Effects of diabetes on protein synthesis in fast- and slow-twitch rat skeletal muscle

The effects of acute (2-day) and long-term (7-day) diabetes on rates of protein synthesis, peptide-chain initiation, and levels of RNA were examined in rat skeletal muscles that are known to have differing proportions of the three fiber types: fast-twitch white, fast-twitch red, and slow-twitch red. Short-term diabetes resulted in a 15% reduction in the level of RNA in all the muscles studied and an impairment in peptide-chain initiation in muscles with mixed fast-twitch fibers. In contrast, the soleus, a skeletal muscle with high proportions of slow-twitch red fibers, showed little impairment in initiation. When the muscles were perfused as a part of the hemicorpus preparation, addition of insulin to the medium caused a rapid reversal of the block in initiation in mixed fast-twitch muscles but had no effect in the soleus. The possible role of fatty acids in accounting for these differences is discussed. Long-term diabetes caused no further reduction in RNA, but resulted in the development of an additional impairment to protein synthesis that also affected the soleus and that was not corrected by perfusion with insulin. The defect resulting from long-term diabetes may involve elongation or termination reactions.     

Effects of chronic sympathectomy on muscle fibre composition ISO-myosin patterns, protein synthesis and calcium content in canine gracilis muscle

We have previously reported functional and histological studies in five beagle dogs with unilateral lumbar sympathectomy. Three months later, fatiguability in the gracilis muscles was increased on the denervated sides, and this was associated with an increase in the relative distribution of FT fibres. Biochemical studies now show that these changes were associated with an increase in cytosolic protein without change in DNA content; this is consistent with an increase in cell size. There was a reduction in the proportion of slow myosin light chain isoforms from 50 +/- 7 to 34 +/- 6%. Noradrenaline levels were increased on the denervated sides but this may reflect greater vascularity. Calcium content did not correlate with fibre type but there was a positive relation with both noradrenaline content (r = 0.73; P less than 0.05) and DNA content (r = 0.84; P less than 0.05). It is concluded that sympathectomy induces several biochemical changes in skeletal muscle which constitute a change and increase in fast myosin light chain synthesis and a corresponding fibre type transformation.     

The effect of unloading on protein synthesis in human skeletal muscle

Atrophy of skeletal muscle is observed in response to immobilization and lack of weight-bearing. The aim of this study was to investigate the effect of immobilization on muscle protein synthesis and associated biochemical parameters in skeletal muscle of healthy volunteers employing a standardized model of lower limb unloading. One leg was unloaded for 10 days, and percutaneous muscle biopsies were taken before and at the end of the unloading period. The capacity for protein synthesis, as reflected by the concentration of RNA, decreased by 16% (P < 0.05) although the fractional synthesis rate (FSR) of protein was not significantly changed after 10 days of unloading. Furthermore there was an increase in the concentration of the free branched chain amino acids in muscle by 48% (P < 0.05).     

Effects of contraction and insulin on protein synthesis, AMP-activated protein kinase and phosphorylation state of translation factors in rat skeletal muscle

In rat epitrochlearis skeletal muscle, contraction inhibited the basal and insulin-stimulated rates of protein synthesis by 75 and 70%, respectively, while increasing adenosine monophosphate-activated protein kinase (AMPK) activity. Insulin, on the other hand, stimulated protein synthesis (by 30%) and increased p70 ribosomal protein S6 kinase (p70S6K) Thr389, 40S ribosomal protein S6 (rpS6) Ser235/236, rpS6 Ser240/244 and eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) Thr37/46 phosphorylation over basal values. Electrical stimulation had no effect on mammalian target of rapamycin complex 1 (mTORC1) signalling, as reflected by the lack of reduction in basal levels of p70S6K, rpS6 Ser235/236, rpS6 Ser240/244 and 4E-BP1 phosphorylation, but did antagonize mTORC1 signalling after stimulation of the pathway by insulin. Eukaryotic elongation factor-2 (eEF2) Thr56 phosphorylation increased rapidly on electrical stimulation reaching a maximum at 1 min, whereas AMPK Thr172 phosphorylation slowly increased to reach threefold after 30 min. Eukaryotic elongation factor-2 kinase (eEF2K) was not activated after 30 min of contraction when AMPK was activated. This could not be explained by the expression of a tissue-specific isoform of eEF2K in skeletal muscle lacking the Ser398 AMPK phosphorylation site. Therefore, in this skeletal muscle system, the contraction-induced inhibition of protein synthesis could not be attributed to a reduction in mTORC1 signalling but could be due to an increase in eEF2 phosphorylation independent of AMPK activation.     

