DIZE-mediated Endogenous Activation Of ACE2 Regulates Lipid Deposition In Adipose Tissue Of High-fat Diet Rats And Its Mechanism

ACE2 is a key enzyme in the renin-angiotensin system(RAS),which can promote lipolysis through the ACE2/Ang1-7/Mas axis,inhibit fat synthesis,and improve obesity and related metabolic disorders.Diminazene(DIZE)as an endogenous activator of ACE2 has been studied in the treatment of cardiovascular diseases and other diseases,but its role and mechanism in adipose tissue have not been reported yet.This study explored the effect and mechanism of DIZE on fat deposition in rat adipose tissue induced by high-fat diet.The experiment includes the following aspects:1.The alleviating effect of DIZE on lipid deposition in adipose tissue of high-fat diet rats and its relationship with ACE2Including two parts in vivo and in vitro.32 SD rats were selected for the in vivo experiment.After the adaptation period,8 rats were fed with normal diet as normal control group(CON group),and 24 rats were fed with high-fat diet.After 4 weeks of continuous feeding,24 high-fat diet rats were randomly divided into a high-fat model group(MOD group,high-fat diet),Lovastatin group(LOV group,high-fat diet+15 mg/kg/d lovastatin gavage)and DIZE group(high-fat diet+15 mg/kg/d DIZE gavage).Continue feeding for another 6 weeks.After the glucose tolerance test,all the experimental rats are weighed,blood is collected,dissected,subcutaneous and other adipose tissues are taken,and the fat coefficient is calculated by weighing.At the same time,some samples were fixed with paraformaldehyde or stored at-80℃,and the following experiments were carried out:1)HE staining to observe the deposition of lipid droplets in adipose tissue;2)Determination of biochemical indicators such as TC and TG in serum;3)RT-qPCR method to analyze the expression level of ACE2 mRNA in each adipose tissue;ELISA method was used to detect the content of ACE2,Ang Ⅱ and Ang1-7.After in vitro experiments select 3T3-L1 cells to induce differentiation,select the appropriate concentration by MTT method,and then treat the differentiated 3T3-L1 cells with a final concentration of 12.5,25μM DIZE for 48 h.Or after pretreatment with siACE2 interference,add 25μM DIZE to continue processing for 48h,The cells were collected to detect the expression of ACE2 and FAS protein by Western blot,and oil red O staining was used to observe the accumulation of lipid droplets.Results:1)The weight of rats fed with high-fat diet increased,the fat coefficient increased,and the glucose tolerance decreased significantly(P<0.05);NEFA in the blood increased(P<0.05),and there was no significant change in TC and TG;The area of lipid droplets in each adipose tissue increased significantly(P<0.05);2)Compared with the model group,rats in the DIZE group had significantly lower body weight gain(P<0.05)and increased glucose tolerance(P<0.05);serum NFFA levels decreased,and there was no significant difference in TC and TG;The lipid droplet area in each adipose tissue was significantly reduced(P<0.05);3)All members of RAS exist in white and brown adipose tissue.After the high-fat diet was fed,the content of ACE2 in each adipose tissue decreased,and the difference between WAT and BAT in the epididymis was significant(P<0.05);the expression of ACE2 mRNA and protein content in iWAT of the DIZE group increased significantly(P<0.05);The content of Ang Ⅱ in each adipose tissue was significantly decreased(P<0.05),and the content of Ang1-7 was significantly increased(P<0.05).4)Treatment of induced differentiation of 3T3-L1 cells with 25 μM DIZE can significantly promote intracellular ACE2 protein expression(P<0.05),and inhibit FAS protein expression and intracellular lipid droplet content.Conclusion:DIZE treatment can inhibit the body weight gain of high-fat diet rats,reduce serum NEFA content,improve glucose tolerance,relieve lipid deposition in white adipose tissue of high-fat diet rats,and inhibit the whitening of BAT.And this effect is related to the high activity or high expression of ACE2 in adipose tissue.DIZE may promote the degradation of Ang Ⅱ and the production of Ang1-7 by activating the expression of ACE2,thereby inhibiting lipid deposition.2.Regulative effect of DIZE on glucose and lipid metabolism in adipose tissue induced by high-fat diet in ratsFrom the perspective of adipose tissue glucose and lipid metabolism,the possible mechanism of DIZE alleviating fat deposition in adipose tissue was discussed.32 SD rats were selected for the study,and the animal breeding and grouping treatment were the same as in the previous chapter.After the test,eWAT,iWAT and BAT were collected,and the following experiments were carried out:1)Determination of NEFA content in each adipose tissue;2)RT-qPCR method to determine the mRNA expression levels of HSL,ATGL,LPL,the key factors of esterification in each adipose tissue;3)RT-qPCR and Western Blot method to determine the expression levels of key ester synthesis factors ACC,FAS,SREBPlc,DGAT1 in adipose tissue;4)Western Blot method to determine the expression levels of glucose uptake and oxidation related factors GLUT4 and CS in adipose tissue;Results:1)Compared with the control group,the content of NEFA in the adipose tissue of the model group did not change significantly.The expression levels of HSL,ATGL and LPL mRNA in white adipose tissue