Insulin fails to enhance mTOR phosphorylation, mitochondrial protein synthesis, and ATP production in human skeletal muscle without amino acid replacement

Abstract

Systemic insulin administration causes hypoaminoacidemia by inhibiting protein degradation, which may in turn inhibit muscle protein synthesis (PS). Insulin enhances muscle mitochondrial PS and ATP production when hypoaminoacidemia is prevented by exogenous amino acid (AA) replacement. We determined whether insulin would stimulate mitochondrial PS and ATP production in the absence of AA replacement. Using l-[1,2-¹³C]leucine as a tracer, we measured the fractional synthetic rate of mitochondrial as well as sarcoplasmic and mixed muscle proteins in 18 participants during sustained (7-h) insulin or saline infusion (n = 9 each). We also measured muscle ATP production, mitochondrial enzyme activities, mRNA levels of mitochondrial genes, and phosphorylation of signaling proteins regulating protein synthesis. The concentration of circulating essential AA decreased during insulin infusion. Mitochondrial, sarcoplasmic, and mixed muscle PS rates were also lower during insulin (2-7 h) than during saline infusions despite increased mRNA levels of selected mitochondrial genes. Under these conditions, insulin did not alter mitochondrial enzyme activities and ATP production. These effects were associated with enhanced phosphorylation of Akt but not of protein synthesis activators mTOR, p70(S6K), and 4EBP1. In conclusion, sustained physiological hyperinsulinemia without AA replacement did not stimulate PS of mixed muscle or protein subfractions and did not alter muscle mitochondrial ATP production in healthy humans. These results support that insulin and AA act in conjunction to stimulate muscle mitochondrial function and mitochondrial protein synthesis.

Fig. 1.

Effects of sustained insulin or saline infusions on transcript levels of mitochondrial genes and nuclear-encoded regulators of mitochondrial biogenesis. See methods for definitions. *P < 0.05, Insulin vs. Saline.

Fig. 2.

Effects of sustained insulin or saline infusions on mitochondrial cytochrome c oxidase (COX; A) and citrate synthase (CS; B) enzyme activity and percent changes in mitochondrial ATP production rate (MAPR) measured using different substrates (see methods for definitions; C); 2-h measurements were not available for ATP production.

Fig. 3.

Effects of sustained insulin or saline infusions (0–7 h or 0–2 and 2–7 h) on skeletal muscle fractional synthetic rate (FSR) of mitochondrial proteins. Values were calculated using leucine isotopic model as described in text. *P < 0.05, Insulin vs. Saline; $P < 0.05, 0–2 h vs. 2–7 h.

Fig. 4.

Time course of arterial plasma [¹³C]ketoisocaproic acid (¹³CKIC) enrichment (A) and plasma concentration (B) before each biopsy.

Fig. 5.

Effects of sustained insulin or saline infusions (0–7 h or 0–2 and 2–7 h) on skeletal muscle FSR of sarcoplasmic (A and C) or mixed (B and D) muscle proteins. Values were calculated using leucine isotopic model as described in text. *P < 0.05, Insulin vs. Saline; $P < 0.05, 0–2 h vs. 2–7 h.

Fig. 6.

Effects of insulin or saline infusions on phosphorylation of skeletal muscle Akt (A), SESN2 (B), mTOR (C), p70 S6 kinase (D), 4EBP1 (E), EIF2α (F). See methods for definitions. Except for SESN2 (total protein/GAPDH), data are expressed as ratio of phosphorylated over total (P/T) protein and as fold changes at 2-h and 7-h infusions. *P < 0.05, vs. Basal (Insulin vs. Saline).

Fig. 7.

Effects of insulin or saline infusions on phosphorylation of skeletal muscle FOXO3A (A), total p62 (B), or total beclin protein levels (C). Data for p62 and beclin are expressed as total protein over GAPDH protein levels; FOXO3A is expressed as ratio of phosphorylated over total protein, and all panels are expressed as fold changes at 2-h and 7-h infusions. *P < 0.05, vs. Basal (Insulin vs. Saline).