HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

Abstract

Background: G protein-coupled receptor 40 (GPR40) acts as a modulator of various physiological functions, including glycaemic lowering, anti-inflammation and antioxidative stress, in several tissues. However, the role of GPR40 in skeletal muscles remains unclear.

Methods: To investigate the roles of muscle GPR40, C2C12 myoblasts and myotubes were stimulated with palmitate and HD6277, a GPR40 agonist. Muscle strength and myofiber thickness were measured in obese and aged mice fed HD6277.

Results: In C2C12 myoblasts, the addition of HD6277 induced phosphorylated Akt levels and expression of the myogenic factors, myogenin (MyoG), myocyte enhancer factor 2C (Mef2c) and myosin heavy chain (MyHC, p < 0.05). These changes resulted in accelerated muscle differentiation from myoblasts to myotubes (MyHC-positive area +56.52%; myotube width +34.08% vs. Veh, p < 0.05). In C2C12 myotubes, a palmitate-mediated decrease in the phosphorylation of forkhead box protein O1A (FOXO1A) and increase in the expression of E3 ubiquitin ligases, atrogin-1 and muscle RING-finger protein 1 (MuRF1) were reversed by HD6277 (p < 0.05). Additionally, HD6277 inhibited palmitate-induced apoptotic events such as the Bcl-2 (Bcl2)-associated X protein (Bax)/Bcl-2 ratio, caspase 3 cleavage and nuclear fragmentation in C2C12 myoblasts and myotubes (p < 0.05). These beneficial HD6277-mediated actions disappeared after the addition of an Akt inhibitor (p < 0.05). Similar to in vitro studies, HD6277 administration in obese and aged mice increased myogenic factors and decreased E3 ubiquitin ligase expression and apoptotic events (p < 0.05). HD6277 increased muscle strength (+9.88% vs. Aged, p < 0.05) and myofiber thickness (+29.01% vs. Aged, p < 0.05) in aging mice but only improved myofiber thickness (+11.84% vs. HFD, p < 0.05) in obese mice.

Conclusion: HD6277 can increase myogenic factors and reduce E3 ligase-mediated proteolysis to inhibit muscle atrophy in aged mice. Our results suggest that GPR40 agonists may have potential as therapeutic agents for sarcopenia.

Keywords: GPR40; muscle atrophy; myoblast; myogenic factors; myotube; sarcopenia.

FIGURE 1

HD6277 accelerated myotube formation in an Akt‐dependent manner in C2C12 myoblasts. (A,B) The cells were differentiated with various doses of HD6277 (0, 10 or 25 μM) or HD6277 plus GW1100 (10 μM) for 2 days, and then, the phosphorylated Akt level was determined by Western blotting. (C) The cells were differentiated with HD6277 (25 μM) with or without an Akt inhibitor (1 or 2 μM) for 2 days, and then, the transcript level of Mef2c was analysed by qPCR. (D) MyoG and MyHC levels were determined by Western blotting. (E) The MyHC‐positive area and myotube width were calculated in MyHC‐stained cells (green fluorescence). Scale bar: 275 μm. Akti, Akt inhibitor; GW, GW1100; HD, HD6277; Veh, vehicle. Error bars indicate the mean ± SD (*p < 0.05; ANOVA with post hoc t test).

FIGURE 2

HD6277 inhibited the palmitate‐mediated reduction in myotube formation in an Akt‐dependent manner in C2C12 myoblasts. (A,B) The cells were stimulated with palmitate (200 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with GW1100 (10 μM). The phosphorylated Akt level was analysed by Western blotting. (C) The cells were stimulated with palmitate (200 μM), palmitate plus HD6277 (25 μM) or palmitate plus HD6277 with Akt inhibitor (2 μM) and then differentiated for 2 days. The transcript levels of Mef2c and Myf5 were analysed by qPCR. (D) MyoG and MyHC levels were analysed by Western blotting. (E) MyHC‐stained cells (green fluorescence) were observed under a fluorescence microscope to calculate the MyHC‐positive area and myotube width. Scale bar: 275 μm. Akti, Akt inhibitor; GW, GW1100; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (*p < 0.05; ANOVA with post hoc t test).

