Physical exercise attenuates age-related muscle atrophy and exhibits anti-ageing effects via the adiponectin receptor 1 signalling

Abstract

Background: Although the adiponectin signalling exerts exercise-mimicking effects, whether this pathway contributes to the anti-ageing benefits of physical exercise has not been established yet.

Methods: Swim exercise training and wheel running were used to measure lifespan in the nematode Caenorhabditis elegans and skeletal muscle quality in mice, respectively. Muscle weight, muscle fibre cross-sectional area (CSA) and myonuclei number were used to evaluate muscle mass. RNA sequencing (RNA-Seq) analysis of skeletal muscle in exercised mice was used to study the underlying mechanisms. Western blot and immunofluorescence were performed to explore autophagy- and senescence-related markers.

Results: The C. elegans adiponectin receptor PAQR-1/AdipoR1, but not PAQR-2/AdipoR2, was activated (3.55-fold and 3.48-fold increases in p-AMPK on Days 1 and 6, respectively, P < 0.001), which was involved in lifespan extension in exercised worms. Exercise training increased skeletal muscle mass index (1.29-fold, P < 0.01), muscle weight (1.75-fold, P < 0.001), myonuclei number (1.33-fold, P < 0.05), muscle fibre CSA (1.39-fold, P < 0.05) and capillary abundance (2.19-fold, P < 0.001 for capillary density; 1.58-fold, P < 0.01 for capillary number) in aged mice. Physical exercise reduced protein (2.94-fold, P < 0.001) and mRNA levels (1.70-fold, P < 0.001) of p16^INK4a , a marker for cellular senescence, in skeletal muscle of aged mice. These beneficial effects of exercise on skeletal muscle of mice were dependent on AdipoR1. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for differentially expressed genes in skeletal muscle between exercised mice with and without AdipoR1 knockdown by RNA-Seq analysis revealed that several KEGG pathways, such as 'AMPK signalling pathway' (P < 0.001), 'FOXO signalling pathway' (P < 0.001) and 'autophagy' (P < 0.001) were overrepresented. Knockdown of FoxO3a inhibited exercise-mediated beneficial effects on skeletal muscle quality of mice by inhibiting autophagy/mitophagy (3.81-fold reduction in LC3-II protein, P < 0.001; 1.53-fold reduction in BNIP3 protein, P < 0.05). Knockdown of daf-16, the FoxO homologue in C. elegans, reduced autophagy (2.77-fold and 2.06-fold reduction in GFP::LGG-1 puncta in seam cells and the intestine, respectively, P < 0.05) and blocked lifespan extension by exercise in worms.

Conclusions: Our findings provide insights into how the AdipoR1 pathway has an impact on the anti-ageing benefits of exercise and implicate that activation of the AdipoR1 signalling may represent a potential therapeutic strategy for reducing age-related loss of skeletal muscle.

Keywords: AdipoR1; C. elegans; FOXO; autophagy; exercise; rodent; skeletal muscle loss.

Figure 1

The PAQR‐1/AdipoR1 pathway is involved in lifespan extension in swim‐exercised worms. (A) AMPK was activated by swim exercise in worms. The phosphorylation of AMPK (Thr172) was measured by western blotting (left panel). Quantification of the ratio of p‐AMPK to actin (right panel). (B) Knockdown of paqr‐1 by RNAi significantly reduced the phosphorylation of AMPK (left panel). Quantification of the ratio of p‐AMPK to actin (right panel). (C) The expression of fat‐7::gfp was not altered in swim‐exercised worms. Representative images of FAT::GFP in Day 0 worms after swim exercise (left panel). Quantification of GFP levels (right panel). Scale bars: 100 μm. (D) Swim exercise significantly extended the lifespan in worms. Swim exercise failed to extend the lifespan in paqr‐1(tm3262) (E) and aak‐2(ok524) (F) mutants. ^*** P < 0.001, swim‐exercised worms versus control worms. ns, not significant.

Figure 2

Exercise improves muscle quality via AdipoR1 in mice. (A) Exercise paradigm in mice. Mice at 16 months of age in exercised groups were performed voluntary wheel running for 4 months. (B) The protein levels of AdipoR1 in skeletal muscle were upregulated by exercise. The protein levels of AdipoR1 and AdipoR2 were measured by western blotting (left panel). Quantification of the ratio of either AdipoR1 or AdipoR2 to GAPDH (right panel). (C) Knockdown of Adipor1 inhibited the activation of AMPK induced by exercise. The phosphorylation of AMPK (Thr172) was measured by western blotting (left panel). Quantification of the ratio of p‐AMPK to AMPK (right panel). (D) Muscle mass index of gastrocnemius muscles (n = 5 per group). (E) Muscle weight in gastrocnemius muscles of mice (n = 5 per group). (F) Myonuclei number in gastrocnemius muscles of mice (n = 5 per group). *P < 0.05, ^** P < 0.01 and ^*** P < 0.001. Ad, AdipoR1; NC, negative control; ns, not significant.

