Adiponectin receptors activation performs dual effects on regulating myogenesis and adipogenesis of young and aged muscle satellite cells

Abstract Objectives Skeletal muscle mass and function deteriorate with ageing. Adiponectin receptors (APNrs), mainly activated by adiponectin, participate in various physiological activities and have varying signalling pathways at different ages. This study aimed to explore whether discrepant performance exists in APNr activation regulating young and aged muscle satellite cells (MUSCs) and whether age‐related muscle dysfunction could be alleviated upon APNr activation. Methods The gastrocnemius muscle phenotype was observed in male mice aged 2 and 18 months. An APNr agonist (AdipoRon) was used in vitro and in vivo to investigate the changes in cell biological behaviours and whether muscle dysfunction could be retarded after APNr activation. Results Aged mice exhibited decreased muscle mass and increased fat infiltration. APNr activation inhibited C2C12 cells and young MUSCs (YMUSCs) proliferation but showed no obvious effect on aged MUSCs (AMUSCs). Moreover, APNr activation inhibited the migration of both YMUSCs and AMUSCs. Interestingly, APNr activation hampered the myogenic differentiation but advanced the adipogenic differentiation of YMUSCs, yet exact opposite results were presented in AMUSCs. It was demonstrated that Wnt and PI3K signalling pathways may mediate the phenotypic differences. Furthermore, in vivo experiments verified that APNr activation ameliorated age‐related muscle atrophy and excessive fat infiltration. Conclusions APNr activation exerted dual effects on the regulation of myogenesis and adipogenesis of YMUSCs and AMUSCs and rescued age‐related skeletal muscle dysfunction.

commonly referred to as skeletal muscle atrophy. 4,5 Moreover, muscles are associated with adipose tissue; the distribution of which changes with age in both physiological and pathological processes.
With increasing age, the levels of intramuscular fat increase within and around the fibre bundles, leading to muscle dysfunction. 6,7 Clinical and preclinical evidence has confirmed that enhancing muscle performance in elderly individuals is achieved by reducing intramuscular lipids content. 8,9 Pharmacological therapeutic interventions that simultaneously alleviate skeletal muscle atrophy and improve the corresponding lipid metabolism would be an ideal drug therapies for agerelated muscle dysfunction but have not yet been identified.
Skeletal muscle can recover its structural integrity and function in a short time after certain injuries because of its strong regeneration ability. Moreover, the regulation of skeletal muscle maintenance, repair and regeneration is mainly mediated by muscle stem cells, of which muscle satellite cells (MUSCs) play a key role. 4,10 MUSCs are flat and protuberant cells in skeletal muscle adjacent to the plasma membrane of muscle fibres and lie beneath the basal lamina. MUSCs are generally quiescent and are activated to proliferate and migrate upon injury or external signal intervention. 11 Proliferating cells finally differentiate into myoblasts and fuse with each other or with existing muscle fibres to form new muscle fibres; this process is regulated by a series of myogenic regulatory factors, including myogenin (MyoG) and MyoD. 12 Other identified internal or external transcription factors are also involved in regulating muscle regeneration via the interaction of MUSCs with inflammatory cells, stromal cells, nutritional signals and the extracellular matrix. 13 However, in the aged state, MUSCs lack sufficient regulatory signals, and impaired function weakens their potential for muscle regeneration. 14 Moreover, ageing is associated with the accumulation of intracellular lipids, and MUSCs have the potential for adipogenic differentiation. Notably, adipocytes and myoblasts are derived from a common pool of progenitor cells that express the early myogenic regulatory factor, Myf5. Genes that monitor myogenesis and adipogenesis are regulated by epigenetic mechanisms, suggesting that either process could be chosen under certain conditions. 15 Thus, impaired muscle regeneration and increased infiltration of adipose tissue with age cause skeletal muscle dysfunction.
Adiponectin is an important adipocytokine synthesized and secreted by adipocytes. Generally, adiponectin executes multiple metabolic activities, such as improving insulin sensitivity, anti-inflammatory effects and promoting energy utilization by binding to and activating cell surface receptor-adiponectin receptors (APNrs), namely AdipoR1 and AdipoR2. 16,17 As an important target of adiponectin, skeletal muscle can express APNrs, and muscle metabolism is regulated via APNr activation in certain aspects such as muscle regeneration and lipid distribution. The decreased regeneration ability of age-related muscle stem cells and muscle metabolic disorders could be improved through the adiponectin/ AdipoR1 axis-mediated AMP-activated protein kinase (AMPK) pathway in senescence-accelerated mice. 18 APNr activation via globular adiponectin could inhibit the expression of muscle atrophy marker proteins Atrogin-1 and muscle ring finger protein-1 (MuRF-1), improving muscle atrophy induced by dexamethasone. 19 In addition, APNr activation can induce goat skeletal MUSCs to differentiate into adipocytes and alleviate the diabetic phenotype. 20,21 Furthermore, circulating levels and functions of adiponectin differ in multiple physiological and pathological states. However, the mechanism by which APNr activation affects muscle function through downstream signals at different ages remains unknown.
Given the close link between APNr-mediated signalling and muscle metabolism, we aimed to test the efficacy of APNr activation on MUSC function at different ages. AdipoRon, a newly discovered small molecule activator of APNrs, 22  Gibco BRL, Grand Island, New York) in a 37% CO 2 humidified incubator.
Isolation and culture of MUSCs were performed as previously described. 23 Briefly, the hindlimb muscle tissues were isolated and fully cut into pieces. Then, the pieces were then digested with collagenase I for 1 h and centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the precipitate was digested with trypsin for 15 min. The pellet was resuspended in a culture flask after centrifugation. The suspension was transferred to a new flask coated with Matrigel for 2.5 h, and AdipoRon (Selleck Chemicals, China) was used to activate APNrs.

