Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Effect of plasma insulin and branched-chain amino acids on skeletal muscle protein synthesis in fasted lambs

The increase in fractional rate of protein synthesis (K-s) in the skeletal muscle of growing rats during the transition from fasted to fed state has been explained by the synergistic action of a rise in plasma insulin and branched-chain amino acids (BCAA). Since growing lambs Also exhibit an increase in K-s with level of feed intake, the objective of the present study was to determine if this synergistic relationship between insulin and BCAA also occurs in ruminant animals. Six 30 kg fasted (72 h) lambs (8 months of age) received each of four treatments, which were based on continuous infusion into the jugular vein for 6 h of: (1) saline (155 mmol NaCl/l); (2) a mixture of BCAA (0.778 mumol leucine, 0.640 mumol isoleucine and 0.693 mumol valine/min.kg); (3) 18.7 mumol glucose/min.kg (to induce endogenous insulin secretion): (4) co-infusion of BCAA and glucose. Within each period all animals received the same isotope of phenylalanine, (Phe) as follows: (1) L-[1-C-13]Phe; (2) L-phenyl-[ring H-2(5)]-alanine; (3) L-[N-15]Phe; (4) L-[ring 2,6-H-3]Phe. Blood was sampled serially during infusions to measure plasma concentrations of insulin, glucose and amino acids, and plasma free Phe isotopic activity; biopsies were taken 6 h after the beginning of infusions to determine K-s in in. longissimus dorsi and vastus muscle. Compared with control (saline-infused) lambs, K-s was increased by an average of 40% at the end of glucose infusion, but this effect was not statistically significant in either of the muscles sampled. BCAA infusion, alone or in combination with glucose, also had no significant effect on K-s compared with control sheep. K-s was approximately 60% greater for vastus muscle than for m. longissimus dorsi (P<0.01), regardless of treatment. It is concluded that there are signals other than insulin and BCAA that are responsible for the feed-induced increase in K-s in muscle of growing ruminant animals.

Effect of heat treatment on changes in texture, structure and properties of Thai indigenous chicken muscle

Changes in texture, microstructure, colour and protein solubility of Thai indigenous and broiler chicken Pectoralis muscle stripes cooked at different temperatures were evaluated. The change in shear value of both chicken muscles was a significant increase from 50 to 80 degrees C but no change from 80 to 100 degrees C. A significant decrease in fibre diameter was obtained in samples heated to an internal temperature of 60 degrees C and the greatest shrinkage of sarcomeres was observed with internal temperatures of 70-100 and 80-100 C for broiler and indigenous chicken muscles, respectively (P < 0.05). Cooking losses of indigenous chicken muscles increased markedly in the temperature range 80-100 C and were significantly higher than those of the broiler (P < 0.001). With increasing temperature, from 50 to 70 degrees C, cooked chicken muscle became lighter and yellower. Relationships between changes in sarcomere length, fibre diameter, shear value, cooking loss and solubility of muscle proteins were evaluated. It was found that the solubility of muscle protein was very highly correlated with the texture of cooked broiler muscle while sarcomere length changes and collagen solubility were important factors influencing the cooking loss and texture of cooked indigenous chicken muscle. (c) 2004 Elsevier Ltd. All rights reserved.

High temperature reduction of metmyoglobin in aqueous muscle extracts

Oxymyoglobin in aqueous extracts of fresh beef longissimus dorsi muscles was initially oxidised to metmyoglobin during heat treatments at temperatures in the range 50-70 degreesC. The metmyoglobin then underwent reduction to a red pigment that was shown spectrally to be identical to oxymyoglobin. The formation of oxymyoglobin involved a heat induced precipitate that when removed from the solution, allowed oxidation to metmyoglobin to occur. However, on re-addition of the precipitate further reduction to oxymyoglobin took place. Dialysis of the muscle extract prior to heating markedly inhibited the reduction but addition of NADH to the dialysate permitted further reduction. The precipitate plus NADH caused oxymyoglobin formation in the presence of metmyoglobin but neither the precipitate nor NADH alone induced this formation. It is concluded that the initial conversion of oxymyoglobin to metmyoglobin on heating fresh beef muscle extracts was reversible and that the reverse reaction depended on the presence of both NADH and a muscle protein.

