DarrenW29
06-11-2021, 11:45 AM
Dulaglutide improved muscle strength and attenuated muscle loss in disuse condition. Ten-week-old C57BL/6 male mice were subjected to spiral wire immobilization for 4 d and administrated dulaglutide (Trulicity, 600 µg/kg, subscutaneous). Mice were maintained for additional 7 d. (A) Grip strength was measured on day 11 and grip strength value was normalized to final body weight. (B) Body weights on day 0 and 11 were recorded. (C) The total muscle weight and weight of various muscle types, including gastrocnemius (GA), soleus, tibialis anterior (TA), extensor digitorum longus (EDL), and quadriceps (QD), were measured right after sacrifice and normalized to the final body weight. Data are shown as means ± S.E.M, n = 6–7/group; *p < 0.05, **p < 0.01, ***p < 0.001. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle, ID; immobilization + dulaglutide.
Dulaglutide treatment increased muscle fiber size in disuse-induced skeletal muscle atrophy. (A) GA muscle tissue sections were stained with H&E and examined under microscope. (B) The cross-sectional area (CSA) of muscle fiber was measured using ImageJ program and the average CSA is shown. (C) Distribution of myofiber size. Data are shown as means ± S.E.M, n = 5; *p < 0.05, **p < 0.01, ***p < 0.001 versus control + vehicle or immobilization + vehicle group. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide.
Dulaglutide treatment decreased the mRNA and protein expression levels of degradation-related genes under disuse-induced muscle atrophy. (A) The mRNA levels of the genes encoding MuRF-1, atrogin-1, and myostatin were analyzed with RT-qPCR in GA muscle tissue, n = 5. GAPDH mRNA served as an internal control. (B) Immunoblotting and (C) quantification analyses of MuRF-1, atrogin-1, and myostatin protein expression in GA muscle. Beta-actin was used as a loading control to ensure equal protein loading, n = 3. (D) The mRNA expression of myosin heavy chain isoforms, including myosin heavy chain type I, type IIa, and type IIb, was evaluated with RT-qPCR, n = 5. GAPDH mRNA was used as an internal control. (E) Immunoblotting and (F) quantification analyses of myosin heavy chain protein expression in GA muscle. Beta-actin was used as a loading control, n = 5. Data are shown as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with control + vehicle or immobilization + vehicle group. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide.
Dulaglutide treatment reduced the expression of proinflammatory cytokines and p50 NF-κB in disuse-induced muscle atrophy. (A) The mRNA expression levels of TNF-α, IL-1β, and IL-6 were analyzed with RT-qPCR in GA muscle tissue. GAPDH mRNA was used as an internal control. (B) Immunoblotting and (C) quantification analyses of p50 NF-κB and p-IκBα protein expression in GA muscle. Beta-actin was used as a loading control. Data are shown as mean ± S.E.M, n = 5; **p < 0.01 or ***p < 0.001 as compared with control + vehicle or disuse + vehicle group. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide.
Dulaglutide prevents apoptosis in disuse condition. (A) Immunoblotting and (B) quantification analyses of caspase-3, cleaved PARP, and Bax proteins in GA muscle. Beta-actin was used as the loading control. Data are showed as mean ± S.E.M, n = 5; *p < 0.05, **p < 0.01, ***p < 0.001. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide; ns, not significant.
GLP-1 receptor agonist treatment increased Hsp72 protein expression through the regulation of AMPK signaling. (A) Immunoblotting and (B) quantification analyses of Hsp72 protein expression in GA muscle. (C) Immunoblotting and (D) quantification analyses of Hsp72 protein expression in soleus muscle. Beta-actin was used as a loading control. Data are shown as mean ± S.E.M, n = 5; *p < 0.05, **p < 0.01, ***p < 0.001. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide. (E) Immunoblotting and (G) quantification analyses of Hsp72 and p-AMPK proteins in C2C12 myotubes treated with dulaglutide (1.5 µg/ml, 12 h) or Ex-4 (20 nM, 30 min or 3 h). (F) Immunoblotting and (H) quantification analysis of Hsp72 and p-AMPK proteins in C2C12 myotubes pre-treated with compound C at 20 µM for 1 h and then exposed to dulaglutide (1.5 µg/ml, 3 h) or Ex-4 (20 nM, 3 h). Beta-actin was used as the loading control. Data are shown as mean ± S.E.M, n = 3; *p < 0.05, **p < 0.01.