| Senescence-associated β-galactosidase staining
A senescence β-galactosidase staining kit (Solarbio, Beijing, China) was used to test the expression of senescence-associated β-galactosidase in cells from young and aged mice.

| Cell proliferation and migration
To test the effect of AdipoRon on C2C12 cells, YMUSCs and AMUSCs, cells were seeded in 96-well plates at 5 Â Transwell assays were performed to assess the effect of Adi-poRon on MUSC migration. The cells were seeded into the upper chamber at a concentration of 2 Â 10 4 /well. Media (containing gradient AdipoRon) with or without 10% serum were added to the lower and upper chambers, respectively. After 18 h, the upper chamber was gently wiped off with cotton swabs, and the cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet dye.

| Myogenic and adipogenic induction
For myogenic differentiation, the medium was composed of DMEM with 2% (C2C12 cells) or 3% (MUSCs; vol/vol) horse serum (Solarbio, China). Cells were exposed to induction and differentiation media for adi-   Table S1.  Table S2). An ECL kit was used for visualization.

| SiRNA transfection
After cell adherence, AdipoR1 siRNA, AdipoR2 siRNA, AdipoR1 siRNA plus AdipoR2 siRNA and scramble siRNA were prediluted in serum-free Opti-MEM medium with 5% EndoFectin (Gene Pharma, China) per well (the siRNA sequences were listed in Table S3). Myogenic and adipogenic induction was performed for another 3 days after 8 h of treatment.

| Immunofluorescence
For immunofluorescence, cells were fixed with 4% paraformaldehyde solution for 10 min. After washing three times with phosphate-buffered saline, the cells were incubated with 0.5% Triton X-100 for 15 min and blocked with 1% bovine serum albumin for 0.5 h. The primary antibody was added and incubated at 4 C overnight, and the secondary antibody was successively incubated the next day. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI).

| Animal preparation and processing
All animal procedures were approved by the Animal Care Committee of Sichuan University. Male C57BL/6 mice aged 2 months (young mice) and 18 months (aged mice) were both obtained from the West China Medical Laboratory Animal Center of Sichuan University. Gastrocnemius muscles from young and aged mice were isolated and weighed for later analysis. Sixty aged mice (18 months old) were randomly divided into three groups. AdipoRon was dissolved in corn oil and intra-gastrically administered to (0, 5 and 50 mg/kg body weight [BW]) every alternate day. At 2 and 4 weeks, gastrocnemius muscles were obtained, weighed and then subjected to qRT-PCR, western blot and histological detection.

