Muscle myoblast isolation and culture

Primary myoblasts were isolated using type I collagenase and dispase B digestion (33, 34). Briefly, hind-limb skeletal muscles from adult WT and Mstn^−/− mice were collected, minced, and digested. The digestions were stopped with Ham's F10 medium containing 20% fetal bovine serum (FBS), filtered (100 μm), and centrifuged at 450 g for 5 min. The resulting pellets were resuspended, and cells were seeded on collagen-coated dishes and cultured in growth medium containing Ham's F10 medium with 20% FBS, 4 ng/ml basic fibroblast growth factor, and 1% penicillin–streptomycin at 37°C with 5% CO₂. The medium was changed every 2 d. To prepare myotube CM, myoblasts from WT and Mstn^−/− mice were induced to differentiate in DMEM with 2% horse serum and 1% penicillin/streptomycin on 70% confluence. After 3 d of differentiation, cells were incubated in DMEM plus 10% FBS for 24 h; the resulting medium was then collected, filtered, and used as myotube CM. To inhibit Mstn signaling, cells were treated with anti-Mstn (kindly provided by Dr. Yong-Soo Kim, University of Hawaii, Manoa, HI, USA) or follistatin (SRP3045, 200 ng/ml; Sigma, St. Louis, MO, USA). To inhibit AMPK, cells were treated with compound C (5 µM; Millipore, Billerica, MA, USA).

Adipose SVF cell isolation and culture

The WAT SVF cells were isolated using collagenase digestion followed by density separation (35, 36). Briefly, forelimb subcutaneous WAT depots were collected and minced into 2- to 5-mm² pieces. WAT pieces were then digested in 1.5 mg/ml collagenase at 37°C for 1.5–2 h. The digestions were stopped with DMEM containing 10% FBS, filtered (100 μm), and centrifuged at 450 g for 5 min. The freshly isolated SVF cells from the WAT were seeded and cultured in growth medium containing DMEM, 20% FBS, and 1% penicillin/streptomycin at 37°C with 5% CO₂ for 3 d, followed by feeding with fresh medium every 2 d. On confluence, the cells were induced with induction medium containing DMEM, 10% FBS, 2.85 μM insulin, 0.3 μM dexamethasone, and 0.63 mM 3-isobutyl-methylxanthine (IBMX) for 3 d and then differentiated in differentiation medium containing DMEM, 200 nM insulin, and 10 nM T3 for 4 d until adipocytes matured. For fibronectin type III domain containing 5 (Fndc5) treatment, SVF cells were induced to differentiate, and Fndc5 peptide (ab117436, 20 nM; Abcam, Cambridge, MA, USA) was added in the differentiation medium during the last 2 d. For the CM treatment, SVF cells were induced to differentiate with 50% CM (1:1 CM:fresh induction medium) for 3 d and then maturated in differentiation medium containing 50% CM for 4 d until adipocytes matured. For the Fndc5 antibody-neutralizing experiments, 1.25 μg/ml of Fndc5 antibody (ab93373; Abcam) was used.

Plasma and muscle extract

Blood samples of anesthetized WT and Mstn^−/− mice were collected into EDTA-containing tubes to prevent coagulation. Samples were then centrifuged for 15 min at 3000 rpm, and the supernatant plasma were collected. For muscle extract, the gastrocnemius muscles of WT and Mstn^−/− mice were isolated and incubated in PBS with constant shaking for 1–2 h. Protein concentrations were measured using Pierce BCA Protein Assay Reagent (Pierce Biotechnology, Rockford, IL, USA), and equal amounts of plasma or muscle extracts from the WT and Mstn^−/− mice were used for all experiments.

Total RNA extraction, cDNA synthesis, and real-time PCR

Total RNA extraction, cDNA synthesis, and real-time PCR were performed as described previously (35, 36). Briefly, total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. RNA was treated with RNase-free DNase l to remove contaminating genomic DNA. The purity and concentration of total RNA were measured by a spectrophotometer (Nanodrop 3000; Thermo Fisher, Waltham, MA, USA) at 260 and 280 nm. Ratios of absorption (260/280 nm) of all samples were between 1.8 and 2.0. Then 5 μg of total RNA was reversed transcribed using random hexaprimers and Moloney murine leukemia virus (M-MLV) reverse transcriptase. Real-time PCR was carried out with a Roche LightCycler 480 PCR system with SYBR Green Master Mix and gene-specific primers (Roche Diagnostics, Indianapolis, IN, USA). Primer sequences are from published papers (17, 35, 36). The 2^−ΔΔCT method was used to analyze the relative expression of genes using 18S rRNA as an internal control.