Food restriction reverses the hyper-muscular phenotype and force generation capacity deficit of the myostatin null mouse

Food restriction has a great impact on skeletal muscle mass by inducing muscle protein breakdown to provide substrates for energy production through gluconeogenesis. Genetic models of hyper-muscularity interfere with the normal balance between protein synthesis and breakdown which eventually results in extreme muscle growth. Mutations or deletions in the myostatin gene result in extreme muscle mass. Here we evaluated the impact of food restriction for a period of 5 weeks on skeletal muscle size (i.e., fibre cross-sectional area), fibre type composition and contractile properties (i.e., tetanic and specific force) in myostatin null mice. We found that this hyper-muscular model was more susceptible to catabolic processes than wild type mice. The mechanism of skeletal muscle mass loss was examined and our data shows that the myostatin null mice placed on a low calorie diet maintained the activity of molecules involved in protein synthesis and did not up-regulate the expression of genes pivotal in ubiquitin-mediated protein degradation. However, we did find an increase in the expression of genes associated with autophagy. Surprisingly, the reduction on muscle size was followed by improved tetanic and specific force in the null mice compared to wild type mice. These data provide evidence that food restriction may revert the hyper-muscular phenotype of the myostatin null mouse restoring muscle function.

Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein

CSRP3 or muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with alpha-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerase I and II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative nuclear localization signal (NLS) of MLP, nuclear import of the protein in cultured rat neonatal myocytes was inhibited. The NLS of MLP also localizes to the nucleolus. Inhibition of nuclear translocation prevented the increased protein accumulation in response to phenylephrine. Furthermore, cyclic strain of myocytes after prior NLS treatment to remove nuclear MLP resulted in disarrayed sarcomeres. Increased protein synthesis and brain natriuretic peptide expression were also prevented suggesting that MLP is required for remodeling of the myo filaments and gene expression. These findings suggest that nucleocytoplasmic shuttling MLP plays an important role in the regulation of the myocyte remodeling and hypertrophy and is required for adaptation to hypertrophic stimuli. (C) 2009 Elsevier Inc. All rights reserved.

Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2

Oxidized low-density lipoproteins (LDL) play a central role in atherogenesis and induce expression of the antioxidant stress protein heme oxygenase 1 (HO-1). In the present study we investigated induction of HO-1 and adaptive increases in reduced glutathione (GSH) in human aortic smooth muscle cells (SMC) in response to moderately oxidized LDL (moxLDL, 100 mu g protein/ml, 24 h), a species containing high levels of lipid hydroperoxides. Expression and activity of HO-1 and GSH levels were elevated to a greater extent by moxLDL than highly oxidized LDL but unaffected by native or acetylated LDL. Inhibitors of protein kinase C (PKC) or mitogen-activated protein kinases (MAPK) p38(MAPK) and MEK or c-jun-NH2-terminal kinase (JNK) significantly attenuated induction of HO-1. Phosphorylation of p38(MAPK), extracellular signal-regulated kinase (ERK1/2), or JNK and nuclear translocation of the transcription factor Nrf2 were enhanced following acute exposure of SMC to rnoxLDL (100 mu g proteiri/ml, 1-2 h). Pretreatment of SMC with the antioxidant vitamin C (100 mu M, 24 h) attenuated the induction of HO-1 by moxLDL. Native and oxidized LDL did not alter basal levels of intracellular ATP, mitochondrial dehydrogenase activity, or expression of the lectin-like oxidized LDL receptor (LOX-1) in SMC. These findings demonstrate for the first time that activation of PKC, p38(MAPK), JNK, ERK1/2, and Nrf2 by oxidized LDL in human SMC leads to HO-1 induction, constituting an adaptive response against oxidative injury that can be ameliorated by vitamin C. (C) 2005 Elsevier Inc. All rights reserved.

Symmorphosis through dietary regulation: a combinatorial role for proteolysis, autophagy and protein synthesis in normalising muscle metabolism and function of hypertrophic mice after acute starvation

Animals are imbued with adaptive mechanisms spanning from the tissue/organ to the cellular scale which insure that processes of homeostasis are preserved in the landscape of size change. However we and others have postulated that the degree of adaptation is limited and that once outside the normal levels of size fluctuations, cells and tissues function in an aberant manner. In this study we examine the function of muscle in the myostatin null mouse which is an excellent model for hypertrophy beyond levels of normal growth and consequeces of acute starvation to restore mass. We show that muscle growth is sustained through protein synthesis driven by Serum/Glucocorticoid Kinase 1 (SGK1) rather than Akt1. Furthermore our metabonomic profiling of hypertrophic muscle shows that carbon from nutrient sources is being channelled for the production of biomass rather than ATP production. However the muscle displays elevated levels of autophagy and decreased levels of muscle tension. We demonstrate the myostatin null muscle is acutely sensitive to changes in diet and activates both the proteolytic and autophagy programmes and shutting down protein synthesis more extensively than is the case for wild-types. Poignantly we show that acute starvation which is detrimental to wild-type animals is beneficial in terms of metabolism and muscle function in the myostatin null mice by normalising tension production.

