GLP‑1 receptor agonist protects palmitate-induced insulin resistance in skeletal muscle cells by up-regulating sestrin2 to promote autophagy

Abstract

In this study, we aimed to determine whether liraglutide could effectively reduce insulin resistance (IR) by regulating Sestrin2 (SESN2) expression in L6 rat skeletal muscle cells by examining its interactions with SESN2, autophagy, and IR. L6 cells were incubated with liraglutide (10-1000 nM) in the presence of palmitate (PA; 0.6 mM), and cell viability was detected using the cell counting kit-8 (CCK-8) assay. IR-related and autophagy-related proteins were detected using western blotting, and IR and autophagy-related genes were analyzed using quantitative real-time polymerase chain reaction. Silencing SESN2 was used to inhibit the activities of SESN2. A reduction in insulin-stimulated glucose uptake was observed in PA-treated L6 cells, confirming IR. Meanwhile, PA decreased the levels of GLUT4 and phosphorylation of Akt and affected SESN2 expression. Further investigation revealed that autophagic activity decreased following PA treatment, but that liraglutide reversed this PA-induced reduction in autophagic activity. Additionally, silencing SESN2 inhibited the ability of liraglutide to up-regulate the expression of IR-related proteins and activate autophagy signals. In summary, the data showed that liraglutide improved PA-induced IR in L6 myotubes by increasing autophagy mediated by SESN2.

Figure 1

Effects of liraglutide on palmitate (PA)-induced cell viability in L6 myotubes. (A) Cell viability was analyzed by cell counting kit 8 (CCK-8) assay. The cells were treated with various concentrations of liraglutide (10, 100, 1000 nM) for 16 h. (B) Viability of L6 myotubes in response to different doses of PA (0.2, 0.4, 0.6, 0.8 mM) for 16 h measured by CCK-8 assay. (C) Cells were incubated with 0.6 mM PA for 16 h with or without liraglutide at different concentrations. *P < 0.05 vs control; #P < 0.05 vs PA; △P < 0.05 vs PA + Lir10.

Figure 2

Liraglutide improves insulin resistance (IR) in PA-induced L6 myotubes. (A) The glucose oxidase–peroxidase micromethod was used to determine the glucose level in the cell supernatant. (B) The mRNA levels of GLUT4 in L6 myotubes were determined by quantitative reverse transcriptase-polymerase chain reaction. (C) and (D) The protein levels of GLUT4 and p-Akt/Akt in L6 myotubes were determined using western blotting. The results are expressed as means ± S.D. of three independent experiments. *P < 0.05 vs con; #P < 0.05 vs PA; △P < 0.05 vs Lir10.

Figure 3

Liraglutide activates PA-induced L6 myotubes autophagy and up-regulates SESN2 expression. (A) and (B) The mRNA levels of LC3II and P62 in L6 myotubes were determined by RT-qPCR. (C) and (D) The protein levels of LC3II/LC3I and P62 in L6 myotubes were determined using western blotting. The (E) mRNA and (F) protein levels of SESN2 in L6 myotubes were determined using RT-qPCR and western blotting. The results are expressed as means ± S.D. of three independent experiments. *P < 0.05 vs con; #P < 0.05 vs PA; △P < 0.05 vs Lir10.

Figure 4

Liraglutide attenuated IR by inducing autophagy activation in myotubes. The protein levels of LC3II/LC3I (A), P62 (B), p-Akt/Akt (C) and GLUT4 (D), in L6 myotubes were determined using western blotting. The results are expressed as means ± S.D. of three independent experiments. *P < 0.05 vs CON, #p < 0.05 vs 3-MA treatment alone.

Figure 5

Lilarutide ameliorates palmitate PA-induced IR and autophagy via SESN2. The (A) mRNA and (B) protein levels of SESN2 in L6 myotubes were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot. (C) and (D) The protein levels of LC3B and P62 in L6 myotubes were determined using RT-qPCR. (E) and (F) The mRNA levels of LC3II/LC3I and P62 in L6 myotubes were determined using western blotting. The results are expressed as means ± S.D. of three independent experiments. *P < 0.05 vs siCON.

Figure 6

Liraglutide regulated L6 myotube IR by activating SESN2. (A) The glucose oxidase–peroxidase micromethod was used to determine the level of glucose in the cell supernatant. (B) The mRNA levels of GLUT4 in L6 myotubes were determined using quantitative reverse transcriptase-polymerase chain reaction. (C) and (D) The protein levels of GLUT4 and p-Akt/Akt in L6 myotubes were determined using western blotting. The results are expressed as means ± S.D. of three independent experiments. *P < 0.05 vs siCON.

Figure 7

Schematic diagram of the mechanism of liralutide in reducing IR in skeletal muscle. PA suppresses SESN2 expression, leading to downregulation of autophagy which further impaired phosphorylation of Akt and expression of GLUT4. As a result, causing L6 cells IR. Liraglutide improved PA-induced IR in L6 myotubes by increasing autophagy-mediated by SESN2.