The Role Of AMPK Signaling Pathway In ER Stress Induced By Saturated Fatty Acids

Objective: Using palmitate to treat differentiated mature myotubes in vitro and construct a model of endoplasmic reticulum stress(ER stress)in muscle induced by saturated fatty acid,we studied the relationship between the activation of AMPK pathway and ER stress in the early stage of skeletal muscle,and the effects of different degrees of activation of AMPK pathway on ER stress,thus providing a theoretical basis for pathological changes induced by obesity.Methods:(1)We used mature myotubes as research object which had been induced in virto.The myotubes were treated with palmitate,the main component of saturated fatty acids,for different time(3,6,12,24h)to extract total protein and RNA.Western blotting analysis of dynamic changes of p-AMPK and ER stress PERK pathway downstream proteins expression in myotubes after treatment with different time of palmitate.(2)The time at which the activation of AMPK pathway was most correlated with ER stress was selected,and using Compound C to inhibit AMPK signaling pathway.Real-time PCR and Western blotting were used to detect the effect of AMPK blockade on the expression of ER stress PERK pathway downstream genes and proteins.(3)The myotubes were treated with different concentrations of AICAR,Western blotting and Real-time PCR was used to detect effect of different degrees of activation of AMPK on the expression of ER stress PERK pathway downstream proteins and genes,in order to investigate the does-dependence of the activation of AMPK pathway on ER stress;The AICAR does which mostly active the ER stress was selected to treat with the cells in different times,Western blotting and Real-time PCR was used to detect the effect of different degrees of activation of AMPK on the expression of ER stress PERK pathway downstream proteins and genes,in order to investigate the time-dependence of the activation of AMPK pathway on endoplasmicreticulum stress.Results:(1)After treatment with palmitate 3,6,12,24 h,the activation of p-AMPK protein(P<0.001)and ER stress PERK pathway downstream proteins p-eIF2?,CHOP,ATF4 were dramatically upregulation at 12h(P<0.001).(2)Treatment of myotubes with palmitate and AMPK inhibitor Compound C for 12 h,which reduced the p-AMPK protein activation induced by palmitate(P<0.05),and the ER stress PERK pathway downstream proteins p-eIF2?,CHOP,ATF4 were also downregulation(P<0.001).At the same time,the endoplasmic reticulum stress chaperone BiP mRNA and ER stress PERK pathway downstream genes ATF4,CHOP,GADD34 mRNA also decreased by Compound C incubated with palmitate(P<0.001).(3)The myotubes were treated with 0.125-2mM AICAR,when the concentration of AICAR was 1-2 mM,the p-AMPK was dramatically activate(P<0.001),and when the concentration of AICAR was 0.5-1mM,ER stress PERK pathway downstream protein ATF4 obviously upregulate(P<0.01),ER tress chaperone BiP mRNA and its PERK pathway downstream genes ATF4,CHOP,GADD34 mRNA also dramatically increased(P<0.05).Conclusions: Saturated fatty acids treatment of cells at different stages,AMPK signaling pathway was activated in 12 h,and highly correlated with endoplasmic reticulum stress,while p-AMPK expression was significantly inhibited after 24 h;Blocking the AMPK pathway with the specific inhibitor Compound C can significantly ameliorate the saturated fatty acids-induced ER stress response.In this way,it proved that activation of the AMPK pathway is necessary to saturated fatty acids-induced early ER stress;AICAR has a significant time-and dose-dependent effect of ER stress.This suggest that at lower concentrations(0.5-1mM)AICAR cansignificantly activate skeletal muscle ER stress.In order to demonstrate that moderately activate AMPK signaling pathway also can activate ER stress.