Aldosterone Induced Up-expression Of ICAM-1 And ET-1 In Pancreatic Islet Endothelium May Associate With Islet Inflammation And Fibrosis

BackgroundHigh incidence of glucose intolerance or diabetes has been proved in primary aldosteronism.Adrenalectomy can reverse glucose intolerance in primary aldosteronism.Prospective cohort studies have also shown that high level of serum aldosterone is associated with increased incidence of T2D.So it is ascertained that excess aldosterone can damage glucose metabolism.Excess aldosterone contributes insulin resistance in adipose tissue,skeletal muscle and vasculature,and inhibits insulin secretion,which are two major features of impaired glucose metabolism.One reason why aldosterone reduces insulin secretion is that it directly stimulates the generation of reactive oxygen species in islet beta cells.Besides,aldosterone is also a risk factor of fibrosis,inflammation and endothelial dysfunction in heart and kidney.But to date,there is no relative study to investigate the effects of aldosterone on pancreatic islet inflammation and fibrosis.The histology of islets in T2D displays an inflammatory process.Macrophages and lymphocytes infiltration,amyloid deposition,and fibrosis are main characteristics of islet inflammation in T2D.Aggravated islet inflammation involves in islet beta cell dysfunction,and contributes to the development of T2D.Islet endothelium is essential for pancreatic development,endocrine cell differentiation,and maintenance of islet function.The endothelium also plays a significant role in inflammation and fibrosis.ICAM-1 in endothelium is involved in the adhesion of monocyte.In fact,ICAM-1 is considered to be the most important adhesion molecule for leukocyte recruitment to inflamed sites.Adherence of lymphocytes and other inflammatory cells to the islet vascular endothelium,followed by transendothelial migration into the endocrine tissue,is essential for islet inflammation which may lead to the development of diabetes.ET-1,mainly from vascular endothelium,is a potent vasoconstrictor peptide and plays a vital role in diabetic vascular complications.Excess ET-1 consistently and markedly decreases pancreatic circulation,especially islet blood flow.ET-1 has been shown to promote the induction of the myofibroblast phenotype from resident fibroblasts,epithelial/endothelial cells,stellate cells;circulating bone marrow-derived monocytes(fibrocytes),and vascular pericytes.And the synthesis and deposition of extracellular matrix components by myofibroblast result in fibrosis in kidney,pulmonary vascular,skin,and heart.Therefore,we proposed and tested the hypothesis that impaired glucose metabolism inT2D is partly associated with elevated aldosterone which may cause islet inflammation and fibrosis.We detected serum aldosterone level and islet macrophages infiltration and fibrosis in diabetic db/db mice.We mainly focused on the expression of ICAM-1 and ET-1 in diabetic islet.And we also investigated the mechanism that aldosterone induced up-expression of ICAM-1 and ET-1 in islet endothelium MS1 cells in vitro.PurposesTo investigate the relationship between aldosterone and islet inflammation and fibrosis in type 2 diabetes.To verify the expression of mineralocorticoid receptor in islet endothelium and the effect of aldosterone on the expression of ICAM-1 and ET-1 in islet endothe ium.Methods(1)Construction of diabetic db/db mouse model,serum aldosterone level was detected by ELISA,islet morphology was observed by HE staining,islet macrophage infiltration was observed by F4/80 staining,and islet fibrosis was detected by Masson staining.Immunohistochemistry was used to detect the expression of endothelial markers CD31,ICAM-1 and ET-1 in islets.(2)MS1 cells were treated with ALD at concentrations of 0,10,25,50,75,100,500,and 1000 nmol/L for 4 days.Then total RNA were extracted and qPCR were proceed for the follow indicators:adhesion molecules ICAM-1,VCAM,MCP1,CD62E,CD62P;endothelial function and oxidative stress related indicators ET-1,iNOS,eNOS,NOX4,G6PD and inflammation indicators IL-1β,IL-6,TNF-α.Cell viability was measured by M-TT and CCK8.