SMAD3 negatively regulates serum irisin and skeletal muscle FNDC5 and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) during exercise

Abstract

Beige adipose cells are a distinct and inducible type of thermogenic fat cell that express the mitochondrial uncoupling protein-1 and thus represent a powerful target for treating obesity. Mice lacking the TGF-β effector protein SMAD3 are protected against diet-induced obesity because of browning of their white adipose tissue (WAT), leading to increased whole body energy expenditure. However, the role SMAD3 plays in WAT browning is not clearly understood. Irisin is an exercise-induced skeletal muscle hormone that induces WAT browning similar to that observed in SMAD3-deficient mice. Together, these observations suggested that SMAD3 may negatively regulate irisin production and/or secretion from skeletal muscle. To address this question, we used wild-type and SMAD3 knock-out (Smad3(-/-)) mice subjected to an exercise regime and C2C12 myotubes treated with TGF-β, a TGF-β receptor 1 pharmacological inhibitor, adenovirus expressing constitutively active SMAD3, or siRNA against SMAD3. We find that in Smad3(-/-) mice, exercise increases serum irisin and skeletal muscle FNDC5 (irisin precursor) and its upstream activator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) to a greater extent than in wild-type mice. In C2C12 myotubes, TGF-β suppresses FNDC5 and PGC-1α mRNA and protein levels via SMAD3 and promotes SMAD3 binding to the FNDC5 and PGC-1α promoters. These data establish that SMAD3 suppresses FNDC5 and PGC-1α in skeletal muscle cells. These findings shed light on the poorly understood regulation of irisin/FNDC5 by demonstrating a novel association between irisin and SMAD3 signaling in skeletal muscle.

Keywords: Exercise; Irisin; Obesity; SMAD Transcription Factor; Skeletal Muscle; Transforming Growth Factor Beta (TGF-B).

FIGURE 1.

Serum irisin and muscle FNDC5 are regulated by SMAD3. A, serum irisin in lean sedentary male WT and Smad3^−/− mice (n = 5–25 mice). B, protein expression and quantification in quadriceps muscle of lean sedentary male WT and Smad3^−/− mice (n = 5–25 mice). C, serum irisin following 1 week of treadmill exercise in lean WT and Smad3^−/− male and female mice (n = 6–8 mice). D, protein expression and quantification in quadriceps muscle of exercised mice from C. The results represent the means ± S.E. (versus WT mice when not indicated). E–G, C2C12 myoblasts were differentiated into myotubes for 3 days prior to treatment with TGF-β or TGF-βR1 inhibitor (SB431543) for 24 h (the results are from at least three experiments). Cells were harvested on day 4 for mRNA and protein analysis. F and G, effects of TGF-β or SB431543 treatments (24 h) on gene (F) and protein (G) expression in C2C12 myotubes. The samples from the PGC-1α representative image were run on the same gel but were noncontinuous. The results represent the means ± S.E. (versus differentiation (Diff.) media vehicle). #, <0.08; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

TGF-β suppresses FNDC5 expression in cultured skeletal muscle cells in a time-dependent manner. A and B, C2C12 myoblasts were differentiated into myotubes for 3 days prior to treatment with TGF-β or TGF-βR1 inhibitor (SB431543) for 24 h. The cells were harvested on day 4 for mRNA and protein analysis. A, time course of TGF-β treatments on gene expression in C2C12 myotubes. B, time course of SB431543 treatments on gene expression in C2C12 myotubes (the results are from at least three experiments). The results represent the means ± S.E. (versus differentiation (Diff.) media vehicle). #, <0.08; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 3.

SMAD3 binds the promoter regions of FNDC5 and PGC-1α in cultured skeletal muscle cells. A–E, C2C12 myoblasts were differentiated into myotubes for 4 days in the continued presence of TGF-β or TGF-βR1 inhibitor (SB431543). The cells were harvested on day 4 for mRNA and protein analysis. Undiff., undifferentiated. B and C, effects of TGF-β or SB431543 treatments (96 h) on gene (B) and protein (C) expression in differentiating C2C12 myoblasts. The samples from the FNDC5 representative image were run on the same gel but were noncontinuous (the results are from at least three experiments). D and E, effects of TGF-β treatments (96 h) on SMAD3 occupancy of the PGC-1α and FNDC5 promoter in differentiating C2C12 myoblasts. Graphs represent relative expression of PGC-1α and FNDC5 promoter DNA following ChIP. D, PGC-1α promoter occupancy by SMAD3. The right graph represents promoter region −125 to +32, and the left graph represents promoter region −7280 to −7083. E, FNDC5 promoter occupancy by SMAD3. The right graph represents promoter region −805 to −675, and the left graph represents promoter region −3197 to −3061 (the results are from two experiments). The results represent the means ± S.E. (versus differentiation (Diff.) media vehicle). #, <0.08; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

