Preexercise aminoacidemia and muscle protein synthesis after resistance exercise

PURPOSE We have previously shown that the aminoacidemia caused by the consumption of a rapidly digested protein after resistance exercise enhances muscle protein synthesis (MPS) more than the amino acid (AA) profile associated with a slowly digested protein. Here, we investigated whether differential feeding patterns of a whey protein mixture commencing before exercise affect postexercise intracellular signaling and MPS. METHODS Twelve resistance-trained males performed leg resistance exercise 45 min after commencing each of three volume-matched nutrition protocols: placebo (PLAC, artificially sweetened water), BOLUS (25 g of whey protein + 5 g of leucine dissolved in artificially sweetened water; 1× 500 mL), or PULSE (15× 33-mL aliquots of BOLUS drink every 15 min). RESULTS The preexercise rise in plasma AA concentration with PULSE was attenuated compared with BOLUS (P < 0.05); this effect was reversed after exercise, with two-fold greater leucine concentrations in PULSE compared with BOLUS (P < 0.05). One-hour postexercise, phosphorylation of p70 S6K and rpS6 was increased above baseline with BOLUS and PULSE, but not PLAC (P < 0.05); furthermore, PULSE > BOLUS (P < 0.05). MPS throughout 5 h of recovery was higher with protein ingestion compared with PLAC (0.037 ± 0.007), with no differences between BOLUS or PULSE (0.085 ± 0.013 vs. 0.095 ± 0.010%•h, respectively, P = 0.56). CONCLUSIONS Manipulation of aminoacidemia before resistance exercise via different patterns of intake of protein altered plasma AA profiles and postexercise intracellular signaling. However, there was no difference in the enhancement of the muscle protein synthetic response after exercise. Protein sources producing a slow AA release, when consumed before resistance exercise in sufficient amounts, are as effective as rapidly digested proteins in promoting postexercise MPS.

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state. J Appl Physiol 112: 1805-1813, 2012. First published March 1, 2012; doi:10.1152/japplphysiol.00170.2012.- We made sex-based comparisons of rates of myofibrillar protein synthesis (MPS) and anabolic signaling after a single bout of high-intensity resistance exercise. Eight men (20 ± 10 yr, BMI = 24.3 ± 2.4) and eight women (22 ± 1.8 yr, BMI = 23.0 ± 1.9) underwent primed constant infusions of L-[ring-13C6]phenylalanine on consecutive days with serial muscle biopsies. Biopsies were taken from the vastus lateralis at rest and 1, 3, 5, 24, 26, and 28 h after exercise. Twenty-five grams of whey protein was ingested immediately and 26 h after exercise. We also measured exercise-induced serum testosterone because it is purported to contribute to increases in myofibrillar protein synthesis (MPS) postexercise and its absence has been hypothesized to attenuate adaptative responses to resistance exercise in women. The exercise-induced area under the testosterone curve was 45-fold greater in men than women in the early (1 h) recovery period following exercise (P < 0.001). MPS was elevated similarly in men and women (2.3- and 2.7-fold, respectively) 1-5 h postexercise and after protein ingestion following 24 h recovery. Phosphorylation of mTORSer2448 was elevated to a greater extent in men than women acutely after exercise (P = 0.003), whereas increased phosphorylation of p70S6K1Thr389 was not different between sexes. Androgen receptor content was greater in men (main effect for sex, P = 0.049). Atrogin-1 mRNA abundance was decreased after 5 h recovery in both men and women (P < 0.001), and MuRF-1 expression was elevated in men after protein ingestion following 24 h recovery (P = 0.003). These results demonstrate minor sex-based differences in signaling responses and no difference in the MPS response to resistance exercise in the fed state. Interestingly, our data demonstrate that exerciseinduced increases in MPS are dissociated from postexercise testosteronemia and that stimulation of MPS occurs effectively with low systemic testosterone concentrations in women.

Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise

Background: Ingestion of whey or casein yields divergent patterns of aminoacidemia that influence whole-body and skeletal muscle myofibrillar protein synthesis (MPS) after exercise. Direct comparisons of the effects of contrasting absorption rates exhibited by these proteins are confounded by their differing amino acid contents. Objective: Our objective was to determine the effect of divergent aminoacidemia by manipulating ingestion patterns of whey protein alone on MPS and anabolic signaling after resistance exercise. Design: In separate trials, 8 healthy men consumed whey protein either as a single bolus (BOLUS; 25-g dose) or as repeated, small, "pulsed" drinks (PULSE; ten 2.5-g drinks every 20 min) to mimic a more slowly digested protein. MPS and phosphorylation of signaling proteins involved in protein synthesis were measured at rest and after resistance exercise. Results: BOLUS increased blood essential amino acid (EAA) concentrations above those of PULSE (162% compared with 53%, P < 0.001) 60 min after exercise, whereas PULSE resulted in a smaller but sustained increase in aminoacidemia that remained elevated above BOLUS amounts later (180-220 min after exercise, P < 0.05). Despite an identical net area under the EAA curve, MPS was elevated to a greater extent after BOLUS than after PULSE early (1-3 h: 95% compared with 42%) and later (3-5 h: 193% compared with 121%) (both P < 0.05). There were greater changes in the phosphorylation of the Akt-mammalian target of rapamycin pathway after BOLUS than after PULSE. Conclusions: Rapid aminoacidemia in the postexercise period enhances MPS and anabolic signaling to a greater extent than an identical amount of protein fed in small pulses that mimic a more slowly digested protein. A pronounced peak aminoacidemia after exercise enhances protein synthesis.

Nutrient provision increases signalling and protein synthesis in human skeletal muscle after repeated sprints

The effect of nutrient availability on the acute molecular responses following repeated sprint exercise is unknown. The aim of this study was to determine skeletal muscle cellular and protein synthetic responses following repeated sprint exercise with nutrient provision. Eight healthy young male subjects undertook two sprint cycling sessions (10 × 6 s, 0.75 N m torque kg -1, 54 s recovery) with either pre-exercise nutrient (24 g whey, 4.8 g leucine, 50 g maltodextrin) or non-caloric placebo ingestion. Muscle biopsies were taken from vastus lateralis at rest, and after 15 and 240 min post-exercise recovery to determine muscle cell signalling responses and protein synthesis by primed constant infusion of L-[ring- 13C 6] phenylalanine. Peak and mean power outputs were similar between nutrient and placebo trials. Post-exercise myofibrillar protein synthetic rate was greater with nutrient ingestion compared with placebo ( ? 48%, P<0.05) but the rate of mitochondrial protein synthesis was similar between treatments. The increased myofibrillar protein synthesis following sprints with nutrient ingestion was associated with coordinated increases in Akt-mTOR-S6KrpS6 phosphorylation 15 min post-exercise (?200-600%, P<0.05), while there was no effect on these signalling molecules when exercise was undertaken in the fasted state. For the first time we report a beneficial effect of nutrient provision on anabolic signalling and muscle myofibrillar protein synthesis following repeated sprint exercise. Ingestion of protein/carbohydrate in close proximity to high-intensity sprint exercise provides an environment that increases cell signalling and protein synthesis.

Alcohol ingestion impairs maximal post-exercise rates of myofibrillar protein synthesis following a single bout of concurrent training

Introduction The culture in many team sports involves consumption of large amounts of alcohol after training/competition. The effect of such a practice on recovery processes underlying protein turnover in human skeletal muscle are unknown. We determined the effect of alcohol intake on rates of myofibrillar protein synthesis (MPS) following strenuous exercise with carbohydrate (CHO) or protein ingestion. Methods In a randomized cross-over design, 8 physically active males completed three experimental trials comprising resistance exercise (8×5 reps leg extension, 80% 1 repetition maximum) followed by continuous (30 min, 63% peak power output (PPO)) and high intensity interval (10×30 s, 110% PPO) cycling. Immediately, and 4 h post-exercise, subjects consumed either 500 mL of whey protein (25 g; PRO), alcohol (1.5 g·kg body mass−1, 12±2 standard drinks) co-ingested with protein (ALC-PRO), or an energy-matched quantity of carbohydrate also with alcohol (25 g maltodextrin; ALC-CHO). Subjects also consumed a CHO meal (1.5 g CHO·kg body mass−1) 2 h post-exercise. Muscle biopsies were taken at rest, 2 and 8 h post-exercise. Results Blood alcohol concentration was elevated above baseline with ALC-CHO and ALC-PRO throughout recovery (P<0.05). Phosphorylation of mTORSer2448 2 h after exercise was higher with PRO compared to ALC-PRO and ALC-CHO (P<0.05), while p70S6K phosphorylation was higher 2 h post-exercise with ALC-PRO and PRO compared to ALC-CHO (P<0.05). Rates of MPS increased above rest for all conditions (~29–109%, P<0.05). However, compared to PRO, there was a hierarchical reduction in MPS with ALC-PRO (24%, P<0.05) and with ALC-CHO (37%, P<0.05). Conclusion We provide novel data demonstrating that alcohol consumption reduces rates of MPS following a bout of concurrent exercise, even when co-ingested with protein. We conclude that alcohol ingestion suppresses the anabolic response in skeletal muscle and may therefore impair recovery and adaptation to training and/or subsequent performance.

