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Protein Breakdown (protein + breakdown)
Selected AbstractsForearm and leg amino acid metabolism in the basal state and during combined insulin and amino acid stimulation after a 3-day fastACTA PHYSIOLOGICA, Issue 3 2009J. Gjedsted Abstract Aim:, Fasting is characterized by a progressive loss of protein, but data on protein kinetics are unclear and few have studied the effects of re-feeding. The present study was designed to test the hypothesis that a combined infusion of insulin and amino acids after fasting would induce compensatory increases in protein synthesis and reductions in protein breakdown at the whole body level and in muscle. Methods:, We included 10 healthy male volunteers and studied them twice: (1) in the post-absorptive state and (2) after 72 h of fasting. Amino acid kinetics was measured using labelled phenylalanine and tyrosine, whole body energy expenditure was assessed and urea nitrogen synthesis rates were calculated. Results:, After fasting we observed an increase in arterial blood concentration of branched chain amino acids and a decrease in gluconeogenic amino acids (P < 0.05). Isotopically determined whole body, forearm and leg phenylalanine fluxes were unaltered apart from a 30% decrease in phenylalanine-to-tyrosine conversion (2.0 vs. 1.4 ,mol kg,1 h,1, P < 0.01). During infusion of insulin and amino acids, amino acid concentrations increased. Conclusion:, Our data indicate that after a 72-h fast basal and insulin/amino acid-stimulated regional phenylalanine fluxes in leg and forearm muscle are unaltered. During fasting concentrations of gluconeogenic amino acids decrease and hepatic and/or renal phenylalanine-to-tyrosine conversion decreases. Thus, as opposed to glucose and lipid metabolism, fasting does not induce insulin resistance as regards amino acid metabolism. [source] Molecular mechanisms activating muscle protein degradation in chronic kidney disease and other catabolic conditionsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2005J. Du Abstract Muscle atrophy is a prominent feature of chronic kidney disease (CKD) and is frequent in other catabolic conditions. Results from animal models of these conditions as well as patients indicate that atrophy is mainly owing to accelerated muscle proteolysis in the ubiquitin-proteasome (Ub-P'some) proteolytic system. The Ub-P'some system, however, rapidly degrades actin or myosin but cannot breakdown actomyosin or myofibrils. Consequently, another protease must initially cleave the complex structure of muscle. We identified caspase-3 as an initial and potentially rate-limiting proteolytic step that cleaves actomyosin/myofibrils to produce substrates degraded by the Ub-P'some system. In rodent models of CKD and other catabolic conditions, we find that caspase-3 is activated and cleaves actomyosin to actin, myosin and their fragments. This initial proteolytic step in muscle leaves a characteristic footprint, a 14-kDa actin band, providing a potential diagnostic tool to detect muscle catabolism. We also found that stimulation of caspase-3 activity depends on inhibition of IRS-1-associated phosphatidylinositol 3-kinase (PI3K) activity; inhibiting PI3K in muscle cells also leads to expression of a critical E3-ubiquitin-conjugating enzyme involved in muscle protein breakdown: atrogin-1/MAFbx. Thus, protein breakdown by caspase-3 and the ubiquitin-proteasome system in muscle are stimulated by the same signal: a low PI3K activity. These responses could yield therapeutic strategies to block muscle atrophy. [source] AMP-activated protein kinase enhances the expression of muscle-specific ubiquitin ligases despite its activation of IGF-1/Akt signaling in C2C12 myotubesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Jun F. Tong Abstract Two muscle-specific ubiquitin ligases (UL), muscle atrophy F box (MAFbx) and muscle RING finger 1 (MuRF1), are crucial for myofibrillar protein breakdown. The insulin like growth factor-1 (IGF-1) pathway inhibits muscle UL expression through Akt-mediated inhibition of FoxO transcription factors, while AMP-activated protein kinase (AMPK) promotes UL expression. The underlying cellular mechanism, however, remains obscure. In this study, the effect of AMPK and its interaction with IGF-1 on ubiquitin ligases expression was investigated. C2C12 myotubes were treated with 0, 0.1, 0.3, and 1.0,mM 5-aminoimidazole-4-carboxamide-1-,- D -ribofuranoside (AICAR) in the presence or absence of 50,ng/ml IGF-1. IGF-1 activated Akt, which enhanced phosphorlytion of FoxO3a at Thr 318/321 and reduced the expression of UL. Intriguingly, though activation of AMPK by 0.3 and 1.0,mM AICAR synergized IGF-1-induced Akt activation, the expression of UL was not attenuated, but strengthened by AMPK activation. AICAR treatment decreased FoxO3a phosphorylation at 318/321 in the cytoplasm and induced FoxO3 nuclear relocation. mTOR inhibition increased basal MAFbx expression and reversed the inhibitory effect of IGF-1 on UL expression. In conclusion, our data show that AMPK activation by AICAR stimulates UL expression despite the activation of Akt signaling, which may be due to the possible antagonistic effect of FoxO phosphorylation by AMPK on phosphorylation by Akt. In addition, AMPK inhibition of mTOR may provide an additional explanation for the enhancement of UL expression by AMPK. J. Cell. Biochem. 