Effects of thyroxine on protein turnover in rat skeletal muscle

The effects of thyroxine (T4) on protein turnover in skeletal muscle were studied using normal, thyroidectomized (thyrex), and hypophysectomized (hypox) rats. Thyrex rats had a depressed growth rate that was accompanied by 50% reductions in the level of RNA and the rate of protein synthesis in gastrocnemius muscle, as determined in the perfused hemicorpus. Protein synthetic efficiency (protein synthesis per unit RNA) was decreased by 18%. Daily treatment of thyrex rats with T4 at different dose levels for up to 16 days led to improved growth rates, elevated RNA concentrations, and increased protein synthesis rates. The primary effect of T4 was to increase the protein synthetic capacity of muscle. Protein degradation, determined in the perfused hemicorpus, and activity of a lysosomal protease, determined in unperfused muscle, were reduced in the thyrex condition. Treatment of thyrex rats with T4 increased protein degradative rates, but not protease activity. Hypox rats, which also exhibited depressed skeletal muscle protein synthesis, responded to T4 and combined T4 and growth hormone with marked improvements in protein synthesis.     

Nutrient signalling in the regulation of human muscle protein synthesis

The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are important nutrient- and energy-sensing and signalling proteins in skeletal muscle. AMPK activation decreases muscle protein synthesis by inhibiting mTOR signalling to regulatory proteins associated with translation initiation and elongation. On the other hand, essential amino acids (leucine in particular) and insulin stimulate mTOR signalling and protein synthesis. We hypothesized that anabolic nutrients would be sensed by both AMPK and mTOR, resulting in an acute and potent stimulation of human skeletal muscle protein synthesis via enhanced translation initiation and elongation.
We measured muscle protein synthesis and mTOR-associated upstream and downstream signalling proteins in young male subjects ( n = 14) using stable isotopic and immunoblotting techniques. Following a first muscle biopsy, subjects in the 'Nutrition' group ingested a leucine-enriched essential amino acid–carbohydrate mixture (EAC). Subjects in the Control group did not consume nutrients. A second biopsy was obtained 1 h later. Ingestion of EAC significantly increased muscle protein synthesis, modestly reduced AMPK phosphorylation, and increased Akt/PKB (protein kinase B) and mTOR phosphorylation ( P < 0.05). mTOR signalling to its downstream effectors (S6 kinase 1 (S6K1) and 4E-binding protein 1 (4E-BP1) phosphorylation status) was also increased ( P < 0.05). In addition, eukaryotic elongation factor 2 (eEF2) phosphorylation was significantly reduced ( P < 0.05). Protein synthesis and cell signalling (phosphorylation status) was unchanged in the control group ( P > 0.05).
We conclude that anabolic nutrients alter the phosphorylation status of both AMPK- and mTOR-associated signalling proteins in human muscle, in association with an increase in protein synthesis not only via enhanced translation initiation but also through signalling promoting translation elongation.     

