Differential Responses of Blood Essential Amino Acid Levels Following Ingestion of High-Quality Plant-Based Protein Blends Compared to Whey Protein-A Double-Blind Randomized, Cross-Over, Clinical Trial.

This study assessed the bio-equivalence of high-quality, plant-based protein blends versus Whey Protein Isolate (WPI) in healthy, resistance-trained men. The primary endpoint was incremental area under the curve (iAUC) of blood essential Amino Acids (eAAs) 4 hours after consumption of each product. Maximum concentration (Cmax) and time to maximum concentration (Tmax) of blood leucine were secondary outcomes. Subjects (n = 18) consumed three plant-based protein blends and WPI (control). An analysis of Variance model was used to assess for bio-equivalence of total sum of blood eAA concentrations. The total blood eAA iAUC ratios of the three blends were [90% CI]: #1: 0.66 [0.58-0.76]; #2: 0.71 [0.62-0.82]; #3: 0.60 [0.52-0.69], not completely within the pre-defined equivalence range [0.80-1.25], indicative of 30-40% lower iAUC versus WPI. Leucine Cmax of the three blends was not equivalent to WPI, #1: 0.70 [0.67-0.73]; #2: 0.72 [0.68-0.75]; #3: 0.65 [0.62-0.68], indicative of a 28-35% lower response. Leucine Tmax for two blends were similar to WPI (#1: 0.94 [0.73-1.18]; #2: 1.56 [1.28-1.92]; #3: 1.19 [0.95-1.48]). The plant-based protein blends were not bio-equivalent. However, blood leucine kinetic data across the blends approximately doubled from fasting concentrations, whereas blood Tmax data across two blends were similar to WPI. This suggests evidence of rapid hyperleucinemia, which correlates with a protein's anabolic potential.

Consuming Lower-Protein Nutrition Bars with Added Leucine Elicits Postprandial Changes in Appetite Sensations in Healthy Women.

BACKGROUND: Higher-protein meals (>25 g protein/meal) have been associated with enhanced satiety but the role of amino acids is unclear. Leucine has been proposed to stimulate satiety in rodents but has not been assessed in humans. OBJECTIVE: We assessed the acute effects of lower-protein nutrition bars, enhanced with a leucine peptide (LP), on postprandial appetite sensations in combination with plasma leucine and peptide YY (PYY) in healthy women. METHODS: Utilizing a double-blind randomized crossover design, 40 healthy women [28 ± 7.5 y; body mass index (BMI, in kg/m2): 23.5 ± 2.4] consumed the following isocaloric (180 kcal) pre-loads on 3 separate visits: control bar [9 g protein with 0 g added LP (0-g LP)] or treatment bars [11 g protein with 2 g added LP (2-g LP) or 13 g protein with 3 g added LP (3-g LP)]. Pre- and postprandial hunger, desire to eat, prospective food consumption (PFC), fullness, and plasma leucine were assessed every 30 min for 240 min. Plasma PYY was assessed hourly for 240 min (n = 24). RESULTS: Main effects of time (P < 0.0001) and treatment (P < 0.03) were detected for postprandial hunger, desire to eat, PFC, and fullness. Post hoc analyses revealed that the 2-g and 3-g LP bars elicited greater increases in fullness and greater decreases in PFC compared with 0-g LP (all, P < 0.05) with no differences between the 2-g and 3-g LP bars. The 2-g bar elicited greater decreases in hunger and desire to eat compared with the 0-g LP bar (both, P ≤ 0.01), whereas 3-g LP did not. Appetite incremental areas under the curves (iAUCs) and PYY outcomes were not different between bars. A treatment × time interaction was detected for plasma leucine with increases occurring in a leucine-dose-dependent manner (P < 0.0001). CONCLUSION: Despite the dose-dependent increases in plasma leucine following the consumption of lower-protein bars enhanced with LP, only the 2-g LP bar elicited consistent postprandial changes in select appetite sensations compared with the 0-g LP bar. This study was registered on clinicaltrials.gov as NCT02091570.

