The Study On The Role And Mechanism Of PTEN And Upstream Proteins In Pathological Cardiac Hypertrophy And Non-Alcoholic Fatty Liver Disease

1.BackgroundHeart failure(HF)is a group of syndromes characterized by abnormal function or structure of the heart,which leads to impaired ventricular ejection or filling,impaired organs and tissues metabolism and systemic or pulmonary circulation congestion.Heart failure is a leading cause of morbidity and mortality in modern society.According to the Atherosclerosis Risk in Communities Study of the US National Heart,Lung,and Blood Institute,the 30-day,1-year,and 5-year case fatality rates after hospitalization for heart failure were 10.4%,22%,and 42.3%.Pathological cardiac hypertrophy is a key risk factor for heart failure.It mainly includes cardiac geometric changes,extracellular matrix fibrosis and the re-expression of embryonic genes such as brain natriuretic peptide(BNP),atrial natriuretic peptide(ANP)and β-myosin heavy chain(β-MHC).At first,these adaptive compensatory mechanisms could maintain circulation.With the aggravation of primary diseases and the excessive activation of neurohumoral cytokines,pathological myocardial hypertrophy developed from compensatory to decompensated,and finally developed into heart failure.The main drugs used in clinic are β-blockers,aldosterone receptor antagonists and renin aldosterone system inhibitors.However,some patients cannot tolerate these drugs,and the disease of patients who can tolerate these drugs are still in progress.Therefore,it is of great clinical significance to continue to explore how to delay and reverse the progress of pathological myocardial hypertrophy.Multiple signaling pathways have been demonstrated to positively regulate protein synthesis and cardiac hypertrophy,including PTEN and PI3K/AKT signaling pathway,Ca2+/calmodulin dependent protein kinase signaling pathway,mitogen activated protein kinase(MAPK)signaling pathway,Wnt signaling pathway and JAK/STAT signaling pathway.PTEN and PI3K/AKT signaling pathway has a direct impact on the development of cardiac hypertrophy.PTEN(phosphatase and tensin homology deleted on chromosome 10)is a tumor suppressor gene which mutations exist in many malignant tumors such as glioblastoma,melanoma,breast cancer,endometrial cancer and prostate cancer.Cardiac specific PTEN knockout mice exhibited cardiac hypertrophy and decreased myocardial contractility.LKB1IP(liver kinase B1 interacting protein 1)was first discovered in 2001 as liver kinase B1(LKB1)binding protein,also known as STK11IP.At present,the literatures about LKB1IP were very few,and the role of LKB1IP in vivo has not been studied.Our previous study found that the expression of LKB1IP was significantly up-regulated in pathological cardiac hypertrophy,and the expression of LKB1IP was highly positively correlated with p-AKT,suggesting that LKB1IP may play an important role in the development of pathological cardiac hypertrophy.Taken together,we put forward the following scientific hypothesis:Neurohumoral factors such as isoproterenol(ISO)binded to G protein-coupled receptor on the membranes of cardiomyocytes and activate PI3K/AKT signaling pathway,which lead to the increased protein synthesis and pathological cardiac hypertrophy.At the same time,ISO stimulation could promote the binds of LKB1IP and PTEN.As a result,the phosphatase activity of PTEN was inhibited and the AKT signaling pathway was activated,which aggravated pathological cardiac hypertrophy.Therefore,LKB1IP knock out mice and neonatal rat cardiomyocytes(NRCMs)were used to explore the role of LKB1IP/PTEN pathway in pathological cardiac hypertrophy in vivo and in vitro.2.Objectives(1)Explore the expression of LKB1IP in myocardial tissue and cardiomyocytes challenged with pathological stimuli.(2)Explore the pathological effect of LKB1IP knockout on myocardial hypertrophy.