Turnover In Muscle Research Articles

Inverse Regulation of Protein Turnover and Amino Acid Transport in Skeletal Muscle of Hypercatabolic Patients

We have investigated the relationships between the rates of muscle protein synthesis and degradation and of transmembrane transport of selected amino acids in leg skeletal muscle of 19 severely burned patients and 18 normal controls in the postabsorptive state. Patients were studied on the 14 ± 5 postburn day, and their mean burn size was 66% ± 18% of total body surface area. Methods were based on the leg arteriovenous balance technique in combination with biopsies of the vastus lateralis muscle and infusions of isotopic tracers of amino acids. Net muscle protein breakdown was greater in the patients because of an 83% increase in the rate of muscle protein degradation. The rate of muscle protein synthesis was also increased in the patients but to a lesser extent than protein degradation, i.e. by 50% with the arteriovenous phenylalanine balance technique and by 49% with the direct tracer incorporation method. The absolute values of inward transport of phenylalanine, leucine, and lysine were not significantly different in the two groups. However, the ability of transport systems to take up amino acids from the bloodstream, as assessed by dividing inward transport by amino acid delivery to leg muscle, were 50–63% lower in the patients. In contrast, outward phenylalanine and lysine transport were 40% and 67% greater in the patients than in the controls, respectively. We conclude the primary alteration in muscle protein metabolism is an acceleration of protein breakdown, and the increase in protein synthesis likely is due to increased intracellular amino acid availability as a result of accelerated breakdown. Transmembrane transport in the outward direction is accelerated, presumably to facilitate the export of amino acids from muscle to other tissues. In contrast, transmembrane transport in the inward direction is impaired relatively to the increased delivery of circulating amino acid to skeletal muscle secondary to accelerated blood flow.

Effects of high doses of glucocorticoids on free amino acids, ribosomes and protein turnover in human muscle.

Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanisms responsible for this catabolic effect are controversial. We investigated the effects of 60 mg day(-1) prednisolone on protein synthesis and degradation in human skeletal muscle. Healthy adults (n = 9) were studied in the postabsorptive state, before and after 3 days of prednisolone treatment. The L-[ring 2,6(-3)H(5)]-phenylalanine tracer technique, concentration and size distribution of the ribosomes, mRNA content of the ubiquitin-proteasome pathway components in muscle, phenylalanine flux across the leg, and the free amino acid concentrations in skeletal muscle were used to study muscle protein metabolism. The concentrations of most amino acids in arterial blood increased after prednisolone. There were also increased effluxes of phenylalanine, asparagine, arginine, alanine, methionine and isoleucine from the leg. The rate of protein degradation, as measured by the appearance rate (Ra) of phenylalanine, increased by 67% (P = 0.023) which, together with a doubling of the net release of phenylalanine from the leg (P = 0.007), indicated accelerated protein degradation. The pathway was not identified but there was no significant increase in mRNAs' encoding components of the ubiquitin-proteasome pathway. There was a 6% reduction in polyribosomes (P = 0.007), suggesting a decrease in the capacity for protein synthesis, although there was no measured decrease in the rate of protein synthesis. These findings indicate that high doses of prednisolone lead to a sharp increase in net protein catabolism, which depends more on enhanced protein breakdown, and an uncertain effect on protein synthesis. The mechanisms stimulating proteolysis and the pathway stimulated to increase muscle protein degradation should be explored.

Capillary electrophoretic analysis of µ- and m-calpain using fluorescently labeled casein substrates

Calpains are unique calcium-dependent thiol proteases that have been proposed to participate in a number of physiological processes including signal transduction and protein turnover in skeletal muscle. Calpains exist in two major forms. Interestingly, the two forms of protease show no significant difference in their action on various substrates. The only demonstrable difference in their activity involves the concentration of calcium required for activation. Both mu- and m-calpains typically achieve half maximal activation at 50 microM and 0.7 mM calcium, respectively. The focus of this study was to examine the action of both forms of calpain on casein substrates and assess whether any differences could be observed in the resulting peptide finger print using capillary electrophoresis. Purified mu- and m-calpain were incubated for various lengths of time with Oregon Green labeled alphas- and beta-casein. The reactions were stopped with sodium dodecyl sulfate (SDS) and products separated by capillary electrophoresis in micellar electrokinetic capillary chromatography (MEKC) mode using laser-induced fluorescence (LIF) detection. Comparison of the electropherograms showed no difference in the peptide profile for either enzyme. However, it was found that beta-casein was hydrolyzed more extensively than alphas-casein, by both enzymes. Capillary electrophoresis was found to be a very sensitive technique for detection of calpain activity. Using beta-casein as substrate, the CE approach was able to detect 2-3 ng of calpain activity. The results also suggest that capillary electrophoresis is a useful tool for proteolytic investigations of protein structure.

