Study On The Role Of NPY Y1 Receptor In The Activation Of Ca²⁺-AMPK Signaling Pathway In Glucose Uptake Of Cardiomyocytes

Background:Heart failure（HF）is a common,disabling and potentially fatal disease.It is the main cause of morbidity and mortality worldwide and the final course of all cardiovascular diseases.The basic pathophysiological process of HF is myocardial remodeling,and energy remodeling is found to exist at the same time.The energy metabolism of heart failure is in a relatively negative equilibrium state,which shows that the ratio of ATP and ADP/ATP increases,while glucose oxidation dominates,while fatty acid oxidation decreases significantly.Neuropeptide Y（NPY）is the most abundant peptide substance expressed in brain and heart.It mainly plays a role by binding with its receptor.In the nervous system,NPY plays a regulatory role in energy metabolism and food intake.Y1,Y2 and Y5 are the key factors for NPY to regulate animal feeding.In the hypothalamus,negative energy balance induces NPY release,and promotes body feeding through Y1 and Y5 receptors,giving priority to carbohydrate intake.Y2 plays the opposite role.Based on the similarity between the characteristics of energy metabolism of pressure overload myocardium and the characteristics of energy metabolism of hypothalamus by NPY and its receptor subtypes,we found that NPY can promote cardiomyocyte hypertrophy through Y1 and Y5 receptors,and inhibit cardiomyocyte hypertrophy through Y2 receptors.Whether NPY affects myocardial glycometabolism has not been reported yet.We hypothesize that NPY participates in the process of glycometabolism and cardiac hypertrophy in cardiomyocytes through its receptors.The aim of this study is to establish a pressure overload cardiomyocyte model to observe the effects of NPY Y1 receptor subtypes on myocardial glycometabolism,and further reveal the molecular mechanism of NPY Y1 receptor-Ca²⁺-AMPK pathway involved in the regulation of myocardial glycometabolism.The successful implementation of this project will provide more sufficient scientific basis for NPY receptor subtypes as a new target for the treatment of heart failure.Objectives:1、The effect of NPY Y1 receptor subtype on glucose uptake of cardiomyocytes.2、To explore the mechanism of NPY Y1 receptor regulating the glucose uptake of cardiomyocytes through Ca²⁺-AMPK pathway.Methods:1.Using rat cardiomyocyte cell line H9C2 as the research object,using different concentrations of NPY Y1 receptor agonist(10^-8～10^-6mol·L^-1)and NPY Y1 receptor inhibitor(10^-8～10^-6mol·L^-1)stimulated H9C2 cardiomyocytes at different times（0h,6h,12h,24h,48h）,observed the effect of NPY Y1 receptor agonists and inhibitors on cells,and detected the uptake of 2-NBDG by H9C2 cells using fluorescence microscopy.The effect of cellular glucose uptake was observed.The m RNA and protein expressions of ANF,β-MHC,GLUT-1 and GLUT-4 in H9C2 cells were detected by real-time PCR and Western Blot.2.Stimulation of H9C2 cardiomyocytes with 10-6mol·L-1NPY Y1 receptor agonist and 10^-6mol·L-1NPY Y1 receptor inhibitor for 24h,determination of intracellular Ca²⁺ion concentration and regulation of AMPK and p AMPK.Results:1.After different concentrations of NPY Y1 receptor agonists(10^-8～10^-6mol·L^-1)stimulated H9C2 cardiomyocytes for different times（0h,6h,12h,24h,48h）,the effects of cell sugar uptake were detected.Compared with the normal control group,the fluorescence intensity of 2-NBDG in NPY Y1 receptor agonist group increased with the increase of NPY Y1 agonist concentration,and increased with the increase of npy1 agonist concentration;the fluorescence intensity in npy1 receptor agonist group increased at all times,and reached the peak at 24h,and decreased at 48h.The results showed that the activation of NPY Y1 receptor could stimulate H9C2 cardiomyocytes to promote glucose absorption（n=6,P<0.05）.The higher the concentration of NPY Y1 agonist and the longer the stimulation time were,the more obvious the effect of promoting glucose absorption was.When the concentration was 10^-6mol·L^-1 and the stimulation time was 24hours,the most obvious effect was that NPY Y1 receptor agonist stimulated H9C2cardiomyocytes to absorb glucose in a concentration and time-dependent manner.2.The expression of ANF,β-MHC,GLUT-4 m RNA and protein in cardiomyocytes was up-regulated in a concentration dependent manner in the range of 10^-8-10^-6mol·L-1after 24 hours stimulation with NPY Y1 receptor agonists（10-8-10-6mol·L-1）（P<0.05）The higher the concentration of NPY Y1 receptor agonist was,the less the expression of GLUT-1 protein was.There was no significant difference in the expression of GLUT-1protein（P>0.05）.3.Different concentrations of NPY Y1 receptor inhibitors(10^-8～10^-6mol·L^-1)were used for different time（0h,6h,12h,24h,48h）to detect the effect of cell sugar uptake.Compared with the control group,the fluorescence intensity of cardiomyocytes in different concentration groups and time points decreased（n=6,P<0.05）,and the inhibition effect of NPY Y1 receptor inhibitor was the most obvious when the concentration of NPY Y1 receptor inhibitor was 10^-6mol·L^-1 and the time was 24h,suggesting that inhibition of NPY Y1 receptor can inhibit the glucose absorption of cardiomyocytes,the inhibition effect was dose-dependent and time-dependent.4.The expression ofβ-MHC and GLUT-4 protein decreased with the increase of NPY Y1 receptor inhibitor concentration（P<0.05）Compared with the control group,the expression of ANF and GLUT-1 had no significant change（P>0.05）.It is suggested that inhibition of NPY Y1 receptor expression can inhibit the expression of GLUT-4 m RNA and protein in H9C2 cardiomyocytes,but has no significant effect on the expression of ANF,β-MHC,GLUT-1 m RNA and protein.5.When H9C2 cells were stimulated with 10^-6mol·l^-1NPY Y1 receptor agonist and10^-6mol·l-1npyy1 receptor inhibitor for 24 hours,compared with the normal control group,the Ca²⁺concentration in H9C2 cells stimulated by NPY Y1 receptor agonist increased（P<0.001）,and the p-AMPK/AMPK ratio was significantly increased（P<0.05）The Ca²⁺concentration in H9C2 cardiomyocytes stimulated by NPY Y1 receptor inhibitor decreased（P<0.01）,and the p-AMPK/AMPK ratio in H9C2 cardiomyocytes stimulated by NPY Y1 receptor inhibitor decreased.Conclusions:.1.NPY Y1 receptor promotes GLUT-4 expression in cardiomyocytes and increases the effect of glucose uptake.2.Ca²⁺-AMPK pathway may be a pathway that NPY Y1 receptor mediates glucose uptake of cardiomyocytes through GLUT-4.