(3)Explore the specific mechanism of LKB1IP to regulate PTEN activity.3.Methods3.1 Animal model constructionLKB1IP knockout mice(LKB1IP-/-)in a C57BL/6J background were purchased from the Jackson Laboratory.Mice of 8 to 10 weeks old were chanlleged with transverse aortic constriction(TAC)or subcutaneously injection of β-adrenoceptor(β-AR)agonist ISO(10 mg/kg per day,14 days)for 14 days.3.2 Cell modelsNRCMs were isolated from neonatal 3-day-old rats.(1)NRCMs were stimulated with ISO for 24 hours before extracting RNA and protein.(2)NRCMs were stimulated by ISO for 24h,48h and 72h before extracting total protein.(3)NRCMs were stimulated with ISO,PE and AngⅡ for 24 hours before extracting total protein.(4)After transfecting with LKB1IP overexpression adenovirus or LKB1IP siRNA,the NRCMs were stimulated with ISO for 24 hours and labeled with FITC labeled phalloidin.(5)CoIP:After transfecting with LKB1IP overexpression adenovirus and PTEN overexpression adenovirus at the same time,the NRCMs total protein was extracted to detect the binding of LKB1IP and PTEN.NRCMs were stimulated with ISO for 15 minutes.Then the total protein was extracted and the binding of LKB 1 IP to PTEN was detected.3.3 Human heart samplesHeart slices of patients with heart failure were obtained from patients with end-stage heart failure who received heart transplantation.Control tissue was obtained from healthy heart donors.The use of hearts samples from patients was approved by scientific research ethics committee of Qilu Hospital of Shandong University.3.4 Detection of cardiac function in miceEchocardiography and hemodynamic analyses were performed before mice were sacrificed,then the hearts were harvested to analyze the hypertrophic response.3.5 Histopathology and immunohistochemistryAfter the heart was removed,the middle part of the heart was fixed in 4%paraformaldehyde.After 24 hours,the heart was washed with running water for 2 hours.Then the heart was dehydrated with alcohol gradient and embedded in paraffin.The thickness of the section was 5 μM.After that,H&E staining,WGA staining,immunohistochemical staining and double immunofluorescence staining were performed.3.6 Western blot analysisThe protein of myocardial tissue and cells was extracted,separated by SDS-PAGE,and then transferred to PVDF membrane.After blocking in 5%skim milk,the imprinting was detected with LKB1IP,p-AKT,AKT,PTEN and GAPDH antibodies.After overnight at 4℃,the PVDF membrane was washed three times with TBST.The PVDF membrane was incubated with horseradish peroxidase binding secondary antibody and observed by chemiluminescence method.3.7 Quantitative PCR(qPCR)Total RNA was extracted from heart tissue or NRCMs with Trizol reagent.1 μg RNA was reverse transcribed into cDNA with the corresponding kit.SYBR was used for PCR amplification to detect the RNA levels of LKB1IP,ANP,BNP,β-MHC and GAPDH.3.8 Statistical analysisAll data are expressed as mean±SEM.Analyses involved use of SPSS v23(SPSS Inc.,Chicago,IL)and data passed normality and equal variance tests.Statistical comparisons between 2 groups involved Student’s t test and otherwise one-way ANOVA and Bonferroni post-tests.All statistical tests were two-tailed,and P<0.05 was considered statistically significant.4.Results4.1 LKB1IP expression was upregulated in the hypertrophic heart and cellsImmunohistochemistry revealed significantly greater expression of LKB1 IP in the human failing heart than in normal controls.Immunohistochemistry,Western blot and qPCR showed that the levels of LKB1IP were significantly elevated in hypertrophic mouse heart sections as compared with corresponding controls.Similarly,LKB1IP protein and mRNA levels were increased in hypertrophic cardiac cells,that is,NRCMs stimulated with ISO.Furthermore,the expression of LKB1IP was upregulated under pathological hypertrophic stimuli such as AngⅡ or PE for 24 hr.Collectively,these data demonstrate elevated LKB1IP expression under pathological cardiac hypertrophy.4.2 Establishment of LKB1IP knockout miceTo