Effect of starvation on initiation of protein synthesis in skeletal muscle and heart

Psoas muscle of rats starved for 2 or 4 days contained increased levels of ribosomal subunits and exhibited reduced rates of protein synthesis in vitro, demonstrating a starvation-induced inhibition of peptide-chain initiation. The activity of an eIF-2-like initiation factor, assayed in postribosomal supernatants, decreased in psoas during starvation, parallel to a 25% reduction in the RNA level. Reduced eIF-2 activity did not result from nucleotide depletion or increased deacylation of initiator tRNA, nor was it abolished by extensive dialysis. Perfusion of psoas muscle in the presence of insulin reversed the starvation-induced block in peptide-chain initiation, but did not alter the activity of eIF-2 or level of RNA. Furthermore, heart muscle did not manifest a starvation-induced block in peptide-chain initiation even though the activity of eIF-2 and the level of RNA decreased as a result of food deprivation. Thus loss of eIF 2 activity in psoas and heart did not parallel changes in peptide-chain initiation but was associated with a reduction in tissue RNA. These results indicate that the level of eIF-2 is not rate-limiting for peptide-chain initiation under the conditions tested in this study.     

The effects of acute passive stretch on muscle protein synthesis in humans.

We examined the effect of an isolated bout of maximal tolerated passive stretch on fractional muscle protein synthetic rate in human soleus muscle. Eight healthy males performed two separate trials with the same leg: one session of passive stretch and one of intermittent active isometric contraction at a force equivalent to that which occurred during the passive stretch trial. This force was approximately 40% of maximum voluntary contraction force and produced volitional fatigue in approximately 27 min. Intermittent passive stretch, for the same duration, elicited a 6.1 degrees increase in joint angle (P<.0005) with silent electromyography. Fractional protein synthetic rate from experimental and control soleus in each trial was assessed from biopsy samples over the period 10-22 hr postexercise by the incorporation rate of L-[1-13C] leucine into muscle. Protein synthesis was elevated in the soleus of the exercised leg following the active contraction trial by 49% (P<.05) but not following the passive stretch trial. Results indicate that a single bout of maximal passive stretch does not significantly elevate fractional muscle protein synthetic rate in humans and thus suggests that muscle stretch per se is not the stimulus for the muscle hypertrophy that occurs with resistance training.     

Human soleus muscle protein synthesis following resistance exercise

AIM: It is generally believed the calf muscles in humans are relatively unresponsive to resistance training when compared with other muscles of the body. The purpose of this investigation was to determine the muscle protein synthesis response of the soleus muscle following a standard high intensity bout of resistance exercise. METHODS: Eight recreationally active males (27 +/- 4 years) completed three unilateral calf muscle exercises: standing calf press/heel raise, bent-knee calf press/heel raise, and seated calf press/heel raise. Each exercise consisted of four sets of 15 repetitions (approximately 15 repetition maximum, RM, or approximately 70% 1RM). Fractional rate of muscle protein synthesis (FSR) was determined with a primed constant infusion of [2H5]phenylalanine coupled with muscle biopsies immediately and 3 h following the exercise in both the exercise and non-exercise (resting control) leg. RESULTS: FSR was elevated (P < 0.05) in the exercise (0.069 +/- 0.010) vs. the control (0.051 +/- 0.012) leg. Muscle glycogen concentration was lower (P < 0.05) in the exercise compared with the control leg (Decrease from control; immediate post-exercise: 54 +/- 5; 3 h post-exercise: 36 +/- 4 mmol kg(-1) wet wt.). This relatively high amount of glycogen use is comparable with previous studies of resistance exercise of the thigh (i.e. vastus lateralis; approximately 41-49 mmol kg(-1) wet wt.). However, the exercise-induced increase in FSR that has been consistently reported for the vastus lateralis (approximately 0.045-0.060% h(-1)) is on average approximately 200% higher than reported here for the soleus (0.019 +/- 0.003% h(-1)). CONCLUSIONS: These results suggest the relatively poor response of soleus muscle protein synthesis to an acute bout of resistance exercise may be the basis for the relative inability of the calf muscles to respond to resistance training programs.     

Effects of starvation and diabetes on protein synthesis in lung.