Leucine, Not Total Protein, Content of a Supplement Is the Primary Determinant of Muscle Protein Anabolic Responses in Healthy Older Women.

Background: Older adults show a blunted muscle protein synthesis (MPS) response to postprandial hyperaminoacidemia relative to younger adults. Evidence suggests that this anabolic resistance can be overcome by consuming greater quantities of leucine. Objective: The purpose of this trial was to determine whether the addition of leucine to a smaller dose (10 g) of milk proteins would, when compared with a larger dose (25 g) of whey protein isolate (WPI), result in similar increases in acute (hourly) and integrated (daily) myofibrillar protein synthesis (myoPS). Methods: Healthy older (mean ± SD age: 69 ± 1 y) women (n = 11/group) were randomly assigned with the use of a single-blind, parallel-group design to twice-daily consumption of either WPI [25 g WPI (3 g l-leucine)] or leucine (LEU; 10 g milk protein with 3 g total l-leucine) for 6 d. Participants performed unilateral resistance exercise to allow assessment of the impact of the supplement alone and with resistance exercise. We determined acute (13C6-phenylanine) and integrated [using deuterated water (D2O)] rates of myoPS in the fasting (acute), basal (integrated), nonexercised, and exercised states. Results: Acute myoPS increased in both legs in response to LEU (fed: 45%; fed+exercise: 71%; P < 0.001) and WPI (fed: 29%; fed+exercise: 47%; P < 0.001) compared with fasting; the increase was greater with LEU than with WPI in the exercised leg (46%; P = 0.04) but not in the rested leg (P = 0.07). The acute myoPS response was greater in the exercised leg than in the rested leg for both WPI (63%) and LEU (58%) (P < 0.001). Integrated myoPS increased with WPI and LEU in the exercised leg (both 9%; P < 0.001) during supplementation, and with WPI (3%; P = 0.02) but not LEU (2%, P = 0.1) in the rested leg compared with the basal state. Conclusions: A lower-protein (10 compared with 25 g/dose), leucine-matched beverage induced similar increases in acute and integrated myoPS in healthy older women. Lower-protein supplements with added leucine may represent an advantageous approach in older adults to maintain skeletal muscle anabolic sensitivity and attenuate muscle loss; however, further work is needed using longer-term interventions to substantiate these findings. This trial was registered at www.clinicaltrials.gov as NCT02282566.

Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial.

Background: Older women may not be consuming enough protein to maintain muscle mass. Augmentation of protein intake with leucine may enhance the muscle protein synthetic response in older women to aid in maintaining muscle mass. Objective: We measured the acute (hourly) and integrated (daily) myofibrillar protein synthesis (myoPS) response to consumption of a high-quality mixed protein beverage compared with an isonitrogenous protein beverage with added leucine. Design: In a parallel design, free-living, healthy older women (aged 65-75 y, n = 11/group) consumed a fixed, weight-maintaining diet with protein at 1.0 g · kg-1 · d-1 and were randomly assigned to twice-daily consumption of either 15 g milk protein beverage containing 4.2 g leucine (LEU) or 15 g mixed protein (milk and soy) beverage containing 1.3 g leucine (CON). Unilateral leg resistance exercise allowed a determination of acute ([13C6]-phenylalanine infusion, hourly rate) and integrated (deuterated water ingestion, daily rate) exercised and rested myoPS responses. Results: Acute myoPS increased in response to feeding in the rested (CON: 13% ± 4%; LEU: 53% ± 5%) and exercised (CON: 30% ± 4%; LEU: 87% ± 7%) leg in both groups, but the increase was greater in LEU (P < 0.001). Integrated myoPS increased during the supplementation period in both legs (rested: 9% ±1%; exercised: 17% ± 2%; P < 0.001) in LEU, but in the exercised leg only (7% ± 2%; P < 0.001) in CON. Conclusions: A 15-g protein-containing beverage with ∼4 g leucine induced greater increases in acute and integrated myoPS than did an isonitrogenous, isoenergetic mixed-protein beverage. Declines in muscle mass in older women may be attenuated with habitual twice-daily consumption of a protein beverage providing 15 g protein and higher (4.2 g/serving) amounts of leucine. This trial was registered at clinicaltrials.gov as NCT02282566.

