Effects Of AMPK On Its Downstream Targets And Insulin Secretion In INS-1 Cells

Background:Type 2 diabetes is characterized by pancreaticβcells dysfunction and insulin resistance.Metformin,an oral hypoglycemics usually used in clinic to treat diabetes, can activate AMPK(AMP-activated protein kinase),and increase glucose intake and reduce blood glucose level,which leads to improve insulin resistance.AMPK is a key regulator of cellular energy metabolism and promotes fatty acid oxidation by sensing the changes of AMP/ATP ratio.Up to now,most of studies about AMPK focused on its effects on peripheral tissues,however,in pancreatic isletβcells under "physiological condition",effects of AMPK activation on its downstream targets and insulin secretion are remain largely elusive.Pancreatic/duodenal homeobox-1(PDX-1),aβ-cell-specific transcriptor, maintains the function of pancreaticβ-cell in development,differentiation,maturation and regeneration.PDX-1 promotes transcriptional expression of insulin and insulin related genes,and is necessary for maintaining pancreaticβ-cell functions.However, the present reports concerning a relationship between AMPK and PDX-1 are lacking, and whether AMPK could regulate PDX-1 expression is still not clear.The peroxisome proliferator-activated receptors(PPARs)are ligand-regulated transcriptors,and belong to the nuclear receptor superfamily.PPARαand PPARγare two members of PPARs subfamily,whose agonists are mainly used to adjust lipid metabolism and improve insulin sensitivity.In pancreaticβ-cell lines and skeletal muscles,it has been reported AMPK had effects on the expression and activity of PPARα.Therefore there exists possibility that PPARαis a downstream molecular of AMPK besides PDX-1.Considering PPARαand PPARγhas the same PPRE sequence, and the similar sequence has been also found in PDX-1promoter,we supposed that AMPK regulates expression of PDX-1,PPARαand PPARγinβcells.If so,whether PPARαand PPARγare involved in the process of AMPK regulating PDX-1?Objectives:1.To observe whether PDX-1,PPARαand PPARγare downstream moleculars of AMPK in INS-1 cells;2.To explore whether PPARαand PPARγare involved in the regulation of PDX-1 by AMPK;3.To observe effects of AMPK activation on intracellular insulin content and glucose-stimulated insulin secretion(GSIS)under "physiological conditions".Methods:Cells were divided into eight groups:group C:control group;group A:0.5 mM AICAR(AMPK agonist),group CC:10μM Compound C(AMPK antagonist);group AC:0.5 mM AICAR + 10μM Compound C;group M:5μM MK886(PPARαantagonist);group AM:0.5 mM AICAR + 5μM MK886;group B:50μM BADGE (PPARγantagonist);group AB:0.5 mM AICAR + 50μM BADGE.The transcriptional levels of PDX-1,PPARαand PPARγwere detected by RT-PCR and real-time quantitative RT-PCR respectively;PDX-1 protein expression from cytoplasm and nuclear was measured by western blot while the PPARαprotein expression in total protein was determined by immunoprecipitation.Insulin concentration was measured by radioimmunoassay.Results:1.Changes of PDX-1,PPARαand PPARγexpression:Compared to control group,AMPK activation induced by AICAR markedly elevated the mRNA content of PDX-1,PPARαand PPARγ;While Compound C-induced AMPK inactivation reduced the mRNA content of them relative to group A.Nuclear PDX-1 protein and PPARαfrom total protein in group A were significantly increased compared to group C,but decreased in group AC relative to group A.2.Effects of PPARα/PPARγantagonists on nuclear PDX-1 protein expression: Compared to group A,nuclear PDX-1 protein expression levels both in group AM and in group AB reduced markedly,and we failed to detect significant difference of PDX-1 protein between group AM and group M,between group AB and group B.3.Changes of intracellular insulin content and GSIS:Compared to control group, AICAR-induced AMPK activation decreased intracellular insulin content,GSIS and insulin secretion index(ISI)in INS-1 cells.Conclusions:①AMPK activation could regulate the expression of not only PDX-1 but also PPARαas well as PPARγ,which implies that PDX-1,PPARαand PPARγare downstream moleculars of AMPK;②PPARα/PPARγare involved in the regulation of PDX-1 by AMPK,which suggests an AMPK-PPARα/PPARγ-PDX-1 signal pathway in INS-1 cells;③Under "physiological conditions",AICAR-induced AMPK activation decreases intracellular insulin content and GSIS in INS-1 cells.