Metabolism of lung proteins was investigated in rats starved 3 days or made diabetic with streptozotocin. Body weight was below normal in both groups, but lung weight decreased only in starved animals. Total lung protein and RNA (mg/lung) decreased during starvation and diabetes. Protein concentration (mg/g) was unchanged in either group of animals; RNA concentration decreased only during starvation. Protein synthesis, estimated in lungs perfused in situ, was reduced 22% in starvation, but remained unchanged in diabetes. Inhibition of protein synthesis was accounted for by loss of RNA. Ribosomal profiles were unchanged by starvation, suggesting an unaltered relationship between rates of peptide-chain initiation and elongation in vivo. Activity of an eIF-2-like initiation factor decreased during starvation in proportion to the loss of RNA. In diabetes, factor activity remained normal. Thus, starvation but not streptozotocin-induced diabetes, reduced the capacity of the lung to synthesize protein. No evidence for reduced efficiency of synthesis was observed.     

Time course of changes in protein synthesis in marcaine-induced skeletal muscle regeneration

The time course of the regeneration of rat skeletal muscle has been examined after injection of the myotoxic drug, Marcaine, to induce regeneration. Muscle wet weight decreases during the initial phase of the regeneration process while the ability of the regenerating muscle to incorporate [35S]methionine into protein, the yield and activity of muscle polysomes and the yield of total and poly(A) + RNA all increase initially. Following the initial changes, these parameters return to near control values by 30 days after Marcaine injection. Theoretical calculations suggest that the changes in polysome yield and activity are sufficient to account for the changes in the ability of muscle fragments to synthesize protein during the regeneration process. The specific activity of total muscle RNA in the wheat germ cell-free system decreases initially during the early stages of the regeneration process. This decrease may reflect the fact that while the yields of both total and poly(A) + RNA increase during the early stages of regeneration, the percentage of the total RNA which is poly(A) + decreases initially.     

Mechano-transduction to muscle protein synthesis is modulated by FAK

We examined the involvement of focal adhesion kinase (FAK) in mechano-regulated signalling to protein synthesis by combining muscle-targeted transgenesis with a physiological model for un- and reloading of hindlimbs. Transfections of mouse tibialis anterior muscle with a FAK expression construct increased FAK protein 1.6-fold versus empty transfection in the contralateral leg and elevated FAK concentration at the sarcolemma. Altered activation status of phosphotransfer enzymes and downstream translation factors showed that FAK overexpression was functionally important. FAK auto-phosphorylation on Y397 was enhanced between 1 and 6 h of reloading and preceded the activation of p70S6K after 24 h of reloading. Akt and translation initiation factors 4E-BP1 and 2A, which reside up- or downstream of p70S6K, respectively, showed no FAK-modulated regulation. The findings identify FAK as an upstream element of the mechano-sensory pathway of p70S6K activation whose Akt-independent regulation intervenes in control of muscle mass by mechanical stimuli in humans.     

Influence of anabolic agents on protein synthesis and degradation in muscle cells grown in culture

Muscle cell culture (L6) studies were conducted to determine whether anabolic agents have a direct effect on the muscle cell. The effects of zeranol, testosterone propionate, estradiol benzoate, progesterone, dexamethasone and anabolic agent-dexamethasone combinations on protein synthesis and degradation were measured. Myoblast and myotube cultures were pretreated with 1 microM compounds for 12, 24 and 48 h before a 6-h synthesis or degradation measuring period. Protein synthesis was determined as cpm of [3H] leucine incorporated per mg cell protein. Protein degradation was measured by a pulse-chase procedure using [3H] leucine and expressed as the percentage labeled protein degraded in 6 h. Progesterone slightly increased (P less than .05) protein synthesis in myoblast cultures. Testosterone propionate had no effect on synthesis. Protein synthesis was decreased by estradiol benzoate (P less than .01) in myotube cultures. Protein degradation was not altered appreciably by anabolic agents. Protein synthesis was initially inhibited in myotubes (P less than .05) by dexamethasone, but increased (P less than .01) in myoblasts and myotubes in the extended incubation time. Dexamethasone also consistently increased protein degradation, but this required several hours to be expressed. Anabolic agents did not interfere with dexamethasone-induced increases in protein synthesis and degradation. The magnitude of response and sensitivity were similar for both the myoblast and the more fully differentiated myotube for all compounds tested. These results indicate that anabolic agents at the 1 microM level do not have a direct anabolic effect on muscle or alter glucocorticoid-induced catabolic response in muscle.     