Figure 3

FOXO3a functions as a downstream molecule of AdipoR1 and improves muscle quality induced by exercise in mice. (A) Enrichment analysis of KEGG pathways was identified using the DAVID annotation tool. (B) Genes related to the FOXO signalling pathway, autophagy and the AMPK signalling pathway were regulated by AdipoR1, and exercise is shown in grey circles. 1, autophagy; 2, the FOXO signalling pathway; 3, the AMPK signalling pathway. (C) AdipoR1 regulated FOXO3a‐dependent genes. (D) Muscle mass index of gastrocnemius muscles (n = 5 per group). (E) Muscle weight in gastrocnemius muscles of mice (n = 5 per group). (F) Myonuclei number in gastrocnemius muscles of mice (n = 5 per group). *P < 0.05 and ^** P < 0.01. Fo, FoxO3a; NC, negative control; ns, not significant; siAd, knockdown of AdipoR1 by RNAi.

Figure 4

The AdipoR1–FOXO3a signalling induces autophagy in skeletal muscle of mice after exercise. (A, B) Exercise significantly increased the levels of the LC3B‐II and reduced the levels of p62 in skeletal muscle of exercised mice. The blot is typical of three independent experiments (A). Quantification of the ratio of LC3B‐II to β‐actin (B). Quantification of the ratio of p62 to β‐actin (B). (C, D) Knockdown of either AdipoR1 or FoxO3a by shRNA reduced the levels of the LC3B‐II in skeletal muscle of exercised mice. The blot is typical of three independent experiments (C). Quantification of the ratio of LC3B‐II to β‐tubulin (D). (E, F) Exercise upregulated the protein expression of BNIP3 in the skeletal muscle of mice. Knockdown of either AdipoR1 or FoxO3a by shRNA reduced the levels of the BNIP3 in the skeletal muscle of exercised mice. Representative images of immunohistochemical staining for BNIP3 (E). Scale bars: 50 μm. Quantification of BNIP3 expression in gastrocnemius muscles of mice (F). *P < 0.05, ^** P < 0.01 and ^*** P < 0.001. Ad, AdipoR1; Fo, FoxO3a; NC, negative control; ns, not significant.

Figure 5

DAF‐16 is involved in lifespan extension in swim‐exercised worms. (A, B) Swim exercise significantly increased the nuclear accumulation of DAF‐16::GFP. Knockdown of paqr‐1 by RNAi did not affect the nuclear localization of DAF‐16 induced by swim exercise. Representative images of DAF‐16::GFP expression pattern in worms (A). Scale bars: 100 μm. Quantification of DAF‐16 distribution (B). (C, D) Swim exercise upregulated the expressions of hsp‐16.2::nCherry, dod‐3::gfp and mtl‐1p::bfp. However, RNAi knockdown of paqr‐1 or aak‐2 reduced the expressions of these three genes. Representative images of dod‐3p::gfp, hsp‐16.2p::nCherry and mtl‐1p::bfp in worms after swim exercise (C). Scale bars: 200 μm. Quantification of fluorescent intensity of hsp‐16.2p::nCherry, dod‐3p::gfp and mtl‐1p::bfp (D) in worms. (E) Swim exercise failed to extend the lifespan in daf‐16(mu86) worms. These results are means ± SD of three independent experiments (n = 30 worms per experiment). *P < 0.05, ^** P < 0.01 and ^*** P < 0.001. ns, not significant.

Figure 6

DAF‐16 induces autophagy, which is required for lifespan extension in swim‐exercised worms. (A) Representative images of autophagosomes (GFP::LGG‐1 puncta) in the seam cells and intestine of 0‐day‐old worms subjected to swim exercise. The arrow denotes a representative autophagosome. Knockdown of DAF‐16 by RNAi reduced autophagy induced by swim exercise, which was detected by GFP::LGG‐1 puncta in the seam cells and intestine of swim‐exercised worms. Scale bars: 10 μm. (B) The numbers of GFP::LGG‐1 puncta were counted. These results are means ± SD of three independent experiments (n = 30–35 worms per experiment). (C, D) Autophagy was required for lifespan extension in swim‐exercised worms. Swim exercise failed to extend the lifespan in worms subjected to bec‐1 (C) or unc‐51 RNAi (D). (E, F) Mitophagy was involved in lifespan extension in swim‐exercised worms. Swim exercise failed to extend the lifespan in worms subjected to dct‐1 (E) or pink‐1 RNAi (F). *P < 0.05 and ^** P < 0.01. ns, not significant.

Figure 7

Autophagy/mitophagy is required for exercise‐induced healthy longevity in worms. Autophagy was involved in delaying the appearance of the ageing markers, including body bending (A), pharyngeal pumping (B) and relative collagen levels (C), in swim‐exercised worms. These results are means ± SD of three or five independent experiments (n = 30–35 worms per experiment). (D) Swim exercise restored age‐related muscle deterioration in an autophagy‐dependent manner. Representative images of myo‐3p::GFP::myo‐3 in exercised worms. Scale bars: 10 μm. *P < 0.05 and ^** P < 0.01. ns, not significant.