| Histology staining
Gastrocnemius samples were used to prepare paraffin and frozen sections. For paraffin sections, samples were embedded in paraffin, and 4-μm-thick sections were acquired. After de-waxing and rehydration, the sections were subjected to haematoxylin and eosin (HE) staining (Solarbio, China). Frozen sections were also acquired after dehydration, embedded with optimal cutting temperature compound, and subjected to oil red O staining (Solarbio, China). The cross-sectional area (CSA) and Feret's diameter of the muscle fibres were determined using ImageJ software. For immunofluorescence staining of paraffin sections, antigen retrieval was performed after deparaffinization, and the subsequent procedures were similar to those used for immunofluorescence staining.

| Statistical analysis
Quantitative data are presented as the means ± SD. Comparisons between groups were performed using one-way ANOVA. The IBM SPSS Statistical software (version 20.0, IBM Corp., Armonk, New York) was used to analyse the experimental results. The significant p-value was set to <0.05.

| Skeletal muscle atrophy and intermuscular adipose tissue infiltration appeared in aged mice
Phenotypic characteristics of skeletal muscle from 2-month-old and 18-month-old mice were evaluated. HE staining indicated that both CSA and Feret's diameter of the muscle fibres were reduced in aged mice compared with young mice, and aged mice also showed an increase in fat mass according to the oil red O staining ( Figure 1A,B). In addition, muscle weight relative to BW (MW/BW) significantly decreased with age ( Figure 1C). The results of qRT-PCR showed that Atrogin-1 and MuRF-1, two representative proteins of muscle atrophy, were highly expressed in aged muscle samples ( Figure 1D). Moreover, the results also suggested that myogenic factors (MyoD, MyoG) declined with age, while higher expression of adipogenic factors (CEBP-α and LPL) was observed in aged mice ( Figure 1D). Consistently, results of western blot and the subsequent quantitative analysis further confirmed these findings ( Figure 1E,F).
In addition, a greater amount of inguinal fat was observed in aged mice compare with young mice ( Figure S1).
F I G U R E 1 Skeletal muscle atrophy and intermuscular adipose tissue infiltration were discovered in aged mice. (A) Haematoxylin and eosin (HE) and oil red O staining of gastrocnemius muscles from young and aged mice. Bar =100 μm. (B), (C) Cross-sectional area (CSA), Ferret's diameter, and muscle weight/body weight ratio of muscles in young and aged mice. (D), (E) Gene and protein expression in gastrocnemius muscles isolated from young and aged mice. (F) Quantitative analysis of protein expression levels between young and aged mice. *p < 0.05, compared with the young group. Data are presented as the mean ± SD (n = 10 male C57BL/6 mice/group).

| AMUSCs showed a senescent phenotype and impaired myogenesis compared with YMUSCs
YMUSCs and AMUSCs were isolated from young and aged mice, respectively. SA-β-gal staining showed that more senescent cells presenting blue staining were found in AMUSCs (Figure 2A). In addition, immunofluorescence detection verified that MuRF-1 was highly expressed in AMUSCs compare with YMUSCs, and less myosin staining in AMUSCs hinted its impaired capability to form myotubes ( Figure 2B). Furthermore, the expression of AdipoR1 and AdipoR2 was observed in both YMUSCs and AMUSCs and the results of immunofluorescence staining showed that they were both distributed in the cytoplasm and cytomembrane ( Figure 2C). Activation of AMPK and peroxisome proliferator-activated receptor-α (PPAR-α) signalling pathways upon AdipoRon treatment were confirmed by western blot, suggesting that APNr activation was achieved in both YMUSCs and AMUSCs ( Figure 2D).
APNr activation upon AdipoRon treatment was further testified via activated AMPK and PPAR-α signalling pathways ( Figure 3D). The effect of APNr activation on C2C12 cell differentiation was then explored. Immunofluorescence staining after 5 days of myogenic induction showed that myogenesis of C2C12 cells was suppressed in a concentration-dependent manner ( Figure 3E). The qRT-PCR and western blot assays showed that the expression of myogenic markers, such as muscle creatine kinase

