Therapeutic Effect Of Sirtuin 3 On Ameliorating Nonalcoholic Fatty Liver Disease:the Role Of The ERK-CREB Pathway And Bnip3-mediated Mitophagy

Background:Nonalcoholic fatty liver disease(NAFLD)is now the most prevalent form of chronic liver disease.Pathogenically increased free fatty acid levels have been associated with the development and progression of NAFLD.Excessive free fatty acids enhance the oxidation of fatty acids in the liver producing excessive superoxide.Subsequently uncontrolled oxidative stress disrupts mitochondrial function.Damaged mitochondria fail to produce enough ATP to ensure normal hepatocyte function,resulting in disorders in lipid metabolism regulation,blood glucose management and protein synthesis and output.Poorly structured mitochondria could liberate proapoptotic factors into the cytoplasm/nucleus,initiating mitochondria-dependent hepatocyte death.Accordingly,understanding the mechanisms by which hyperlipidaemia impose damage on mitochondria is significant for designing a novel approach to treat fatty liver disease.Sirtuin 3,a type of NAD dependent deacetylase expressed mainly in mitochondria,has recently been reported to have multiple effects on mitochondrial protection in response to several types of stress,such as oxidative stress,fatty acid composition and myocardial infarction.The effect of Sirtuin 3 on Nonalcoholic fatty liver disease is not clear.Although mitochondria are susceptible to hyperlipidaemia-trigger damage,mitochondria can employ lysosomes to remove damaged mitochondrial fragments,which is termed mitophagy.Mitophagic activity in liver cells is primarily regulated by Bnip3.So we are wondering whether Bnip3-mediated mitophagy is regulated by Sirtuin 3 in the setting of fatty liver disease.Aim:Our study is to explore the role of Sirtuin 3 in nonalcoholic fatty liver disease with a focus on mitophagy and the ERK-CREB pathway.Methods:In vivo,WT mice and Sirtuin 3 transgenic mice were selected for the high fat group and low fat group to establish the NAFLD animal model(WT&Sirt3-TG)and control(WT&Sirt3-TG).ELISA,Immunohistochemistry,Western Blot were used to evaluate the body weight,liver pathology,glucose and lipid metabolism,liver inflammation and fibrosis between HFD(WT&Sirt3-TG)and LFD(WT&Sirt3-TG).Besides,primary hepatocytes were isolated from WT mice and Sirt3 transgenic mice and underwent Palmitic acid injury.In cells experiments,MTT assay,TUNEL assay,ELSIA,mitochondrial extraction,western blot and immunofluorescence colocalization were used to evaluate cell viability,cell apoptosis,mitochondrial energy metabolism,mitochondrial ROS overproduction,mitochondrial Cyt-c leakage,and mitochondrial apoptotic pathway activation between WT and Sirt3 overexpressing cells when treated with PA.Bnip3 siRNA was used in PA treated Sirt3 expressing cells to analyze the relationship between Bnip3 and Sirtuin 3.PD98059 was added to PA treated Sirt3 expressing cells to observe the role of ERK-CREB in the mechanism in which Sirtuin 3 regulates Bnip3 mediated mitochondrial autophagy.Results:1.Compared to LFD,HFD increased the body weights and liver weights(P<0.05).The levels of fasting blood glucose,c-peptide,HbA1c and glucagon were upregulated in HFD-treated WT mice(P<0.05).The levels of triglycerides,total cholesterol,leptin,adiponectin,alamine transaminase and aspartate transaminase were increased in HFD-treated WT mice(P<0.05).These were attenuated in Sirt3-TG mice(P<0.05).HFD treatment increased the size of hepatocytes(P<0.05),as evaluated via H&E staining.However,this alteration was reversed by Sirt3 overexpression(P<0.05).HFD treatment promotes lipid accumulation in the liver,and this effect was reversed by Sirt3 overexpression(P<0.05).Additionally,liver fibrosis was measured via Sirius Red staining,and the results indicated that HFD-triggered liver fibrosis was attenuated in Sirt3-TG mice(P<0.05).This finding was further verified via analyzing fibrosis parameters using Western Blotting Collagen ?