Antioxidant alpha-lipoic acid and protein turnover in insulin-resistant rat muscle

We have shown previously that the antioxidant alpha-lipoic acid (ALA) can stimulate glucose transport and can enhance the stimulation of this process by insulin in skeletal muscle from insulin-resistant obese Zucker rats. As insulin can also acutely activate general protein synthesis and inhibit net protein degradation in skeletal muscle, we hypothesized that ALA could directly affect protein turnover and also increase the effect of insulin on protein turnover in isolated skeletal muscle from developing obese Zucker rats. In epitrochlearis muscles isolated from obese Zucker rats, insulin (2 mU/ml) significantly ( p < 0.05) increased in vitro protein synthesis (phenylalanine incorporation into protein) and decreased net protein degradation (tyrosine release), whereas a racemic mixture of ALA (2 mM) had no effect on either process. Interestingly, rates of protein synthesis in muscle from obese Zucker rats were substantially lower compared to those values observed in age-matched insulin-sensitive Wistar rats, whereas rates of protein degradation were comparable. Obese Zucker rats were also treated chronically with either vehicle or ALA (50 mg/kg/d for 10 d). Again, insulin significantly increased net protein synthesis and decreased net protein degradation in epitrochlearis muscles isolated from vehicle-treated obese Zucker rats; however, this stimulatory effect of insulin was not improved by prior in vivo ALA treatment. These results indicate that the previously described effect of the antioxidant ALA to increase insulin-stimulated glucose transport in skeletal muscle of obese, insulin-resistant rats does not apply to another important insulin-regulatable process, protein turnover. These findings imply that the cellular mode of action for ALA is restricted to signaling factors unique to the activation of glucose transport, and does not involve the pathway of stimulation of general protein synthesis and net protein degradation.

Molecular mechanisms regulating protein turnover in muscle

Loss of muscle mass is a risk factor for mortality in chronic renal failure (CRF). Catabolic signals (eg, acidosis, glucocorticoids, insulin resistance) present in CRF stimulate the ubiquitin-proteasome proteolytic pathway in muscle but the activation mechanism(s) have been elusive. We have identified distinct mechanisms that may work in concert to increase the degradation of muscle proteins. Glucocorticoids increase the transcription of genes encoding components of the ubiquitin-proteasome pathway, thereby increasing the proteolytic capacity of muscle cells. Another signal could be a decreased response to insulin because acute diabetes is a potent stimulus for protein degradation by the ubiquitin-proteasome pathway and CRF impairs insulin signaling in muscle. Together, these responses increase the breakdown of muscle, contributing to protein malnutrition in CRF.

Early and selective disappearance of telethonin protein from the sarcomere in neurogenic atrophy.

Mutations of the human telethonin gene have recently been shown to cause limb girdle muscular dystrophy type 2G in three Brazilian families. The mRNA has been shown to be dynamically regulated in animals, however, the fate of the protein in human muscle is unknown. In order to assess the expression of telethonin in more frequently encountered myopathological conditions we generated and characterized a rabbit antiserum raised against the C-terminal end of telethonin by immunoblotting and immunogold EM. Indirect immunofluorescence analysis of a wide variety of neuromuscular disorders including dystrophinopathies, metabolic myopathies, denervation disorders, congenital and inflammatory myopathies revealed that the characteristic Z-band staining of telethonin was preserved in all disease entities included in our study. However, a reduced telethonin immunoreactivity was observed in up to 10% of type II fibers in 10 cases of neurogenic atrophy. A decreased telethonin staining was more frequently observed in early stages of fiber atrophy than in type II fibers displaying normal or highly atrophic fiber diameters. Hence, not only the telethonin transcript is rapidly downregulated in denervated muscle but the protein itself undergoes dynamic changes while its known sarcomeric binding partner titin remains unaltered. Beyond its role as a static component of Z-bands, these findings indicate that telethonin protein levels seems to be at least in part regulated by neuronal activity and is thus linked to the dynamic control of myofibrillogenesis and muscle turnover in human skeletal muscle.