Daily intake of dairy products in Brazil and contributions to nutrient intakes: a cross-sectional study.

OBJECTIVE: Dairy products are sources of protein and micronutrients important in a healthy diet. The purpose of the present analysis was to estimate consumption of dairy products by Brazilians and identify contributions of dairy products to nutrient intakes. DESIGN: Dairy consumption data were obtained from 24 h dietary records. Dairy products were defined as milk (including flavoured), cheese and yoghurt. Estimates of dairy product intakes were generated for all individuals, individuals in urban and rural households and for age groups 10-18 years, 19-59 years and ≥60 years. Contributions to nutrient intakes were estimated for the total sample and sub-populations. SETTING: Nationwide cross-sectional survey, 2008-2009. SUBJECTS: Nationally representative sample of individuals aged ≥10 years in the Individual Food Intake survey, a component of the Brazilian Household Budget Survey (n 34 003). RESULTS: Among individuals aged ≥10 years, per capita intake of dairy products was 142 (se 2.1) g/d. Dairy product intake was higher among individuals in urban compared with rural areas and among groups 10-18 years and ≥60 years compared with adults aged 19-59 years. Dairy products accounted for 6.1% of daily energy intake, 7.3% of protein, 16.9% of saturated fat, 11.1% and 4.3% of total and added sugars, respectively, and 10.2-37.9% of daily Ca, vitamin D, P, vitamin A and K. CONCLUSIONS: Dairy products were substantial contributors to daily intakes of selected nutrients of concern in Brazil, although mean daily dairy product consumption was less than a typical portion. Education efforts in Brazil to raise awareness about the nutritional role of dairy foods may serve to improve overall diet quality.

Level of dietary protein intake affects glucose turnover in endurance-trained men.

BACKGROUND: To examine the effects of higher-protein diets on endogenous glucose metabolism in healthy, physically active adults, glucose turnover was assessed in five endurance-trained men (age 21.3 ± 0.3 y, VO2peak 70.6 ± 0.1 mL kg-1 min-1) who consumed dietary protein intakes spanning the current dietary reference intakes. FINDINGS: Using a randomized, crossover design, volunteers consumed 4 week eucaloric diets providing either a low (0.8 g kg-1 d-1; LP), moderate (1.8 g kg-1 d-1; MP), or high (3.6 g kg-1 d-1; HP) level of dietary protein. Glucose turnover (Ra, glucose rate of appearance; and Rd glucose rate of disappearance) was assessed under fasted, resting conditions using primed, constant infusions of [6,6-2H2] glucose. Glucose Ra and Rd (mg kg-1 min-1) were higher for MP (2.8 ± 0.1 and 2.7 ± 0.1) compared to HP (2.4 ± 0.1 and 2.3 ± 0.2, P < 0.05) and LP (2.3 ± 0.1 and 2.2 ± 0.1, P < 0.01) diets. Glucose levels (mmol/L) were not different (P > 0.05) between LP (4.6 ± 0.1), MP (4.8 ± 0.1), and HP (4.7 ± 0.1) diets. CONCLUSIONS: Level of protein consumption influenced resting glucose turnover in endurance athletes in a state of energy balance with a higher rate of turnover noted for a protein intake of 1.8 g kg-1 d-1. Findings suggest that consumption of protein in excess of the recommended dietary allowance but within the current acceptable macronutrient distribution range may contribute to the regulation of blood glucose when carbohydrate intake is reduced by serving as a gluconeogenic substrate in endurance-trained men.

AMPK represses TOP mRNA translation but not global protein synthesis in liver.