Aside from the blood glucose-lowering effects, GLP-1 receptor agonists also exert beneficial effects on the skeletal muscle by increasing glucose uptake (Thompson and Kanamarlapudi, 2013), fat oxidation, and thermogenic gene expression (Choung et al., 2017). In addition, GLP-1 receptor agonist, Ex-4, imparts therapeutic effects in muscle atrophy induced by dexamethasone (Hong et al., 2019). In the present study, we investigated the effect of dulaglutide, a GLP-1 receptor agonist, on disuse-induced muscle atrophy and evaluated the underlying mechanisms.
Disuse-induced skeletal muscle atrophy is closely related to inflammatory process (Hunter et al., 2002). In addition, GLP-1-based therapies have been shown to exert anti-inflammatory effects in chronic inflammatory diseases (Kim et al., 2017). We evaluated the expression of inflammatory cytokines and found that the mRNA levels of TNF-α, IL-1β, and IL-6 in the GA muscle were upregulated following 10 days from immobilization and that dulaglutide treatment inhibited this increase (Figure 4A). NF-κB activation is important for the induction of inflammatory cytokines, while p50 NF-κB, not p65, is activated during disuse condition (Hunter et al., 2002). IκBα is an inhibitory factor for NF-κB activation (Yamamoto and Gaynor, 2004). We found that p50 NF-κB level increased in immobilized mice while dulaglutide treatment ameliorated this effect. We also examined the expression of p-IκBα, a negative regulator (Yamamoto and Gaynor, 2004), and found that its expression was downregulated in immobilized mice and that dulaglutide treatment restored the levels (Figures 4B, C). These results suggest that GLP-1 receptor agonist may inhibit p-IκBα degradation and decrease p50 NF-κB, thereby contributing to the amelioration of inflammation during disuse conditions in the skeletal muscle.
In conclusion, we demonstrate that treatment with dulaglutide, a GLP-1 receptor agonist, could recover muscle strength, muscle mass, and muscle fiber size, which were reduced during immobilization. Dulaglutide treatment attenuated the induction of atrophic genes, such as those encoding MuRF-1, atrogin-1, and myostatin, and enhanced MHC expression. In addition, dulaglutide treatment inhibited the expression of inflammatory cytokines and apoptotic genes through the induction of heat shock protein 72 (Hsp72) expression via AMPK activation, contributing to the amelioration of disuse-induced muscle atrophy.
Dulaglutide treatment increased muscle fiber size in disuse-induced skeletal muscle atrophy. (A) GA muscle tissue sections were stained with H&E and examined under microscope. (B) The cross-sectional area (CSA) of muscle fiber was measured using ImageJ program and the average CSA is shown. (C) Distribution of myofiber size. Data are shown as means ± S.E.M, n = 5; *p < 0.05, **p < 0.01, ***p < 0.001 versus control + vehicle or immobilization + vehicle group. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide.
Dulaglutide treatment decreased the mRNA and protein expression levels of degradation-related genes under disuse-induced muscle atrophy. (A) The mRNA levels of the genes encoding MuRF-1, atrogin-1, and myostatin were analyzed with RT-qPCR in GA muscle tissue, n = 5. GAPDH mRNA served as an internal control. (B) Immunoblotting and (C) quantification analyses of MuRF-1, atrogin-1, and myostatin protein expression in GA muscle. Beta-actin was used as a loading control to ensure equal protein loading, n = 3. (D) The mRNA expression of myosin heavy chain isoforms, including myosin heavy chain type I, type IIa, and type IIb, was evaluated with RT-qPCR, n = 5. GAPDH mRNA was used as an internal control. (E) Immunoblotting and (F) quantification analyses of myosin heavy chain protein expression in GA muscle. Beta-actin was used as a loading control, n = 5. Data are shown as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with control + vehicle or immobilization + vehicle group. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide.
Dulaglutide treatment reduced the expression of proinflammatory cytokines and p50 NF-κB in disuse-induced muscle atrophy. (A) The mRNA expression levels of TNF-α, IL-1β, and IL-6 were analyzed with RT-qPCR in GA muscle tissue. GAPDH mRNA was used as an internal control. (B) Immunoblotting and (C) quantification analyses of p50 NF-κB and p-IκBα protein expression in GA muscle. Beta-actin was used as a loading control. Data are shown as mean ± S.E.M, n = 5; **p < 0.01 or ***p < 0.001 as compared with control + vehicle or disuse + vehicle group. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide.