Mechanism of increased tolerance to hypothermia after composite Indian herbal preparation II administration.

Investigation of the mechanism of increased tolerance to stress induced hypothermia after the administration of composite Indian herbal preparation II (CIHP II), a combination of several plant ingredients and minerals. The effect of oral CIHP II administration (1 mg/g of body weight), prior to cold (5 degrees C)-hypoxia (428 mm Hg)-restraint (C-H-R) exposure in rats on cardiac and skeletal muscle oxidation was studied in vitro by estimating conversion of glucose-U-14C and Palmitate-1-(14)C to 14CO2. In vitro adipose tissue lipolysis and incorporation of glucose-U-14C into skeletal muscle glycogen was also studied. A single dose of CIHP II-enhanced resistance to hypothermia (rectal temperature [T(rec)] 23 degrees C) during C-H-R exposure as evidenced by increased glucose turnover rate in heart and skeletal muscle tissue. The blood glucose and skeletal muscle glycogen were conserved. Cardiac free fatty acid oxidation was also increased. During recovery from hypothermia (T(rec) 37 degrees C) blood glucose and muscle glycogen levels were conserved. Five doses of CIHP II increased resistance to cold by increased adipose fat mobilization and cardiac oxidation. Glucose oxidation was spared. During recovery from hypothermia, the glucose turnover and oxidation in skeletal muscle was increased as was fat mobilization from adipose tissue and its oxidation by heart muscle. CIHP II intake prior to C-H-R exposure resulted in increased glucose turnover rate and fat utilization. This perhaps helped increase the resistance to C-H-R-induced hypothermia and speeded recovery.

Mitochondria as ATP consumers: cellular treason in anoxia.

In anoxia, mitochondria change from being ATP producers to potentially powerful ATP consumers. This change occurs, because the mitochondrial F(1)F(0)-ATPase begins to hydrolyze ATP to avoid the collapse of the proton motive force. Species that can survive prolonged periods of O(2) lack must limit such ATP use; otherwise, this process would dominate glycolytic metabolism and threaten ATP delivery to essential ATP-consuming processes of the cell (e.g., ion-motive ATPases). There are two ways to limit ATP hydrolysis by the F(1)F(0)-ATPase, namely (i) reduction of the proton conductance of the mitochondrial inner membrane and (ii) inhibition of the enzyme. We assessed these two possibilities by using intact mitochondria isolated from the skeletal muscle of anoxia-tolerant frogs. Our results show that proton conductance is unaltered between normoxia and anoxia. However, ATP use by the F(1)F(0)-ATPase is limited in anoxia by a profound inhibition of the enzyme. Even so, ATP use by the F(1)F(0)-ATPase might account for approximately 9% of the ATP turnover in anoxic frog skeletal muscle.

Temperature effects on protein turnover in crustacean muscle

Temperature effects on protein turnover in crustacean muscle

Anabolic and catabolic mediators of intestinal protein turnover: a new experimental approach.

Studies on regulation of protein turnover in skeletal muscle have revealed the important contributions of protein synthesis and catabolism to tissue protein balance, and have identified a host of specific anabolic and catabolic stimuli and biochemical mechanisms that regulate these processes. This knowledge is critical to current efforts designed to promote anabolism and limit atrophy. Of the tissues with a potentially large contribution to whole-body amino acid metabolism, protein turnover of the intestine stands out as being poorly understood. The intestine is subject to complexities in regulation of its metabolism that are not apparent for other tissues. The study of intestinal protein turnover also entails some important technical challenges. We recently developed an in-situ experimental system for study of intestinal mucosal protein synthesis with the following unique features: multiple observations within an animal; controlled delivery of nutritional stimuli to the apical side, basolateral side, or both; and luminal delivery of tracer in a flooding dose for determination of protein synthesis. We have begun to use the system to test the specific roles of individual luminal nutrients in regulation of mucosal protein synthesis. We have also used protease gene expression as an index of potential regulation of catabolic pathways.

