Resistance exercise increases human skeletal muscle AS160/TBC1D4 phosphorylation in association with enhanced leg glucose uptake during postexercise recovery

Abstract

Akt substrate of 160 kDa (AS160/TBC1D4) is associated with insulin and contraction-mediated glucose uptake. Human skeletal muscle AS160 phosphorylation is increased during aerobic exercise but not immediately following resistance exercise. It is not known whether AS160 phosphorylation is altered during recovery from resistance exercise. Therefore, we hypothesized that muscle AS160/TBC1D4 phosphorylation and glucose uptake across the leg would be increased during recovery following resistance exercise. We studied 9 male subjects before, during, and for 2 h of postexercise recovery. We utilized femoral catheterizations and muscle biopsies in combination with indirect calorimetry and immunoblotting to determine whole body glucose and fat oxidation, leg glucose uptake, muscle AMPKalpha2 activity, and the phosphorylation of muscle Akt and AS160/TBC1D4. Glucose oxidation was reduced while fat oxidation increased ( approximately 35%) during postexercise recovery (P <or= 0.05). Glucose uptake increased during exercise and postexercise recovery (P <or= 0.05). Akt phosphorylation was increased at 1 h and AMPKalpha2 activity increased at 2 h postexercise (P <or= 0.05). Phospho(Ser/Thr)-Akt substrate (PAS) phosphorylation (often used as a marker for AS160) was unchanged immediately postexercise and increased at 1 h (P <or= 0.05) and 2 h postexercise (P = 0.07). The PAS antibody is not always specific for AS160/TBC1D4 and can detect proteins at a similar molecular weight. Therefore, we immunoprecipitated AS160/TBC1D4 and then blotted with the PAS antibody, which confirmed that PAS phosphorylation is occurring on AS160/TBC1D4. There was also a positive correlation between PAS phosphorylation and leg glucose uptake during recovery (P < 0.05). We conclude that resistance exercise increases AS160/TBC1D4 phosphorylation in association with an increase in leg glucose uptake during postexercise recovery.

Figures

Fig. 1.

Study design and timeline in hours. Schematic displaying the study design used to measure the effect of resistance exercise on the regulation of whole body fat and glucose oxidation, glucose uptake, and cell signaling in male subjects. The study design consisted of a baseline, exercise, 1-h postexercise, and 2-h postexercise period. Continuous breath sampling was obtained with a Sensor Medics Vmax series metabolic cart. Indocyanine green (ICG) was infused to measure blood flow during each period. Blood samples were collected to measure plasma glucose and insulin and blood flow and to calculate glucose uptake. Muscle biopsies were used to measure muscle cell signaling, specifically components upstream and partially responsible for the activation of Akt substrate of 160 kDa (AS160).

Fig. 2.

Whole body glucose oxidation and postexercise fat oxidation. Breath samples were continuously obtained with the Vmax series metabolic cart during each period of the study. Whole body glucose and fat oxidation during exercise are not reported due to a respiratory quotient (RQ) being greater than 1. A: whole body glucose oxidation. B: whole body fat oxidation. Data are expressed as means ± SE. 1 h Post, 1 h postexercise; 2 h Post, 2 h postexercise. *P ≤ 0.05 vs. baseline.

Fig. 3.

Blood insulin and glucose concentrations. Blood samples were obtained 4 times at regular intervals (approximately every 10 min.) during each period of the study: baseline, exercise, 1 h postexercise, and 2 h postexercise. Venous insulin and glucose concentrations. Data are expressed as means ± SE. *P ≤ 0.05 vs. baseline.

Fig. 4.

ACC phosphorylation. Data are from each biopsy taken at baseline, immediately following resistance exercise (Exercise), 1 h following exercise, and 2 h following resistance exercise. Data are expressed as means ± SE. *P ≤ 0.05 vs. baseline (n = 7). Insets: representative blots for each time point: baseline (B), exercise (Ex), 1 h postexercise (1 h), and 2 h postexercise (2 h).

Fig. 5.

Phospho(Ser/Thr)-Akt substrate (PAS) phosphorylation (A) and immunoprecipitation of AS160/TBC1D4 (B). A: data are from each biopsy taken at baseline, immediately following resistance exercise, 1 h following exercise, and 2 h following resistance exercise for PAS160 phosphorylation. *P ≤ 0.05 vs. baseline; n = 7. B: AS160/TBC1D4 was first immunoprecipitated and then blotted with the PAS antibody. Data are expressed as means ± SE. *P ≤ 0.05 time effect; n = 6. Insets: representative blots for each time point [baseline (B), exercise (Ex), 1 h postexercise (1 h), and 2 h postexercise (2 h)].

Fig. 6.

Correlation of PAS phosphorylation and glucose uptake across the leg. Data points from baseline and 1 h and 2 h post-resistance exercise are included in the Pearson product-moment correlation (24 data points). PAS phosphorylation was positively correlated with glucose uptake across the leg (R = 0.41; P ≤ 0.05).