Skeletal Muscle Protein Metabolism Research Articles

Role of epinephrine in the control of protein degradation during fasting

The present work investigated the physiological role of epinephrine in the control of muscle protein degradation during fasting. For this, 3‐week‐old rats, previously submitted to adrenodemedullation (ADMX; 10 d after the surgical removal of adrenal medulla) were fasted for 2 days. ADMX abolished the plasma epinephrine concentration in both fed and fasted animals but did not affect plasma corticosterone levels. In sham rats, fasting increased overall proteolysis, the activity of ubiquitin‐proteasome system (UPS), the mRNA levels of atrogenes (Atrogin‐1 and MuRF1) and autophagy‐related genes (GABARAPl1 and LC3b). ADMX in fasted animals reduced the cAMP content (30%) in extensor digital longus (EDL) muscles and induced an additional increase of overall proteolysis which was associated with a higher activity of lysosomal (50%) and UPS (2x) systems and expression of atrogenes (~3x) and autophagic genes (~5x). pAkt was reduced (40%) by ADMX in muscles from fed rats. The lower phosphorylation levels of Akt were further decreased by ADMX. The data suggest that depletion of plasma epinephrine exacerbates the catabolic effects of fasting on skeletal muscle protein metabolism probably due to a decrease in cAMP/Akt signaling pathway. Supported by FAPESP (10/11015‐0; 08/06694‐6).

Rapamycin administration does not impair basal protein metabolism in human skeletal muscle

We have previously shown that administration of the mTORC1 inhibitor, rapamycin, to humans blocks the increase in skeletal muscle protein synthesis (MPS) in response to resistance exercise and amino acid ingestion. Whether rapamycin administration impairs basal MPS in human skeletal muscle is unknown. Six young (26±2y) subjects were studied during two separate 4h trials, in which each trial was divided into two, 2h basal periods. The trials were identical except during one trial a single oral dose (16mg) of rapamycin was administered immediately prior to the second basal period. Muscle biopsies (vastus lateralis) were obtained at 0, 2 and 4h, marking the beginning and end of each basal period, to examine MPS, mTORC1 signaling, and markers of autophagy (LC3BI and LC3BII protein). During the control trial, MPS, whole body protein breakdown (phenylalanine Ra), mTORC1 signaling and markers of autophagy were similar between both basal periods (p>0.05). During the rapamycin trial, these variables were similar to the control trial (p>0.05) and were unaltered by rapamycin administration (p>0.05). Thus, basal protein metabolism and mTORC1 signaling were not affected by rapamycin administration. We conclude that rapamycin administration only impairs mTORC1 signaling and MPS in human skeletal muscle when combined with a stimulus such as resistance exercise or increased amino acid availability.NIAMS R01AR049877, NIA P30AG024832, NIH T32HD07539, NCRR 1UL1RR029876

Skeletal muscle responses to negative energy balance: effects of dietary protein.

Sustained periods of negative energy balance decrease body mass due to losses of both fat and skeletal muscle mass. Decreases in skeletal muscle mass are associated with a myriad of negative consequences, including suppressed basal metabolic rate, decreased protein turnover, decreased physical performance, and increased risk of injury. Decreases in skeletal muscle mass in response to negative energy balance are due to imbalanced rates of muscle protein synthesis and degradation. However, the underlying physiological mechanisms contributing to the loss of skeletal muscle during energy deprivation are not well described. Recent studies have demonstrated that consuming dietary protein at levels above the current recommended dietary allowance (0.8 g · kg(-1) · d(-1)) may attenuate the loss of skeletal muscle mass by affecting the intracellular regulation of muscle anabolism and proteolysis. However, the specific mechanism by which increased dietary protein spares skeletal muscle through enhanced molecular control of muscle protein metabolism has not been elucidated. This article reviews the available literature related to the effects of negative energy balance on skeletal muscle mass, highlighting investigations that assessed the influence of varying levels of dietary protein on skeletal muscle protein metabolism. Further, the molecular mechanisms that may contribute to the regulation of skeletal muscle mass in response to negative energy balance and alterations in dietary protein level are described.

