Metformin attenuates angiotensin Ⅱ induced cardiac fibrosis and transforming growth factor-β1 production through the inhibition of hepatocyte nuclear factor4

Aim In diabetic patients, mefformin appears to provide cardiovascular protection that cannot be attributed only to its antihyperglycemic effects.Metformin is also known as the AMPactivated protein kinase (AMPK) activator.Our previous study suggested that metformin inhibits transforming growth factorβ1 (TGFβ1) production in a mouse heart failure model of pressure overload.TGFβ1 is a key factor in cardiac fibrosis and is usually induced by Angiotensin Ⅱ (Ang Ⅱ) in the pressure overload mouse models.This study investigated the effect of metformin on cardiac fibrosis and TGFβ production induced by Angl and the underlying mechanisms.Methods C57/BL6wildtype and AMPKα2 knockout mice were used.AngⅡ (3 mg · kg1 · d1) was infused subcutaneously into mice for 7 days.Adult mouse cardiac fibroblasts were isolated and treated with AngⅡ(1 μmol · L1) and/or metformin (1 mmol · L1).Results In C57/BL6 mice, metformin inhibits AngⅡinduced cardiac fibrosis.In cardiac fibroblasts, metformin inhibits TGFβ1 expression and production induced by AngⅡ.AMPK inhibitor, compound C, reversed the effects of metformin.In vivo, AMPKα2 deficiency further increases AngⅡinduced TGFβ1production.In cardiac fibroblasts, metformin inhibited AngⅡ induced hepatocyte nuclear factor4 (HNF4α protein level increase and HNF4α binding with TGFβ1 promoter using chromatin immunoprecipitation assay.In vivo,AMPKα2 deficiency further increased AngⅡinduced HNF4o protein level.Using HNF4α adenovirus, overexpressing HNF4α led to a 1.5fold increase in TGFβ1 mRNA expression.HNF4a siRNA blocked AngⅡ induced TGFβ1 production.Luciferase reporter with deleted HNF4a binding sites showed decreased TGFbl transcriptional activity induced by Angl.In AMPK α2/heart, the inhibition of metformin on HNF4a protein was attenuated.Conclusion Metformin inhibits AngⅡ induced cardiac fibrosis and TGFβ1 production through AMPK activation.The underlying mechanism is that AMPK activation inhibits AngⅡ induced HNF4α and then decreases TGFβ1 expression.184-18