108: 458,468, 2009. © 2009 Wiley-Liss, Inc. [source] Influence of the Coagulant Level on Early Proteolysis in Ovine Cheese-like Systems Made with Sterilized Milk and Cynara cardunculusJOURNAL OF FOOD SCIENCE, Issue 7 2004S.V. Silva ABSTRACT: The effect of coagulant level on the quality and quantity of protein breakdown during the first 24 h of ripening of cheese-like systems, manufactured with sterilized ovine milk using crude aqueous extracts of Cynara cardunculus as coagulant, was experimentally assessed. Urea-polyacrylamide gel electrophoresis was performed on both water-soluble and water-insoluble cheese extracts to monitor the casein degradation pattern; the ripening extension index and the ripening depth index were thus calculated. Peptides from the water-soluble fraction were isolated by reverse-phase, high-performance liquid chromatography and partially sequenced by Edman degradation. Higher residual coagulant levels in curdled milk led to earlier breakdown of caseins, as expected. The primary cleavage sites were Phe105-Met106 in k-casein, Phe23-Val24 in ,s1 -casein, and Leu127-Thr128, Ser142-Trp143, Leu165-Ser166, and Leu190-Tyr191 in ,-casein. [source] Abnormalities of whole body protein turnover, muscle metabolism and levels of metabolic hormones in patients with chronic heart failureJOURNAL OF INTERNAL MEDICINE, Issue 1 2006H. NŘRRELUND Abstract. Objective., It is well known that chronic heart failure (CHF) is associated with insulin resistance and cachexia, but little is known about the underlying substrate metabolism. The present study was undertaken to identify disturbances of basal glucose, lipid and protein metabolism. Design., We studied eight nondiabetic patients with CHF (ejection fraction 30 ± 4%) and eight healthy controls. Protein metabolism (whole body and regional muscle fluxes) and total glucose turnover were isotopically assayed. Substrate oxidation were obtained by indirect calorimetry. The metabolic response to exercise was studied by bicycle ergometry exercise. Results., Our data confirm that CHF patients have a decreased lean body mass. CHF patients are characterised by (i) decreased glucose oxidation [glucose oxidation (mg kg,1 min,1): 1.25 ± 0.09 (patients) vs. 1.55 ± 0.09 (controls), P < 0.01] and muscle glucose uptake [a , v diffglucose (,mol L,1): ,10 ± 25 (patients) vs. 70 ± 22 (controls), P < 0.01], (ii) elevated levels of free fatty acids (FFA) [FFA (mmol L,1): 0.72 ± 0.05 (patients) vs. 0.48 ± 0.03 (controls), P < 0.01] and 3-hydroxybutyrate and signs of elevated fat oxidation and muscle fat utilization [a , v diffFFA (mmol L,1): 0.12 ± 0.02 (patients) vs. 0.05 ± 0.01 (controls), P < 0.05] and (iii) elevated protein turnover and protein breakdown [phenylalanine flux (,mol kg,1 h,1): 36.4 ± 1.5 (patients) vs. 29.6 ± 1.3 (controls), P < 0.01]. Patients had high circulating levels of noradrenaline, glucagon, and adiponectin, and low levels of ghrelin. We failed to observe any differences in metabolic responses between controls and patients during short-term exercise. Conclusions., In the basal fasting state patients with CHF are characterized by several metabolic abnormalities which may contribute to CHF pathophysiology and may provide a basis for targeted intervention. [source] Gene and protein expression associated with protein synthesis and breakdown in paraplegic skeletal muscleMUSCLE AND NERVE, Issue 4 2008Micah J. Drummond PhD Abstract Spinal cord injury reduces the rate of skeletal muscle protein synthesis and increases protein breakdown, resulting in rapid muscle loss. The purpose of this study was to determine whether long-term paraplegia would eventually result in a downregulation of muscle mRNA and protein expression associated with both protein synthesis and breakdown. After 10 weeks of spinal cord transection, soleus muscle from 12 rats (6 sham-control, 6 paraplegic) was studied for mRNAs and proteins associated with protein synthesis and breakdown using real-time polymerase chain reaction and immunoblotting techniques. Protein kinase B (PKB/Akt), ribosomal S6 kinase 1 (S6K1), and myogenin mRNA were downregulated, whereas muscle ring finger 1 (MuRF1) and phospho-forkhead transcription factor 4 (FoxO4) protein were increased in paraplegic rats. We conclude that gene and protein expression of pathways associated with protein synthesis are reduced, whereas some markers of protein breakdown remain elevated following chronic paraplegia. Clinical interventions designed to increase muscle protein synthesis may be helpful in preventing excessive muscle loss during long-term paraplegia. Muscle Nerve, 2008 [source] Not All Injury-induced Muscle Proteolysis Is Due to Increased Activity of the Ubiquitin/Proteasome System: Evidence for Up-Regulation of Macrophage-associated Lysosomal Proteolysis in a Model of Local TraumaNUTRITION REVIEWS, Issue 1 2003Article first published online: 16 SEP 200 A characteristic response to injury is a dramatic loss of skeletal muscle protein owing to increased muscle protein breakdown. Over the past decade, numerous studies have indicated that up-regulaton of the ubiquitin-proteasome system is a common mechanism underlying such injury-induced muscle proteolysis. However, a recent study using a single-impact trauma to the gastrocnemius