Protein ingestion increases myofibrillar protein synthesis after concurrent exercise

PURPOSE: We determined the effect of protein supplementation on anabolic signaling and rates of myofibrillar and mitochondrial protein synthesis after a single bout of concurrent training. METHODS: Using a randomized cross-over design, 8 healthy males were assigned to experimental trials consisting of resistance exercise (8 × 5 leg extension, 80% 1-RM) followed by cycling (30 min at ~70% VO2peak) with either post-exercise protein (PRO: 25 g whey protein) or placebo (PLA) ingestion. Muscle biopsies were obtained at rest, 1 and 4 h post-exercise. RESULTS: Akt and mTOR phosphorylation increased 1 h after exercise with PRO (175-400%, P<0.01) and was different from PLA (150-300%, P<0.001). MuRF1 and Atrogin-1 mRNA were elevated post-exercise but were higher with PLA compared to PRO at 1 h (50-315%, P<0.05), while PGC-1α mRNA increased 4 h post-exercise (620-730%, P<0.001) with no difference between treatments. Post-exercise rates of myofibrillar protein synthesis increased above rest in both trials (75-145%, P <0.05) but were higher with PRO (67%, P<0.05) while mitochondrial protein synthesis did not change from baseline. CONCLUSION: Our results show that a concurrent training session promotes anabolic adaptive responses and increases metabolic/oxidative mRNA expression in skeletal muscle. Protein ingestion after combined resistance and endurance exercise enhances myofibrillar protein synthesis and attenuates markers of muscle catabolism and thus is likely an important nutritional strategy to enhance adaptation responses with concurrent training.

Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit

The myofibrillar protein synthesis (MPS) response to resistance exercise (REX) and protein ingestion during energy deficit (ED) is unknown. We determined, in young men (n=8) and women (n=7), protein signaling, resting post-absorptive MPS during energy balance [EB: 45 kcal∙(kg FFM∙d)-1] and after 5d of ED [30 kcal∙(kg FFM∙d)-1] as well as MPS while in ED after acute REX in the fasted state and with the ingestion of whey protein (15 and 30 g). Post-absorptive rates of MPS were 27% lower in ED than EB (P<0.001), but REX stimulated MPS to rates equal to EB. Ingestion of 15 and 30 g of protein after REX in ED increased MPS ~16 and ~34% above resting EB, (P<0.02). p70 S6Kthr389 phosphorylation increased above EB only with combined exercise and protein intake (~2-7 fold; P<0.05). In conclusion, short-term ED reduces post-absorptive MPS, however, a bout of REX in ED restores MPS to values observed at rest in EB. The ingestion of protein after REX further increases MPS above resting EB in a dose-dependent manner. We conclude that combining REX with increased protein availability after exercise enhances rates of skeletal muscle protein synthesis during short term ED and could, in the long term, preserve muscle mass.

Fluctuations in protein synthesis from a single RNA template: Stochastic kinetics of ribosomes

Proteins are polymerized by cyclic machines called ribosomes, which use their messenger RNA (mRNA) track also as the corresponding template, and the process is called translation. We explore, in depth and detail, the stochastic nature of the translation. We compute various distributions associated with the translation process; one of them-namely, the dwell time distribution-has been measured in recent single-ribosome experiments. The form of the distribution, which fits best with our simulation data, is consistent with that extracted from the experimental data. For our computations, we use a model that captures both the mechanochemistry of each individual ribosome and their steric interactions. We also demonstrate the effects of the sequence inhomogeneities of real genes on the fluctuations and noise in translation. Finally, inspired by recent advances in the experimental techniques of manipulating single ribosomes, we make theoretical predictions on the force-velocity relation for individual ribosomes. In principle, all our predictions can be tested by carrying out in vitro experiments.