/?/? were increased in response to HFD treatment(P<0.05)and were reduced in Sirt3-TG mice(P<0.05).Furthermore,the pathways related to liver fibrosis,such as TGF ? and MMP 9 were also upregulated in HFD-treated mice and were downregulated in Sirt3-TG mice(P<0.05).The liver inflammatory response was also evaluated.Through ELISA and Western Blotting analysis we demonstrated that inflammatory factors such as TNF a,IL-6 and MCP 1 were increased in HFD-fed mice(P<0.05)and were reduced to near-normal levels in Sirt3-TG mice(P<0.05).2.Primary hepatocytes were isolated from WT and Sirt3-TG mice and were then treated with palmitic acid(PA,75?mol/L for 24h)in vitro.Subsequently,cellular viability was measured via MTT assays.PA treatment significantly reduced hepatocyte viability(P<0.05)and this effect was reversed by Sirt3 overexpression(P<0.05).To understand the mechanism by which PA reduced cellular viability,we performed TUNEL assays to investigate the apoptotic index of hepatocytes in response to PA treatment.The number of TUNEL-positive cells was increased in the PA-loaded cells(P<0.05)and was reduced to near-normal levels in the Sirt3-overexpressing cells(P<0.05).3.ROS was increased by PA treatment(P<0.05)as determined using flow cytometry analysis.The concentrations of antioxidants such as GSH,SOD and GPx were downregulated in PA-treated cells(P<0.05).However,Sirt3 overexpression repressed the ROS generation and reversed antioxidants to near-normal levels(P<0.05).PA-suppressed ATP production was reversed by Sirt3 overexpression(P<0.05).The mitochondrial potential was reduced in response to PA treatment(P<0.05)and was reversed to near-normal levels in the Sirt3-overexpressing cells(P<0.05).At the molecular levels,mitochondrial potential was stabilized via the mitochondrial respiratory complex.However,PA treatment reduced the expression of the mitochondrial respiratory complex(P<0.05),and this effect was reversed by Sirt3 overexpression(P<0.05).Mitochondrial potential reduction promotes pro-apoptotic factor leakage from the mitochondria into the cytoplasm/nucleus.PA treatment promotes the Cyt-c leakage into the cytoplasm/nucleus when compared to that of the control group(P<0.05).However,the PA-triggered Cyt-c leakage was strongly inhibited by Sirt3 overexpression(P<0.05).As a consequence of Cyt-c liberation,pro-apoptotic proteins such as Caspase3,Caspase9,and Bax were increased in PA-treated cell(P<0.05).By comparison,the contents of anti-apoptotic proteins such as Bcl-2 were correspondingly downregulated(P<0.05).4.Compared to the control group,PA treatment reduced the expression of LC3II and increased the expression of LC3I(P<0.05),indicative of the impairment of autophagosomes.Subsequently,mitochondria were isolated,and the mitochondrial LC3?(mito-LC3?)was measured.Similarly,the content of mito-LC3? was also repressed by PA when compared to that of the control group(P<0.05).Additionally,the mitophagy parameters,such as Beclinl and Atg5,were also downregulated in response to PA treatment(P<0.05).Altogether,these data indicate that mitophagy is inactivated by PA in hepatocytes(P<0.05).Interestingly,regaining Sirt3 reversed the expression of mito-LC3?,Atg5 and Beclin1(P<0.05),suggestive of mitophagy activation in response to Sirt3 overexpression.Bnip3 expression was downregulated by PA treatment(P<0.05)and was upregulated in the Sirt3-overexpressing cells(P<0.05).Numerous mitochondria were co-located with lysosomes in the control group;however,PA treatment disrupted the fusion between mitochondria and lysosomes(P<0.05).Interestingly,overexpression of Sirt3 re-activated the interaction between mitochondria and lysosomes(P<0.05),and this effect was abrogated by deleting Bnip3(P<0.05),indicating that Sirt3 maintained mitophagy via Bnip3.5.Loss of Bnip3 negated the protective