Rheb REGULATES CRAB MUSCLE ATROPHY

Moulting their shells and misplacing limbs, crabs continually depend on their versatile musculature to reshape their bodies. According to Donald Mykles and colleagues from Colorado State University and the University of Wisconsin – Stevens Point, USA, the massive claw muscle of blackback land crabs withers by as much as 78% before they can slip their shells off. However, Mykles and his colleagues made an interesting discovery in 2010. Instead of shutting down protein synthesis to allow the muscle to wither, the crabs ramp it up 11-fold by shutting down myostatin expression – which usually keeps protein synthesis in check. Knowing that myostatin regulates protein expression in mammals via the mTOR (metazoan target of rapamycin) signalling cascade, which controls mRNA translation, Mykles decided to investigate the mTOR pathway in blackback land crabs to find out whether it also regulates muscle rebuilding in crabs during moulting (p. 590).Stimulating crabs to moult, the team collected muscles from the animals and searched for the mRNA of four key mTOR signalling proteins – Rheb, mTOR, Akt and s6k. The crabs expressed all four genes, confirming that the mTOR pathway is active in moulting crab’s atrophied muscle.Next, the team measured the expression levels of each gene in crabs that had been induced to moult using methods that raise the animals’ moulting hormone (ecdysteroid, which regulates myostatin levels) over different time scales. They found that Rheb levels increased in the moulting crabs’ claws muscles and in the atrophied thoracic muscles of crabs that had lost a limb. Mykles says ‘Rheb is a key activator of mTOR. Its up-regulation by moulting indicates that Rheb plays a role in the stimulation of mTOR-mediated protein synthesis by ecdysteroids’.However, when the team investigated the mechanism by which Rheb is upregulated in the two muscles, there were differences. They found a strong correlation between the levels of Rheb in the crabs’ claws and the ecdysteroid levels, suggesting that ecdysteroid does regulate protein synthesis via mTOR in the claw muscle. Yet, when they compared the ecdysteroid levels and Rheb levels in the thoracic muscles – that wither in response to limb loss – the team found no correlation. Ecdysteroid hormone does not control muscle atrophy in response to limb loss.Analysing the expression patterns of Rheb and myostatin in atrophied claw and thoracic muscles, Mykles and colleagues also saw different expression relationships between the proteins in the two tissues. ‘If blackback land crab myostatin plays a role in both types of atrophy, any unifying mechanism must reconcile the differences between myostatin and Rheb expression in the two muscles’, the team says.Having shown that Rheb is a key component of the signalling pathway that regulates muscle atrophy in crabs, the team says that it is keen to build, ‘a mechanistic understanding of the interactions between ecdysteroids and the signalling pathways that control protein metabolism in crustacean skeletal muscle’.

Skeletal muscle protein balance and metabolism in the elderly.

The loss of lean muscle mass occurring with advancing age is termed sarcopenia. This condition often leads to a concomitant loss of strength, increased frailty and risk of falls and an overall loss of functional independence in the elderly. Muscle protein metabolism is a dynamic process characterized by the balance between the synthesis and breakdown of muscle proteins. A disturbance of this equilibrium can lead to the loss of muscle mass, and a perturbation of muscle protein turnover with aging has been proposed to play a role in the development of sarcopenia. However, basal muscle protein synthesis and breakdown rates do not differ between young and old adults, which has led to the hypothesis that older adults are resistant to anabolic stimuli. In support of this hypothesis, older adults have either no response or a blunted response to nutrients, insulin and resistance exercise, and this anabolic resistance is likely a key factor in the loss of skeletal muscle mass with aging. Recent studies have investigated potential interventions to overcome this anabolic resistance. In particular, combining resistance exercise with essential amino acid supplementation restores the muscle protein anabolic response in older men. The novel rehabilitation technique of performing light resistance exercise during blood flow restriction was also successful in overcoming the anabolic resistance to exercise. Future research is needed to determine whether these novel interventions will be successful in preventing sarcopenia and improving muscle strength and function in older adults.

Exercise, Amino Acids, and Aging in the Control of Human Muscle Protein Synthesis