The AMP-activated protein kinase (AMPK) represses signaling through the mammalian target of rapamycin complex 1 (mTORC1). In muscle, repression of mTORC1 leads to a reduction in global protein synthesis. In contrast, repression of mTORC1 in the liver has no immediate effect on global protein synthesis. In the present study, signaling through mTORC1 and translation of specific mRNAs such as those bearing a 5'-terminal oligopyrimidine (TOP) tract and were examined in rat liver following activation of AMPK after treadmill running. Activation of AMPK repressed translation of the TOP mRNAs encoding rpS6, rpS8, and eEF1alpha. In contrast, neither global protein synthesis nor translation of mRNAs encoding GAPDH or beta-actin was changed. Basal phosphorylation of the mTORC1 target 4E-BP1, but not S6K1 or rpS6, was reduced following activation of AMPK. Thus, in liver, AMPK activation repressed translation of TOP mRNAs through a mechanism distinct from downregulated phosphorylation of S6K1 or rpS6.

Postexercise whole-body protein turnover response to three levels of protein intake.

PURPOSE: This investigation examined the effect of variations in protein intake on whole-body protein turnover (WBPTO) after exercise in endurance-trained males. METHODS: Five male runners (21.3 +/- 0.3 yr, 179 +/- 2 cm, 70.6 +/- 0.1 kg, 8.7 +/- 0.4% body fat, 70.6 +/- 0.1 VO2peak) participated in a randomized, crossover-design diet intervention, where they consumed either a low- (0.8 g.kg(-1); LP), moderate- (1.8 g.kg(-1); MP), or high-protein (3.6 g.kg(-1); HP) diet for 4 wk. WBPTO (Ra, leucine rate of appearance; NOLD, nonoxidative leucine disposal; and Ox, leucine oxidation) were assessed after a 75-min run at 70% VO2peak after each diet-intervention period. RESULTS: Leucine Ra (indicator of protein breakdown) and leucine Ox were greater on the HP diet than on the LP diet (Ra, 123.4 +/- 6.9 vs 97.9 +/- 6.0 micromol.kg(-1).h(-1); Ox, 23.9 +/- 0.5 vs 17.0 +/- 0.8 micromol.kg(-1).h(-1), P < 0.05). No differences were noted in NOLD (an indicator of protein synthesis) across diets. Plasma branched chain amino acids (BCAA) at rest were greater for MP and HP than for LP, and nonessential amino acids (NEAA) were greater for LP than MP at rest and greater than MP and HP after exercise. CONCLUSION: Findings from this study show that variations in protein intake can alter plasma amino acid levels and modulate rates of WBPTO after exercise. Additionally, a lower protein intake was associated with decreased rates of WBPTO after exercise.

Time course changes in signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation by AICAR.

The role of the AMP-activated kinase (AMPK) as a metabolic sensor in skeletal muscle has been far better characterized for glucose and fat metabolism than for protein metabolism. Therefore, the studies presented here were designed to examine the effects of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR)-induced AMPK signaling on effector mechanisms of mRNA translation and protein synthesis in cultures of C(2)C(12) myotubes. The findings show that, following AICAR (2 mM) treatment, AMPK phosphorylation was increased within 15 min and remained elevated throughout a 60-min time course. In association with the increase in AMPK phosphorylation, global rates of protein synthesis declined to 90, 70, and 63% of the control values at the 15-, 30-, and 60-min time points, respectively. By 60 min, polysomes disaggregated into free ribosomal subunits, suggesting an inhibition of initiation of mRNA translation. However, phosphorylation of eukaryotic elongation factor 2 was increased at 15 and 30 min but then declined to control values by 60 min, suggesting a transient inhibition of translation elongation. The decline in protein synthesis and changes in mRNA translation were associated with a repression of the mammalian target of rapamycin (mTOR) signaling pathway, as indicated by increased association of Hamartin with Tuberin, increased association of regulatory associated protein of mTOR with mTOR, and dephosphorylation of the downstream targets ribosomal protein S6 kinase-1 and eukaryotic initiation factor 4E-binding protein-1. They were also associated with activation of the MAPK signaling pathway, as indicated by increased phosphorylation of MEK1/2 and ERK1/2 and the downstream target eIF4E. Overall, the data support the conclusion that AICAR-induced AMPK activation suppresses protein synthesis through concurrent repression of mTOR signaling and activation of MAPK signaling, the combination of which modulates transient changes in the initiation and elongation phases of mRNA translation.