| APNr activation presented dual effects on myogenesis and adipogenesis of YMUSCs and AMUSCs
Immunofluorescence staining of myosin indicated that YMUSCs reacted to APNr activation similarly to C2C12 cells: myotube formation was repressed by AdipoRon in a concentration-dependent manner, while a significantly advanced effect on myotube formation was found in AMUSCs ( Figure 5A). The qRT-PCR and western blot assays  and AdipoR2 knockdown ( Figure 6D).  results of MW/BW showed the same tendency ( Figure 8C). Oil red O staining showed that fat infiltration was alleviated by AdipoRon treatment ( Figure 8B). Moreover, AdipoRon administration caused no significant change in BW ( Figure S3A). In addition, the results of immunofluorescence staining, qRT-PCR and western blot showed that AdipoRon inhibited the expression of muscle atrophy markers (Atrogin-1 and MuRF-1) and adipogenic markers (CEBP-α and LPL) and promoted the expression of myogenic markers (MyoD and MyoG) as the concentration increased ( Figure 8D-F). Moreover, the AMPK and PPAR-α signalling pathways were both activated after AdipoRon treatment ( Figure 8F). Furthermore, inguinal fat was reduced after the administration of AdipoRon, and no significant drug toxicity-related injuries were found in the heart, kidney or liver samples ( Figure S3B,C).