(3)Verify the expression of mineralocorticoid receptor in the islet endothelium.The MR mRNA of MS1 cells and positive control kidneys were detected by PCR and agarose gel electrophoresis.WB was used to detect MR protein expression and 11 β-HSD2 in human umbilical vein endothelial cells(HUVEC),human aortic endothelial cells(HAEC)and kidney tissues.Immunofluorescence was proceed to detect the expression of MR and endothelial marker CD31 in MS1 cells and pancreatic islet.(4)MS1 cells were treated with ALD at a concentration of 0,10,25,50,75,100 nmol/L or divided into four groups:control group,ALD(aldosterone),EPL(Eplerenone)and ALD + EPL for 4 days.The mRNA levels of ICAM-1 and ET-1 were detected by qPCR.ICAM-1 protein levels were detected by Western blot.ET-1 levels in the supernatant were assayed by ELISA.Results1.db/db diabetic mice have elevated serum aldosterone levels and increased expression of ICAM-1 and ET-1 in islets.Serum aldosterone levels in db/db diabetic mice increased by 3.31 times.HE staining showed that the islet structure was disordered and there were more irregular and dilated capillary.Excess macrophages infiltration can be observed intra-islet or peri-islet and islet fibrosis was remarkably increased in diabetic db/db mice.The results of immunohistochemistry showed that the mean density of endothelium marker CD31 in islet was decreased to 57.8 Y%while the mean densities of ICAM-1 and ET-1 were increased to 228%and 214%respectively in diabetic db/db mice.2.MS1 cells were treated with ALD at concentrations of 0,10,25,50,75,100,500 and 1000 nmol/L for 4 days.The results of qPCR showed that the adhesion molecule ICAM-1 increased while other adhesion molecules(VCAM,MCP1,CD62E,CD62P)did not change significantly;We also found that aldosterone could improve ET-1 transcription.There was no significant change in iNOS and NOX4.Inflammatory cytokines IL-1β,IL-6 and TNF-a were increased.Aldosterone hardly had any effect on cell proliferation and cytotoxicity detected by MTT and CCK8.3.The mineralocorticoid receptor is expressed in the islet endothelium.MR mRNA was expressed in MS1 cells as revealed by Agarose gel electrophoresis.MR protein and 11 p-HSD2 also expressed in MS1 cells when detected by WB.Immunofluorescence revealed that MR mainly located in the cytoplasm in MS1 cells and collocated with CD31,a representative marker of endothelium,in mice islets.When MS1 cells were exposed to aldosterone for 24 hours,MR nuclear translocation can be observed increasingly.4.Aldosterone promotes the transcription and expression of ICAM-1 and ET-1 in MS1 cells in a MR dependent manner.Aldosterone could boost ICAM-1 transcription in a concentration dependent manner from 0 nM to 100 nM in MS1 cells.The mRNA levels of ET-1 also increased in accordance with the concentration of aldosterone.Western blot analysis showed that aldosterone also increased ICAM-1 protein expression.The concentrations of ET-1 in the supernatant also showed the same inclination.Even at the minimum concentration tested of 25 nmol/L,aldosterone significantly improved the level of ET-1 in the supernatant.Then we sought to determine whether these effects depend on MR or not.We discovered that MR antagonist eplerenone completely withdrew the effect of aldosterone on ET-1 expression.Eplerenone also completely reverse the effect of aldosterone on ICAM-1 mRNA level.When we fixed the concentration of aldosterone(75 nM),we found that eplerenone(from 0 μM to 50 μM)reduced ICAM-1 protein expression in a concentration-dependent manner.Eplerenone also undermined the influence of aldosterone on the protein expression of ICAM-1.ConclusionAldosterone up-regulates the expression of ICAM-1 and ET-1 in islet endothelial cells through mineralocorticoid receptor,and this may associate with the progression of diabetes.