Free essential amino acid feeding improves endurance during resistance training via DRP1-dependent mitochondrial remodelling

Abstract

Background: Loss of muscle strength and endurance with aging or in various conditions negatively affects quality of life. Resistance exercise training (RET) is the most powerful means to improve muscle mass and strength, but it does not generally lead to improvements in endurance capacity. Free essential amino acids (EAAs) act as precursors and stimuli for synthesis of both mitochondrial and myofibrillar proteins that could potentially confer endurance and strength gains. Thus, we hypothesized that daily consumption of a dietary supplement of nine free EAAs with RET improves endurance in addition to the strength gains by RET.

Methods: Male C57BL6J mice (9 weeks old) were assigned to control (CON), EAA, RET (ladder climbing, 3 times a week), or combined treatment of EAA and RET (EAA + RET) groups. Physical functions focusing on strength or endurance were assessed before and after the interventions. Several analyses were performed to gain better insight into the mechanisms by which muscle function was improved. We determined cumulative rates of myofibrillar and mitochondrial protein synthesis using ²H₂O labelling and mass spectrometry; assessed ex vivo contractile properties and in vitro mitochondrial function, evaluated neuromuscular junction (NMJ) stability, and assessed implicated molecular singling pathways. Furthermore, whole-body and muscle insulin sensitivity along with glucose metabolism, were evaluated using a hyperinsulinaemic-euglycaemic clamp.

Results: EAA + RET increased muscle mass (10%, P < 0.05) and strength (6%, P < 0.05) more than RET alone, due to an enhanced rate of integrated muscle protein synthesis (19%, P < 0.05) with concomitant activation of Akt1/mTORC1 signalling. Muscle quality (muscle strength normalized to mass) was improved by RET (i.e., RET and EAA + RET) compared with sedentary groups (10%, P < 0.05), which was associated with increased AchR cluster size and MuSK activation (P < 0.05). EAA + RET also increased endurance capacity more than RET alone (26%, P < 0.05) by increasing both mitochondrial protein synthesis (53%, P < 0.05) and DRP1 activation (P < 0.05). Maximal respiratory capacity increased (P < 0.05) through activation of the mTORC1-DRP1 signalling axis. These favourable effects were accompanied by an improvement in basal glucose metabolism (i.e., blood glucose concentrations and endogenous glucose production vs. CON, P < 0.05).

Conclusions: Combined treatment with balanced free EAAs and RET may effectively promote endurance capacity as well as muscle strength through increased muscle protein synthesis, improved NMJ stability, and enhanced mitochondrial dynamics via mTORC1-DRP1 axis activation, ultimately leading to improved basal glucose metabolism.

Keywords: Metabolic flux; Mitochondrial dynamics; Muscle mass; Neuromuscular junction stability; Physical performance; Protein synthesis rate.

Figure 1

EAA + RET increases not only muscle mass and strength but endurance capacity. (A) Schematic representation of the experimental design. This figure was created with BioRender.com. (B) Total hindlimb muscle mass (sum of soleus, plantaris, flexor hallucis longus, gastrocnemius, extensor digitorum longus, and tibialis anterior weight) (n = 8 per group). (C) Laminin staining of muscle cross‐sectional area and distribution of myofiber size in gastrocnemius (n = 7–8 per group). Scale bar, 100 μm. (D) Muscle strength is representative of maximal carrying capacity (n = 8 per group). (E) Relative muscle quality in maximal carrying capacity (maximal carrying capacity normalized by hindlimb muscle mass). (F) Total running distance during the treadmill exhaustion test. Data are presented as mean ± SE. *Significant difference between labelled groups (*P < 0.05). EAA + RET, essential amino acids + resistance exercise training; EAA, essential amino acids; RET, resistance exercise training; SED, sedentary; Veh, vehicle.