Protein extraction and Western blot analysis

The protein extraction and Western blot procedures were conducted as described previously (36). Briefly, total protein was isolated from cells using RIPA buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS. Protein concentrations were determined using Pierce BCA protein assay reagent (Pierce Biotechnology). Proteins were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore Corp., Billerica, MA, USA), and blocked in 5% fat-free milk for 1 h at room temperature, then incubated with primary antibodies in 5% milk overnight at 4°C. The Ucp1 and Fndc5 antibodies were from Abcam, and Pgc1α, pAMPK, GAPDH, and α-tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies (anti-rabbit IgG or anti-mouse IgG; Jackson ImmunoResearch, West Grove, PA, USA) were diluted 1:8000. Immunodetection was performed using enhanced chemiluminescence (ECL) Western blotting substrate (Pierce Biotechnology) and detected with a Gel Logic 2200 imaging system (Carestream, Rochester, NY, USA).

Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining

WAT tissues from the WT and Mstn^−/− mice were fixed in 10% formalin for 24–48 h at room temperature. Fixed tissues were embedded in paraffin and cut at 3–10 μm for H&E and IHC staining. For H&E staining, the sections were deparaffinized, rehydrated, and stained with hematoxylin for 15 min. Sections were then rinsed in running tap water and stained with eosin for 1 min. H&E-stained sections were dehydrated and mounted. For IHC staining, tissue sections were blocked with PBS containing 5% goat serum, 2% bovine serum albumin (BSA), 0.2% Triton X-100, and 0.1% sodium azide for 1 h after antigen retrieval. Then, the samples were incubated with Ucp1 primary antibodies diluted in blocking buffer overnight. After washing with PBS, the samples were incubated with secondary antibodies for 45 min at room temperature, followed by reactions with ABC and DAB reagents. Images were captured with a Nikon D90 digital camera installed on a Nikon (Diaphot) inverted microscope (Nikon, Tokyo, Japan).

Inhibition of Mstn signaling fails to induce browning of white adipocytes in vitro

We next sought to determine whether browning is an autonomous effect of inhibition of Mstn signaling in the adipocytes. We induced adipogenic differentiation of WAT SVF cells isolated from WT and Mstn^−/− mice, and assessed expression of BAT marker genes. The Mstn^−/− SVF cells were able to differentiate normally into lipid-filling adipocytes without obvious morphological defects. Surprisingly, adipocytes differentiated from WT and Mstn^−/− SVF cells expressed similar levels of Ucp1, Prdm16, Cidea, Pgc1α, and Pparα (Supplemental Fig. S2A). To confirm these results, we induced adipogenic differentiation of WAT SVF cells from WT mice in the presence of soluble ActIIB (sActRIIB; 2000 ng/ml) or a neutralizing antibody against Mstn (anti-Mstn; 2000 ng/ml), agents that have been shown to inhibit Mstn signaling (24). Neither treatment affected the overall differentiation of the SVF cells or induced BAT-specific genes after differentiation (Supplemental Fig. S2B, C). Taken together, these data demonstrate that loss of Mstn or inhibition of Mstn signaling in preadipocytes does not account for the browning phenotype observed in the WAT of Mstn^−/− mice.

Skeletal muscle-derived factor drives WAT browning in Mstn^−/− mice

The non-cell-autonomous effect of Mstn on the browning of WAT adipocytes suggests that one or more circulating factors might be responsible for this process. Indeed, we found that plasma extracted from Mstn^−/− mouse blood potently unregulated Ucp1 expression in adipocytes differentiated from WAT SVF cells of WT mice (Fig. 2A). We hypothesize that a muscle-derived factor (myokine) regulates the browning of WAT in the Mstn^−/− mice, based on the inversely correlated increases in muscle mass and decreases in fat mass in these mice. As expected from this hypothesis, Mstn^−/− skeletal muscle extracts significantly up-regulated Ucp1 expression in adipocytes differentiated from WAT SVF cells of WT mice (Fig. 2B). As whole-muscle extracts contain many non-muscle-derived factors, we next directly tested the effect of CM collected from myotube cultures differentiated from WT and Mstn^−/− myoblasts (designated as WT-CM and Mstn^−/−-CM). Compared with the WT-CM, Mstn^−/−-CM significantly increased expression of BAT marker genes Ucp1, Prdm16, Cidea, and Pparα, but not WAT marker genes adiponectin and leptin, in subcutaneous WAT SVF-derived adipocytes (Fig. 2C). Similar changes were observed using visceral WAT-derived SVF cells treated with WT-CM or Mstn^−/−-CM (Fig. 2D). These results indicate that browning of WAT in Mstn^−/− mice is muscle driven.

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Figure 2.