Insights into muscle atrophy and recovery pathway based on genetic models.

PURPOSE OF REVIEW: Muscle atrophy is a pervasive problem that occurs with disuse, aging, and a myriad of disease conditions. The purposes of this review are to describe recent advances in studying muscle atrophy that have elucidated pathways involved at the molecular level; to compare different types of atrophy--primary (e.g. bed rest, immobilization) and secondary (when the atrophy is related to pathology as well as disuse, e.g. injury, sepsis etc.) and their multiple common features; to review progress in studying the recovery process and clinical status. RECENT FINDINGS: Major advances have been made at the molecular level. There are two phenotypes for muscle atrophy--primary, which is mainly related to disuse (e.g. bed), and secondary, when the atrophy is related to pathology as well as disuse. It appears that the two forms have multiple elements in common. Studies on the recovery process reveal a very complex sequence of events that are not the simple reverse of the muscle loss process. In contrast to the progress at the molecular level, progress in treating muscle atrophy or accelerating recovery has been disappointing. SUMMARY: Although nutritional supplementation and pharmacological agents continue to have the potential to minimize muscle atrophy, given its minimal risks, exercise sets a very high standard for treatment options when medically appropriate. Identification of pathways and control points offers the potential for new approaches.

Level of dietary protein impacts whole body protein turnover in trained males at rest.

The current investigation examined the effect of variations in protein intake on Whole body protein turnover (WBPTO) at rest in endurance-trained males. Whole body protein turnover is influenced by both diet and exercise. Whether endurance athletes require more protein than the non-exerciser remains equivocal. Five male runners (21.3 +/- 0.3 years, 179 +/- 2 cm, 70.6 +/- 0.1 kg, 8.7% +/- 0.4% body fat, 70.6 +/- 0.1 VO(2)max) participated in a randomized, crossover design diet intervention where they consumed either a low-protein (LP; 0.8 g/kg), moderate-protein (MP; 1.8 g/kg), or high-protein (HP; 3.6 g/kg) diet for 3 weeks. Whole body protein turnover (Ra, leucine rate of appearance; NOLD, nonoxidative leucine disposal; and Ox, leucine oxidation), nitrogen balance, and substrate oxidation were assessed at rest following each dietary intervention period. The HP diet increased leucine Ra (indicator of protein breakdown; 136.7 +/- 9.3, 129.1 +/- 7.4, and 107.8 +/- 3.1 micromol/[kg . h] for HP, MP, and LP diets, respectively) and leucine Ox (31.0 +/- 3.6, 26.2 +/- 4.3, and 18.3 +/- 0.6 micromol/[kg . h] for HP, MP, and LP diets, respectively) compared with LP diet (P < .05). No differences were noted in nonoxidative leucine disposal (an indicator of protein synthesis) across diets. Nitrogen balance was greater for HP diet than for MP and LP diets (10.2 +/- 0.7, 1.8 +/- 0.6, and -0.3 +/- 0.5 for HP, MP, and LP diets, respectively). Protein oxidation increased with increasing protein intake (54% +/- 6%, 25% +/- 1%, and 14% +/- 2% for HP, MP, and LP diets, respectively). Findings from this study show that variations in protein intake can modulate WBPTO and that protein intake approximating the current recommended dietary allowance was not sufficient to achieve nitrogen balance in the endurance-trained males in this investigation. Our results suggest that a protein intake of 1.2 g/kg or 10% of total energy intake is needed to achieve a positive nitrogen balance. This is not a concern for most endurance athletes who routinely consume protein at or above this level.