FNDC5 and PGC-1α are regulated by SMAD3 in cultured skeletal muscle cells. A–H, C2C12 myoblasts were transfected with nontargeting control siRNA or siRNA against SMAD3 or transduced with control adenovirus harboring green fluorescent protein or adenovirus harboring CA-SMAD3 during differentiation into myotubes and then treated (24 h) with TGF-β or TGF-βR1 inhibitor (SB431543) continuously during the last day of differentiation. Diff., differentiated; Undiff., undifferentiated. B and C, mRNA and protein expression in differentiated C2C12 myoblasts transfected with siRNA for 72 h. D, relative luciferase activity normalized to protein levels in differentiated C2C12 myoblasts transfected with a GAGA-Luc expression plasmid, transfected with siRNA and treated (24 h) with TGF-β or SB431543. E, effects of TGF-β treatment (24 h) on gene expression in C2C12 myotubes. F, relative luciferase activity normalized to protein levels in differentiated C2C12 myoblasts transfected with a GAGA-Luc expression plasmid, transduced with adenovirus, and treated (24 h) with TGF-β or SB431543. G, mRNA expression in differentiated C2C12 myoblasts transduced with adenovirus for 72 h. H, effects of TGF-β and SB431543 treatments (24 h) on gene expression in C2C12 myotubes (the results are from at least three experiments). The results represent the means ± S.E. (versus differentiation media vehicle when not indicated). #, <0.08; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Exercise increases serum irisin and skeletal muscle FNDC5 and induces browning in subcutaneous adipose tissue of lean mice. A, percent increase in serum irisin during 3 weeks of treadmill or swimming exercise in wild-type male mice. *, treadmill versus sedentary; §, swimming versus sedentary. B, area under the curve (AUC) of the absolute serum irisin values (ng/ml) over 3 weeks of exercise. C, gene expression in quadriceps muscle of mice from A following 3 weeks of exercise. D and E, protein expression and quantification in quadriceps muscle of mice from A plus an additional group of mice exercised by free running for 3 weeks. F, gene expression of brown adipose tissue markers in subcutaneous fat of mice from A (n = 8 mice per group). The results represent the means ± S.E. (versus sedentary mice). #, <0.08; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

Irisin is negatively correlated with pSMAD3 in exercised obese mice. A, body weight measured in age matched male wild-type mice fed a NCD and male wild-type mice fed a HFD at the indicated time points. B and C, fat mass (B) and fasting blood glucose (C) in mice from A measured at 9 weeks. D, area under the curve (AUC) of the absolute serum irisin values (ng/ml) over 2 weeks of exercise. E, percent increase in serum irisin during 2 weeks of treadmill exercise in mice from A. *, NCD treadmill versus NCD sedentary; §§, HFD treadmill versus HFD sedentary. F, gene expression in quadriceps muscle of mice from A following 2 weeks of treadmill exercise. G, serum TGF-β levels in mice from A following 1 week of treadmill exercise. H, percent decrease in serum TGF-β in mice from A following 1 week of treadmill exercise. I, protein expression and quantification in quadriceps muscle of mice from A (n = 6–8 mice per group). The results represent the means ± S.E. (versus NCD sedentary mice when not indicated). #, <0.08; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

SMAD3 suppresses irisin production and/or secretion from skeletal muscle by inhibiting FNDC5 and PGC-1α expression. TGF-β binds the TGF-βR1/TGF-βR2 complex, SMAD3 is phosphorylated and translocates into the nucleus to bind the promoters of PGC-1α and FNDC5 to suppress their transcription. Suppression of FNDC5 transcription and protein leads to decrease in circulating irisin.