FIGURE 3

HD6277 suppressed palmitate‐mediated cytotoxicity in an Akt‐dependent manner in C2C12 myoblasts. (A,D) The cells were stimulated with palmitate (200 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with Akt inhibitor (1 or 2 μM), after which the Bax/Bcl2 ratio and cleaved caspase 3 level were determined by Western blotting. (B,E) Cell viability was measured using the EZ‐Cytox solution. (C,F) DNA fragmentation was visualized using a TUNEL assay kit. Scale bar: 125 μm. TUNEL‐positive cells (red fluorescence) were observed and counted under a fluorescence microscope. Akti, Akt inhibitor; C.Cas‐3, cleaved caspase 3; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (*p < 0.05; ANOVA with post hoc t test).

FIGURE 4

HD6277 reduced the palmitate‐induced proteolytic processes in an Akt‐dependent manner in C2C12 myotubes. (A,B) Fully differentiated cells were stimulated with palmitate (400 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with GW1100 (10 μM), and then, the phosphorylated Akt level was determined by Western blotting. (C–F) Fully differentiated cells were stimulated with palmitate (400 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with Akt inhibitor (2 μM). Western blotting was then performed to analyse the levels of phosphorylated FOXO1A, atrogin‐1, MuRF1 and ubiquitination. Akti, Akt inhibitor; GW, GW1100; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (*p < 0.05; ANOVA with post hoc t test).

FIGURE 5

HD6277 inhibited palmitate‐induced cytotoxicity in an Akt‐dependent manner in C2C12 myotubes. (A,D) Fully differentiated cells were incubated with palmitate (400 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with Akt inhibitor (2 μM), and then, Western blotting was performed to determine the levels of the Bax/Bcl2 ratio and cleaved caspase 3. (B,E) Cell viability was measured using EZ‐Cytox solution. (C,F) DNA fragmentation was analysed by the TUNEL assay. Scale bar: 125 μm. TUNEL‐positive cells (red fluorescence) were calculated under a fluorescence microscope. Akti, Akt inhibitor; C.Cas‐3, cleaved caspase 3; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (*p < 0.05; ANOVA with post hoc t test).

FIGURE 6

HD6277 induced the transcription of myogenic genes and reduced the expression of proteolytic proteins in obese mice. (A) The phosphorylated Akt levels were determined by Western blotting. (B) Transcript levels of PAX3, PAX7, Mef2c, Myf5, MyoD and MyoG were calculated by qPCR. (C,D) Western blotting was performed to analyse the levels of phosphorylated FOXO1A, atrogin‐1, MuRF, ubiquitination, Bax, Bcl2 and cleaved caspase 3. (E) The cross‐sectional area (CSA) of muscle fibres was measured in images stained with H&E dyes. Scale bar: 20 (400×) and 10 μm (1000×). (F) A grip strength device was used to determine four‐limb muscle strength. C.Cas‐3, cleaved caspase 3; HFD, high‐fat diet–fed mice; HFD + HD, HFD mixed with HD6277‐fed mice; ND, normal diet–fed mice; ns, no significant. Error bars indicate the mean ± SD (*p < 0.05; ANOVA with post hoc t test).

FIGURE 7

HD6277 delayed muscle atrophy in aged mice. (A) Western blotting showed the phosphorylated Akt level. (B) PAX3, PAX7, Mef2c, Myf5, MyoD and MyoG transcripts were determined by qPCR. (C,D) The levels of phosphorylated FOXO1A, atrogin‐1, MuRF, ubiquitination, Bax, Bcl2 and cleaved caspase 3 were analysed by Western blotting. (E) The CSA of the muscle fibres was calculated in images stained with H&E dyes. Scale bar: 20 (400×) and 10 μm (1000×). (F) The grip strength device was used to analyse four‐limb muscle strength. Aged, normal diet–fed aged mice; Aged + HD, normal diet mixed with HD6277‐fed aged mice; C.Cas‐3, cleaved caspase 3. Error bars indicate the mean ± SD (*p < 0.05; two‐tailed t tests assuming unequal variance).

FIGURE 8

Schematic diagram of HD6277 functions in muscles. The HD6277‐Akt axis enhanced myogenic factors, reduced proteolytic processes and inhibited cytotoxicity in C2C12 cells and muscle tissues from obese and aged mice. Furthermore, HD6277 administration improved muscle strength and the CSA of muscle fibres in aged mice.