Effects of energy supplements on the differentiation of skeletal muscle satellite cells

Abstract To investigate the effects of the activator of AMPK and high glucose on the differentiation of mouse SMSCs, primary SMSCs were isolated from mouse extensor digitorum longus muscle and grown to near confluence (80%). Postconfluent cells were cultured in a growth medium with different inductors: AICAR, glucose, and AICAR mixed with glucose. The specific protein expressions of SMSCs, myoblasts, adipocytes, and brown adipocytes were analyzed on days 0, 3, 5, 7, and 10. The results showed treatment with AICAR in SMSCs markedly activated AMPK phosphorylation (p < .05) and increased protein expression of Pax7 and MyoD (p < .05), high concentrations of intracellular glucose upregulated UCP‐1 protein expression and enhanced lipid accumulation (p < .05), the cowork of AICAR and glucose affected a decrease on MyoD, PPARg, and UCP‐1 expression (p < .05) and an increase on Pax7. The present study indicated that the certain energy supplements influence the direction of SMSC differentiation which may contribution on the structure of muscle and meat quality, sequentially.

For example, it was observed that knocking out the α1 isoform of AMPK resulted in a high, self-renewal rate, and Warburg-like glycolysis in SMSCs (Fu et al., 2015). A variety of adenosine triphosphate (ATP)-consuming stimuli can activate AMPK, such as AICAR, exercise, electrical stimulation, and glucose deprivation (Gordon et al., 2008;Hutber et al., 1997;Salt et al., 1998;Winder & Hardie, 1996). Recently, it is suggested that the AMPK phosphorylation level, as a indicator of energy variation, may altered by exercise, and AICAR as an AMPK activator is involved in the differentiation of SMSCs in vitro. Therefore, it is the purpose of this study to investigate the effects of energy alteration on SMSC differentiation.
Alternatively, a high concentration of glucose not only increases extracellular energy but also has been confirmed to induce adipogenic differentiation in mesenchymal stem cells (Aguiari et al., 2008;Ronningen et al., 2015;Tao et al., 2010). Aguiari et al. (2008) found that primary cells derived from adipose tissue or skeletal muscle can differentiate into adipocytes when cultured in high glucose to form viable and vascularized adipose tissue when implanted in vivo.
Therefore, it is possible to induce the direct conversion of myoblasts to adipocytes by increasing the concentration of plasma glucose. This would increase intramuscular adipogenic differentiation of SMSCs resulting in marbling (Guillet-Deniau et al., 2004). It has been demonstrated that both AMPK activity and plasma glucose concentration contribute to muscle mass in humans and rodents (Guillet-Deniau et al., 2004;Theret et al., 2017). 5-Amino-1-β-d-ribofuranosylimidazole-4-carboxamide (AICAR) is one of the mainly activators which can be used as an experimental tool to activate AMPK (Merrill et al., 1997). In this respect, chronic treatment with AICAR to activate AMPK in muscle was reported to eventually result in glycogen accumulation (Winder & Holmes, 2000). Furthermore, glucose suppresses AMPK activation in isolated rat skeletal muscle and attenuation of exercise-induced AMPK-α2 activation in human muscle by oral ingestion has been reported (Akerstrom et al., 2006). Thus, the objective of this study was to assess the singular effects of AMPK activity and high glucose induction and their combination on the differentiation of SMSCs. In this respect, this study intended to explore the influence of energy supplementation on the multipotency differentiation of SMSCs, which may contribute to the development of postnatal muscle mass and intramuscular fat and thus improve meat quality.

| Isolation of primary mouse muscle satellite cells
Satellite cells were isolated from the hind limb muscles of three 2-week-old mice as previously described (Pasut et al., 2013) with some modifications. The muscles were dissected and minced to release cells by digestion in buffer containing a collagenase/dispase solution. Fast-attaching nonmyogenic cells were depleted by repeated plating. Primary satellite cells displayed as fusiform shape and were plated in growth medium (GM) that consisted of DMEM/F-12 (Gibco, Thermo Fisher) with 20% fetal bovine serum (Sijiqing), 10% horse serum (Solarbio), and 1% antibiotic mixture (Gibco, Thermo Fisher).
The cells were incubated in a 37℃ standard cell culture incubator with 5% CO 2 (Thermo Fisher).

| Differentiation assay
The SMSCs were grown in GM, which was changed every day until confluence to 80%. The postconfluent cells were cultured in four differentiation media: GM as control group (CG); GM + 300 µM AICAR (Ark Pharm, Montluçon, France) as the AICAR group (AG); GM + 25 mM glucose as the glucose group (HG); and GM + 300 µM AICAR + 25 mM glucose as the mixture group (AG + HG). Each group was prepared in triplicate, and the medium was changed every 48 hr until day 10. The cell samples were harvested on 0 day, 3 days, 5 days, 7 days, and 10 days for immunohistochemistry, Western blot, and Oil Red O analyses.