Inhibition of denervation changes in skeletal muscle by blockers of protein synthesis

1. A study was made of the effects of inhibitors of protein synthesis (actinomycin D, chloramphenicol and cycloheximide) on the in vivo development of extrajunctional cholinergic receptors, tetrodotoxin-resistant action potentials, slowing of the time course of the action potential, and on the fall in the resting membrane potential in denervated mouse skeletal muscle.2. Actinomycin D (0.5 mg/kg, I.P.) reduced the incorporation of uridine into skeletal muscle by 50-60% for 4-5 days. There was no simultaneous reduction in L-leucine incorporation.3. Actinomycin D (0.5 mg/kg I.P., 1 day after denervation) inhibited the development of extrajunctional cholinergic receptors, tetrodotoxin-resistant action potentials and the fall in resting membrane potential for approximately 4 days.4. The post-denervation fall in the maximum rate of rise and in the amplitude of the overshoot of the muscle fibre action potential was unaffected by actinomycin D treatment.5. Actinomycin D failed to inhibit the appearance of the membrane changes if given later than 2 days after denervation.6. Chloramphenicol (6 g/kg in divided doses) and cycloheximide (40 g/kg every 12 hr) were also able to inhibit the appearance of the membrane changes.7. It is concluded that some denervation induced changes in the muscle fibre membrane depend on the synthesis of new proteins. The results imply that the motor nerve cell normally exerts a regulatory influence on the genome of the muscle cell.     

Short-term starvation decreases skeletal muscle protein synthesis rate in man.

The rate of protein synthesis in skeletal muscle was determined in the post-absorptive state and after 3 days of starvation in healthy volunteers. The flooding dose technique employing intravenous injection of (1-13C)leucine (0.05 g kg-1) was used and incorporation of isotope into muscle protein was measured by taking percutaneous biopsies at 0 and 90 min. Blood samples were taken during the incorporation period for assessment of the enrichment of the free amino acid precursor of protein synthesis. The median (25,75 quartiles) rate of muscle protein synthesis after an overnight fast was 2.03 (2.00,2.23) % days-1 when the precursor enrichment was obtained by measurement of the plasma alpha-ketoisocaproate, taken to be representative of muscle free leucine. Repeat measurements in the same subjects after 3 days of total starvation showed a decrease to 1.82 (1.57,2.05) % days-1. Rates calculated on the basis of the plasma leucine as precursor were 5% lower at both times. An interindividual variation in response to starvation was observed, but the median decrease of 13% in the rate of protein synthesis was statistically significant (P less than 0.01).     

Insulin action on rates of muscle protein synthesis following eccentric, muscle-damaging contractions

The purpose of this study was to determine whether eccentric, muscle-damaging contractions affect insulin action on muscle protein synthesis. Male Wistar rats (n = 28) were anaesthetized either once or twice separated by 7 days' rest, and one limb was electrically stimulated to contract eccentrically, while the contralateral limb served as a non-stimulated control. Twenty-four and 48 h after contractions, rates of protein synthesis were assessed in soleus and red or white gastrocnemius muscles during a hindlimb perfusion with or without insulin (20 000 microU mL(-1)). Rates of protein synthesis were not different in non-stimulated muscle, with or without insulin (P > 0.05). In red or white gastrocnemius without insulin, rates of protein synthesis were significantly reduced (P < 0.05) 24 and 48 h after a single session and 48 h after a double session of muscle contractions. However, protein synthesis was normalized with insulin 24 and 48 h after contractions in red, and 48 h after contractions in white gastrocnemius. In soleus muscle, protein synthesis was impaired only 48 h after the second session, but partially restored by insulin (P < 0.05). These results indicate that muscle becomes more sensitive to insulin action on rates of protein synthesis after muscle-damaging contractions.