evaluate the hypertrophic effects of LKB 1 IP in vivo,we first used a mouse model with global knockout of LKB1IP(LKB1IP-/-).The absence of LKB1IP in the heart tissues was first confirmed by qPCR.LKB 1 IP expression in the main heart tissues was further detected by western blot analysis,thus indicating effective LKB1IP deletion in LKB1IP-/-mice.WT and LKB1IP-/-mice did not differ in body weight.The blood pressure of WT and LKB1IP-/-mice were measured.There were no significant differences between WT and LKB1IP-/-mice.To clarify the effect of LKB1IP knockout on LKB1 and its downstream AMP-activated protein kinase(AMPK),we assessed protein levels by western blot analysis,which showed no significant differences in LKB1,phosphorylated AMPKα(T172)and total AMPKa levels between WT and LKB1IP-/-mice.4.3 LKB1IP deficiency alleviates ISO-induced hypertrophy in vivoThe pathological cadiac hypertrophy model was induced by subcutaneous injection of isoprenaline(ISO),and the thickness and cardiac function of the ventricular wall were measured by the ultrasonic instrument.ISO-induced cardiac hypertrophy was significantly attenuated in LKB1IP-/-mice,as evidenced by lower IVSd,IVSs,LVPWd,LVPWs,ratios of HW/BW and HW/TL,the area of myocardial cells,myocardial fibrosis,and the expression of ANP and β-MHC.4.4 LKB1IP deficiency alleviates TAC-induced hypertrophy in vivoIn order to evaluate whether LKB1IP knockout could alleviate pressure overload-induced cardiac hypertrophy in vivo,TAC operation was performed.Compared with WT mice undergoing TAC operation,the pathological myocardial hypertrophy of LKB1IP-/-mice was significantly reduced.Therefore,LKB1IP knockout significantly reduced the pathological myocardial hypertrophy caused by TAC.4.5 LKB1IP Overexpression aggravated ISO induced cardiac hypertrophy in vitroISO treatment significantly increased the mean cross-sectional area of NRCMs and the expression of ANP,BNP and β-MHC,which was further enhanced by LKB1 IP overexpression.4.6 LKB1IP knockdown alleviated ISO induced cardiac hypertrophy in vitroISO treatment significantly increased the mean cross-sectional area of NRCMs and the expression of ANP,BNP and β-MHC,which was further alleviated by LKB1IP knockdown.4.7 LKB1IP promotes AKT phosphorylationIn the mouse models of cardiac hypertrophy,ISO or TAC significantly increased AKT phosphorylation at Thr 308 as compared with their controls in WT mice,which was attenuated in LKB1IP-/-mice.Consistently,ISO-induced AKT phosphorylation in NRCMs was enhanced with LKB1IP overexpression and suppressed with LKB1IP knockdown.4.8 LKB1IP directly combined with PTENTo investigate the way that LKB1IP regulates Akt phosphorylation,we detected whether LKB1IP could interact with PTEN,the negative regulator of PI3K/AKT signaling.NRCMs were infected with adenovirus expressing Flag-tagged LKB1IP and HA-tagged PTEN followed by immunoprecipitation,which showed that LKB1IP could bind to PTEN.Meanwhile,we further verified the interaction between LKB1IP and PTEN by immunofluorescence detection in pathological hypertrophic myocardium.4.9 LKB1IP promoted the phosphorylation of AKT by inhibiting PTEN phosphatase activityThe results of PTEN phosphatase activity test showed that overexpression of LKB1IP significantly reduced the activity of PTEN phosphatase.To further confirm LKB1IP regulates AKT phosphorylation by PTEN,NRCMS were transfected with LKB1IP and PTEN adenovirus,and stimulated with ISO for 24 hours.Western blot showed that LKB1IP overexpression enhanced AKT phosphorylation.The effect of LKB1IP on AKT phosphorylation was inhibited when PTEN was overexpressed.Taken together,these data demonstrated that LKB1IP may positively activate AKT signaling by directly targeting PTEN and subsequently inhibit its phosphatase activity.5.Conclusions(1)LKB1IP was up-regulated in pathological hypertrophy cardiac tissues and cells.(2)Knockout of LKB1IP could alleviate the ISO and TAC induced pathological cardiac hypertrophy.