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle.

Dystrophin is the product of the Duchenne muscular dystrophy (DMD) gene. Dystrophin-related protein (utrophin), an autosomal homologue of dystrophin, was studied in skeletal muscle from normal fetuses aged 9-26 weeks and one stillbirth of 41 weeks' gestation, and compared with low- and high-risk DMD fetuses aged 9-20 weeks. Utrophin was present at the sarcolemma from before 9 weeks' gestation, although there was variability in intensity both within and between myotubes. Sarcolemmal immunolabelling became more uniform, and levels of utrophin increased to a maximum at approximately 17-18 weeks. Levels then declined, until by 26 weeks sarcolemmal labelling was negligible and levels were similar to adult control muscle. By 41 weeks there was virtually no sarcolemmal labelling, although immunolabelling of capillaries was bright. Similar results were obtained with normal and DMD fetal muscle. Utrophin is therefore expressed in the presence and absence of dystrophin and down-regulated before birth in normal fetal muscle fibres. Samples were not available to determine whether or when, utrophin levels decline in DMD fetal muscle. On Western blots, utrophin was shown to have a smaller relative molecular mass than adult dystrophin, but similar to the fetal isoform. Blood vessels were brightly immunolabelled at all ages, although utrophin immunolabelling of peripheral nerves increased with gestational age.

Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure

Impaired mechanosensing leads to heart failure and we have previously shown that a decreased ratio of cytoplasmic to nuclear CSRP3/Muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. Here we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 μM blebbistatin resulted in a ∼3 fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signaling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme-oxygenase1 (HO-1) activity with PPZII blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea pig model of angiotensin II infusion (400 ng/kg/min) over 12 weeks. Using subcellular fractionation we showed that the MLP ratio increased 88% (n = 4, P < 0.01) during compensated hypertrophy, but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01 n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signaling.

Effect of combined heat and high-pressure treatments on the texture of chicken breast muscle (Pectoralis fundus)

Commercially supplied chicken breast muscle was subjected to simultaneous heat and pressure treatments. Treatment conditions ranged from ambient temperature to 70 °C and from 0.1 to 800 MPa, respectively, in various combinations. Texture profile analysis (TPA) of the treated samples was performed to determine changes in muscle hardness. At treatment temperatures up to and including 50 °C, heat and pressure acted synergistically to increase muscle hardness. However, at 60 and 70 °C, hardness decreased following treatments in excess of 200 MPa. TPA was performed on extracted myofibrillar protein gels that after treatment under similar conditions revealed similar effects of heat and pressure. Differential scanning calorimetry analysis of whole muscle samples revealed that at ambient pressure the unfolding of myosin was completed at 60 °C, unlike actin, which completely denatured only above 70 °C. With simultaneous pressure treatment at >200 MPa, myosin and actin unfolded at 20 °C. Unfolding of myosin and actin could be induced in extracted myofibrillar protein with simultaneous treatment at 200 MPa and 40 °C. Electrophoretic analysis indicated high pressure/temperature regimens induced disulfide bonding between myosin chains.

Intracellular signalling pathways regulating the adaptation of skeletal muscle to exercise and nutritional changes

The focus of the present review is to assimilate current knowledge concerning the differing signalling transduction cascades that control muscle mass development and affect skeletal muscle phenotype following exercise or nutritional uptake. Effects of mechanical loading on protein synthesis are discussed. Muscle growth control is regulated by the interplay of growth promoting and growth suppressing factors, which act in concert. Much emphasis has been placed on understanding how increases in the rate of protein synthesis are induced in skeletal muscle during the adaptive process. One key point to emerge is that protein synthesis following resistance exercise or increased nutrient availability is mediated through changes in signal transduction involving the phosphorylation of mTOR and sequential activation of downstream targets. On the other hand, AMPK activation plays an important role in the inhibition of protein synthesis by suppressing the function of multiple translation regulators of the mTOR signalling pathway in response to cellular energy depletion and low metabolic conditions. The effects of exercise and/or nutritional uptake on the activation of signalling molecules that regulate protein synthesis are highlighted, providing a better understanding of the molecular changes in the cell.

Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells

What is already known about this subject center dot Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins. center dot In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets. center dot Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid-TP interaction inhibits signalling downstream TP has not been shown. What this study adds center dot This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells. center dot Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TP alpha- and TP beta-transfected HEK 293T cells was explored using binding assays and the TP antagonist H-3-SQ29548. Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC50 10-30 mu M). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by > 50% H-3-SQ29548 binding to different cell types. These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.