In this review, we discuss recent research in the field of human skeletal muscle protein metabolism characterizing the acute regulation of mammalian target of rapamycin complex (mTORC) 1 signaling and muscle protein synthesis (MPS) by exercise, amino acid nutrition, and aging. Resistance exercise performed in the fasted state stimulates mixed MPS within 1 h after exercise, which can remain elevated for 48 h. We demonstrate that the activation of mTORC1 signaling (and subsequently enhanced translation initiation) is required for the contraction-induced increase in MPS. In comparison, low-intensity blood flow restriction (BFR) exercise stimulates MPS and mTORC1 signaling to an extent similar to traditional, high-intensity resistance exercise. We also show that mTORC1 signaling is required for the essential amino acid (EAA)-induced increase in MPS. Ingestion of EAAs (or protein) shortly after resistance exercise enhances MPS and mTORC1 signaling compared with resistance exercise or EAAs alone. In older adults, the ability of the skeletal muscle to respond to anabolic stimuli is impaired. For example, in response to an acute bout of resistance exercise, older adults are less able to activate mTORC1 or increase MPS during the first 24 h of postexercise recovery. However, BFR exercise can overcome this impairment. Aging is not associated with a reduced response to EAAs provided the EAA content is sufficient. Therefore, we propose that exercise combined with EAA should be effective not only in improving muscle repair and growth in response to training in athletes, but that strategies such as EAA combined with resistance exercise (or BFR exercise) may be very useful as a countermeasure for sarcopenia and other clinical conditions associated with muscle wasting.

Protein metabolism and gene expression in skeletal muscle of critically ill patients with sepsis

Muscle wasting negatively affects morbidity and mortality in critically ill patients. This progressive wasting is accompanied by, in general, a normal muscle PS (protein synthesis) rate. In the present study, we investigated whether muscle protein degradation is increased in critically ill patients with sepsis and which proteolytic enzyme systems are involved in this degradation. Eight patients and seven healthy volunteers were studied. In vivo muscle protein kinetics was measured using arteriovenous balance techniques with stable isotope tracers. The activities of the major proteolytic enzyme systems were analysed in combination with mRNA expression of genes related to these proteolytic systems. Results show that critically ill patients with sepsis have a variable but normal muscle PS rate, whereas protein degradation rates are dramatically increased (up to 160%). Of the major proteolytic enzyme systems both the proteasome and the lysosomal systems had higher activities in the patients, whereas calpain and caspase activities were not changed. Gene expression of several genes related to the proteasome system was increased in the patients. mRNA levels of the two main lysosomal enzymes (cathepsin B and L) were not changed but, conversely, genes related to calpain and caspase had a higher expression in the muscles of the patients. In conclusion, the dramatic muscle wasting seen in critically ill patients with sepsis is due to increased protein degradation. This is facilitated by increased activities of both the proteasome and lysosomal proteolytic systems.

Suppression of 5′-Nucleotidase Enzymes Promotes AMP-activated Protein Kinase (AMPK) Phosphorylation and Metabolism in Human and Mouse Skeletal Muscle

The 5'-nucleotidase (NT5) family of enzyme dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. We hypothesized that gene silencing of NT5 enzymes to increase the intracellular availability of AMP would increase AMP-activated protein kinase (AMPK) activity and metabolism. We determined the role of cytosolic NT5 in metabolic responses linked to the development of insulin resistance in obesity and type 2 diabetes. Using siRNA to silence NT5C2 expression in cultured human myotubes, we observed a 2-fold increase in the AMP/ATP ratio, a 2.4-fold increase in AMPK phosphorylation (Thr(172)), and a 2.8-fold increase in acetyl-CoA carboxylase phosphorylation (Ser(79)) (p < 0.05). siRNA silencing of NT5C2 expression increased palmitate oxidation by 2-fold in the absence and by 8-fold in the presence of 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside. This was paralleled by an increase in glucose transport and a decrease in glucose oxidation, incorporation into glycogen, and lactate release from NT5C2-depleted myotubes. Gene silencing of NT5C1A by shRNA injection and electroporation in mouse tibialis anterior muscle reduced protein content (60%; p < 0.05) and increased phosphorylation of AMPK (60%; p < 0.05) and acetyl-CoA carboxylase (50%; p < 0.05) and glucose uptake (20%; p < 0.05). Endogenous expression of NT5C enzymes inhibited basal lipid oxidation and glucose transport in skeletal muscle. Reduction of 5'-nucleotidase expression or activity may promote metabolic flexibility in type 2 diabetes.