(3)LKB IIP overexpression aggravated ISO-induced cardiac hypertrophy,while LKB1IP knockdown attenuated ISO-induced cardiac hypertrophy in vitro.(4)LKB1IP inhibited PTEN phosphatase activity by directly binding to PTEN.1.BackgroundDuring the past decades,with the rapid development of social economy,the lifestyle and diet structure of the people have changed significantly.The incidence rate of non-alcoholic fatty liver disease(NAFLD)has been increasing year by year,and it has become the main reason of chronic liver disease in the world.With the increase of NAFLD incidence rate,the number of patients with cirrhosis and end-stage liver disease caused by NAFLD has increased dramatically.There was a prospective study that NAFLD will become the leading cause of end-stage liver disease and liver transplantation in the next ten years,which will bring serious burden to the social economy.NAFLD is a pathological process that encompasses a spectrum of liver metabolic disorders starting with non-inflammatory liver steatosis,defined as greater than 5%triglyceride(TG)accumulation in hepatocytes,which then leads to inflammation,fibrosis and cirrhosis.At present,there are many theories about the mechanism of NAFLD,and the"second strike" theory is the most cited one.Although this view has great reference value,it remains controversial.However,the pathogenic factors of NAFL and NASH have one thing in common:the ability of liver dealing with major energy metabolic substrates(carbohydrates and fatty acids)is decreased,leading to lipid accumulation in hepatocytes.Therefore,elucidation of energy metabolism in hepatocytes is essential for NAFLD.The phosphatase and tensin homolog deleted on the chromosome 10(PTEN)/phosphoinositide 3 kinase(PI3K)/AKT pathway has an important role because it acts downstream of the insulin receptor.PTEN is a bispecific phosphoinositide and protein phosphatase that dephosphorylates phosphatidylinositol-3,4,5-trisphosphate(PIP3),terminating signaling downstream of phosphatidylinositol 3-kinase(PI3K),then decreases AKT activity.Liver specific PTEN knockout could promote the development of NAFLD and tumor,but improve glucose tolerance.PTEN is an important regulator of hepatic adipogenesis,glucose metabolism and homeostasis.Therefore,understanding the expression and activity regulation of PTEN is very important for exploring the mechanism of NAFLD.However,the upstream regulatory pathway of PTEN has not been fully elucidated.Human antigen R(HuR)is an RNA binding protein and selectively binds to adenylate uridylate-rich elements(AREs),which are usually found in the 3’untranslated regions(UTR)of its targets via its RNA recognition motifs.Global HuR-deficient mice show embryonic lethality due to extraembryonic placental defects.In addition,HuR regulates cellular cholesterol homeostasis through modulating ATP-binding cassette transporter A1(ABCA1)expression.However,the specific role of HuR in hepatic steatosis related glucose metabolism has not been explicitly explored.At the same time,we found that the 3’-UTR of mouse PTEN mRNA contains ARE sites,suggesting that HuR may affect the development of NAFLD by affecting the expression of PTEN.Therefore,this study proposed the following scientific hypothesis:HuR could bind to the 3’ UTR of Pten mRNA and increased its stability and translation,thus leading to changes in liver glucose and lipid metabolism and affecting the metabolic balance of the body.2.Objectives(1)Explore the expression of HuR and PTEN induced by high-fat diet.(2)Explore the metabolic phenotype of liver specific HuR knockout mice fed with high-fat diet.(3)Explore the molecular mechanism of HuR regulating lipid metabolism.3.Methods3.1 Experimental model constructionThe Alb-Cre/loxP system was used to construct the liver-specific HuR-knockout(HuRLKO)mice.The male mice aged 8 weeks were given normal diet or high-fat diet for 24 weeks.3.2 Cell experiments(1)The primary hepatocytes were extracted and treated with BSA and PO.The protein and RNA were extracted and the expression level of HuR was detected.