3-Amino-1,2,4-triazole is an inhibitor of protein synthesis on mitoribosomes in Neurospora crassa

The effects of the herbicide, 3-amino-1,2,4-triazole, an inhibitor of heme synthesis in rat liver, have been examined in the mold Neurospora crassa. The drug is a potent inhibitor of the growth of the mold and produces biochemical changes identical to those produced by chloramphenicol. 3-Amino-1,2,4-triazole, like chloramphenicol, is a direct and specific inhibitor of protein synthesis on mitoribosomes. A decrease in the levels of mitochondrial proteins which are completely or partly made on mitoribosomes and an accumulation in the levels of mitochondrial proteins of cytosolic origin have been observed. Both drugs depress porphyrin and heme levels, but there is actually an elevation in the levels of δ-aminolevulinate dehydratase, the rate-limiting enzyme of the heme-biosynthetic pathway in Neurospora crassa. In liver the enzyme is present in non-limiting amounts and the levels are depressed under conditions of 3-amino-1,2,4-triazole treatment. In Neurospora crassa the ‘derepression’ of δ-aminolevulinate dehydratase under conditions of 3-amino-1,2,4-triazole or chloramphenicol treatment is only partial because the drugs inhibit protein synthesis on mitoribosomes. It is concluded that an optimal rate of protein synthesis on mitoribosomes is necessary to maintain an adequate rate of heme synthesis.

Stimulation by adenosine 3′,5′-cyclic monophosphate of protein synthesis by adenohypophyseal polyribosomes

Addition of dibutyryl 3′,5′-cyclic AMP to slices of bovine pituitary stimulated incorporation of [3H]leucine into protein, whether or not actinomycin D was present; therefore the influence of 3′,5′-cyclic AMP on protein synthesis by bovine pituitary polysomes was studied. If the cyclic nucleotide was added to the complete protein-synthesizing system (including pH 5.0 enzyme), stimulation of [3H]leucine incorporation occurred only with pH 5.0 enzyme from rat liver; there was no stimulation when homologous enzyme, i.e., from bovine pituitary, was used. Addition of 3′,5′-cyclic AMP to the polysomes, before addition of pH 5.0 enzyme, resulted in stimulation of protein synthesis with either source of enzyme, but stimulation was facilitated to a greater degree, over the range 0.5-2 mM 3′,5′-cyclic AMP, when rat liver was the source. The stimulation of protein synthesis was prevented by the addition of cycloheximide. With rat liver pH 5.0 enzyme the product of hydrolysis of 3′,5′-cyclic AMP was mainly 5′-AMP whereas with pituitary pH 5.0 enzyme there was also dephosphorylation and deamination resulting in production of hypoxanthine and other bases. However, using either source of pH 5.0 enzyme and the complete protein-synthesizing system (i.e., including an ATP-regenerating mechanism) most of the 3H from hydrolysis of [3H]3′,5′-cyclic AMP was incorporated into ATP. The data are seen as compatible with a stimulation by 3′,5′-cyclic AMP of translation by pituitary polysomes; the significance of the importance of the source of pH 5.0 enzyme used in the system is obscure.

Important role of the amino acid attached to tRNA in formylation and in initiation of protein synthesis in Escherichia coli

In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation. This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase. Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine. By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis. Thus, the G4:C69 base pair contributes toward formylation of the tRNA and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recognition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.

The requirements for Ca2+, protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L

We have previously reported that both Ca2+ and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induction of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exogenous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumulation of CACHT mRNA was inhibited by a plasma membrane Ca2+ channel blocker verapamil. Treatment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of staurosporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involvement of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca2+ influx from extracellular space, protein phosphorylation, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation.

Protein phosphatase inhibition assay for detection of diarrhetic shellfish poison in oyster

A method based on protein phosphatase enzyme activity inhibition for the detection of diarrhetic shellfish poison (DSP) was used to analyze the DSP toxicity in three oyster samples. Based on the standard dose-effect curve developed with a series of okadaic acid (OA) standard solutions, the DSP toxicity of the three oyster samples collected were screened, and the results showed that there were no OA and dinophysis toxins ( DTXs) in the samples without hydrolization. However, the OA toxicity could be detected in two of the hydrolyzed samples, and the OA toxicity of the two samples were 1.81 and 1.21 mu g OA eq./kg oyster, respectively.