ORM (Cat. No. G9885) was purchased from Sigma (St. Louis, MO). CCR5 antagonist Maraviroc was generously provided by Professor Xin Xie (Shanghai Institute of Materia Medica, CAS). Antibodies specific to AMPK (Cat. No. 2603), p-AMPK (at T172, Cat. No. 4188), glycogen synthase (GS, Cat. No. 3893), p-GS (Cat. No. 3891), and GAPDH (Cat. No. 2118) were obtained from Cell Signaling (Danvers, MA).

C57BL/6 mice (18–22 g) were purchased from Sino-British SIPPR/BK Laboratory Animals (Shanghai, China). The CCR5-deficient mice (B6.129P2-Ccr5tm1Kuz/J, Stock Number: 005427) were obtained from Jackson Laboratory (Bar Harbor, MA). AMPKα2 knockout mice were generated by S. Vaulont as described previously (Viollet et al., 2003). Mice were 6–8 weeks of age at the start of the experiments. All animals were maintained at 22°C on a 12-h light/dark cycle with free access to water and a standard rodent diet. All animal experiments were undertaken in accordance with the National Institute of Health's “Guide for the Care and Use of Laboratory Animals,” with the approval of the Scientific Investigation Board of the Second Military Medical University.

C2C12 cells (mouse muscle; myoblast; Cat. No. GNM26) were obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and cultured in DMEM high glucose supplemented with 10% fetal calf serum at 37°C and 5% CO₂. In a dose-effect experiment, C2C12 cells were exposed to ORM at the doses of 0, 0.1, 1, or 10 μg/ml for 2 h (Sörensson et al., 2000), then cells were harvested to detect the protein expression of p-AMPK and AMPK.

Mice were placed individually in a cylindrical glass tank (46 cm tall, 20 cm in diameter) of water at 21–23°C. A load consisting of a steel ring weighing 8% of each body weight was attached to the proximal end of the tail. The swimming time was measured from the time the mouse began swimming to the time it could not return to the surface of the water 10 s after sinking (Lei et al., 2016). This experiment was performed double-blindedly.

Muscle fibers group into two major categories: slow twitch type I fibers with high oxidative capacity adapted to endurance exercise, fast twitch type II fibers with low oxidative capacity adapted to sprint (Vogel et al., 2015). Due to the role of ORM in enhancing muscle endurance, slow twitch soleus muscle was adopted in the present study to explore the mechanism of ORM. Muscle fatigue was induced as previously reported (Lenman et al., 1989). Briefly, mice were sacrificed and soleus muscles were quickly isolated. One end of muscle was fixed, and the other was linked to the sensor of a biotic signal collection and processing system (MedLab-U/4CS, MeiYi, Nanjing, China). The muscle contraction was electrically evoked by trains of stimuli at 10 V lasting 5 ms and delivered each second for consecutive 3 min. The ratio of tension at 1, 2, or 3 min to the initial tension (average of the first five contractions) was expressed as the fatigue index.

Immunoblotting analyses were performed using Odyssey Infrared Fluorescent Imaging system. Samples were lysed with M-PER protein extraction reagent (Pierce, Rockford, IL, Cat. No. 78501) supplemented with protease inhibitor mixture (CalBiochem, San Diego, Cat. No. 539137). Protein concentration was estimated using Bradford reagent and bovine serum albumin as standard. Proteins were separated through SDS-PAGE and transferred onto PVDF membranes. The membranes were then incubated in primary antibodies prepared in TBST solutions (20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween-20). Detection was performed using appropriate secondary antibodies conjugated with infrared dyes 680 and 800. Equal loading of the samples was confirmed by re-probing the blots for GAPDH.

The glycogen content from mouse soleus muscle was determined using a glycogen assay kit (Biovision, USA, Cat. No. K646). In brief, 10 mg tissue was homogenized with 200 μl dH₂O on ice and boiled for 10 min to inactivate enzymes. The boiled sample was spun at 18,000 × g for 10 min to collect the supernatant. 25 μl supernatant was added to a 96-well plate, and the volume was adjusted to 50 μl each well with hydrolysis buffer. 2 μl hydrolysis enzyme mix was added to each well and the mixture was incubated for 30 min at room temperature. Then 50 μl reaction mix was added and the mixture was incubated for another 30 min at room temperature. Absorbance at OD 570 nm was then measured. Glycogen standard curve was concurrently prepared.