| Immunohistochemistry analysis
The cells on day 0 (undifferentiated) and day 10 (differentiated) were trypsinized and grown in six-well plates for immunohistochemistry analysis. Briefly, after the culture medium was completely removed, the cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, blocked with BSA (Bovine Serum Albumin) at 5 mg/ml, and incubated with the primary antibody Pax7 (monoclonal goat anti-mouse, 1:1,000; Abcam) overnight at 4°C. The cells were stained with corresponding secondary antibodies (mouse secondary antibody, 1:100; ProteinTech Group, Wuhan, China) for 1 hr at room temperature. The nuclei were counterstained with 10 µl DAPI (Thermo Fisher) for 5 min at room temperature. Images were taken with a fluorescence microscope (Olympus, Tokyo, Japan) and presented with Image-Pro Plus software (Media Cybernetics, Inc.).

| Oil Red O staining of intracellular lipids
The accumulation of triglycerides on days 0, 3, 5, 7, and 10 in three groups was visualized by staining the cells with Oil Red O (Sigma). The cells were transplanted to a 24-well plate, cultured in 37°C under 5% CO2 incubator for 2 hr, and then fixed with 10% formalin solution (Sinopharm Chemical Reagent Co.) for 20 min. The cells were stained with Oil Red O working solution for 10 min, rinsed with 60% isopropyl alcohol (Sinopharm Chemical Reagent Co.), and microphotographed.
Oil Red O concentration was measured spectrophotometrically at 510 nm using a microplate reader (Synergy H1; BioTek, Thermo Fisher).

| Statistical analysis
Treatment time intervals were analyzed by SPSS software (IBM Corp.).
The normality of data distribution and the homogeneity of variance were tested with the Shapiro-Wilk test and Levene's test, respectively. If the ANOVA assumptions were violated, a nonparametric test or a Welch correction was applied when appropriate. The results were expressed as means ± SE. Differences among means with p < .05 represented statistically significant differences.

| Effects of AMPK activator on differentiation of skeletal muscle satellite cells
The effects of AMPK activator on the differentiation of SMSCs were assessed using a specific protein expression. As shown in Figure 1a, the phosphorylation level of the AMPK protein was significantly increased (p < .05) during the process, indicating that it was efficiently activated by AICAR. Pax7 expression (Figure 1b

| Effects of glucose inductor on skeletal muscle satellite cell differentiation
Dietary carbohydrates are the main energy supplements that livestock need for maintenance, growth, and production; overall glucose is the primary energy source for many animals (Nafikov & Beitz, 2007). Unlike the contribution of acetate to subcutaneous adipose, glucose was found to be preferred in intramuscular adipose synthesis, which suggests that a high concentration of intercellular glucose may increase marbling potential (Smith & Crouse, 1984). The effects of glucose inductor on differentiation of SMSCs were tested by the specific protein expression. As shown in Figure 5a and Figure  high express for whole process. As shown in Figure 4 oil red O in HG, the lipid droplet was accumulated with the process, which means that glucose indeed induces satellite cell commit adipogenesis. Thus, we test the expression of UCP-1, the specific protein of other type of adipocytes, brown adipocytes, which showed in Figure 5d that was F I G U R E 5 The cellular morphology of SMSCs differentiation in AG, HG, AG + HG, and CG was observed for 10 days. The images of cells were taken in day 0 and day 10 of differentiation (magnification, ×20). AG: growth medium with 300 μM AICAR group; HG: growth medium with 25 mM glucose group; AG + HG: growth medium with 300 μM AICAR and 25 mM glucose group; and CG: growth medium as control group F I G U R E 4 Immunofluorescence staining for Pax7 on day 0, day 5, and day 10 in AG, HG, and AG + HG, respectively. Nuclei were stained with DAPI (Magnification, ×20). AG: growth medium with 300 μM AICAR group; HG: growth medium with 25 mM glucose group; and AG + HG: growth medium with 300 μM AICAR and 25 mM glucose group increased instantly in the process (p < .05 in day 5). Which in agree with Pasut et al. (2016), that Pax7-null satellite cells entered brown adipogenesis other than myogenic program by downregulating MyoD and miR-133 expression which was associated with Notch signaling rescuing. The present results suggested that the energy variation intracellular caused by high concentration of glucose may alter the fate transdifferentiation of myoblasts into brown adipocytes of skeletal muscle satellite cells.

| Effects of glucose and AICAR cowork on skeletal muscle satellite cell differentiation
To further explore the effects of the two energy sources on the preadipocytes (Pasut et al., 2016;Tong et al., 2008). And on the other side, Isabelle et al. (2004) found that high concentration of plasma glucose stimulated SREBP-1c upregulated leading to an increased intracellular lipid accumulation in contracting myotubes and satellite cells. Similar treatment with glucose, Yue et al. (2016) found that satellite cells differentiate into adipogenic program by activating mTOR, while Paola et al. (2008) suggested that the oxidizing agents of reactive oxygen species (ROS) are involved in the adipogenesis of satellite cells. These evidences implied that high glucose is able to induce adipogenic differentiation of satellite

| CON CLUS ION
In conclusion, the present study suggested that AMPK activitymediated satellite cell myogenic differentiation in vitro and physical exercise may activate AMPK phosphorylation to induce myogenesis of satellite cells in vivo. Furthermore, satellite cells committed adipogenesis program by increasing the concentration of intracellular glucose, which propose to enhance lipid accumulation in skeletal muscle. On the other side, variation of these two energy supplements in the meantime affected satellite cells commit to proliferation other than multipotential differentiation.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

E TH I C A L A PPROVA L
The animal experiments were approved by the Committee of Animal  Commission (1997) to minimize the suffering of animals.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.