Effects of dietary protein intake on indexes of hydration.

This study aims to characterize the relationship between increased protein intake and hydration indexes. Five men participated in a 12-week, randomized, crossover, controlled diet intervention study. Subjects consumed eucaloric diets containing 3.6 (high protein), 1.8 (moderate protein), and 0.8 (low protein) g/kg/day of protein for 4 weeks each. Energy intakes were based on requirements established relative to resting energy expenditure and activity at baseline. Assessments included blood urea nitrogen, plasma osmolality, urine-specific gravity, and estimates of fluid balance. Repeated-measures analyses of variance and paired t tests were used to determine effects of treatment and time. Fluid intake and fluid balance were unaffected. Blood urea nitrogen was higher for high protein vs low protein and vs moderate protein, and urine-specific gravity was higher for high protein vs moderate protein. Baseline plasma osmolality was greater for high protein vs low protein and vs moderate protein. The effect of increasing dietary protein on fluid status was minimal.

Dietary protein intake impacts human skeletal muscle protein fractional synthetic rates after endurance exercise.

This investigation evaluated the physiological impact of different dietary protein intakes on skeletal muscle protein synthesis postexercise in endurance runners. Five endurance-trained, male runners participated in a randomized, crossover design diet intervention, where they consumed either a low (0.8 g/kg; LP)-, moderate (1.8 g/kg; MP)-, or high (3.6 g/kg; HP)-protein diet for 4 wk. Diets were designed to be eucaloric with carbohydrate, fat, and protein approximating 60, 30, and 10%; 55, 30, and 15%; and 40, 30, and 30% for LP, MP, and HP, respectively. Substrate oxidation was assessed via indirect calorimetry at 3 wk of the dietary interventions. Mixed-muscle protein fractional synthetic rate (FSR) was measured after an endurance run (75 min at 70% V(O2 peak)) using a primed, continuous infusion of [(2)H(5)]phenylalanine. Protein oxidation increased with increasing protein intake, with each trial being significantly different from the other (P < 0.01). FSR after exercise was significantly greater for LP (0.083%/h) and MP (0.078%/h) than for HP (0.052%/h; P < 0.05). There was no difference in FSR between LP and MP. This is the first investigation to establish that habitual dietary protein intake in humans modulates skeletal muscle protein synthesis after an endurance exercise bout. Future studies directed at mechanisms by which level of protein intake influences skeletal muscle turnover are needed.

Resistance exercise increases muscle protein synthesis and translation of eukaryotic initiation factor 2Bepsilon mRNA in a mammalian target of rapamycin-dependent manner.

The contribution of mammalian target of rapamycin (mTOR) signaling to the resistance exercise-induced stimulation of skeletal muscle protein synthesis was assessed by administering rapamycin to Sprague-Dawley rats 2 h prior to a bout of resistance exercise. Animals were sacrificed 16 h postexercise, and gastrocnemius protein synthesis, mTOR signaling, and biomarkers of translation initiation were assessed. Exercise stimulated the rate of protein synthesis; however, this effect was prevented by pretreatment with rapamycin. The stimulation of protein synthesis was mediated by an increase in translation initiation, since exercise caused an increase in polysome aggregation that was abrogated by rapamycin administration. Taken together, the data suggest that the effect of rapamycin was not mediated by reduced phosphorylation of eukaryotic initiation factor 4E (eIF4E) binding protein 1 (BP1), because exercise did not cause a significant change in 4E-BP1(Thr-70) phosphorylation, 4E-BP1-eIF4E association, or eIF4F complex assembly concomitant with increased protein synthetic rates. Alternatively, there was a rapamycin-sensitive decrease in relative eIF2Bepsilon(Ser-535) phosphorylation that was explained by a significant increase in the expression of eIF2Bepsilon protein. The proportion of eIF2Bepsilon mRNA in polysomes was increased following exercise, an effect that was prevented by rapamycin treatment, suggesting that the increase in eIF2Bepsilon protein expression was mediated by an mTOR-dependent increase in translation of the mRNA encoding the protein. The increase in eIF2Bepsilon mRNA translation and protein abundance occurred independent of similar changes in other eIF2B subunits. These data suggest a novel link between mTOR signaling and eIF2Bepsilon mRNA translation that could contribute to the stimulation of protein synthesis following acute resistance exercise.