were significantly increased(P<0.05),while the results in BAT were the opposite(P<0.05);Compared with the model group,the NEFA content of eWAT in the DIZE group increased significantly(P<0.05),The content of NEFA in iWAT and BAT was significantly decreased(P<0.05);the expression of HSL,ATGL,LPL mRNA in the white adipose tissue was significantly decreased(P<0.05);2)The expression levels of ACC,FAS,SREBPlc,DGAT1 mRNA and protein in each adipose tissue of rats in the model group were significantly increased(P<0.05);Compared with the model group,the mRNA expression and protein expression of the above key fat synthesis factors in each adipose tissue of the DIZE group were significantly decreased(P<0.05);3)The expression levels of GLUT4 and CS protein in each adipose tissue of rats in the model group were significantly increased(P<0.05);The expression levels of GLUT4 and CS protein in the white adipose tissue of rats in the DIZE group were higher than that in the model group(P<0.05).Conclusion:The lipid anabolism in the adipose tissue of rats fed with high-fat diet is superior to catabolism,and the increase in lipid synthesis leads to fat deposition.DIZE treatment has a certain regulatory effect on the lipid metabolism disorder of adipose tissue induced by high-fat diet rats.The mechanism on the one hand inhibits lipid synthesis by inhibiting the expression of ester synthesis-related factors;on the other hand,it inhibits the de novo synthesis of fatty acids by promoting the oxidative decomposition of glucose uptake by adipose tissue,thereby inhibiting fat deposition.3.Molecular mechanism of inhibitory effect of DIZE on lipid deposition in adipose tissue of rats fed with high-fat dietIn the experiments in the previous chapter,we confirmed that DIZE can inhibit fat deposition in high-fat diet rats by inhibiting fat synthesis and glucose metabolism in adipose tissue.This chapter uses subcutaneous white fat in the groin and brown adipose tissue in the scapular area as the test objects.By studying the changes in factors related to thermogenesis and mitochondrial biological function in rat adipose tissue,focusing on the energy-sensing factor AMPK and its signaling pathways.To explore the molecular mechanism of DIZE inhibiting fat deposition in high-fat diet rats.32 SD rats were selected for the study,and the animals were raised and processed in groups as in the previous chapter.After the experiment,iWAT and BAT were collected,and the following experiments were performed:1)Determine the expression levels of key factors for thermogenesis(Dio2,Otopl,Zicl)and uncoupling protein UCP1 in two types of adipose tissue;2)Analyze the mitochondrial copy number(mtDNA)and the expression levels of mitochondrial activity and function-related proteins(OPA1 and SDHA)in the two types of adipose tissues;3)Determine the expression levels of key proteins in the AMPK/SIRT1-PGC-1α signaling pathway in adipose tissue.Results:1)Compared with the control group,the expression levels of Dio2,Otopl,and Zicl mRNA in the two adipose tissues of the model group were significantly down-regulated(P<0.05),and the protein expression of UCP1 in iWAT was down-regulated,BAT was significantly up-regulated(P<0.05);The expressions of Dio2,Otop1 and Zic1 mRNA and UCP1 protein in brown adipose tissue of the DIZE group were significantly higher than those in the model group(P<0.05).There was no significant change in thermogenesis factors in the subcutaneous adipose tissue of the groin,and the expression of UCP1 protein was significantly down-regulated(P<0.05);2)Compared with the control group,the copy number of mtDNA in brown adipose tissue of rats in the model group increased significantly(P<0.05);the expression of mitochondrial fusion protein OPA1 in subcutaneous adipose tissue of the inguinal increased significantly,but the expression of respiratory chain protein SDHA decreased significantly(P<0.05),the results were opposite in brown adipose tissue;after DIZE treatment,the expression of OPA1 and SDHA protein in each adipose tissue increased significantly(P<0.05);3)The expressions of AMPK,SIRT1 and PGC-1α protein in the two adipose tissues of the model group were significantly decreased(P<0.05);the expressions of the three proteins in the DIZE group were significantly up-regulated.The results suggest that DIZE treatment activates the functional activity of mitochondria in adipose tissue,and at the same time promotes mitochondrial fusion,it can also enhance the function of mitochondrial respiratory chain,showing enhanced heat production and uncoupling.Cells prevent fat synthesis in adipose tissue by enhancing energy consumption,thereby alleviating fat deposition induced by high-fat diet,and this effect is carried out through the AMPK/SIRT1-PGC-1α pathway.Our research results found that,as in other tissues,ACE2 exists in both white and brown adipose tissue.As a major member of the local renin-angiotensin system(RAS)in adipose tissue,it produces Ang1-7 by degrading Ang Ⅱ.It plays an important role in adipose tissue glucose and lipid metabolism and thermogenesis,and has a certain inhibitory effect on fat deposition induced by high fat.DIZE indirectly plays a role in inhibiting fat deposition by activating ACE2.The results suggest that DIZE as an ACE2 activator can be used as a new idea to improve and treat obesity and related metabolic disorders.