Muscle protein metabolism responds similarly to exogenous amino acids in healthy younger and older adults during NO-induced hyperemia

The combination of increasing blood flow and amino acid (AA) availability provides an anabolic stimulus to the skeletal muscle of healthy young adults by optimizing both AA delivery and utilization. However, aging is associated with a blunted response to anabolic stimuli and may involve impairments in endothelial function. We investigated whether age-related differences exist in the muscle protein anabolic response to AAs between younger (30 ± 2 yr) and older (67 ± 2 yr) adults when macrovascular and microvascular leg blood flow were similarly increased with the nitric oxide (NO) donor, sodium nitroprusside (SNP). Regardless of age, SNP+AA induced similar increases above baseline (P ≤ 0.05) in macrovascular flow (4.3 vs. 4.4 ml·min(-1)·100 ml leg(-1) measured using indocyanine green dye dilution), microvascular flow (1.4 vs. 0.8 video intensity/s measured using contrast-enhanced ultrasound), phenylalanine net balance (59 vs. 68 nmol·min(-1)·100 ml·leg(-1)), fractional synthetic rate (0.02 vs. 0.02%/h), and model-derived muscle protein synthesis (62 vs. 49 nmol·min(-1)·100 ml·leg(-1)) in both younger vs. older individuals, respectively. Provision of AAs during NO-induced local skeletal muscle hyperemia stimulates skeletal muscle protein metabolism in older adults to a similar extent as in younger adults. Our results suggest that the aging vasculature is responsive to exogenous NO and that there is no age-related difference per se in AA-induced anabolism under such hyperemic conditions.

Involvement of Skeletal Muscle Protein, Glycogen, and Fat Metabolism in the Adaptation on Early Lactation of Dairy Cows

During early lactation, high-yielding dairy cows cannot consume enough feed to meet nutrient requirements. As a consequence, animals drop into negative energy balance and mobilize body reserves including muscle protein and glycogen for milk production, direct oxidation, and hepatic gluconeogenesis. To examine which muscle metabolic processes contribute to the adaptation during early lactation, six German Holstein cows were blood sampled and muscle biopsied throughout the periparturient period. From pregnancy to lactation, the free plasma amino acid pattern imbalanced and plasma glucose decreased. Several muscle amino acids, as well as total muscle protein, fat, and glycogen, and the expression of glucose transporter-4 were reduced within the first 4 weeks of lactation. The 2-DE and MALDI-TOF-MS analysis identified 43 differentially expressed muscle protein spots throughout the periparturient period. In early lactation, expression of cytoskeletal proteins and enzymes involved in glycogen synthesis and in the TCA cycle was decreased, whereas proteins related to glycolysis, fatty acid degradation, lactate, and ATP production were increased. On the basis of these results, we propose a model in which the muscle breakdown in early lactation provides substrates for milk production by a decoupled Cori cycle favoring hepatic gluconeogenesis and by interfering with feed intake signaling.

MTor Signaling in Skeletal Muscle During Sepsis and Inflammation: Where Does It All Go Wrong?

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that exquisitely regulates protein metabolism in skeletal muscle. mTOR integrates input from amino acids, growth factors, and intracellular cues to make or break muscle protein. mTOR accomplishes this task by stimulating the phosphorylation of substrates that control protein translation while simultaneously inhibiting proteasomal and autophagic protein degradation. In a metabolic twist of fate, sepsis induces muscle atrophy in part by the aberrant regulation of mTOR. In this review, we track the steps of normal mTOR signaling in muscle and examine where they go astray in sepsis and inflammation.

Effect of a chronic intake of high dietary glycated proteins on skeletal muscle protein metabolism during aging in normal rats

Chronic exposure of advanced glycation end products (AGEs) is associated with development of insulin resistance in mammals and in L6 skeletal muscle cells. AGEs increase oxidative stress and inflammation through receptor for AGEs (RAGE). We hypothesized that a chronic intake of dietary high AGEs may affect skeletal muscle protein metabolism and muscle mass in aging. To elucidate this hypothesis, phosphorylation of anabolic signaling pathway (Akt, mTOR, p70S6K, 4E-BP1) and RAGE expression were measured by Western-blot in tibialis in young (5month old), adult (13month old) and old (25month old) rats fed a diet containing heated casein (high-AGEs) or non heated casein (low-AGE) for 3 months. Insulin sensitivity was also evaluated by OGTT. Muscle loss and insulin resistance were observed in old (P<0.05, 0.57±0.08, 2.66±1.32 fold from young, respectively), without any effect of high-AGEs. Phosphorylation of mTOR, p70S6K increased in old rats (P<0.05, 1.59±0.06, 10.59±2.58 fold from young rats, respectively). Interestingly, phosphorylation of p70S6K and RAGE expression were enhanced by high-AGEs in old rats (P<0.05, 4.19±1.02, 1.86±0.28 fold, respectively). Glycated proteins might stimulate signaling pathway of muscle protein synthesis through RAGE. Consequently, chronic intake of glycated proteins does not affect muscle anabolic response during aging.