(2)L02 cells were treated with BSA and PO.The RNA was extracted and the expression level of HuR was detected.(3)The primary hepatocytes of CTR and HuRLKO mice were extracted and treated with BSA and PO.The lipid deposition in hepatocytes was detected by oil red O staining.(4)The primary hepatocytes were extracted and transfected with HuR overexpression adenovirus.The cells were treated with BSA and PO.The lipid deposition in hepatocytes was detected by oil red O staining.(5)The primary hepatocytes of CTR and HuRLKO mice were extracted and transfected with PTEN overexpression lentivirus.The cells were treated with BSA and PO.The lipid deposition in hepatocytes was detected by oil red O staining.(6)RNA binding protein immunoprecipitation(RIP)assay was used to detect the direct binding of HuR to Pten mRNA.(7)The effect of HuR on the stability of Pten mRNA was detected by half-life assay.3.3 Growth curveThe weight of CTR and HuRLKO mice was recorded once a week for 24 weeks from high-fat feeding.3.4 Glucose tolerance(GTT)and insulin tolerance(ITT)For GTT,mice were fasted for 14-16 h and then administered an intraperitoneal(i.p.)injection of glucose(0.75 g/kg body weight).For ITT,mice were fasted for 4-6 h and then administered an i.p.injection of insulin(1.5 U/kg body weight).At 0,15,30,60,90,and 120 min after injection,blood glucose concentrations were measured.3.5 Liver histopathologyParaffin sections were stained with H&E,PAS and immunohistochemistry.Frozen sections were stained with oil red O to detect liver lipid deposition.3.6 Western blotThe protein of myocardial tissue and cells was extracted,separated by SDS-PAGE,and then transferred to PVDF membrane.After blocking in 5%skim milk,the imprinting was detected with HuR,AKT,phosphorylated AKT(Ser473),PTEN,TUBA1A,and GAPDH antibodies.After overnight at 4℃,the PVDF membrane was washed three times with TBST.The PVDF membrane was incubated with horseradish peroxidase binding secondary antibody and observed by chemiluminescence method.3.7 Quantitative PCR(qPCR)Total RNA was extracted from liver tissue or cells with Trizol reagent.1μg RNA was reverse transcribed into cDNA with the corresponding kit.SYBR was used for PCR amplification to detect the RNA level of corresponding genes.3.8 Statistical analysisAll data are expressed as mean ± SEM and passed normality and equal variance tests.SPSS v23(SPSS Inc.,Chicago,IL,USA)was used for all analyses.The comparison of two groups was determined using Student’s t-tests and the comparison of multiple groups by one-way ANOVA with Bonferroni post-hoc tests.All statistical tests were two-tailed,and P<0.05 was considered statistically significant.4.Results4.1 HuR expression was down regulated under high fat dietFirst,C57BL/6J mice were given a high-fat diet for 24 weeks to establish a nonalcoholic fatty liver disease(NAFLD)model.qPCR,Western blot analysis,and immunohistochemistry showed that HuR levels were significantly decreased in the livers of mice fed a HFD as compared with the controls.To detect HuR expression in the early stage of hepatic steatosis,C57BL/6J mice were fed a HFD for 2,4 and 6 weeks.The levels of HuR mRNA and protein were significantly downregulated at the 6th week.HuR may play a potential role in the development of NAFLD.4.2 HuR expression was down regulated in hepatocytes stimulated by POIn L02 cells and primary hepatocytes stressed with palmitic acid and oleic acid(PO),HuR protein levels were also downregulated.However,HuR mRNA levels were not significantly regulated in primary hepatocytes stimulated with PO,which suggests that the downregulation of HuR protein might be a post-translational regulatory mechanism.Downregulation of HuR under PO stimulation was prevented by CQ treatment,suggesting that PO induced HuR reduction via lysosomal degradation