Muscle-derived factors induce browning of white adipocytes. A) Relative expression of BAT (Ucp1) and WAT (adiponectin, leptin) marker genes in white adipocytes differentiated from ingWAT and treated with plasma derived from WT or Mstn^−/−mice; n = 4. B) Relative expression of BAT and WAT marker genes in white adipocytes differentiated from ingWAT and treated with WT or Mstn^−/− muscle extracts; n = 6. C, D) Relative expression of BAT and WAT marker genes in asWAT-derived (C) and visceral WAT-derived (D) adipocytes treated with WT and Mstn^−/− myotube CM; n = 6. *P < 0.05; ** P < 0.01.

Muscle-derived Fndc5 (irisin) mediates browning of WAT in Mstn^−/− mice

A newly identified myokine, irisin (encoded by the Fndc5 gene), has been reported to mediate exercise-induced browning of WAT (3). We confirmed that Fndc5 is highly expressed in muscle compared to WAT (>200-fold higher in muscle). To investigate whether the browning in Mstn^−/− mice is mediated by irisin, we first checked the expression of Fndc5 in WT and Mstn^−/− muscles. The mRNA levels of Fndc5, along with its upstream activators Pgc1α and Pgc1β, were up-regulated ~3 fold in the Mstn^−/− than WT muscles (Fig. 3A). In addition, the protein levels of Fndc5 and Pgc1α were also elevated in the Mstn^−/− compared to WT muscles (Fig. 3B, C). Furthermore, inhibition of Mstn signaling with anti-Mstn antibody or follistatin (200 ng/ml) both up-regulated the expression of Pgc1α and Fndc5 in cultured myotubes (Fig. 3D, E). Collectively, these results show that Mstn^−/− increases Pgc1α and Fndc5 expression in the skeletal muscle.

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Figure 3.

Inhibition of Mstn increases the expression of Pgc1α and Fndc5 in muscle. A) Relative expression of Pgc1α, Fndc5, Pgc1β, and Cpt1β in WT and Mstn^−/− tibialis anterior (TA) muscles. B, C) PGC1α and Fndc5 protein levels in the gastrocnemius (gastoc; B) and TA (C) muscles of WT and Mstn^−/− mice. D, E) Relative expression of Pgc1α and Fndc5 in myotubes treated with follistatin (D) or Mstn antibody (E). **P < 0.01; n = 4.

We further investigated whether irisin is the key active component in Mstn^−/− myotube conditioned medium that induce browning of WAT adipocytes. We first confirmed that Fndc5 recombinant peptide robustly induces expression of BAT marker genes (Ucp1, Prdm16, Cidea, and Pgc1α) in adipocytes differentiated from WAT SVF cells (Fig. 4A). We next used Fndc5 antibody to neutralize the irisin in Mstn^−/−-CM. The addition of anti-Fndc5 to WT myotube-conditioned medium (WT-CM) had no effect on the expression of Ucp1, Prdm16, and Cidea, but moderately reduced the expression of Pgc1α and Pparα (Fig. 4B). This result suggests that WT-CM contains little irisin. Consistent with our earlier results in Fig. 2C, Mstn^−/−-CM up-regulated all the BAT signature genes of cultured adipocytes (Fig. 4B). Most importantly, anti-Fndc5 abolished the browning effects of Mstn^−/−-CM (Fig. 4B), demonstrating that irisin is the active component in the Mstn^−/−-CM that induces browning. Furthermore, anti-Fndc5 also abolished the inductive effect of Mstn^−/−-CM on beige adipocyte markers Tmem26, CD137 and Tbx1 (Fig. 4C). These results provide compelling evidence that Mstn^−/− drives browning of WAT through muscle-specific irisin.

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Figure 4.

Muscle-derived Fndc5 mediates browning of WAT in the Mstn^−/− mice. A) Fndc5-recombinant protein (20 nM) induces expression of BAT marker genes in white adipocyte cultures; n = 4. *P < 0.05, **P < 0.01. B) Relative expression of BAT marker genes in white adipocytes treated with WT myotube CM (WT-CM), WT myotube CM plus Fndc5 antibody (1.25 μg/ml, WT-CM + anti-Fndc5), Mstn^−/− myotube CM (MSTN-CM), and Mstn^−/− myotube CM plus Fndc5 antibody (1.25 μg/ml, MSTN-CM + anti-Fndc5); n = 6. Note that Fndc5 antibody abolishes the effect of Mstn^−/− myotube CM on browning. C) Relative expression of beige adipocyte-specific genes in white adipocytes treated as indicated in B; n = 6. ^a,b,cBars with no letter in common differ significantly.

This project is partially supported by funding from the Muscular Dystrophy Association, the U.S. National Institutes of Health, and the U.S. Department of Agriculture to S.K.

The authors thank Yong-Soo Kim (University of Hawaii, Manoa, HI, USA) for providing anti-Mstn and ActRIIB. The authors are grateful to Jun Wu and Pengpeng Zhang for mouse colony and technical support. The authors declare no conflicts of interests.