Testosterone modulates gene expression pathways regulating nutrient accumulation, glucose metabolism and protein turnover in mouse skeletal muscle

Testosterone regulates energy metabolism and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. This study investigated the response of skeletal muscle to castration and testosterone replacement in 8-week-old male mice. Using microarray analyses of mRNA levels in gastrocnemius muscle, 91 genes were found to be negatively regulated by testosterone and 68 genes were positively regulated. The mRNA levels of the insulin signalling suppressor molecule Grb10 and the glycogen synthesis inhibitors, protein phosphatase inhibitor-1 and phosphorylase kinase-γ, were negatively regulated by testosterone. The insulin-sensitive glucose and amino acid transporters, Glut3 and SAT2, the lipodystrophy gene, Lpin1 and protein targeting to glycogen were positively regulated. These changes would be expected to increase nutrient availability and sensing within skeletal muscle, increase metabolic rate and carbohydrate utilization and promote glycogen accumulation. The observed positive regulation of atrogin-1 (Fbxo32) by testosterone could be explained by the phosphorylation of Akt and Foxo3a, as determined by Western blotting. Testosterone prevented the castration-induced increase in interleukin-1α, the decrease in interferon-γ and the atrophy of the levator ani muscle, which were all correlated with testosterone-regulated gene expression. These findings identify specific mechanisms by which testosterone may regulate skeletal muscle glucose and protein metabolism.

Skeletal muscle protein metabolism in the elderly: Interventions to counteract the 'anabolic resistance' of ageing

Age-related muscle wasting (sarcopenia) is accompanied by a loss of strength which can compromise the functional abilities of the elderly. Muscle proteins are in a dynamic equilibrium between their respective rates of synthesis and breakdown. It has been suggested that age-related sarcopenia is due to: i) elevated basal-fasted rates of muscle protein breakdown, ii) a reduction in basal muscle protein synthesis (MPS), or iii) a combination of the two factors. However, basal rates of muscle protein synthesis and breakdown are unchanged with advancing healthy age. Instead, it appears that the muscles of the elderly are resistant to normally robust anabolic stimuli such as amino acids and resistance exercise. Ageing muscle is less sensitive to lower doses of amino acids than the young and may require higher quantities of protein to acutely stimulate equivalent muscle protein synthesis above rest and accrue muscle proteins. With regard to dietary protein recommendations, emerging evidence suggests that the elderly may need to distribute protein intake evenly throughout the day, so as to promote an optimal per meal stimulation of MPS. The branched-chain amino acid leucine is thought to play a central role in mediating mRNA translation for MPS, and the elderly should ensure sufficient leucine is provided with dietary protein intake. With regards to physical activity, lower, than previously realized, intensity high-volume resistance exercise can stimulate a robust muscle protein synthetic response similar to traditional high-intensity low volume training, which may be beneficial for older adults. Resistance exercise combined with amino acid ingestion elicits the greatest anabolic response and may assist elderly in producing a 'youthful' muscle protein synthetic response provided sufficient protein is ingested following exercise.

Protein catabolism and impairment of skeletal muscle insulin signalling in heart failure

Derangements in systemic and local metabolism develop in patients with CHF [chronic HF (heart failure)] and contribute to the progression of the disease. Impaired skeletal muscle metabolism, morphology and function leading to exercise intolerance are hallmarks of the syndrome of CHF. These changes result in abnormal glucose and lipid metabolism, and the associated insulin resistance, which contribute to progression of skeletal muscle catabolism and development of muscle atrophy in patients with advanced HF. In the present issue of Clinical Science, Toth and co-workers demonstrate the impairment of skeletal muscle protein metabolism in patients with HF, and specifically show an impaired anabolic response in the skeletal muscle of these patients following a period of nutritional deficiency.

