Muscle full effect after oral protein: time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 signaling

Journal Title (Medline/Pubmed accepted abbreviation): Am J Clin Nutr
Year: 2010
Volume: 92
Page numbers: 1080-1088
doi: 10.3945/ajcn.2010.29819

Summary of Background and Research Design

Background: Human muscle protein synthesis (MPS) increases following ingestion or infusion of amino acids (AAs) followed by a plateau at ~120 minutes and return to postabsorptive rates. This return to postabsorptive rates occurs despite continued availability of AAs, in what is called the “muscle full” state, for up to 4 hours. The muscle full hypothesis states that once muscle reaches maximal AA uptake, any additional AAs would not be a substrate for MPS, but would be diverted instead towards oxidation. Blood concentration of AAs following ingestion or intravenous administration of protein, AAs, and essential AAs (EAAs [especially leucine]), stimulates MPS in a dose-dependent and saturable manner. Amino acids not only provide a substrate for MPS, they stimulate initiation and elongation of messenger RNA (mRNA) translation within myocytes—a process potentially mediated by the mammalian target of rapamycin complex 1 (mTORC1). Activation of mTORC1 increases phosphorylation of ribosomal S6 kinase (S6K1) and 4E-binding protein 1 (4EBP1). Researchers have not yet characterized the time course of muscle protein synthesis relative to amino acid availability from a meal of nutritionally meaningful size.

Hypothesis/purpose of study:This study was conducted to determine: 1) if a protein meal (48 g whey) would stimulate a short-lived MPS response despite causing longer and more sustained elevation of plasma and intramuscular amino acid concentrations; and 2) if molecular signaling responses that regulate mRNA translation would prefigure the molecular signaling of MPS responses.

Subjects:Recreationally active, healthy young men (N = 8, mean ± standard error of the mean [SEM] age 21 ± 2 yr; body mass index [BMI] 22.9 ± 0.9 kg/m2) participated in the study.

Experimental design:Single-arm, muscle physiology study

Treatments and protocol:Subjects were studied during 8.5 hours of primed continuous infusion of [1,2?13C2] leucine tracer with intermittent blood samples and quadriceps biopsies for determination of MPS and anabolic signaling. After 2.5 hours to establish baseline MPS, subjects consumed 48 g whey protein isolate (equivalent to ~20 g EAA) in 500 mL water. Glucose, AA, and insulin concentrations were assessed from plasma samples. Muscle samples were analyzed for de novo MPS based on incorporation of [1,2?13C2] leucine tracer into muscle protein as measured by gas chromatography–combustion-isotope ratio mass spectrometry. Protein synthesis in response to protein meal was determined based on the increase in incorporation of [1,2 13C2] leucine between muscle biopsies by using the labeling of plasma
α?ketoisocaproate as a surrogate of leucyl-t-RNA (the transfer RNA specific to leucine). Immunoprecipitation and immunoblotting of phosphorylated muscle proteins (indicating activation) was conducted for mTOR (Ser2448), S6K1 (Thr389), 4EBP1 (Thr37/46), eukaryotic initiation factor (eIF)-4E (Ser209), extracellular regulated kinase (ERK)-1/2 (Tyr202/204), eukaryotic elongation factor (eEF)-2 (Thr56), eIF4G (Ser1108), adenosine monophosphate (AMP)-activated protein kinase (AMPK; Thr172), eIF2Bε (Ser535), eIF2a (Ser51), and pan-actin as a loading control. Kinase assays were conducted for protein kinase B (PKB), which was immunoprecipitated and incubated with glycogen synthase kinase 3b (GSK3b) and adenosine triphosphate (ATP) followed by immunoblotting for GSK3b. Interactions of components of the eIF4F complex were assessed via immunoprecipitation of Sepharose-bound eIF4E followed by immunoblotting for total 4EBP1, eIF4G, and eIF4E (to determine if translation machinery [and therefore de novo protein synthesis] assembled in response to treatment).
Summary of research findings:
  • After oral protein bolus, plasma insulin concentrations increased sharply and were elevated at 30 and 60 minutes (+296% and +303%, respectively; P < .01) falling to postabsorptive values after 90 minutes.
  • Plasma glucose concentrations remained at typical postabsorptive values (~5.5 mmol/L) throughout the study.
  • Plasma EAA concentrations were significantly increased after 30 minutes, peaked at 60 minutes (+131%; P < .01), and remained elevated for 180 minutes.
    • Nonessential AA concentrations increased at 30 minutes (+31%; P =.05), returned to basal values by 120 minutes, and were significantly decreased by 360 minutes, compared with postabsorptive AA concentrations.
  • Plasma α-ketoisocaproate concentrations were increased by 60 minutes (+70%; P <.01) and remained elevated throughout the study (ie, 6 hr after oral protein bolus).
  • Mean (± SEM) MPS increased from 0.03% ± 0.003% to 0.10% ± 0.01%/hour at 45 to 90 minutes after oral protein bolus.
    • Thereafter, MPS returned to baseline rates even though EAA concentrations remained elevated (+130% at 120 min, +80% at 180 min).
    • Although MPS decreased thereafter, all signals, with the exception of PKB activity, remained elevated.
    • The phosphorylation of eIF2a increased only at 180 minutes.
  • PKB activity and phosphorylation of eIF4G preceded the rise in MPS and increases in phosphorylation of ribosomal S6K1, and 4EBP1 mirrored MPS responses until 90 minutes.
  • Phosphorylation of AMPK and eEF2 did not increase; phosphorylation of ERK1/2, eIF4E, mTOR, and eIF2Be were also unchanged.

Interpretation of findings/Key practice applications:

Similar to observations with AA administration, MPS after oral protein supplementation was delayed ~30 to 45 minutes but was then almost tripled between 60 and 90 minutes before rapidly returning to baseline despite continued availability of leucine and EAAs. Before MPS increase, phosphorylation (activation) of eIF4G (via PKB) occurred along with phosphorylation of S6K1 and 4EBP1. In contrast to the study’s hypothesis, the return of MPS to basal values occurred before dephosphorylation (essentially deactivation) of S6K1, 4EBP1, and eIF4G and prior to disassembly of the eIF4F protein translation complex. With the observed decline of MPS in the presence of ample activating stimulus (ie, plasma and intramuscular leucine), it appears likely that skeletal muscle can gauge its ability to synthesize new proteins. Elevated mTORC1 signaling may only occur during the initial increase in MPS, whereas MPS signaling remains high even after MPS has returned to basal levels. The significance of prolonged presence of protein-activating signals outlasting the MPS response remains unexplained. Taken together, these results suggest that protein and AA supplementation may induce short-term stimulation of MPS by ~1.5 hours after ingestion; however, the MPS response is saturable. As a result, strategies for protein supplementation that favor periodic or pulse dosing of protein or AAs should be favored over continuous supply of these supplements.
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