Professor Yan Chen (Institute for Nutritional Sciences, Chinese Academy of Sciences) helped to detect the activity of glycogen synthase. Briefly, mouse soleus muscle extracts were prepared by homogenizing 90–100 mg powdered muscles on ice in 400 μl of homogenization buffer (50 mM Tris-HCl pH 7.8), 100 mM NaF, 10 mM EDTA, 2 mM EGTA, and protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO, Cat. No. P8340]). The homogenates were centrifuged for 5 min at 3000 rpm, and the supernatants were retained to analyze as previously described (Zhang et al., 2014). Briefly, the supernatants were measured and adjusted to a concentration of 10 mg/ml by adding homogenization buffer. Muscle extracts (3 ul) were added to reaction cocktail I (27 ul) containing 25 mM Tris-HCl (pH 7.4), 100 mM KCl, 5 mM MgCl₂, 0.5% Glycogen (Sigma-Aldrich, St. Louis, MO, Cat. No. G8876), 10 mM glucose-6-phosphate (G-6P), and incubated with or without 5 mM 5′-urdiphosphate-glucose (UDPG, Sigma-Aldrich, St. Louis, MO, Cat. No. U4625) at 37°C for 30 min. Then the reaction was terminated at 100°C for 3 min. After centrifugation for 5 min at 10,000 rpm, supernatants (20 ul) were mixed with reaction cocktail II (10 ul) containing 6 mM HEPES (pH 7.4), 100 mM KCl, 5 mM MgCl₂, 2 mM PEP (Sigma-Aldrich, St. Louis, MO, Cat. No. 860077), 0.4 mM NADH (Roche Applied Science, Penzberg, Germany, Cat. No. 10107735001), 4 units pyruvate kinase/ml (Sigma-Aldrich, St. Louis, Cat. No. P1506) and 2 units lactate dehydroxylase (LDH)/ml (Sigma-Aldrich, St. Louis, MO, Cat. No. L3916). Reaction product was immediately detected by spectrophotometry at 340 nm, using a microtiter plate reader (Tecan). Then GS activity was calculated as followed: U/L = ΔA/min^*(V^*10⁶)/(ε^*v^*L). ΔA: variation of the absorbance; V: volume of the reaction system (ml); ε: Molar extinction coefficient (cm.mol); v: sample amount (ml); L: the light path length of colorimetric cuvette (cm).

Data were expressed as mean ± s.d. Statistical analyses were performed using Student's t-test when comparing two samples with equal variance (Figure 1) or one-way ANOVA with Dunnett's post-hoc test for the comparison of multiple treatments to controls (Figure 2). In Figures 3–6, statistical evaluation was performed by two-way ANOVA. When ANOVA revealed significant differences, a post hoc test was used to correct for multiple comparisons (Turkey's test). Differences between groups were considered statistically significant at P < 0.05.

We first testified whether AMPKα2 is involved in the regulation of muscle endurance and glycogen storage. Isolated mouse soleus muscle was utilized to electronically induce fatigue in vitro. As shown in Figure 1A, AMPKα2 knockout significantly decreased the fatigue index at 1, 2, and 3 min of the stimulation, suggesting that AMPK may directly enhance muscle endurance. Consistent with previous reports (Andreelli et al., 2003), AMPKα2 knockout mice have significantly decreased muscle glycogen content when compared to that in their wild littermates (Figure 1B). We suspected that AMPK may mediate the effect of ORM/CCR5 in enhancing muscle endurance and glycogen synthesis.