Type IV collagen and its degradation in paralyzed human muscle: effect of functional electrical stimulation.

The purpose of this study was to evaluate the effects of spinal cord injury (SCI) and functional electrical stimulation (FES) of paralyzed muscles on type IV collagen content and proteins involving its degradation, which is initiated by matrix metalloproteinase (MMP)-2 and -9 and regulated by their tissue inhibitors (TIMPs)-2 and -1. Ten SCI subjects participated in an 18-month program of functional electrical stimulation (FES) of their leg muscles. Needle biopsies were taken from the vastus lateralis muscle before and at various times during the training period, and from able-bodied controls. Type IV collagen concentration was unaltered. ProMMP-2 level of SCI subjects before the training period tended to be higher than able-bodied controls and was significantly above the control level after FES. MMP-9 concentration was unchanged. The results suggest accelerated type IV collagen turnover in skeletal muscle of SCI individuals especially after FES as a part of adaptive process of the muscle.

Skeletal and cardiac muscle protein turnover during cold acclimation in young rats.

The effect of long-term cold exposure on skeletal and cardiac muscle protein turnover was investigated in young growing animals. Two groups of 36 male 28-day-old rats were maintained at either 5 degrees C (cold) or 25 degrees C (control). Rates of protein synthesis and degradation were measured in vivo on days 5, 10, 15, and 20. Protein mass by day 20 was approximately 28% lower in skeletal muscle (gastrocnemius and soleus) and approximately 24% higher in heart in cold compared with control rats (P < 0.05). In skeletal muscle, the fractional rates of protein synthesis (k(syn)) and degradation (k(deg)) were not significantly different between cold and control rats, although k(syn) was lower (approximately -26%) in cold rats on day 5; consequent to the lower protein mass, the absolute rates of protein synthesis (approximately -21%; P < 0. 05) and degradation (approximately -13%; P < 0.1) were lower in cold compared with control rats. In heart, overall, k(syn) (approximately +12%; P < 0.1) and k(deg) (approximately +22%; P < 0.05) were higher in cold compared with control rats; consequently, the absolute rates of synthesis (approximately +44%) and degradation (approximately +54%) were higher in cold compared with control rats (P < 0.05). Plasma triiodothyronine concentration was higher (P < 0.05) in cold compared with control rats. These data indicate that long-term cold acclimation in skeletal muscle is associated with the establishment of a new homeostasis in protein turnover with decreased protein mass and normal fractional rates of protein turnover. In heart, unlike skeletal muscle, rates of protein turnover did not appear to immediately return to normal as increased rates of protein turnover were observed beyond day 5. These data also indicate that increased rates of protein turnover in skeletal muscle are unlikely to contribute to increased metabolic heat production during cold acclimation.

Mechanical strain modulates maximal phosphatidylinositol turnover in airway smooth muscle.

Mechanical strain regulates the maximal level of myosin light chain phosphorylation mediated by muscarinic activation in airway smooth muscle. Accordingly, we tested the hypothesis that mechanical strain regulates maximal phosphatidylinositol (PI) turnover (V(max)) coupled to muscarinic receptors in bovine tracheal smooth muscle. We found that PI turnover was not significantly length dependent in unstimulated tissues. However, carbachol-induced PI turnover was linearly dependent on muscle length at both 1 and 100 microM. The observed linear length dependence of PI turnover at maximal carbachol concentration (100 microM) suggests that mechanical strain regulates V(max). When carbachol concentration-PI turnover relationships were measured at optimal length and at 20% optimal length, the results could be explained by changes in V(max) alone. To determine whether the length-dependent step is upstream from heterotrimeric G proteins, we investigated the length dependence of fluoroaluminate-induced PI turnover. The results indicate that fluoroaluminate-induced PI turnover remained significantly length dependent at maximal concentration. These findings together suggest that regulating functional units of G proteins and/or phospholipase C enzymes may be the primary mechanism of mechanosensitive modulation in airway smooth muscle.

Effects of troglitazone on substrate storage and utilization in insulin-resistant rats.