Insulin Stimulates Human Skeletal Muscle Protein Synthesis via an Indirect Mechanism Involving Endothelial-Dependent Vasodilation and Mammalian Target of Rapamycin Complex 1 Signaling

Abstract Objective: Our objective was to determine whether endothelial-dependent vasodilation is an essential mechanism by which insulin stimulates human skeletal muscle protein synthesis and anabolism. Subjects: Subjects were healthy young adults (n = 14) aged 31 ± 2 yr. Design: Subjects were studied at baseline and during local leg infusion of insulin alone (control, n = 7) or insulin plus the nitric oxide synthase inhibitor NG-monomethyl-l-arginine (L-NMMA, n = 7) to prevent insulin-induced vasodilation. Methods: We measured skeletal muscle protein metabolism with stable isotope tracers, blood flow with indocyanine green, capillary recruitment with contrast enhanced ultrasound, glucose metabolism with stable isotope tracers, and phosphorylation of proteins associated with insulin (Akt) and amino acid-induced mammalian target of rapamycin(mTOR) complex 1 (mTORC1) signaling (mTOR, S6 kinase 1, and eukaryotic initiation factor 4Ebinding protein 1) with Western blot analysis. Results: No basal differences between groups were detected. During insulin infusion, blood flow and capillary recruitment increased in the control (P &amp;lt; 0.05) group only; Akt phosphorylation and glucose uptake increased in both groups (P &amp;lt; 0.05), with no group differences; and mTORC1 signaling increased more in control (P &amp;lt; 0.05) than in l-NMMA. Phenylalanine net balance increased (P &amp;lt; 0.05) in both groups, but with opposite mechanisms: increased protein synthesis (basal, 0.051 ± 0.006%/h; insulin, 0.077 ± 0.008%/h; P &amp;lt; 0.05) with no change in proteolysis in control and decreased proteolysis (P &amp;lt; 0.05) with no change in synthesis (basal, 0.061 ± 0.004%/h; insulin, 0.050 ± 0.006%/h; P value not significant) in l-NMMA. Conclusions: Endothelial-dependent vasodilation and the consequent increase in nutritive flow and mTORC1 signaling, rather than Akt signaling, are fundamental mechanisms by which insulin stimulates muscle protein synthesis in humans. Additionally, these data underscore that insulin modulates skeletal muscle proteolysis according to its effects on nutritive flow.

Insulin Stimulates Human Skeletal Muscle Protein Synthesis via an Indirect Mechanism Involving Endothelial-Dependent Vasodilation and Mammalian Target of Rapamycin Complex 1 Signaling

Our objective was to determine whether endothelial-dependent vasodilation is an essential mechanism by which insulin stimulates human skeletal muscle protein synthesis and anabolism. Subjects were healthy young adults (n=14) aged 31+/-2 yr. Subjects were studied at baseline and during local leg infusion of insulin alone (control, n=7) or insulin plus the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, n=7) to prevent insulin-induced vasodilation. We measured skeletal muscle protein metabolism with stable isotope tracers, blood flow with indocyanine green, capillary recruitment with contrast enhanced ultrasound, glucose metabolism with stable isotope tracers, and phosphorylation of proteins associated with insulin (Akt) and amino acid-induced mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling (mTOR, S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1) with Western blot analysis. No basal differences between groups were detected. During insulin infusion, blood flow and capillary recruitment increased in the control (P<0.05) group only; Akt phosphorylation and glucose uptake increased in both groups (P<0.05), with no group differences; and mTORC1 signaling increased more in control (P<0.05) than in L-NMMA. Phenylalanine net balance increased (P<0.05) in both groups, but with opposite mechanisms: increased protein synthesis (basal, 0.051+/-0.006 %/h; insulin, 0.077+/-0.008 %/h; P<0.05) with no change in proteolysis in control and decreased proteolysis (P<0.05) with no change in synthesis (basal, 0.061+/-0.004 %/h; insulin, 0.050+/-0.006 %/h; P value not significant) in L-NMMA. Endothelial-dependent vasodilation and the consequent increase in nutritive flow and mTORC1 signaling, rather than Akt signaling, are fundamental mechanisms by which insulin stimulates muscle protein synthesis in humans. Additionally, these data underscore that insulin modulates skeletal muscle proteolysis according to its effects on nutritive flow.

Effects of endotoxaemia on protein metabolism in rat fast-twitch skeletal muscle and myocardium.