effect of Sirt3 on mitochondrial potential stabilization(P<0.05).In addition,Sirt3-inhibited Cyt-c liberation was also reversed by Bnip3 deletion in hepatocytes treated with PA(P<0.05).Moreover,the concentration of antioxidants was increased by Sirt3 overexpression(P<0.05)and was reduced in response to Bnip3 deletion(P<0.05).These data indicated that Sirt3 overexpression maintains mitochondrial function.Caspase 9,the key marker of mitochondrial apoptosis,was activated by PA treatment(P<0.05)and was inactivated by Sirt3 overexpression(P<0.05).However,the loss of Bnip3 increased the Caspase 9 activity despite the overexpression of Sirt3(P<0.05).Moreover,the content of LDH was increased in PA-loaded hepatocytes(P<0.05),indicative of the breakage of cell membranes due to cell death.However,Sirt3 overexpression reduced the LDH release(P<0.05),and this effect was dependent on Bnip3 expression(P<0.05).Besides,to observe the role of Bnip3-related mitophagy in lipid accumulation,we performed Oil Red O staining in hepatocyte.Sirt3 overexpression inhibited PA-mediated lipid accumulation(P<0.05),this effect of which was negated by Bnip3 deletion(P<0.05).6.ERK-CREB pathway was inactivated by PA treatment,as evidenced by decreased phosphorylated ERK and CREB expression(P<0.05).Interestingly,Sirt3 overexpression reversed the levels of p-ERK content and p-CREB(P<0.05).PD98059,an inhibitor of the ERK signaling pathway,not only inhibited Sirt3-mediated ERK and CREB activation but also repressed Sirt3-induced Bnip3 upregulation(P<0.05),indicating that the ERK-CREB pathway is responsible for Sirt3-modulated Bnip3 elevation.This finding was further validated via immunofluorescence assays.The fluorescence intensities of p-ERK and Bnip3 were decreased in PA-treated hepatocytes(P<0.05)and were reversed to near-normal levels in Sirt3-overexpressed cells(P<0.05).However,blockade of the ERK pathway significantly repressed Bnip3 expression despite the overexpression of Sirt3(P<0.05).Compared to the control,PA repressed the cooperation between mitochondria and lysosomes(P<0.05),and this effect was reversed by Sirt3 overexpression(P<0.05).Inhibition of the ERK-CREB pathway abrogated the promotive effect of Sirt3 on mitophagy activation.7.TUNEL assays demonstrated that the number of TUNEL-positive hepatocytes was increased in PA-treated cells(P<0.05)and was reduced in response to Sirt3 overexpression(P<0.05).However,blockade of the ERK-CREB signaling pathway re-elevated the ratio of TUNEL-positive cells despite overexpression of Sirt3(P<0.05).Similarly,the inflammatory factors,such as TNF a,IL 6 and MCP1,were also upregulated in PA-treated cells and were decreased to near-normal levels in response to Sirt3 overexpression in the ERK-CREB signaling pathway activation-dependent manner(P<0.05).PA-repressed ATP production was also reversed by Sirt3 overexpression via activating the ERK-CREB axis(P<0.05).In addition,PA-triggered ROS production was also reduced by Sirt3,and this effect was nullified by inhibiting the ERK-CREB signaling pathway(P<0.05).Conclusions:1.Gain-of-function assays of Sirt3 in vitro and in vivo confirmed that Sirt3 played a protective role against fatty liver disease.2.Sirt3 overexpression maintained mitochondrial function,reduced mitochondria oxidative stress,sustained mitochondrial energy metabolism,stabilized mitochondrial potential,repressed mitochondrial pro-apoptotic factor liberation,and blocked mitochondrial apoptosis activation3.Sirt3 activates the ERK-CREB signaling pathway,and the latter upregulates Bnip3-mediated mitophagy,thereby attenuating mitochondrial damage and inhibiting mitochondria-dependent hepatocyte apoptosis.4.High-fat-mediated liver damage is associated with Sirt3 downregulation,which is followed by ERK-CREB pathway inactivation and Bnip3-mediated inhibition of mitophagy,causing hepatocytes to undergo mitochondria-dependent cell death.