Degradation Of ProANP Protein By IDE In Mouse Heart Tissue And Its Pathological Significance

Diabetes is a worldwide chronic metabolic disease that is seriously harmful to human's health.Hyperglycemia and long-term metabolic disorders in patients with type 1 diabetes mellitus(T1DM)will cause damage to tissues and organs of the whole body and lead to serious complications.T1DM often triggers chronic cardiac inflammation,myocardial hypertrophy,cardiomyocyte apoptosis and myocardial fibrosis,which leads to cardiovascular complications such as cardiomyopathy,heart failure,myocardial infarction,ischemic heart disease and so on.The incidence of these complications in T1DM patients is three to eight-folds higher than that in non-diabetic patients,and they remain the leading cause of morbidity and mortality in T1DM.Insulin degrading enzyme(IDE)is an evolutionarily highly conserved zincdependent metalloprotein hydrolase,which was originally named for its degradation of insulin.Subsequently,it was found that IDE can cleave and inactivate several bioactive peptides with diverse sequences and structures,such as glucagon and ?-amyloid protein(A?),preventing the formation of peptide aggregates in subcellular compartments,thus participating in a variety of physiological and pathological processes.Due to the degradation of insulin and A? by IDE,IDE has been proved to be associated with the occurrence and development of diabetes and Alzheimer's disease(AD).Subsequent studies have shown that IDE can also degrade atrial natriuretic peptide(ANP).ANP is an endogenous active polypeptide of cardiac origin that provide natriuresis,diuresis,vasodilation,antiproliferation,antihypertrophy,antifibrosis and other cardiometabolic protection.In fact,our pre-experimental results show that IDE may be involved in the degradation of intracellular precursor ANP(proANP).Since ANP is involved in controlling water and sodium metabolism and regulating blood pressure by regulating angiotensin,we speculate that IDE may be involved in the occurrence and development of diabetic cardiomyopathy/cardiovascular disease by regulating/degrading ANP/proANP.In this study,eukaryotic expression plasmids pcDNA3.1-HA-IDE and pEGFP-N1ANP/BNP,containing target genes IDE,ANP or BNP were cotransfected into HEK293T cells in vitro,or siRNA that specifically interfered with the expression of IDE was transfected into H9C2 cells.The effect of IDE on intracellular proANP/proBNP expression was tested by Western blot,RNA interference and other molecular biological techniques.Secondly,We observed the expression of IDE in several tissues of C57BL/6 mice and the effects of different levels of glucose and insulin on IDE/proANP expression under physiological conditions.Thirdly,The T1DM mice model(STZ mice)induced by streptozotocin were established,and the regulation of proANP by IDE in the heart of STZ mice was further verified by Western blot,qRT-PCR,IDE enzyme activity and ELISA.Finally,We explore the effect of proinflammatory factor IL-6 on the expression of 1DE in H9C2 cardiomyocytes and the underlying signal pathways.Result:1.In HEK293T cells,overexpression of IDE can lead to the decrease of proANP protein in a dose-dependent manner.Overexpression of IDE did not change the level of proBNP protein in HEK293T cells.In H9C2 cells,after specifically knockdown of IDE,the expression of proANP increased and the level of proBNP did not change.After H9C2 cells were treated with different concentrations of IDE inhibitor 6bK,the expression of IDE did not change,the expression of proANP increased and the level of proBNP remains unchanged.Proteasome inhibitor MG 132 did not increase the level of proANP in HEK293T cells,while lysosome inhibitor CQ could significantly inhibit the degradation of proANP by IDE.2.IDE is a universal expressed protein,with moderate expression in the heart and low expression in the small intestine.Under starvation,the protein expression of IDE in the heart did not change.After injection of exogenous insulin,the levels of IDE protein and mRNA in the heart were continuously up-regulated,reached the peak within 2 hours,and returned to normal level after 4 hours.The proANP protein was significantly down-regulated only at 2 hours after insulin injection.3.Compared with the control group,the expression of IDE in the heart of STZ mice was significantly increased at the protein and mRNA levels,while the expression of proANP at the protein level was significantly decreased,and there was no significant change in the mRNA level.There was no significant change in the activity of IDE enzyme in the heart of STZ mice.The concentration of IDE in serum of STZ mice increased significantly compared with that from their control littermates,while the concentration of ANP decreased significantly.4.Compared with the control group,the expression of IL-6 in the heart of STZ mice was significantly up-regulated at the mRNA level,while the expression of IL-1?,TNF-? and TGF-? at the mRNA level remain unchanged.The results of ELISA showed that the concentration of IL-6 in the heart of STZ mice was significantly higher than that from the control group,but the concentration in serum showed no obvious change compared with the control group.The concentration of TNF-? in serum of STZ mice was significantly higher than that in the control group,but the concentration in the heart had no change compared with the control group.5.IL-6 at concentrations of 10,50 and 100 ng/mL could significantly up-regulate the expression of IDE in H9C2 cells.The expression of proANP in H9C2 cells showed an opposite trend to that of IDE.With the increase of IL-6 incubation time,the expression of IDE in H9C2 cells increased significantly in a time-dependent manner,while the content of proANP protein was continuously down-regulated.6.The expression of IDE in H9C2 cells could be blocked by adding IL-6 antibody in advance.The up-regulation of IDE expression induced by IL-6 in H9C2 cells was significantly inhibited by the addition of selumetinib but not tofacitibib.Conclusion:1.IDE is involved in the degradation of proANP in cells.Inhibition of IDE expression or IDE activity could block the degradation of proANP.The degradation of proANP by IDE is mainly through lysosome.2.Insulin stimulation can up-regulate the expression of IDE and degrade a small amount of proANP in the heart.It is suggested that under physiological conditions,IDE prefers to cleavage insulin first.3.In the heart of STZ mice,the increase of inflammatory factor IL-6 could promote the expression of IDE,resulting in a decline of proANP,which will futher increase the risk of cardiomyopathy/cardiovascular complications in patients with T1DM.Inhibition of ERK/MAPK signaling pathway may alleviate the occurrence and development of the disease.