Bispecific Phosphatase 14 Maintains Glucose And Lipid Homeostasis, Inhibits The Mechanism Of Inflammatory Responses, And Modulates Mitochondrial Unfolded Protein Responses On Myocardial Ischemia/reperfusion Injury

Background and objective:Nonalcoholic fatty liver disease is a manifestation form of metabolic syndrome in the liver,as the most common chronic liver disease,it affects about one third of the population worldwide.Insulin resistance and inflammatory response are not only the pathological characteristics of NAFLD,but also important links in the process of disease progression,they promote each other and then accelerate the progression of NAFLD.NAFLD is not only a progressive disease confined to the liver,but also affects other systems of the body,increasing the risk of cardiovascular diseases,cancer and type Ⅱ diabetes,cardiovascular diseases are the leading cause of death in NAFLD patients.Nevertheless,the insufficient understanding of the pathophysiology of the disease itself has greatly limited the advancement of NAFLD clinical treatment,leading to poor long-term prognosis of NAFLD liver disease and extrahepatic manifestations.Therefore,it is urgent to further explore the mechanism underlying the development and progression of the disease,and to identify key regulators,thus promoting the optimization of NAFLD treatment.Methods:Hepatocyte-specific DUSP14 transgenic(DUSP14-HTG)or knockout(DUSP14-HKO)C57BL/6J mice were constructed(8wks),and then subjected to normal chow(NC)or high-fat diet(HFD)for 12wks.Fasting blood glucose and insulin levels were measured every 2 weeks until 12 weeks.Glucose or insulin tolerance tests were performed at 10 weeks.H&E and oil red O staining were performed to estimate hepatic steatosis.Hepatic glycogen and lipids were measure by commercial kits.Transcription levels of key enzymes involved in glucolipid metabolism,as well as inflammatory mediators were detected by rtPCR.The expression and phosphorylation of molecules in insulin signaling,MAPK and NF-κB pathway were analyzed by Western Blot.Co-IP,GST and mapping experiments were conducted to explore mechanisms;8wks old leptin-deficient mice were injected with recombinant adenovirus for 4 weeks,and then the therapeutic effects of DUSP14 were examined.To clarify the role of TAK1 siganling pathway in DUSP14-mediated protection,TAK1 inhibitor was administrated in 8wks old DUSP14-HKO mice.Results:Reduced expression of DUSP14 in liver samples from NAFLD patients was further confirmed in the liver tissues of HFD-fed mice or ob/ob mice.After 12 weeks of HFD feeding,compared with control mice,the pathological manifestations of NAFLD were significantly alleviated in DUSP14-HTG mice,showing improved glucose and lipid metabolism and insulin sensitivity,reduced steatosis and inflammation,whereas DUSP14-HKO mice developed exacerbated insulin resistance,glucolipid metabolic disorders,liver steatosis and inflammation.In addition,under NC feeding,DUSP14 overexpression or knockout also affected aforementioned metabolic and inflammatory phenotypes.DUSP14 overexpression significantly improved glucolipid metabolic disorders and liver steatosis in ob/ob mice.Mechanistical study showed that DUSP14 directly binds to and dephosphorylates TAK1,resulting in reduced activation of TAK1 and its downstream pathway and molecules JNK,p38,and NF-κB,and thus improving the pathological processes of NAFLD.Disturbing the phosphatase activity site or TAK1 binding domain of DUSP14 abolished its regulatory effect on lipid metabolism.TAK1 inhibitor effectively attenuated the deterioration of glucolipid metabolic phenotype in HFD-fed DUSP14-HKO mice,further confirming the necessity of TAK1 for DUSP14-mediated protective effects.Conclusion:DUSP14 plays an important role in maintaining hepatic glucose and lipid metabolic homeostasis and suppressing inflammation,a novel regulatory function relies on its direct binding and dephosphorylation of TAK1.Background and objective:Mitochondria play a key role in myocardial ischemia/reperfusion(I/R),during which a series of pathological events occur in mitochondria,including mitochondrial electron transport chain complex damage,abnormal energy metabolism,excessive reactive oxygen species,calcium dysregulation and opening of the mitochondrial permeability transition pores,eventually lead to cell death and myocardial injury.Mitochondrial unfolded protein response(UPRmt)is a stress response.A variety of stress stimuli can activate the transcription of nuclear-encoded mitochondrial chaperone proteins and protease genes,to reestablish mitochondrial proteostasis and function.UPRmt has been proved to improve cardiac structural and functional remodeling under pressure overload stress.However,the role of UPRmt in myocardial I/R has not been specified.The purpose of this study is to explore whether UPRmt can exert regulatory effects on different stages of myocardial I/R injury,and further elucidate the underlying mechanism.Methods:Myocardial I/R or sham sugeries were performed on 12-14 week old,male C57BL/6J wild-type mice.Neonatal rat ventricular cardiomyocytes(NRVMs)and H9C2 cells received simulated I/R(sI/R)/simulated ischemia and normoxia treatment,qPCR and WB detected UPRmt markers expressions.Mice treated with oligomycin(500ug/kg)or vehicle(intraperitoneal injection)12 hours before surgeries to activate UPRmt,followed by I/R surgeries;TTC&Evans blue staining evaluated myocardial infarction and ischemic injury.NRVMs and H9C2 cells,treated with oligomycin/doxycycline or DMSO,were subjected to sI/R or simulated ischemia;cell death was estimated by lactate dehydrogenase(LDH)assay and Annexin V-FITC and PI staining followed by flow cytometry for further validation.NRVMs were transfected with siRNA to specifically knockdown the expression of Hsp60 and ClpP or ATF5,cells were then subjected to sI/R or simulated ischemia and control operations;cell death was evaluated by LDH assay and flow cytometry.Results:WB and qPCR results showed that oligomycin(500ug/kg)12h treatment induced maximized activation of UPRmt in murine heart tissues,while oligomycin(NRVMs 2.5ug/ml;H9C2 cells 10ug/ml)or doxycycline(NRVMs 10 ug/ml;H9C2 cells 50 ug/ml)treatment for 12h or 4h is an effective method to induce maximized UPRmt activation in cultured cardiomyocytes.Short-term or long-term I/R injury did not cause any changes in UPRmt level in murine hearts;similarly,sI/R stimulation did not affect the expression levels of UPRmt markers in NRVMs and H9C2 cells.Quantitative results of TTC&Evans blue staining showed that oligomycin-induced UPRmt activation did not affect I/R-induced myocardial ischemia and infarction.At the cellular level,LDH assay and flow cytometry results showed that oligomycin/doxycycline induced UPRmt activation had no influence on cardiomyocyte death caused by sI/R.In addition,down-regulation of important UPRmt functional effectors gene expression did’t not affect sI/R-induced cell death.When the injury was only confined to ischemia stage,the expression levels of UPRmt markers in NRVMs and H9C2 cells were remarkably decreased,and this reduction became more pronounced with the prolongation of ischemia time.Oligomycin-induced UPRmt activation exerted a significant protective effect on simulated ischemia-induced cell death.However,knockdown of Hsp60 and ClpP did not affect oligomycin-induced cellular protection.Addionally,ischemia and hypoxia induced cardiomyocyte death was not affected by down-regulation of Hsp60 and ClpP/ATF5 gene expressions.Conclusion:Myocardial I/R injury did not cause any obvious changes in the activation level of UPRmt.Modulation of UPRmt level exerted no significant regulatory effect on myocardial I/R injury.Simulated ischemia suppressed UPRmt activity in cultured cardiomyocytes,while oligomycin-induced UPRmt activation significantly alleviated cardiomyocyte death caused by simulated ischemic injury.