in hepatocytes.4.3 HuRLKO mice were successfully constructed.In order to explore the role of HuR in NAFLD,we constructed liver specific HuR knockout mice.The results of qPCR,Western blot and immunohistochemistry showed that HuR was completely knocked out in liver,but not in heart,muscle and spleen.4.4 Liver-specific HuR deletion aggravated HFD-induced hepatic steatosis.HuRLKO and CTR mice were given high-fat diet or normal diet for 24 weeks respectively.The weight of mice was monitored weekly and the growth curve was drawn.It was found that there was no significant difference in the weight of HuRLKO and CTR mice in the normal diet group.The weight of the high-fat diet group was significantly higher than that of the normal diet group from the 4th week.At the 24th week,the liver weight/body weight ratio and liver lipid level of the high-fat diet group were significantly higher than that of the normal diet group,indicating that the model of NAFLD was successfully established.From the 12th week,the weight gain of HuRLKO mice was significantly lower than that of CTR mice.The liver weight/body weight ratio of HuRLKO mice was significantly higher than that of CTR mice.In order to find out the causes of the changes of liver weight and body weight,we analyzed the lipid content of liver,oil red O staining of frozen section,H&E staining of paraffin section and qPCR analysis of lipid metabolism related factors in liver.The results showed that HuRLKO significantly increased liver lipid deposition induced by high-fat diet.4.5 Liver-specific HuR deletion aggravated liver function damage induced by HFDThe serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were increased in HuRLKO mice compared with controls fed with HFD,indicating that liver knockout of HuR could aggravate the liver function damage induced by high-fat diet.4.6 Liver-specific HuR deletion improved insulin resistance induced by HFDFasting glucose and insulin levels were significantly decreased in HuRLKO mice compared with controls fed with HFD.Glucose tolerance tests(GTT)and insulin tolerance tests(ITT)also indicated that HuRLKO mice exhibited improved glucose tolerance and insulin resistance when fed HFD.PAS staining showed that liver glycogen content was not affected by HuR knockout.Furthermore,HuR deletion increased the mRNA levels of glycolysis markers,but decreased the expression of gluconeogenesis markers.HuRLKO mice exhibited improved glucose tolerance and insulin resistance when fed HFD was confirmed by insulin-stimulated p-AKT Ser473 analysis in liver,skeletal muscle,and adipose tissue.Taken together,liver-specific HuR deletion improved the HFD-impaired glucose tolerance.4.7 HuR attenuates lipid deposition in hepatocytesTo determine the role of HuR in lipid accumulation in vitro,primary hepatocytes were infected with adenovirus expressing HuR followed by PO treatment.PO-induced lipid accumulation was attenuated by HuR overexpression.Consistently,primary hepatocytes from HuR-knockout mice showed aggravated PO-induced lipid accumulation compared to the controls.In primary hepatocytes stimulated with PO,HuR knockout significantly increased lipogenesis,fatty acid uptake,inflammation,and decreased fatty acid β-oxidation.4.8 HuR reduces the phosphorylation of AKT in hepatocytesConsistent with the in vivo results,insulin-induced AKT phosphorylation in primary hepatocytes was suppressed by HuR overexpression and enhanced with HuR deficiency.Thus,HuR regulates lipid metabolism in hepatocytes in vitro.4.9 PTEN expression was downregulated in fatty liverWestern blot results showed that the expression of PTEN and HuR in HFD group decreased significantly.Compared with the control group,the expression of PTEN in the primary hepatocytes of CTR mice was also significantly decreased in vitro.The results showed that the expression of PTEN and HuR decreased simultaneously in fatty liver.4.10 HuR regulates PTEN expressionThe results of qPCR and Western