The Study Of Role And Mechanism Of HuR In Atherosclerosis And Obesity

1.BackgroundWith the accelerated aging of society and the prevalence of unhealthy lifestyle,the risk factors of cardiovascular diseases have increased significantly.The prevalence rate of cardiovascular diseases in China continues to rise,and the mortality rate of cardiovascular diseases ranks the first place,much higher than that of tumors and other diseases.Atherosclerosis(AS)is the pathophysiological basis of a variety of cardiovascular diseases and the most important cause of acute cardiovascular events.Therefore,it is a significant mission for us to find effective measures for prevention and treatment of AS.Smoking is the main risk factor of cardiovascular diseases,which is closely related to the formation of AS.However,the mechanism of AS caused by smoking is still unclear.Therefore,this study focused on the specific molecular mechanism of nicotine,the main component of tobacco,in promoting AS,and discussed the significance of regulating GTPCH1 in nicotine-induced AS.Since Ross R proposed the "endothelial injury response hypothesis" in 1993,and held that endothelial dysfunction was the initial link and key link of AS,the role of endothelial function in the development of atherosclerosis has attracted much attention.Nitric oxide(NO)is an important vascular homeostasis regulator synthesized by endothelial nitric oxide synthase(eNOS)in endothelial cells.A large amount of evidence suggests that eNOS and NO play an important role in the occurrence of cardiovascular diseases.For example,in eNOS knockout mice,vascular inflammation and endothelial injury are aggravated,which are manifested as hypertension and atherosclerosis.The bioavailability of tetrahydrobiopterin(BH4),the basic cofactor of eNOS,is a key factor in determining eNOS activity.When BH4 level is insufficient,eNOS will reduce molecular oxygen to produce more superoxide anions(O2-·)instead of NO,thus causing vascular oxidative stress and endothelial dysfunction.This phenomenon is called "uncoupling" of eNOS.BH4 level is crucial to determine the catalytic products of eNOS and its effect on endothelial function.GTPCH1 is a key rate-limiting enzyme that catalyzes the synthesis of BH4 from GTP.Inhibition of GTPCH1 will lead to reduced BH4 synthesis,thus promoting the eNOS uncoupling.GTPCH1 deficiency is an important mechanism of cardiovascular diseases and diabetic endothelial dysfunction.Therefore,regulating the level of GTPCH1 is of great significance in alleviating eNOS uncoupling and maintaining normal endothelial function.Human antigen R(HuR)is an RNA-binding protein that binds to adenosine uridine-rich elements(ARE)in the 3’-UTR of target gene mRNA in the cytoplasma,hence increasing the stability of its mRNA and resulting in increased protein level.GTPCH1 can be regulated by a variety of mechanisms,but whether HuR can regulate the stability of GTPCH1 mRNA remains unclear.We identified 9 ARE loci in the 3’-UTR of mouse GTPCH1 mRNA,strongly suggesting that GTPCH1 may be the target gene of HuR.In conclusion,we put forward the following scientific hypothesis:nicotine inhibits the expression of endothelial cell GTPCH1 and interferes with the synthesis of BH4 by affecting HuR activity,leading to eNOS uncoupling and increased atherosclerosis injury.To this end,we supplemented BH4 or improved GTPCH1 expression in the classical AS animal model ApoE-/-mice and human umbilical vein endothelial cells(HUVECs)and explored the role of HuR/GTPCH1 pathway in nicotine-induced atherosclerotic injury in mice.2.Objectives(1)Explore the changes of GTPCH1 and HuR expression in nicotine-induced endothelial functional inj ury.(2)Explore the effects of overexpression of GTPCH1 and supplementation of BH4 on nicotine-induced atherosclerosis in mice.(3)Clarify the specific mechanism of nicotine affecting GTPCH1 expression.3.Methods(1)Animal model construction.Atherosclerosis model was established by feeding 8-week ApoE-/-mice on a high-fat diet for 12 weeks.To explore the effect of BH4 on nicotine-induced atherosclerosis,the following four groups were used:a.ApoE-/-mice+Vehicle;b.ApoE-/-mice+BH4;c.ApoE-/-mice+Vehicle+ Nicotine;d.ApoE-/-mice+BH4+Nicotine.To explore the effect of GTPCH1 on nicotine-induced atherosclerosis,the following four groups were used:a.ApoE-/-mice+LV.GFP;b.ApoE-/-mice+LV.GTPCH1;c.ApoE-/-mice+LV.GFP+Nicotine;d.ApoE-/-mice+ LV.GTPCH1+Nicotine.(2)Cell models.Cultured human umbilical vein endothelial cells were divided into the following groups to explore the effect of BH4 on nicotine-induced endothelial injury,a.HUVECs+Vehicle;b.HUVECs+sepiapterin;c.HUVECs+Nicotine;d.HUVECs+sepiapterin+Nicotine.To investigate the effect of GTPCH1 on icotine-induced atherosclerosis,the following four groups were set.a.HUVECs+LV.GFP+Vehicle;b.HUVECs+LV.GTPCH1+Vehicle;c.HUVECs+LV.GFP+Nicotine;d.HUVECs+LV.GTPCH1+Nicotine.