Figure 2

EAA + RET increases net muscle protein synthesis largely by stimulation of MPS via activation of Akt1/mTORC1 signalling and by suppression of MPB via suppression of myostatin expression. (A) Integrated myofibrillar protein synthesis rate in gastrocnemius over 14 days (n = 5–6 per group). (B) Chronic effect of EAAs and/or RET on relative protein expression of Akt1, mTORC1, p70s6k, rps6, and myostatin in gastrocnemius over 4 weeks and representative image (n = 8 per group). (C) Schematic representation of the experimental design: Mice were sacrificed 1 h after ladder climbing and/or oral EAA injection. This figure was created with BioRender.com. (D) Acute response to EAAs and/or resistance exercise on relative protein expression of Akt1, mTORC1, p70s6k, rps6, and myostatin in gastrocnemius and representative image (n = 6 per group). Data are presented as mean ± SE. *Significant difference between labelled groups (*P < 0.05). Akt1, protein kinase B; EAA, essential amino acids; mTORC1, mammalian target of rapamycin complex 1; myostatin, growth differentiation factor‐8; p70s6k, ribosomal protein S6 kinase beta‐1; RE, resistance exercise; RET, resistance exercise training; rps6, ribosomal protein s6; SED, sedentary; Veh, vehicle.

Figure 3

EAA + RET more robustly enhances neuromuscular junction stability. (A) Acetylcholine receptor cluster size in plantaris (n = 6 per group). Scale bar, 100 μm. (B) Acetylcholine receptor cluster size distribution. (C) Representative image of total and phosphorylated MuSK protein expression in plantaris (n = 8 per group). Data are presented as mean ± SE. *Significant difference between labelled groups (*P < 0.05). AchR, acetylcholine receptor; CON, control; EAA + RET, essential amino acids + resistance exercise training; EAA, essential amino acids; EAA, essential amino acids; MuSK, muscle‐specific tyrosine kinase; RET, resistance exercise training; SED, sedentary; Veh, vehicle; α‐BTX, alpha bungarotoxin.

Figure 4

EAA treatment increases rate of mitochondrial protein synthesis and dynamics. (A) Western blots for COX IV and PGC‐1α in gastrocnemius and representative images (n = 8 per group). (B) The relative mtDNA copy number with qPCR measurement in gastrocnemius. (C) Integrative rate of mitochondrial protein synthesis over 14 days in gastrocnemius. (D) Chronic effect of EAAs and/or resistance exercise training on relative protein expression of DRP1 and OPA1 in gastrocnemius and representative image (n = 8 per group). (E) Acute response to EAAs and/or resistance exercise on relative protein expression of DRP1 and OPA1 in gastrocnemius and representative image (n = 6 per group). Data are presented as mean ± SE. *Significant difference between labelled groups (*P < 0.05). COX IV, cytochrome c oxidase subunit IV; DRP1, dynamin‐related protein 1; EAA, essential amino acids; GAS, gastrocnemius muscle; OPA1, dynamin‐like 120 kDa protein; PGC‐1α, peroxisome proliferator‐activated receptor‐gamma coactivator 1‐alpha; RE, resistance exercise; RET, resistance exercise training; SED, sedentary; Veh, vehicle.

Figure 5

EAA treatment increases mitochondrial abundance and oxidative capacity in DRP1‐dependent manner. (A) Western blot for total and phosphorylated DRP1 after siRNA‐induced DRP1 knockdown and representative images (n = 3 per group). (B) The relative mtDNA copy number with qPCR measurement after siRNA‐induced DRP1 knockdown. (C) Western blot for total and phosphorylated mTORC1 after siRNA‐induced DRP1 knockdown and representative images. (D) Schematic representation of mTROC1‐dependent DRP1 activation by EAA supplementation. This figure was created with BioRender.com. (E) Oxygen consumption rate after siRNA‐induced DRP1 knockdown (n = 5 per group). (F) Maximal respirator capacity after siRNA‐induced DRP1 knockdown. Data are presented as mean ± SE. *Significant difference between labelled groups (*P < 0.05). CON, control; DRP1, dynamin‐related protein 1; EAA, essential amino acids; mTORC1, mammalian target of rapamycin complex 1; OCR, oxygen consumption rate; Veh, vehicle.

Figure 6

EAA + RET improves basal glucose kinetics. (A) Blood glucose concentration during hyperinsulinaemic–euglycaemic clamp (n = 10–11 per group). (B) Glucose infusion rate during the hyperinsulinaemic–euglycaemic clamp. (C) Basal blood glucose concentration. (D) Endogenous glucose production in basal state. (E) Muscle glucose uptake. Data are presented as mean ± SE. *Significant difference between labelled groups (*P < 0.05). CON, control; RET, resistance exercise training; EAA + RET, essential amino acids + resistance exercise training; EGP, endogenous glucose production.