Repression of protein synthesis and mTOR signaling in rat liver mediated by the AMPK activator aminoimidazole carboxamide ribonucleoside.

The studies described herein were designed to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), an activator of the AMP-activated protein kinase (AMPK), on the translational control of protein synthesis and signaling through the mammalian target of rapamycin (mTOR) in rat liver. Effects of AICAR observed in vivo were compared with those obtained in an in situ perfused liver preparation to investigate activation of AMPK in the absence of accompanying changes in hormones and nutrients. AMPK became hyperphosphorylated, as assessed by a gel-shift analysis, in response to AICAR both in vivo and in situ; however, increased relative phosphorylation at the Thr172 site on the kinase was observed only in perfused liver. Phosphorylation of AMPK either in vivo or in situ was associated with a repression of protein synthesis as well as decreased phosphorylation of a number of targets of mTOR signaling including ribosomal protein S6 kinase 1, eukaryotic initiation factor (eIF)4G, and eIF4E-binding protein (4E-BP)1. The phosphorylation changes in eIF4G and 4E-BP1 were accompanied by a reduction in the amount of eIF4E present in the active eIF4E.eIF4G complex and an increase in the amount present in the inactive eIF4E.4E-BP1 complex. Reduced insulin signaling as well as differences in nutrient availability may have contributed to the effects observed in vivo as AICAR caused a fall in the serum insulin concentration. Overall, however, the results from both experimental models support a scenario in which AICAR directly represses protein synthesis and mTOR signaling in the liver through an AMPK-dependent mechanism.

Regulation of protein synthesis associated with skeletal muscle hypertrophy by insulin-, amino acid- and exercise-induced signalling.

Although insulin, amino acids and exercise individually activate multiple signal transduction pathways in skeletal muscle, one pathway, the phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signalling pathway, is a target of all three. Activation of the PI3K-mTOR signal transduction pathway results in both acute (i.e. occurring in minutes to hours) and long-term (i.e. occurring in hours to days) up-regulation of protein synthesis through modulation of multiple steps involved in mediating the initiation of mRNA translation and ribosome biogenesis respectively. In addition, changes in gene expression through altered patterns of mRNA translation promote cell growth, which in turn promotes muscle hypertrophy. The focus of the present discussion is to review current knowledge concerning the mechanism(s) through which insulin, amino acids and resistance exercise act to activate the PI3K-mTOR signal transduction pathway and thereby enhance the rate of protein synthesis in muscle.

Leucine regulates translation initiation in rat skeletal muscle via enhanced eIF4G phosphorylation.

The BCAA, leucine, stimulates protein synthesis in skeletal muscle in part through enhanced initiation of mRNA translation. However, understanding how leucine regulates protein synthesis remains elusive. The intent of the present investigation was to examine the effect of leucine, independent of other regulatory agents, on key events in translation initiation in skeletal muscle and to elucidate the extent to which signaling through the mammalian target of rapamycin (mTOR) accounts for the effect of the amino acid on protein synthesis. Hindlimb preparations from postabsorptive rats were perfused with medium containing food-deprived (1X) or superphysiologic (10X) concentrations of leucine with all other amino acids at 1X concentration. Protein synthesis was significantly greater in both gastrocnemius and soleus perfused with 10X compared with 1X leucine. The stimulatory effects of leucine on protein synthesis were unaffected by a specific inhibitor of PI3-kinase (LY 294002). Moreover, signaling through mTOR, as monitored by the phosphorylation status of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1) or the 70-kDa ribosomal protein S6 kinase (S6K1), was not further enhanced by 10X compared with 1X leucine. However, binding of eIF4E to eIF4G and eIF4G(Ser-1108) phosphorylation in the eIF4E immunoprecipitate were enhanced as was eIF4G(Ser-1108) phosphorylation in the total tissue extract after perfusion with medium containing 10X leucine. Collectively, these observations illustrate an experimental model whereby leucine in the absence of other regulatory agents stimulates eIF4E. eIF4G assembly and protein synthesis directly in skeletal muscle, possibly by augmenting phosphorylation of eIF4G through a signaling pathway independent of mTOR.

Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle.

The purpose of the present investigation was to determine whether mammalian target of rapamycin (mTOR)-mediated signalling and some key regulatory proteins of translation initiation are altered in skeletal muscle during the immediate phase of recovery following acute resistance exercise. Rats were operantly conditioned to reach an illuminated bar located high on a Plexiglass cage, such that the animals completed concentric and eccentric contractions involving the hindlimb musculature. Gastrocnemius muscle was extracted immediately after acute exercise and 5, 10, 15, 30 and 60 min of recovery. Phosphorylation of protein kinase B (PKB) on Ser-473 peaked at 10 min of recovery (282% of control, P < 0.05) with no significant changes noted for mTOR phosphorylation on Ser-2448. Eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) and S6 kinase-1 (S6K1), both downstream effectors of mTOR, were altered during recovery as well. 4E-BP1 phosphorylation was significantly elevated at 10 min (292%, P < 0.01) of recovery. S6K1 phosphorylation on Thr-389 demonstrated a trend for peak activation at 10 min following exercise (336%, P = 0.06) with ribosomal protein S6 phosphorylation being maximally activated at 15 min of recovery (647%, P < 0.05). Components of the eIF4F complex were enhanced during recovery as eIF4E association with eIF4G peaked at 10 min (292%, P < 0.05). Events regulating the binding of initiator methionyl-tRNA to the 40S ribosomal subunit were assessed through eIF2B activity and eIF2 alpha phosphorylation on Ser-51. No differences were noted with either eIF2B or eIF2 alpha. Collectively, these results provide strong evidence that mTOR-mediating signalling is transiently upregulated during the immediate period following resistance exercise and this response may constitute the most proximal growth response of the cell.

Translational control mechanisms modulate skeletal muscle gene expression during hypertrophy.

Understanding the basic mechanisms regulating skeletal muscle hypertrophy is essential to providing strategies for optimizing and maintaining skeletal muscle mass. This review focuses on the importance of mRNA translation in mediating acute increases in protein synthesis after resistance exercise as well as the anabolic response of muscle growth.

Beta -oxidation of free fatty acids is required to maintain translational control of protein synthesis in heart.

The study described herein investigated the role of free fatty acids (FFAs) in the maintenance of protein synthesis in vivo in rat cardiac and skeletal muscle. Suppression of FFA beta-oxidation by methyl palmoxirate caused a marked reduction in protein synthesis in the heart. The effect on protein synthesis was mediated in part by changes in the function of eukaryotic initiation factors (eIFs) involved in the initiation of mRNA translation. The guanine nucleotide exchange activity of eIF2B was repressed, phosphorylation of the alpha-subunit of eIF2 was enhanced, and phosphorylation of eIF4E-binding protein-1 and ribosomal protein S6 kinase was reduced. Similar changes in protein synthesis and translation initiation were not observed in the gastrocnemius following treatment with methyl palmoxirate. In heart, repressed beta-oxidation of FFA correlated, as demarcated by changes in the ATP/AMP ratio and phosphorylation of AMP-activated kinase, with alterations in the energy status of the tissue. Therefore, the activation state of signal transduction pathways that are responsive to cellular energy stress represents one mechanism whereby translation initiation may be regulated in cardiac muscle.