.05). A similar trend was observed in whole-body protein synthesis and breakdown. Plasma glucose levels were 113 +/- 16 and 71 +/- 2 mg/dL, P < 0.05, and insulin levels were 25.3 +/- 9.6 and 7.3 +/- 1.0 uU/mL, for DM versus non-DM, respectively, P < 0.05. No significant differences between DM and non-DM were found in other metabolic hormones. CONCLUSION: The results of this study demonstrate that CHD patients with T2DM under a suboptimal metabolic control display accelerated muscle protein loss compared with a matched group of non-DM CHD patients.     

Myofibrillar protein breakdown in skeletal muscle is diminished in rats with chronic streptozocin-induced diabetes

Previous reports have suggested that insulin may not regulate the breakdown of myofibrillar proteins in skeletal muscle. To further test the role of insulin, insulinopenia was produced by treating rats with streptozocin. After treatment, protein breakdown in skeletal muscle was evaluated with the isolated perfused rat hindquarter preparation. After the inhibition of protein synthesis with cycloheximide, total and myofibrillar protein breakdown were assessed by measuring the release of tyrosine and 3-methylhistidine, respectively, in the perfused hindquarters of diabetic and age-matched control rats. Streptozocin-induced (65 mg/kg) diabetes (3- to 28-day duration) resulted in hyperglycemia, hypoinsulinemia, hyperphagia, increased plasma lipid levels, arrested body and muscle growth, and increased urea and 3-methylhistidine excretion. Despite this, protein breakdown in skeletal muscle diminished. The release of 3-methylhistidine by the perfused hindquarters of diabetic rats decreased, whereas the release of tyrosine remained unchanged, suggesting that the breakdown of myofibrillar proteins was affected specifically. 3-Methylhistidine (unbound) levels in skeletal muscle of unperfused diabetic rats as well as in skin decreased, whereas they increased twofold in the gastrointestinal tract. More severe diabetes (125 mg/kg streptozocin), which resulted in ketoacidosis, augmented protein breakdown in muscle; however, this response was due to a marked fall in food consumption (it was also evident when control rats were pair fed). These data reinforce previous conclusions that insulin does not play a major role in the regulation of myofibrillar protein breakdown in skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin resistance is associated with skeletal muscle protein breakdown in non-diabetic chronic hemodialysis patients

Deranged protein metabolism is known to complicate uremia. Insulin resistance is evident in chronic hemodialysis (CHD) patients. We hypothesized that the degree of insulin resistance would predict protein catabolism in non-diabetic CHD patients. We examined the relationship between Homeostasis Model Assessment (HOMA) and fasting whole-body and skeletal muscle protein turnover in 18 non-diabetic CHD patients using primed-constant infusions of L-(1-(13)C) leucine and L-(ring-(2)H(5)) phenylalanine. Mean+/-s.d. fasting glucose and body mass index were 80.6+/-9.8 mg/dl and 25.4+/-4.4 kg/m(2), respectively. Median (interquartile range) HOMA was 1.6 (1.4, 3.9). Mean+/-s.e.m. skeletal muscle protein synthesis, breakdown, and net balance were 89.57+/-11.67, 97.02+/-13.3, and -7.44+/-7.14 microg/100 ml/min, respectively. Using linear regression, a positive correlation was observed between HOMA and skeletal muscle protein synthesis (R(2)=0.28; P=0.024), and breakdown (R(2)=0.49; P=0.001). An inverse association between net skeletal muscle protein balance and HOMA was also noted (R(2)=0.20; P=0.066). After adjustment for C-reactive protein, only the relationship between HOMA and skeletal muscle protein breakdown persisted (R(2)=0.49; P=0.006). There were no significant associations between components of whole-body protein turnover and HOMA. This study demonstrates that insulin resistance is evident in non-diabetic dialysis patients, is associated with skeletal muscle protein breakdown, and represents a novel target for intervention in uremic wasting.     