BackgroundIt is unclear if the rat myocardium undergoes the same rapid reductions in protein content that are classically observed in fast-twitch skeletal muscle during endotoxaemia.Methodology/Principal FindingsTo investigate this further, and to determine if there is any divergence in the response of skeletal muscle and myocardium in the mechanisms that are thought to be largely responsible for eliciting changes in protein content, Sprague Dawley rats were implanted with vascular catheters and administered lipopolysaccharide (LPS; 150 µg kg−1 h−1) intravenously for 2 h, 6 h or 24 h (saline administered control animals were also included), after which the extensor digitorum longus (EDL) and myocardium were removed under terminal anaesthesia. The protein-to-DNA ratio, a marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P<0.05), but was no different from controls in the myocardium. At the same time point, a significant increase in MAFbx/atrogin-1 and MuRF1 mRNA (3.7±0.7- and 19.5±1.9-fold increase vs. controls, respectively; P<0.05), in addition to protein levels of α1-3, 5–7 subunits of the 20S proteasome, were observed in EDL but not myocardium. In contrast, elevations in phosphorylation of p70 S6K residues Thr421/Ser424, and 4E-BP1 residues Thr37/Thr46 (P<0.05), consistent with an elevation in translation initiation, were seen exclusively in the myocardium of LPS-treated animals.Conclusions/SignificanceIn summary, these findings suggest that the myocardium does not undergo the same catabolic response as skeletal muscle during early endotoxaemia, partly due to the absence of transcriptional and signalling events in the myocardium typically associated with increased muscle proteolysis and the suppression of protein synthesis.

The effect of new proteasome inhibitors, belactosin A and C, on protein metabolism in isolated rat skeletal muscle

The proteasome inhibitors are used as research tools to study of the ATP-dependent ubiquitin-proteasome system. Some of them are at present undergoing clinical trials to be used as therapeutic agents for cancer or inflammation. These diseases are often accompanied by muscle wasting. We herein demonstrate findings about new proteasome inhibitors, belactosin A and C, and their direct effect on protein metabolism in rat skeletal muscle. M. soleus (SOL) and m. extensor digitorum longus (EDL) were dissected from both legs of male rats (40-60 g) and incubated in a buffer containing belactosin A or C (30 microM) or no inhibitor. The release of amino acids into the medium was estimated using high performance liquid chromatography to calculate total and myofibrillar proteolysis. Chymotrypsin-like activity (CTLA) of proteasome and cathepsin B, L activity were determined by fluorometric assay. Protein synthesis and leucine oxidation were detected using specific activity of L-[1-14C] leucine added to medium. Inhibited and control muscles from the same rat were compared using paired t-test. The results indicate that after incubation with both belactosin A and C total proteolysis and CTLA of proteasome decreased while cathepsin B, L activity did not change in both SOL and EDL. Leucine oxidation was significantly enhanced in SOL, protein synthesis decreased in EDL. Myofibrillar proteolysis was reduced in both muscles in the presence of belactosin A only. In summary, belactosin A and C affected basic parameters of protein metabolism in rat skeletal muscle. The response was both muscle- and belactosin-type-dependent.

The Effect of High Glucocorticoid Administration and Food Restriction on Rodent Skeletal Muscle Mitochondrial Function and Protein Metabolism

BackgroundGlucocorticoids levels are high in catabolic conditions but it is unclear how much of the catabolic effects are due to negative energy balance versus glucocorticoids and whether there are distinct effects on metabolism and functions of specific muscle proteins.Methodology/Principal FindingsWe determined whether 14 days of high dose methylprednisolone (MPred, 4 mg/kg/d) Vs food restriction (FR, food intake matched to MPred) in rats had different effects on muscle mitochondrial function and protein fractional synthesis rates (FSR). Lower weight loss (15%) occurred in FR than in MPred (30%) rats, while a 15% increase occurred saline-treated Controls. The per cent muscle loss was significantly greater for MPred than FR. Mitochondrial protein FSR in MPred rats was lower in soleus (51 and 43%, respectively) and plantaris (25 and 55%) than in FR, while similar decline in protein FSR of the mixed, sarcoplasmic, and myosin heavy chain occurred. Mitochondrial enzymatic activity and ATP production were unchanged in soleus while in plantaris cytochrome c oxidase activity was lower in FR than Control, and ATP production rate with pyruvate + malate in MPred plantaris was 28% lower in MPred. Branched-chain amino acid catabolic enzyme activities were higher in both FR and MPred rats indicating enhanced amino acid oxidation capacity.Conclusion/SignificanceMPred and FR had little impact on mitochondrial function but reduction in muscle protein synthesis occurred in MPred that could be explained on the basis of reduced food intake. A greater decline in proteolysis may explain lesser muscle loss in FR than in MPred rats.