Metformin Improves AMP-activated Protein Kinase Expression And Function Both In Vivo And In Vitro

Background and ObjectiveAMP-activated protein kinase(AMPK)plays a key role in energy metabolism,regulates many aspects of cell function,including the glucose and lipid metabolism,the switch between anabolism and catabolism.Metformin,a frontline hypoglycemic agent,can produce multifaceted biological effects by activating AMPK.But whether metformin could increase AMPK expression in physiological condition is still unknown.The present study was to investigate the effects of safe dosage of metformin on AMPK expression and function,and the possible mechanisms in bovine aortic endothelial cells(BAECs)and normal rats.MethodsPart 1Confluent BAECs were treated with 500 ?M metformin for a short-term effect study(0h,0.5h,1h,and 2h)and for a long-term effect study(0h,12 h,24h and 48h).And BAECs were treated with different concentration of metformin(0?M,50?M,100?M,250?M and 500?M)for a dose-response study.At the end of intervention,the expres sion of AMPK and the phosphorylation of AMPK(P-AMPK)was measured by Western blot assay.After treatment with 500?M metformin for 24 h in BAECs,the mRNA of AMPK was also detected via RT-PCR assay.Then BAECs were treated with 500?M metformin for 0h,2h and 24 h,and the AMPK expression,the AMPK phosphorylation and its downstream target the phosphorylation of Co A carboxylase(P-ACC)was tested via Western blot assay.Part 2To further investigate the effects of metformin and the long-term effects of metformin in vivo,SD(Sprague-Dawley)rats were treated with metformin(400mg/kg)per day for 2 weeks,4 weeks and 6 weeks respectively.At the end of treatment,the AMPK,the P-AMPK,the P-ACC and the AMPK mRNA level in tissues of different organs of rats were measured.ResultsPart 1Compared with 0h treatment,metformin treatment for 0.5h,1h and 2h could significantly increase the AMPK phosphorylation in BAECs(P<0.05).But treatment with metformin for 12 h,24h and 48 h in BAECs,the AMPK expression as well as the AMPK phosphorylation was significantly increased(P<0.05).Compared with 0?M metformin treatment,the 50?M,100?M,250?M and 500?M metformin treatment could significantly increase the AMPK phosphorylation(P<0.05),but the AMPK expression and the AMPK phosphorylation was significantly increased together only at the concentration of 250?M and 500?M metformin in BAECs(P<0.05).The AMPK mRNA level was significantly increased after treatment with 500?M metformin for 24 h in BAECs(P<0.05).Compared with 0h treatment,the P-ACC was enhanced significantly in BAECs treated with 500?M metformin for 2h and 24h(P<0.05).And Compared with 2h treatment,the P-ACC was increased significantly after treatment with 500?M metformin for 24 h in BAECs(P<0.05).Part 2Compared with the control group,the AMPK expression,the AMPK phosphorylation,the ACC phosphorylation and the AMPK mRNA level was significantly increased in tissues of thoracic aorta,heart,liver and kidney from rats treated with metformin for 2 weeks,4 weeks and 6 weeks respectively(P<0.05).In tissues of skeletal muscle from the rats treated with metformin,the AMPK phosphorylation and the ACC phosphorylation was significantly enhanced as well(P<0.05),but the AMPK expression and AMPK mRNA level shown no alteration.And these effects of metformin had not shown in tissues of lung.ConclusionMetformin could improve AMPK expression and function both in vivo and vitro.The increased AMPK expression may be mediated by the upregulated AMPK mRNA level caused by metformin.Furthermore,this effect of metformin is a permanent effect in multifold visceral organs.