Effect of the glucocorticoid receptor antagonist RU 38486 on muscle protein breakdown in sepsis

The role of glucocorticoids in muscle catabolism during sepsis was tested with the glucocorticoid receptor antagonist RU 38486. Sepsis was induced in male Sprague-Dawley rats (40 to 60 gm) by cecal ligation and puncture (CLP). Other animals underwent sham operation. Two hours before CLP or sham operation, rats received RU 38486 (5 mg/kg) or a corresponding volume of vehicle by gavage. Sixteen hours after CLP or sham operation, protein synthesis rate was determined by measuring incorporation of 14C-phenylalanine into protein in incubated extensor digitorum longus muscles. Total and myofibrillar protein breakdown rates were determined by measuring net release of tyrosine and 3-methylhistidine, respectively. The protein synthesis rate was approximately 30% lower in rats with sepsis than in sham operated rats and was not affected by treatment with RU 38486. The total protein breakdown rate was increased by approximately 70% and myofibrillar protein degradation was increased more than fivefold in muscle from rats with sepsis. Treatment with RU 38486 resulted in a 28% reduction of total and a 44% reduction of myofibrillar protein breakdown in rats with sepsis but did not affect proteolysis in muscle from sham-operated animals. The results support a role of glucocorticoids in accelerated muscle proteolysis during sepsis. It is not clear whether glucocorticoids are the only required mediator or they interact with other substances to induce muscle protein breakdown during sepsis.     

Calpain activation and cytoskeletal protein breakdown in the corpus callosum of head-injured patients.

Calpain-mediated breakdown of the cytoskeleton has been proposed to contribute to brain damage resulting from head injury. We examined the corpus callosum from patients who died after a blunt head injury in order to determine if there was evidence of these pathophysiological events in a midline myelinated commissure that is susceptible to damage after human head injury. Western blotting revealed marked reductions in the levels of neurofilament triplet proteins 200 and 68kDa in the corpus callosum of head-injured patients compared with control subjects. Neurofilament 200kDa levels were significantly reduced as detected by either phosphorylation-dependent or -independent antibodies. In contrast, there were minimal changes in the levels of beta-tubulin or the microtubule-associated protein, tau, in the head-injured patients, although amyloid precursor protein immunostaining demonstrated axonal damage in 9 of the 10 patients. The inactive 800kDa and active 76kDa subunits of mu-calpain were present in control subjects and head-injured patients. However, there was a significant increase in the levels of calpain-mediated spectrin breakdown products in head-injured patients compared with the control subjects. The results demonstrate that following human blunt head injury, there is a significant degradation of neurofilament proteins and increased levels of calpain-mediated spectrin breakdown products within the corpus callosum. Therefore, our data support the hypothesis that calpain-mediated breakdown of the cytoskeleton may contribute to axonal damage after head injury.     

Increased rates of whole body protein synthesis and breakdown in children recovering from burns.

The rates of whole body protein synthesis and breakdown were determined, with the aid of a constant administration of [15N]glycine, during recovery in 11 acutely burned children, involving a total of 24 studies. Eleven studies were also conducted in seven healthy children before and after reconstructive surgery. Rates of whole body protein synthesis and breakdown, expressed as g protein/kg body weight/day, were significantly (p less than 0.05) and positiviely correlated with per cent body surface area total burn, per cent third-degree burn, and per cent open wound. These rates (synthesis, 7.1 +/- 2.1 g protein/kg/day; breakdown, 6.3 +/- 1.8 g protein/kg/day) were 80 to 100% greater (p less than 0.05) in patients with total burns greater than or equal to 60%, as compared to patients with less than 25% total burns or to the surgical patients. Because of the high energy cost of protein synthesis, it is proposed that an increased whole body protein turnover is partly responsible for the reported elevations in rates of heat production occurring in patients recovering from thermal injury.     