blot showed that HuRLKO significantly decreased the expression of PTEN in liver tissue.qPCR results showed that HuR deficiency reduced the level of mature Pten mRNA but did not affect its pre-mRNA level.This suggests that HuR binds to PTEN mature mRNA rather than pre mRNA.Western blot showed that HuR overexpression could significantly increase the protein level of PTEN.In conclusion,HuR could regulate the expression of PTEN4.11 HuR binds with Pten mRNA and increases its stabilityWestern blot results showed that the expression of PTEN was significantly decreased in the CMLD-2(the inhibitor of HuR)group.The results of RNA immunoprecipitation indicated that HuR could directly bind to Pten mRNA.The results of half-life assay showed that the half-life of AdHuR group was 12 hours,which was significantly longer than that of AdGFP group(8 hours),indicating that overexpression of HuR can significantly increase the half-life of Pten mRNA.Taken together,HuR binds to Pten mRNA and regulates its stability.4.12 HuR regulates hepatic steatosis and glucose metabolism through PTENTo examine whether HuR regulates hepatocyte steatosis through PTEN,CTR and HuRLKO mice were injected with the lentivirus encoding LacZ(Lenti-LacZ)or PTEN(Lenti-PTEN)at week eight of the HFD.PTEN protein levels were significantly increased in the Lenti-PTEN groups.The results of growth curve showed that the weight of HuRLKO mice in PTEN overexpression group increased significantly compared with that in control group at the 16th week.The ratio of liver weight to body weight of HuRLKO mice injected with Lenti-PTEN was significantly lower than that of HuRLKO mice injected with Lenti-LacZ.The levels of TG,NEFA and TC in liver tissue of HuRLKO mice injected with Lenti-PTEN were significantly lower than those of HuRLKO mice injected with Lenti-LacZ.H&E and oil red O staining showed that balloon like degeneration and red lipid droplets deposition in liver tissue of HuRLKO mice injected with Lenti-PTEN were significantly less than those injected with Lenti-LacZ.The results of qPCR showed that the marker of lipogenesis,fatty acid uptake and the synthesis of cholesterol in PTEN overexpression group were significantly lower than those in control group,and the oxidation of fatty acid β was significantly increased.In conclusion,overexpression of PTEN can significantly reduce the lipid deposition induced by HuR knockout.The results of fasting blood glucose and insulin showed that the fasting blood glucose and insulin levels of HuRLKO mice injected with Lenti-PTEN were significantly higher than those of HuRLKO mice injected with Lenti-LacZ,which indicated that PTEN overexpression significantly reversed the increase of blood glucose and insulin resistance induced by liver knockout of HuR.The results of glucose tolerance(GTT)and insulin tolerance(ITT)showed that the glucose tolerance of HuRLKO mice injected with Lenti-PTEN was worse than that of HuRLKO mice injected with Lenti-LacZ.And the response to insulin stimulation of HuRLKO mice injected with Lenti-PTEN was weak than HuRLKO mice injected with Lenti-LacZ.qPCR result showed that overexpression of PTEN could significantly reduce the expression of glycolysis related genes and significantly increase the expression of gluconeogenesis related genes.These results suggest that overexpression of PTEN can significantly reverse the high insulin sensitivity induced by HuR knockout.The result of AST and ALT showed that overexpression of PTEN had no significant effect on liver function.In conclusion,overexpression of PTEN can reverse the changes of glucose and lipid metabolism induced by HuR knockout.5.Conclusions(1)The expression of HuR and PTEN were down regulated in steatosis liver and PO stimulated hepatocytes.(2)HuR knockout aggravates hepatic steatosis induced by HFD,but improves insulin resistance induced by HFD.(3)HuR could bind to the 3’-UTR of Pten mRNA and increase its stability and translation.