(3)Explore the effects of nicotine stimulation on endothelial function and GTPCH1 expression:HUVECs were treated with nicotine at different concentrations or stimulated at different times.Changes in NO,ROS,BH4,GTPCH1 protein and mRNA expression were detected to evaluate endothelial function.Nicotine stimulation was repeated to verify changes in endothelial function and GTPCH1 expression under physiological conditions.(4)Investigate the effect of GTPCH1/BH4 on nicotine-induced endothelial injury.HUVECs were cultured with sepiapterin supplement or GTPCH1 overexpression virus transfection followed by nicotine stimulation.Changes in ROS,BH4 and NO were detected to evaluate whether endothelial function was improved.(5)Explore the effect of GTPCH1/BH4 on nicotine-induced atherosclerosis.8-week ApoE-/-mice were treated with intraperitoneal injection of BH4 or tail vein injection of GTPCH1 overexpressed virus.Samples were taken after 12 weeks of high-fat diet and nicotine in drinking water.Oil red O staining was used to evaluate the atherosclerosis area of aortic root in mice.Serum NO and acetylcholine induced aortic diastolic function were used to evaluate vascular endothelial function.Serum MDA content and 4-HNE staining of aortic root were used to evaluate the degree of lipid peroxidation in plaques.(6)Explore the specific mechanism by which nicotine regulates GTPCH1 expression.Western blot was used to detect the contents of HuR total protein,phosphorylated HuR,nuclear HuR and cytoplasmic HuR in nicotine-stimulated endothelial cells.RNA immunoprecipitation assay confirmed whether HuR could directly bind to GTPCH1 mRNA.The half-life experiment was conducted to observe whether HuR specific inhibitor CMLD-2 affected the stability of GTPCH1 mRNA.4.Results(1)Nicotine stimulation reduced GTPCH1 expression.First,we stimulated HUVECs with nicotine at different concentrations and for different stimulation durations,and found that the expression of GTPCH1 decreased in a concentration-dependent and time-dependent manner.The synthesis of BH4 and NO by endothelial cells decreased and intracellular ROS significantly increased.The synthesis of BH4 and NO and the expression of GTPCH1 showed similar changes after nicotine stimulation in HUVECs cultured under low oxygen condition which is more similar to the human environment.(2)BH4 supplementation or GTPCH1 overexpression reversed nicotine-induced endothelial damage.Endothelial cells were pretreated with BH4 precursor sepiapterin or transfected with GTPCH1 overexpressed virus,and then exposed to nicotine.It was found that the levels of BH4 and NO in HUVECs were significantly increased,while the production of ROS was reduced.These results further confirmed that nicotine may lead to reduced synthesis of BH4 and increased oxidative stress by inhibiting the expression of GTPCH1.(3)BH4 supplementation or GTPCH1 overexpression can improve the progression of nicotine-induced atherosclerosis.After receiving BH4 supplement or injection of GTPCH1 overexpressed virus in ApoE-/-mice fed with high fat,the area of atherosclerotic plaque was significantly reduced,which was accompanied by increased serum NO content,improved endothelial function,decreased lipid peroxidation products 4-HNE and MDA levels,and reduced expression of adhesive molecules in the endothelium.(4)Nicotine regulated the expression of GTPCH1 by inhibiting HuR karyoplasmic translocation.After nicotine stimulation on HUVECs,the expression of total HuR in cells remained unchanged,while the phosphorylation modification affecting the localization of HuR was suppressed.The expression of HuR in the cytoplasm decreased and HuR in the nucleus increased,suggesting that nicotine inhibited the phosphorylation and karyoplasmic translocation of HuR.RNA immunoprecipitation showed that HuR could bind directly to GTPCH1 mRNA while nicotine reduced this binding.The stability of GTPCH1 mRN1 was significantly decreased after CMLD-2 treatment.5.Conclusion(1)This study revealed the important role of GTPCH1 in the process of atherosclerosis by nicotine for the first time,and confirmed that increasing the expression level of GTPCH1 or supplementing BH4 can significantly improve the damage of nicotine to endothelial function,and slow down the process of nicotine-induced atherosclerosis.Targeting GTPCH1 may provide new ideas for clinical improvement of endothelial function in smoking patients with cardiovascular diseases.