Dulaglutide prevents apoptosis in disuse condition. (A) Immunoblotting and (B) quantification analyses of caspase-3, cleaved PARP, and Bax proteins in GA muscle. Beta-actin was used as the loading control. Data are showed as mean ± S.E.M, n = 5; *p < 0.05, **p < 0.01, ***p < 0.001. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide; ns, not significant.
GLP-1 receptor agonist treatment increased Hsp72 protein expression through the regulation of AMPK signaling. (A) Immunoblotting and (B) quantification analyses of Hsp72 protein expression in GA muscle. (C) Immunoblotting and (D) quantification analyses of Hsp72 protein expression in soleus muscle. Beta-actin was used as a loading control. Data are shown as mean ± S.E.M, n = 5; *p < 0.05, **p < 0.01, ***p < 0.001. CV, control + vehicle; CD, control + dulaglutide; IV, immobilization + vehicle; ID, immobilization + dulaglutide. (E) Immunoblotting and (G) quantification analyses of Hsp72 and p-AMPK proteins in C2C12 myotubes treated with dulaglutide (1.5 µg/ml, 12 h) or Ex-4 (20 nM, 30 min or 3 h). (F) Immunoblotting and (H) quantification analysis of Hsp72 and p-AMPK proteins in C2C12 myotubes pre-treated with compound C at 20 µM for 1 h and then exposed to dulaglutide (1.5 µg/ml, 3 h) or Ex-4 (20 nM, 3 h). Beta-actin was used as the loading control. Data are shown as mean ± S.E.M, n = 3; *p < 0.05, **p < 0.01.
Aside from the blood glucose-lowering effects, GLP-1 receptor agonists also exert beneficial effects on the skeletal muscle by increasing glucose uptake (Thompson and Kanamarlapudi, 2013), fat oxidation, and thermogenic gene expression (Choung et al., 2017). In addition, GLP-1 receptor agonist, Ex-4, imparts therapeutic effects in muscle atrophy induced by dexamethasone (Hong et al., 2019). In the present study, we investigated the effect of dulaglutide, a GLP-1 receptor agonist, on disuse-induced muscle atrophy and evaluated the underlying mechanisms.
Disuse-induced skeletal muscle atrophy is closely related to inflammatory process (Hunter et al., 2002). In addition, GLP-1-based therapies have been shown to exert anti-inflammatory effects in chronic inflammatory diseases (Kim et al., 2017). We evaluated the expression of inflammatory cytokines and found that the mRNA levels of TNF-α, IL-1β, and IL-6 in the GA muscle were upregulated following 10 days from immobilization and that dulaglutide treatment inhibited this increase (Figure 4A). NF-κB activation is important for the induction of inflammatory cytokines, while p50 NF-κB, not p65, is activated during disuse condition (Hunter et al., 2002). IκBα is an inhibitory factor for NF-κB activation (Yamamoto and Gaynor, 2004). We found that p50 NF-κB level increased in immobilized mice while dulaglutide treatment ameliorated this effect. We also examined the expression of p-IκBα, a negative regulator (Yamamoto and Gaynor, 2004), and found that its expression was downregulated in immobilized mice and that dulaglutide treatment restored the levels (Figures 4B, C). These results suggest that GLP-1 receptor agonist may inhibit p-IκBα degradation and decrease p50 NF-κB, thereby contributing to the amelioration of inflammation during disuse conditions in the skeletal muscle.
In conclusion, we demonstrate that treatment with dulaglutide, a GLP-1 receptor agonist, could recover muscle strength, muscle mass, and muscle fiber size, which were reduced during immobilization. Dulaglutide treatment attenuated the induction of atrophic genes, such as those encoding MuRF-1, atrogin-1, and myostatin, and enhanced MHC expression. In addition, dulaglutide treatment inhibited the expression of inflammatory cytokines and apoptotic genes through the induction of heat shock protein 72 (Hsp72) expression via AMPK activation, contributing to the amelioration of disuse-induced muscle atrophy.