We further detected whether ORM can induce the activation of AMPK in the skeletal muscles. It has been shown that phosphorylation at T172 significantly increases AMPK activity and may also be used as a readout for its activation (Hawley et al., 1996). We found in myoblast cell C2C12, 0.1–10 μg/ml of ORM treatment for 2 h resulted in AMPK phosphorylation at T172 in a dose-dependent manner (Figure 2A). In mice injected with 200 mg/kg of ORM via tail-vein, serum ORM concentration peaked at 15 min post-injection then gradually declined (Lei et al., 2016, see Figure S5), and soleus muscle AMPK was phosphorylated at 15 min and the amount of phosphorylated AMPK peaked at 120 min post-injection (Figure 2B).

Since ORM could induce AMPK activation and the anti-fatigue effect of ORM was mediated through binding to CCR5 in the skeletal muscle (Lei et al., 2016), we want to figure out whether CCR5 is involved in the ORM-induced activation of AMPK. As shown in Figures 3A,B, tail-vein injection of 200 mg/kg of ORM for 2 h induced the phosphorylation of soleus muscle AMPK, which was inhibited by administration of CCR5 antagonist Maraviroc (200 mg/kg, via gastric gavage, for 3 days) and was largely absent in CCR5^−/−mice, suggesting that activation of AMPK is a downstream event of CCR5 activation.

It is worth noting that, we previously found that the binding of ORM to C2C12 skeletal muscle cells is incompletely inhibited by the CCR5 antibody and antagonist, indicating ORM may bind to other cell surface proteins (Lei et al., 2016). We subsequently found ORM was able to bind directly to LepR in hypothalamus to regulate energy homeostasis. More interestingly, ORM also bound to LepR in C2C12 cells and induced LepR-mediated JAK2-STAT3 activation (Sun et al., 2016, see Figures S7, S8). We therefore further explored whether LepR is also involved in the AMPK activation triggered by ORM in skeletal muscle. As shown in Figure 3C, ORM still remains similar stimulatory effect in AMPK activation in the soleus muscle from LepR deficient db/db mice when compared to that in C57BL/6 mice, indicating ORM-triggered AMPK phosphorylation is not dependent of LepR.

Our previous results revealed that ORM engaged CCR5 to increase the glycogen content in skeletal muscle. Glycogen synthase (GS) is the rate-limiting enzyme of glycogen synthesis, and de-phosphorylated GS is the active form responsible for the synthesis of glycogen (Ferrer et al., 2003). We wondered whether GS was involved in this CCR5-activated glycogen accumulation. Western blot analysis revealed that 200 mg/kg of ORM significantly increased the expression of total GS and decreased the ratio of phosphorylated GS in total GS in the soleus muscles in wild-type mice, but not in CCR5^−/− mice (Figure 4A). Meanwhile, GS activity is markedly increased in soleus muscles from mice treated with ORM, which is inhibited in CCR5^−/− mice, suggesting that ORM promoted the GS activity through CCR5 (Figure 4B). Interestingly, the upregulation of glycogen synthase protein happened at 30 min post ORM-injection, indicating that it may be regulated as an immediate early gene at transcriptional level or affected by protein degradation system at post-transcriptional level, which is worthy of being deeply explored.

We further wondered whether ORM/CCR5-activated-AMPK was also involved in the GS regulation. As shown in Figure 5, vein injection with 200 mg/kg of ORM for 30 min resulted in the significant increase in the expression of total GS (Figure 5A) and its activity (Figure 5B) in skeletal muscle in AMPKα2^+/+ mice, but absent in AMPKα2^−/− mice, indicating this effect was mediated by AMPK pathway.

We have previously reported that administration of purified ORM to the normal mice could significantly extend their swimming time and increase their muscle glycogen storage via CCR5, in which deletion of CCR5 abolished the effect of ORM on fatigue and muscle glycogen (Lei et al., 2016). Since AMPK is the downstream event of ORM/CCR5 activation, we want to make sure that whether AMPK is involved in the regulation of swimming endurance and glycogen storage of ORM. Here we found that in AMPKα2 deficient mice, the ability of ORM to increase the swimming time was completely diminished and it also failed to increase muscle glycogen in these mice (Figures 6A,B). These results indicated that the CCR5-mediated AMPK activation is required for ORM to enhance muscle performance and glycogen storage.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.