(2)In terms of mechanism,nicotine restricts the ability of HuR stabling target gene GTPCH1 mRNA by inhibiting the nucleation of HuR,resulting in reduced GTPCH1 expression,reduced BH4 generation,eNOS uncoupling and accelerated development of atherosclerosis.1.BackgroundWith the progress of social economy,people’s material living standard has been greatly improved.However there are still many health problems,among which obesity has become the most common nutritional imbalance disease.Obesity,as an important risk factor for many diseases,is often associated with type II diabetes,cardiovascular diseases,non-alcoholic fatty liver disease,sleep apnea syndrome,polycystic ovary syndrome and tumors,which seriously affect peopled quality of life.Obesity refers to the state in which adipose tissue,especially white adipose tissue,expands due to the proliferation or hypertrophy of adipocytes.Lipogenesis and lipolysis processes are key factors that regulate the steady-state of the triglyceride-fatty acid cycle and determine the volume of adipose tissue.In excess energy,fat cells synthesize triglycerides(TAG)in large quantities and store them in lipid droplets,known as lipogenesis.During exercise or fasting,triglycerides are hydrolyzed to release fatty acids(FFA)and glycerol into the bloodstream,called lipolysis.Lipolysis process needs the order hydrolysis by adipose triglyceride lipase(ATGL),hormone sensitive lipase(HSL)and monoglyceride lipase(MGL).Since ATGL was first reported in 2004,it has attracted more and more attention because of its important role in the lipolysis process.Recent studies suggest that the function of ATGL in the lipolysis plays an important role in improving obesity,insulin resistance and other phenotj^es.The protein and mRNA levels of ATGL in subcutaneous adipose tissue of obese people were lower than those of normal weight people,and the decrease of ATGL mRNA levels in visceral fat was correlated with insulin resistance.ATGL whole-body knockout mice showed triglyceride overload in white adipose and a variety of non-adipose tissues due to severe lipolysis functional defects,which were manifested as obesity and multi-organ lipid toxicity damage.Adipose tissue specific overexpression of ATGL can significantly reduce obesity induced by high-fat diet and improve insulin resistance.Therefore,the development of therapeutic measures to promote the expression or activity of ATGL is of great significance for the prevention and treatment of obesity and its metabolic related diseases.Human antigen R(HuR)is an RNA-binding protein,which can bind to adenylate-uridylate-rich element(ARE)in the 3’untranslated region(3’-UTR)of the target gene mRNA,thus increasing the stability of its mRNA and resulting in increased protein levels.At present,studies show that HuR is involved in the post-transcriptional regulation of many genes and affects many processes such as inflammation and cancer stress.However,the function and role of HuR in obesity and fat cells have not yet been revealed.At the same time,we found that the mRNA35-UTR of ATGL contains ARE sites,suggesting that HuR may be an intervention target for obesity,dyslipidemia,insulin resistance and other metabolic diseases by affecting the expression of ATGL.In this study,we prepared the adipose tissue specific HuR knockout mice for the first time,and focused on the study of the phenotype and mechanism of the knockout mice fed a high-fat diet to explore the significance of HuR as a new idea and new target for the regulation of obesity and other phenotypes.2.Objectives(1)Explore the phenotype of adipose-specific HuR knockout mice during high-fat feeding.(2)Clarify the function of HuR in adipocytes differentiation and lipolysis.(3)Explore the specific molecular mechanism of HuR in regulating adipose function.3.Methods(1)Experimental model construction.AdipoQ-cre/loxp system was used to construct adipose specific HuR knockout mice,which were given normal diet or high-fat diet after 8 weeks of age.(2)Cell model.The stromal vascular fraction components of white adipose tissue in mice 6-8 weeks old of each genotype were extracted and induced to differentiate into adipocytes.(3)Explore the influence of HuR on fat volume of mice.Monitor mice weight weekly.Total fat content of mice was detected by bone mineral density meter(BMD).The fat content of each part was measured and recorded,including abdominal subcutaneous adipose,epididymal visceral adipose and brown adipose.H&E staining was used to compare the size of adipocytes in different parts of each group.(4)Explore the influence of HuR on mice metabolism.The basal metabolic data of mice were monitored in real time by using the metabolic cage device.IPGTT and IPITT assays,liver oil red O staining,serum and liver lipid content assays were used to evaluate the glucose and lipid metabolism in mice.(5)Explore the influence of HuR on the lipolysis process.Give beta receptor agonist stimulation in vivo or in vitro,and collect serum or cell culture supernatant to detect the fatty acid and glycerol content.Western blot and q-PCR were used to detect the expression of lipolysis related markers.(6)Explore the regulatory effect of HuR on ATGL.The expression of HuR was detected by western blot after the deletion or overexpression of HuR in adipocytes.RNA immunoprecipitation assay confirmed whether HuR could bind to ATGL mRNA directly.The half-life experiment was conducted to observe whether HuR knockout affected the stability of ATGL mRNA.