TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake

Abstract

TNF-related apoptosis-inducing ligand (TRAIL) is a protein that induces apoptosis in cancer cells but not in normal ones, where its effects remain to be fully understood. Previous studies have shown that in high-fat diet (HFD)-fed mice, TRAIL treatment reduced body weight gain, insulin resistance, and inflammation. TRAIL was also able to increase skeletal muscle free fatty acid oxidation. The aim of the present work was to evaluate TRAIL actions on skeletal muscle. Our in vitro data on C2C12 cells showed that TRAIL treatment significantly increased myogenin and MyHC and other hallmarks of myogenic differentiation, which were reduced by Dr5 (TRAIL receptor) silencing. In addition, TRAIL treatment significantly increased AKT phosphorylation, which was reduced by Dr5 silencing, as well as glucose uptake (alone and in combination with insulin). Our in vivo data showed that TRAIL increased myofiber size in HFD-fed mice as well as in db/db mice. This was associated with increased myogenin and PCG1α expression. In conclusion, TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake. These data shed light onto a pathway that might hold therapeutic potential not only for the metabolic disturbances but also for the muscle mass loss that are associated with diabetes.

Fig. 1. TRAIL does not modify lipid accumulation but stimulates myogenic markers.

Experiments were conducted in C2C12 cells on D3 (3^rd day of differentiation), after 24 h exposure to oleic acid or palmitic acid ± TRAIL. A, B Cell viability. C, D Quantification of lipid droplet content (expressed as percentage of Oil red O stained area). E Representative images of C2C12 cells stained with DAPI (left panels) and LipidTOX (right panels) in presence or absence of oleic acid (500 µM) ± TRAIL (100 ng/mL), scale bar 50 μm. F, G Gene expression of Pparg, Pcg1α, Cpt1b, Pepck1, Myod, Myog, and Myh4. Gene expression is reported as mRNA fold induction normalized to the mRNA level of CNT. H Fusion index quantification. The fusion index corresponds to the ratio between the number of nuclei in a MyHC-positive myotube (with > 3 nuclei) and the total number of nuclei. I Representative images of C2C12 cells stained with MitoSox (red staining, top panels) and anti-MyHC (green staining, bottom panels) in the presence or absence of oleic acid (500 µM) ± TRAIL (10 ng/mL), scale bar 50 μm. Results were obtained by 3–5 independent experiments and are presented as median with interquartile range. Significance was assessed with ANOVA and Tukey.

Fig. 2. TRAIL promotes myogenic differentiation.

Experiments were conducted in C2C12 cells during their differentiation. TRAIL treatment was renewed every 24 h between D0 and D4 (D is for day of differentiation). A–B Densitometric analysis and C representative blots of myogenin and MyHC protein expression. D Myotube length. E Fusion index. F Representative images of C2C12 cell morphology. Brightfield (upper panels), MyHC immunofluorescence (middle panels), DAPI staining (lower panels). Scale bar represents 100 µm. G–J Gene expression of Myog, Glut4, Pepck1, and Pgk1. Gene expression is reported as mRNA fold induction normalized to the mRNA level of CNT cells on D1. Results were obtained by 3–4 independent experiments and are presented as median with interquartile range. Significance was assessed with ANOVA and Tukey.

Fig. 3. Dr5 silencing significantly reduces myogenic differentiation.

Experiments were conducted in C2C12 cells during their differentiation (D0–D3). Dr5 was silenced before D0. A, C, E, G Gene expression of Dr5, Myod, Myog, and Myh4. Gene expression is reported as mRNA fold induction normalized to siCTR mRNA level on D0. B, D, F, H Quantification of DR5, MyoD, myogenin, and MyHC protein expression, and I representative blots. J Quantification of myotube length and K the fusion index on D3. Results were obtained by 3–4 independent experiments and are presented as median with interquartile range. Significance was assessed with ANOVA and Tukey tests (A–H), and J, K t-test. L Representative images of C2C12 cell morphology after Dr5 silencing, as assessed on D3. Images are presented in brightfield, and/or after anti-DR5 staining (green staining), and/or after anti-MyHC staining (red staining). Scale bar = 50 µm.

Fig. 4. TRAIL/DR5 pathway regulates AKT phosphorilation.

Experiments were conducted in C2C12 cells during their differentiation (D0–D3). TRAIL treatment was renewed every 24 h. A Representative blots and B–D quantification of LC3B-I and LC3B-II, p62, and myogenin expression on D3. E–F Representative blots and quantification of AMPKα and P-AMPKα expression on D3. G–H Representative blots and quantification of AKT and P-AKT expression on D3. I–J Representative blots and quantification of DR5, AKT, and P-AKT after Dr5 silencing on D3. K–L Representative blots and quantification of AKT and P-AKT in C2C12 cells treated with TRAIL (24 h on D3) ± insulin (20 min on D5 after serum starvation). M Glucose uptake in C2C12 cells treated with TRAIL (24 h on D3) ± insulin (20 min on D5 after serum starvation). Results were obtained by 3–4 independent experiments and are presented as median with interquartile range. Significance was assessed with ANOVA followed by Tukey test, except for J where it was used the t-test.

Fig. 5. TRAIL treatment modulates myofiber size and function in HFD-fed mice.

A Representative images and B–C quantification of quadricep myofiber cross-sectional area (CSA). Scale bar = 50 μm D–E Skeletal muscle gene expression of Mafbx and Murf1. Gene expression is reported as mRNA fold induction normalized to CNT. F Representative blots and G–H quantification of myogenin and PGC1α protein expression. CNT is for standard diet fed mice, HFD is for HFD-fed mice, and HFD + TRAIL is for HFD-fed mice treated with TRAIL. Results are presented as median with interquartile range. Significance was assessed with ANOVA and Tukey tests. n = 5–9 mice per group.