muscle found that, although the rate of muscle proteolysis was dramatically increased, the ubiquitin-proteasome system was not involved. Rather, an increase in lysosomal activity, through infiltration of the damaged tissue by mononuclear macrophages, is responsible for the high rates of protein breakdown. [source] Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racingRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 3 2008Tiia Kuuranne Insulin is a peptide hormone consisting of two peptide chains (A- and B-chain) that are cross-linked by two disulfide bonds. To obtain improved pharmacokinetic onset of action profiles of insulin treatment in diabetic patients, recombinant long-, intermediate-, and rapid-acting insulin analogs are produced, in which the C-terminal end of the B-chain plays an especially important role. A review of the veterinary literature reveals the low prevalence of equine type I diabetes mellitus, which indicates that the therapeutic use of insulin in racing horses is unlikely. Although there is no unequivocal evidence of an overall performance-enhancing effect of insulin, in human sports the misuse of insulin preparations is reported among elite athletes. The desired effects of insulin include the increase of muscular glycogen prior to sports event or during the recovery phase, in addition to a chalonic action, which increases the muscle size by inhibiting protein breakdown. In the present study urinary insulin was detected in equine samples and differences between equine insulin, human insulin, as well as rapidly acting recombinant insulin variants were examined. The method was based on sample purification by solid-phase extraction (SPE) and immunoaffinity chromatography (IAC), and subsequent analysis by microbore liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using top-down sequencing for the determination of various insulins. Product ion scan experiments of intact proteins and B-chains enabled the differentiation between endogenously produced equine insulin, its DesB30 metabolite, human insulin and recombinant insulin analogs, and the assay allowed the assignment of individual product ions, especially those originating from modified C-termini of B-chains. Copyright © 2008 John Wiley & Sons, Ltd. [source] Mechanisms of stretch-induced muscle damage in normal and dystrophic muscle: role of ionic changesTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005D. G. Allen Muscle damage, characterized by prolonged weakness and delayed onset of stiffness and soreness, is common following contractions in which the muscles are stretched. Stretch-induced damage of this sort is more pronounced in the muscular dystrophies and the profound muscle damage observed in these conditions may involve similar pathways. It has been known for many years that damaged muscles accumulate calcium and that elevating calcium in normal muscles simulates many aspects of muscle damage. The changes in intracellular calcium, sodium and pH following stretched contractions are reviewed and the various pathways which have been proposed to allow ion entry are discussed. One possibility is that TRPC1 (transient receptor potential, canonical), a protein which seems to form both a stretch-activated channel and a store-operated channel, is the main source of Ca2+ entry. The mechanisms by which the changes in intracellular ions contribute to reduced force production, to increased protein breakdown and to increased membrane permeability are considered. A hypothetical scheme for muscle damage which incorporates these ideas is presented. [source] ,Senescence-associated vacuoles' are involved in the degradation of chloroplast proteins in tobacco leavesTHE PLANT JOURNAL, Issue 2 2008Dana E. Martínez Summary Massive degradation of photosynthetic proteins is the hallmark of leaf senescence; however the mechanism involved in chloroplast protein breakdown is not completely understood. As small ,senescence-associated vacuoles' (SAVs) with intense proteolytic activity accumulate in senescing leaves of soybean and Arabidopsis, the main goal of this work was to determine whether SAVs are involved in the degradation of chloroplastic components. SAVs with protease activity were readily detected through confocal microscopy of naturally senescing leaves of tobacco (Nicotiana tabacum L.). In detached leaves incubated in darkness, acceleration of the chloroplast degradation rate by ethylene treatment correlated with a twofold increase in the number of SAVs per cell, compared to untreated leaves. In a tobacco line expressing GFP targeted to plastids, GFP was re-located to SAVs in senescing leaves. SAVs were isolated by sucrose density gradient centrifugation. Isolated SAVs contained chloroplast-targeted GFP and the chloroplast stromal proteins Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and glutamine synthetase, but lacked the thylakoid proteins D1 and light-harvesting complex II of the photosystem II reaction center and photosystem II antenna, respectively. In SAVs incubated at 30°C, there was a steady decrease in Rubisco levels, which was completely abolished by addition of protease inhibitors. These results indicate that SAVs are involved in degradation of the soluble photosynthetic proteins of the chloroplast stroma during senescence of leaves. [source] | ||||||||||||||||