(7)Explore the correlation between HuR expression in clinical samples and individual body mass index(BMI).The abdominal subcutaneous adipose tissue of normal weight people and obese people were collected to analyze whether there was a correlation between HuR expression in adipose tissue and individual BMI.4.Results(1)HuRako mice were successfully prepared.In this study,HuRAKO mice were obtained using AdipoQ-cre/loxp system.The mice could survive normally without obvious difference in appearance from the control mice,and the birth rate was in line with Mendelian genetic law.In both white and brown adipose tissues,HuR protein and mRNA levels were significantly decreased,while their expression was unchanged in other tissues.The above conclusion was confirmed by immunohistochemical results.(2)HuR ablation exacerbated high-fat diet induced obesity and insulin resistance.HuRako mice showed more weight gain than the control group since the 6th week of high-fat feeding,and the BMD results suggested that the weight difference was mainly caused by the higher fat content in the knockout group than the control group.After 16 weeks of high fat feeding,the weight of epididymal fat and subcutaneous fat in HuRAK0 mice was significantly higher than that in the control group,while the weight of brown fat was not statistically different.The morphological results showed that the diameter of white fat and brown fat cells in HuRako mice was larger than that in the control group.After 16 weeks of high fat feeding,HuRAK0 mice showed glucose intolerance and insulin resistance.Under insulin stimulation,the phosphorylation level of Akt ser473 in the adipose tissue of HuRako mice was lower than that in the control group.Serum insulin levels increased significantly while adiponectin levels decreased.RT-PCR results showed increased expression of multiple genes associated with inflammation in white adipose tissue and liver.The immunofluroscence staining results showed more F4/80 expression in HuRAK0 adipose tissue.(3)HuR ablation exacerbated high-fat diet induced disturbance of lipid metabolism.HuRako mice developed more obvious hyperlipidemia under the condition of high fat feeding.The serum triglycerides,cholesterol and LDL-C were all higher than the control group.The results of metabolic cage showed that the 〇2 consumption and heat generation of HuRAKO mice were lower than those of the control group under the condition of non-strenuous exercise.The histomorphological and physicochemical results of the liver revealed that the liver morphology of HuRAKO mice was nonnal,while the lipid accumulation was significantly higher than that of the control group.(4)HuR knockout inhibited the lipolysis process.The protein and mRNA levels of lipolysis-related genes were decreased in the adipose tissue of HuRAKO mice After receiving the injection of P3 receptor agonist CL316243,the serum glycerol or fatty acid content of HuR^0 mice were lower than that of the control group.When the adipose tissue or adipocytes were cultured in vitro and stimulated by isoproterenol,the glycerol and fatty acids in the HuR knockout group were significantly lower than those in the control group,wheres HuR overexpression reversed the situation(5)HuR directly controls ATGL.The adipocytes were induced from the SVF components isolated from the epididymal fat of HuRAKO or control mice,and the expression of ATGL in the knockout group was lower than that in the control group.After the treatment of fat cells by the HuR specific inhibitor CMLD-2,ATGL expression was decreased.The results of RNA immunoprecipitation showed that HuR could bind to ATGL mRNA directly,and this binding ability was inhibited by CMLD-2.The half-life results indicated that the stability of ATGL mRNA was significantly inhibited after HuR knockout.(6)The expression of HuR in subcutaneous adipose tissue of obese people was lower than that of normal weight population.We found that compared to control group,the mRNA and protein levels of HuR and ATGL in epiWAT,ingWAT and BAT were significantly lower in obese model mice(C57BL/6J mice fed with high-fat diet for 12 weeks and leptin gene defect ob/ob mice).We collected human abdominal subcutaneous adipose tissue,and western blot results indicated that the expression of HuR and ATGL in adipose tissue of obese population was significantly lower than that of patients with normal weight.According to the standardized HuR expression and BMI data,the scatter plot was drawn,and the analysis showed that the expression level of HuR was negatively correlated with BMI.5.Conclusions(1)In this study,adipose specific HuR knockout(HuRAK0)mice were successfully constructed for the first time.Adipose HuR ablation imapied the lipolysis process and exacerbated high-fat diet induced obesity,insulin resistance and lipid metabolism disorder.(2)In obese people,the HuR expression level of adipose tissue was negatively correlated with BMI.(3)In terms of mechanism,HuR can directly bind with ATGL mRNA,improve the stability of ATGL mRNA,and promote the ATGL expression and lipolysis process.