Kinetic modeling of leucine-mediated signaling and protein metabolism in human skeletal muscle

Abstract

Skeletal muscle protein levels are governed by the relative rates of muscle protein synthesis (MPS) and breakdown (MPB). The mechanisms controlling these rates are complex, and their integrated behaviors are challenging to study through experiments alone. The purpose of this study was to develop and analyze a kinetic model of leucine-mediated mTOR signaling and protein metabolism in the skeletal muscle of young adults. Our model amalgamates published cellular-level models of the IRS1-PI3K-Akt-mTORC1 signaling system and of skeletal-muscle leucine kinetics with physiological-level models of leucine digestion and transport and insulin dynamics. The model satisfactorily predicts experimental data from diverse leucine feeding protocols. Model analysis revealed that total levels of p70S6K are a primary determinant of MPS, insulin signaling substantially affects muscle net protein balance via its effects on MPB, and p70S6K-mediated feedback of mTORC1 signaling reduces MPS in a dose-dependent manner.

Keywords: Biological sciences; Protein.

Graphical abstract

Figure 1

Reaction diagram of the model Reaction diagram for the kinetic model of leucine-mediated signaling and protein metabolism. mTORC2^S2481 and PDK1^P are represented as independent species for the phosphorylation of Akt^S473 and Akt^S473,T308, respectively, but both are subject to the same control as the integrated species. The insulin module is presented as a single species in the model diagram, but it is expanded in the model code to include the three species and four negative feedback loops outlined by Sturis et al. The transfer of masses is denoted by open-headed arrows. Chemical reactions are denoted by solid-headed arrows. Inhibition is denoted by a flat-headed line. Red stars denote the locations of simulated signaling knockdown. Red lines indicate inhibitory links via p70S6K-mediated feedback or rapamycin. Blue lines indicate signaling-mediated control of MPB. P = phospho residue, S = serine, T = threonine, Y = tyrosine.

Figure 2

Model calibration and validation (A) Simulated time courses following model calibration of plasma leucine, intracellular leucine, plasma insulin, the three-pool model parameters F_m,a and F_m,0, Akt (total and serine phosphorylated), p70S6K (total and phosphorylated), and muscle protein balance following a 3.5-gram bolus of leucine. Data points represent experimental data collected from two studies following the ingestion of a 3.5-gram bolus of leucine in human subjects.^, The data points for phospho-Akt^S473 and phopsho-p70S6K^T389 were predicted from spline regression equations obtained from meta-analyzed data. (B and C) Simulated time courses of plasma leucine, plasma insulin, p70S6K, and muscle protein balance following either (B) a single 3.59-gram bolus of leucine or (C) pulsatile leucine feedings (0.59-grams of leucine provided at 0, 45, 90, and 135 min). Data points represent experimental data collected from the Mitchell et al. single bolus intervention (B) or pulsatile feeding intervention (C). Root-mean-square values for each time course are included within each plot. The measured data are presented as means ± SE. FSR = fractional synthetic rate, MPS = muscle protein synthesis, MPB = muscle protein breakdown, NB = net balance. See also Figures S1–S6 and Tables S1–S8.

Figure 3

Knockdown of leucine signaling impairs MPS and net balance Simulated time courses of plasma leucine, intracellular leucine, plasma insulin, Akt, p70S6K, and muscle protein balance following a 3.5-gram bolus of leucine with varying degrees of knockdown to the rate controlling leucine-mediated mTORC1 activity. Signaling knockdown was simulated by decreasing the kinetic rate parameter controlling leucine-mediated mTORC1 activity by 1×, 0.75×, 0.50×, 0.25×, and 0.10× of its calibrated value. F.C. = fold change, FSR = fractional synthetic rate, K.D. = knockdown, MPS = muscle protein synthesis, MPB = muscle protein breakdown. See also Figures S4 and S5.

Figure 4

Increasing post-absorptive p70S6K levels substantially increases MPS Simulated time courses of plasma leucine, intracellular leucine, Akt, p70S6K, and muscle protein balance following a 3.5-gram bolus of leucine with the non-phosphorylated p70S6K concentration at the calibrated value (1×), two times the calibrated value (2×), and four times the calibrated value (4×). FSR = fractional synthetic rate, MPS = muscle protein synthesis, MPB = muscle protein breakdown, NB = net balance.

Figure 5

Enhanced signal activation can compensate for moderate losses in p70S6K levels Simulated time courses of p70S6K, MPS, MPB, and NB following a 3.5-gram bolus of leucine with total p70S6K concentrations (p70S6K + phospho-p70S6K^T389) at (A) the calibrated value (1×), (B) 0.5-times the calibrated value (0.5×), (C) 0.25-times the calibrated value (0.25×), and (D) 0.125-times the calibrated value (0.125×). At each level of p70S6K, the rate controlling the mTORC1 kinase was simulated at 1×, 2×, and 4× of its calibrated value. The 0.25× and 0.125× p70S6K concentration were additionally simulated with the rate controlling mTORC1 kinase set at 32× and 64×, respectively. The baseline MPS time course (1× p70S6K, 1× mTORC1 kinase) in (A) was bolded to serve as a reference line for (B), (C), and (D). FSR = fractional synthetic rate, MPS = muscle protein synthesis, MPB = muscle protein breakdown, NB = net balance.

Figure 6

Knockdown of insulin signaling reduces net protein balance Simulated time courses of plasma leucine, intracellular leucine, plasma insulin, Akt, p70S6K, and muscle protein balance following a 3.5-gram bolus of leucine with varying amounts of knockdown to the rate controlling insulin-mediated insulin receptor (IR_β) phosphorylation. Signaling knockdown was simulated by modulating the kinetic rate parameter by 1×, 0.75×, 0.50×, 0.25×, and 0.10× its calibrated value. F.C. = fold change, FSR = fractional synthetic rate, K.D. = knockdown. MPS = muscle protein synthesis, MPB = muscle protein breakdown.

Figure 7

Contributions of phospho-p70S6K^T389-mediated negative feedback on muscle protein balance Simulated time courses following a 3.5-gram bolus of leucine with the kinetic parameters controlling (A) phospho-p70S6K^T389-mediated phosphorylation of the IRS1 serine residue (k21) and (B) phospho-p70S6K^T389-mediated inhibition of mTORC1 activity (k43) simulated at 0.2×, 0.5×, 1×, 2×, and 5× their calibrated value. Total IRS1 includes the PI3K phospho-IRS1^Y complex and the non-phosphorylated IRS1 protein, neither of which are presented in the IRS1 plot. FSR = fractional synthetic rate, MPS = muscle protein synthesis, MPB = muscle protein breakdown, NB = net balance.