Elevated serum and tissue lipid stores are associated with skeletal muscle insulin resistance and diminished glucose-stimulated insulin secretion, the hallmarks of type 2 diabetes. We studied the effects of 6-wk treatment with the insulin sensitizer troglitazone on substrate storage and utilization in lean control and Zucker diabetic fatty (ZDF) rats. Troglitazone prevented development of diabetes and lowered serum triglycerides (TG) in ZDF rats. Soleus muscle glycogen and TG content were elevated twofold in untreated ZDF rats, and both were normalized by troglitazone to lean control levels (P < 0.05). Troglitazone also normalized insulin-stimulated glucose uptake as well as basal and insulin-stimulated glycogen synthesis, implying increased skeletal muscle glycogen turnover. The proportion of active pyruvate dehydrogenase (PDH) in soleus muscle was reduced in ZDF relative to lean control rat muscle (16 +/- 2 vs. 21 +/- 2%) but was restored by troglitazone treatment (30 +/- 3%). Increased PDH activation was associated with a 70% increase in glucose oxidation. Muscle lipoprotein lipase activity was decreased by 35% in ZDF compared with lean control rats and was increased twofold by troglitazone. Palmitate oxidation and incorporation into TG were higher in ZDF relative to lean control rats but were unaffected by troglitazone treatment. Troglitazone decreased the incorporation of glucose into the acyl group of TG by 60% in ZDF rats. In summary, ZDF rats demonstrate increased skeletal muscle glycogen and TG stores, both of which were reduced by troglitazone treatment. Troglitazone appears to increase both glycogen and TG turnover in skeletal muscle. Normalization of PDH activity and decreased glucose incorporation into acyl TG may underlie the improvements in intracellular substrate utilization and energy stores, which lead to decreased serum TG and glucose.

Role of micronutrients in sport and physical activity.

Many micronutrients play key roles in energy metabolism and, during strenuous physical activity, the rate of energy turnover in skeletal muscle may be increased up to 20-100 times the resting rate. Although an adequate vitamin and mineral status is essential for normal health, marginal deficiency states may only be apparent when the metabolic rate is high. Prolonged strenuous exercise performed on a regular basis may also result in increased losses from the body or in an increased rate of turnover, resulting in the need for an increased dietary intake. An increased food intake to meet energy requirements will increase dietary micronutrient intake, but athletes in hard training may need to pay particular attention to their intake of iron, calcium and the antioxidant vitamins.

Nutritional and hormonal control of muscle and peripheral tissue metabolism in farm species

Alterations in the rate of protein turnover in muscle have marked effects on production efficiency, environmental pollution and perhaps also meat quality. Nutrient and hormonal responses produce differential changes in protein synthesis and degradation; these determine the quality of the end product obtained. Breed selection appears, from limited data, to “protect” the selected characteristic (e.g., muscle gain, wool growth) against nutritional deprivation through maintenance of protein synthesis rates. At intakes below maintenance, the anti-catabolic effects of nutrients appear to operate mainly through inhibition of protein breakdown. In all but the very young animals, this probably involves the anti-proteolytic action of insulin. In contrast, general increases in either protein or total nutrient supply stimulate both protein synthesis and degradation in muscle at intakes above maintenance. These responses are similar to the chronic mechanisms of action of growth hormone (GH) while the majority of acute events reported for insulin-like growth factor-1 (IGF-1) appear to involve primarily changes in protein synthesis. Current data favours IGF-1 action on muscle via an endocrine rather than autocrine (or paracrine) mechanism. Increased protein or energy supply alters the efficiency with which protein is synthesised per unit of cellular RNA, while the GH response involves increases in total RNA but no change in synthetic efficiency. A biphasic model involving insulin and the GH/IGF-1 axis in regulation of nutrient responses is proposed. Muscle sensitivity and responsiveness to insulin, GH and IGF-1 may be regulated by the availability of both total and specific amino acids. Insulin action is specifically influenced by the branch chain amino acids, notably leucine, which may act as signals of nutrient availability to peripheral tissues. The role of individual amino acids in GH and IGF-1 responsiveness is not yet known. Other amino acids, notably those transported into cells by Na +-dependent systems, e.g., glutamine, may also directly effect protein turnover, probably by mechanisms involving changes in cellular hydration.