| DISCUSSION
Skeletal muscle dysfunction is an age-induced decline in muscle strength, accompanied by a loss of muscle mass and accumulation of adipose tissue. 24,25 In this study, we used AdipoRon, an APNr agonist, to explore the effect of APNr activation on skeletal muscle function.
Interestingly, opposite responses were observed in young and aged MUSCs upon APNr activation. AdipoRon impaired the myogenic capability of C2C12 cells and YMUSCs but promoted adipogenesis, whereas AMUSCs showed enhanced myogenesis and inhibited adipogenesis after APNr activation. Different changes in the Wnt and PI3K signalling pathways after APNr activation in the young and aged states may shed light on the aforementioned effects (Figure 9). Since its discovery, the effects of adiponectin on diverse metabolic activities mediated by APNr activation have been widely studied, including bone, lipid and muscle metabolism. [26][27][28] Adiponectin is a systemic bioactive hormone that exerts physiological functions by binding to APNrs (AdipoR1 and AdipoR2). AdipoR1 and AdipoR2 are both abundantly expressed in skeletal muscle and share certain similarities in sequence and ligand-binding sites. 29 They are predicted to have seven transmembrane domains but are distinct from G proteincoupled receptors. 30 AdipoR1 activates the AMPK pathway, and Adi-poR2 activates the PPAR-α pathway; adiponectin binds to the two receptors and regulates glucose uptake, lipid oxidation and other metabolic processes. 31,32 The specific knockout of muscle AdipoR1 leads to a decrease in AMPK activity and involuntary motor ability. 33 Adiponectin intervention in C2C12 cells induced AMPK phosphorylation and PGC1-α activation, which required AdipoR1 signal transduction. 34 However, the large size and complex polymerization characteristics of adiponectin make its extensive use inconvenient, and alternative APNr agonists have been explored in APNr activation. AdipoRon is a small molecule compound that can act precisely on APNrs. As an effective substitute for adiponectin, it was applied in this research to act on APNrs (AdipoR1 and AdipoR2). Similar to previous studies, our study showed that regardless of age, the AMPK and PPAR-α signalling pathways were activated after AdipoRon treatment, suggesting that APNr activation was achieved.
MUSCs have been shown to possess myogenic and adipogenic abilities. 35,36 Numerous studies have shown that the function of MUSCs is impaired with age. 37,38 As part of the ageing process, MUSCs suffer from impaired myogenic ability and decreased regulation of the cell cycle and cell fate, which causes adipose tissue accumulation, leading to muscle dysfunction finally occurs. 39  activation, but the opposite differentiated behaviours were observed in the young and aged states. These data suggest that the effects of APNr activation on MUSC function may be related to age, a prospect that deserves further exploration.
A broader investigation aimed downstream of the adiponectin signalling pathway is also needed to demonstrate further mechanisms that clarify age-related phenotypic differences. In this study, we found that the different manifestations of MUSC differentiation in young and aged states had little to do with the AMPK and PPAR-α signalling pathways; in contrast, the Wnt and PI3K signalling pathways play an important role in adiponectin signalling pathways and are both involved in modulating muscle and adipose metabolism. 40,41 The activated PI3K signalling pathway can attenuate lipid accumulation, rescues myotube formation and ameliorate skeletal muscle atrophy. [42][43][44][45] Wnt signalling participates in the regulation of satellite cell differentiation and self-renewal, and this pathway is poorly activated in mature skeletal muscles. 46 Activated β-catenin signalling can increase myogenin expression to stimulate myoblast differentiation and inhibit adipogenic differentiation. 47 Stabilized β-catenin can govern skeletal muscle fibro/adipogenic progenitor adipogenesis and repress PPAR-γ expression. 41 Moreover, the two signalling pathways are closely connected, and PI3K pathway activation can cause a series of changes in the Wnt pathway. 48 Interestingly, we found that the PI3K/Wnt signalling pathway was inhibited in YMUSCs; in AMUSCs, however, no obvious change was observed in the PI3K pathway, but Wnt pathway activation was evident. Based on the above findings, we inferred that the ageing may disturb the response of the PI3K/Wnt pathway to APNr activation. Although there was no activation of the PI3K pathway, other possible factors could have activated of the Wnt pathway. Studies have verified that changes in sirtuins and mTOR due to ageing may cause changes in the regulatory function of signalling pathways. 49,50 Furthermore, studies have shown that weakening ageinduced ROS levels can decrease the binding of FoxO to β-catenin, transforming FoxO-mediated transcription into original T-cell factor (TCF)-mediated transcription and inhibiting the Wnt pathway. 51 Certainly, the specific factors causing these differences require further exploration.
The activation of APNrs in muscle function has been previously studied. Singh et al. 19 found that activated APNrs could mitigate dexamethasone-induced muscle atrophy in vivo through the AMPK pathway, thereby simulating PPAR-γ coactivator 1α. Balasubramanian et al. 52 observed that APNr activation promoted skeletal muscle repair in aged mice, which was linked to changes in fibre types, especially oxidative fibres. Given that APNr activation could promote osteogenesis and attenuate adipogenesis in AMUSCs, we verified that whether age-induced skeletal muscle dysfunction could be improved via F I G U R E 9 A sketch map of the dual effects of adiponectin receptor (APNr) activation on regulating muscle satellite cell function at different ages. enhanced myogenesis and decreased adipogenesis under APNr activation. As expected, significant increases in MW/BW, CSA and Feret's diameter were observed after AdipoRon administration. The gene and protein expression levels were consistent with the tissue staining results. Furthermore, the levels of marker factors of muscle senescence were significantly decreased.
Although we have provided promising evidence that APNr activation could affect MUSC function and rescue age-related skeletal muscle dysfunction, further in-depth research is required. Owing to the different cellular and internal environments between young and aged states, 53 the influence of certain senescence-associated factors on the regulatory effect of APNr activation has not yet been clarified. For the in vivo studies, whether AdipoRon administration increases the change in adiponectin secretion should also be explored. The complex endocrine and paracrine systems must also be considered. Furthermore, the results we acquired were from the gastrocnemius in male mice, and additional studies may be extended to the muscles of other body parts and female mice to verify the generality of APNr activation.

| CONCLUSIONS
Overall, our study demonstrated that APNr activation had different effects on myogenic and adipogenic differentiation in aged and YMUSCs. In addition, APNr activation could promote the myogenic differentiation of AMUSCs and inhibit their adipogenic differentiation, further rescuing skeletal muscle atrophy, suppressing fat infiltration and alleviating age-related skeletal muscle dysfunction.

CONFLICT OF INTEREST
The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

DATA AVAILABILITY STATEMENT
All data needed to evaluate the conclusions in the manuscript are present in the main figures and the Supplementary Materials. Additional related data are available upon request from the authors.