Muscle

Two important types of muscle are skeletal and smooth muscle. Their similarities and differences are described with regard to general morphology and ultrastructure (arrangement of filaments and T tubules), linking structure to physiological functions. The sliding-filament theory of muscle contraction is explained. Finally, the processes by which nerves excite muscle contraction (excitation–contraction coupling) are outlined, again relating variations in these processes in the two types of muscle to differences between them in their role in the body.     

Fibronectin mRNA and protein accumulation, distribution, and breakdown in rabbit anti-glomerular basement membrane disease

Fibronectin is a multifunctional matrix protein which by immunofluorescence appears to be present in increased amounts during glomerular injury. To examine fibronectin metabolism in glomerular injury, an anti-glomerular basement membrane model that progresses to severe glomerular crescent formation, glomerulosclerosis, and interstitial fibrosis was used. Fibronectin was purified from rabbit plasma, and a monoclonal antibody raised against rabbit fibronectin was used for immunolocalization and quantitation of fibronectin protein. RNA and protein were extracted from isolated glomeruli and whole renal cortex at various times during progression of disease. At day 4, there was a 2.5-fold increase in fibronectin protein which by immunofluorescence appeared to be in the glomerular mesangial area. There was no increase in glomerular fibronectin mRNA at this time. This discrepancy is consistent with the conclusion that, at this early time point, the increased glomerular fibronectin comes predominantly from plasma. By day 7, glomerular fibronectin mRNA and extractable fibronectin protein were increased in association with bright immunofluorescence along the inner aspect of Bowman's capsule where early crescents were forming. Similarly, at day 14, crescents stained very brightly for fibronectin. These results are consistent with the conclusion that, at later time points, fibronectin is synthesized in glomeruli in association with cell division and crescent formation. Degradation of fibronectin in glomerular and cortical extracts was demonstrated under normal and nephritic conditions by finding fibronectin proteolytic fragmentation by Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further evidence that accelerated muscle protein breakdown during sepsis is not mediated by prostaglandin E2.

Prostaglandin E2 (PGE2) reportedly increases protein break-down in skeletal muscle. The role of PGE2 for accelerated muscle proteolysis during sepsis, however, is controversial. In this study, the effect of the prostaglandin synthesis inhibitor indomethacin on PGE2 release and protein breakdown in skeletal muscle from nonseptic and septic rats was evaluated. Sepsis was induced in male Sprague-Dawley rats (40-60 g) by cecal ligation and puncture (CLP). After 16 hours the extensor digitorum longus (EDL) and soleus (SOL) muscles were dissected with intact tendons and incubated in an oxygenated medium, and the release of tyrosine (protein breakdown) and PGE2 into the incubation medium was determined. Paired muscles were incubated in the absence or presence of indomethacin (3 mumol/L or 6 mumol/L). In some experiments the effect of indomethacin was investigated in the presence of different concentrations of insulin (1, 10, or 100 mU/mL) since previous reports suggested an interaction between insulin and prostaglandins on protein turnover in skeletal muscle. In other experiments muscles were incubated in a flaccid or stretched state, which is known to influence the metabolic response to different substances. Protein breakdown rate was 0.210 +/- 0.013 and 0.492 +/- 0.025 mumol Tyr/g X 2 hours in EDL from nonseptic and septic rats, respectively (p less than 0.01). The corresponding values for SOL were 0.480 +/- 0.037 and 0.712 +/- 0.039 mumol Tyr/g X 2 hours (p less than 0.01). Addition of indomethacin to the incubation medium reduced PGE2 release from 29.1 +/- 3.1 to 6.8 +/- 0.7 ng/g X 2 hours in nonseptic SOL and from 50.6 +/- 10.4 to 5.6 +/- 0.7 ng/g X 2 hours in septic SOL. Protein breakdown rate in SOL and EDL from sham-operated or septic rats was unaffected by indomethacin, both when muscles were incubated in a flaccid or stretched state, and when they were incubated in the presence or absence of insulin. The present results do not suggest a role of PGE2 for accelerated muscle proteolysis in the present experimental septic model.