Role of calpain in skeletal-muscle protein degradation.

Although protein degradation is enhanced in muscle-wasting conditions and limits the rate of muscle growth in domestic animals, the proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The goals of this study were to evaluate the roles of the calpains (calcium-activated cysteine proteases) in mediating muscle protein degradation and the extent to which these proteases participate in protein turnover in muscle. Two strategies to regulate intracellular calpain activities were developed: overexpression of dominant-negative m-calpain and overexpression of calpastatin inhibitory domain. To express these constructs, L8 myoblast cell lines were transfected with LacSwitch plasmids, which allowed for isopropyl beta-D-thiogalactoside-dependent expression of the gene of interest. Inhibition of calpain stabilized fodrin, a well characterized calpain substrate. Under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain reduced protein degradation by 30%, whereas calpastatin inhibitory domain expression reduced degradation by 63%. Inhibition of calpain also stabilized nebulin. These observations indicate that calpains play key roles in the disassembly of sarcomeric proteins. Inhibition of calpain activity may have therapeutic value in treatment of muscle-wasting conditions and may enhance muscle growth in domestic animals.

Glycogen turnover in skeletal muscle is stimulated along with glucose uptake in vivo during contraction

We have shown previously that glycogen synthesis in the heart can be stimulated in vivo by epinephrine. Our aim in this study was to determine whether glycogen synthesis in skeletal muscle can be similarly affected during increased energy expenditure. Left sciatic nerves of anesthetized fasted rats were electrically stimulated to allow left hindlimb muscles to contract for 5, 10, and 20 min. Glycogen contents in the contracting muscles at the end of electrical stimulation were found to be ~40% less than resting muscles in the right hindlimbs in all three groups of rats. Accompanying the enhanced glycogenolysis was increased incorporation of the intravenously infused [3- 3H]-glucose into glycogen. The rate of tritium incorporation into glycogen in the contracting muscle was found to be 34-fold greater than resting muscles. Glucose utilization was determined by the phosphorylation of the intravenously injected [ 14C]-2-deoxyglucose in the skeletal muscle. The rate of accumulation of [ 14C]-2-deoxyglucose-6-phosphate in the contracting muscles was found to be 28-fold greater than resting muscles. Glycogen synthesis and glucose uptake indexes, calculated by dividing the radioactivity in [ 3H]-glycogen and [ 14C]-2-DGP by the mean specific activity of their respective precursors in the plasma, were not found to be significantly different in the contracting muscle. In conclusion, our data indicate that: (i) glycogenesis and glycogenolysis can be stimulated concurrently in the skeletal muscle; and (ii) glucose utilization in the skeletal muscle during contraction may be mediated through glycogen turnover.

Protein turnover in skeletal muscle of the diabetic rat: activation of ubiquitin-dependent proteolysis.

Induction of experimental insulin-deficiency by a single administration of streptozotocin to rats resulted in substantial changes in heart and skeletal muscle size and protein content. This was accompanied by a marked loss of total body (carcass) nitrogen and raised concentrations of circulating branched-chain amino acids. These changes were related to alterations in protein turnover in skeletal muscle. Thus, the diabetic animals showed changes in both the fractional protein rates of synthesis (decreased by 37%) and degradation (increased by 141%). The increased protein degradation observed in the muscle of the diabetic animals was associated only with an increase in the expression of the genes controlling ubiquitin-dependent proteolysis. It may be suggested that the hormonal changes associated with the diabetic state play an important role in the regulation of the activity of the ubiquitin-dependent proteolytic system in skeletal muscle, highlighting the major role of this system in the diabetes-related cachexia.

Signalling Pathways Regulating Protein Turnover in Skeletal Muscle

The protein content of skeletal muscle is determined by the relative rates of synthesis and degradation which must be regulated coordinately to maintain equilibrium. However, in conditions such as fasting where amino acids are required for gluconeogenesis, or in cancer cachexia, this equilibrium is disrupted and a net loss of protein ensues. This review, utilising studies performed in several situations, summarizes the current state of knowledge on the possible signalling pathways regulating protein turnover in skeletal muscle and highlights areas for future work.