Increased Expression Of Cyclophilin D Induces Hepatic Steatosis Via Ca²⁺/p38 MAPK/IRE1?/SREBP-1c Pathway

Background:Non-alcoholic fatty liver disease(NAFLD)is one of the most common chronic liver diseases in the world.The underlying mechanism for the development and progression of NAFLD is complex and multifactorial.Fat accumulation in the liver(steatosis)is thought to be the early stage of NAFLD.Evidence has shown that triglyceride(TG)de novo lipogenesis is a prominent abnormality in NAFLD and the key event that leads to massive steatosis.Studies have raised the possibility that NAFLD might be a mitochondrial disease but it is poorly understood how exactly mitochondria function in the initial pathogenesis of steatosis.The opening of the mitochondrial permeability transition pore(mPTP)in the physiological state has a central role in cell metabolism,but excessive opening will lead to mitochondrial stress,which is characterized by impaired mitochondrial respiratory chain function,mitochondrial swelling,reactive oxygen species(ROS)generation and more.Cyclophilin D(CypD),a peptidylprolyl isomerase F(PPIF)residing in the mitochondrial matrix,has been regarded as an initial factor in mPTP opening.CypD deficiency improves mitochondrial functions in the mouse cerebral cortex or skeletal muscle,consequently,it ameliorates learning and memory in Alzheimer's disease or insulin resistance.However,the role of CypD in hepatic steatosis requires further investigation.Sterol regulatory element-binding protein-1c(SREBP-lc)is the key lipogenic transcription factor and our previous study identified that SREBP-lc is indispensable for TG accumulation in the liver.The precursor form of SREBP-lc(p-SREBP-1c)could change into the mature form of SREBP-1c(n-SREBP-lc)catalyzed by SIP and S2P in Golgi.The n-SREBP-lc could translocate into the nucleus and bind to specific sterol regulatory element(SRE)DNA sequences,lead to the upregulation of genes involved in TG biosynthesis.By now the relationship between mitochondrial stress and SREBP-1c is unclear.Researches showed that endoplasmic reticulum(ER)stress can induce expression of SREBP-1c and activation of lipogenesis.Ca2+ metabolism is a connection point between mitochondria and endoplasmic reticulum.CypD-induced ROS accumulation may cause Ca2+ balance disruption and p38 MAPK activation can cause ER stress.In our study,we found that CypD-induced mitochondrial stress triggered hepatic TG accumulation.The mechanism was that CypD stimulated mPTP excessive opening and Ca2+ balance disruption,subsequently causing ER stress via p38 MAPK activation and resulted in enhanced SREBP-1c transcription.Objectives:1.To determine the relationship between CypD expression and hepatic TG accumulation in HFD-induced NAFLD mouse model.2.To determine the effect of CypD on hepatic mitochondrial stress and steatosis in CypD deficiency and AdPPIF(adenoviral overexpression of CypD)mouse models.3.To test the upregulation of SREBP-1c by CypD in vivo and in vitro.4.To determine upregulation of SREBP-1c by CypD dependent on IRE1? in Ire1?-LKO(liver-specific Ire1? knockout)mouse and in vitro.5.To confirm the prevention and therapeutic effect of CypD inhibitor in hepatic steatosis.Methods:1.Animal model(1)High-fat diet mouse model:Eight-week old C57Bl/6j male mice were fed a diet containing 60 kcal%fat or a chow diet.(2)CypD or SREBP-1c deficiency mouse model:All mice housed in SPF environment of the animal center.The knockout mice and wild-type mice were obtained from heterozygous mice breeding and the genotype of the mice was determined by PCR.Eight-week old male mice were used in our study.(3)Irel?-LKO mice:Liver-specific Ire1? knockout(Irela-LKO)mice were produced by intercrossing Irel?flox/flox mice with the Alb-Cre transgenic mice.The genotype of the mice was determined by PCR and eight-week old male mice were used in our study.(4)AdPPIF mouse model:Mouse AdPPIF(CypD overexpression adenovirus)and AdEGFP(empty vector adenovirus)were dissolved in sterile PBS and injected through the caudal vein to mice.The amount of viral particles used in these experiments were 2*108 pfu per mouse and were given three times with a six-day interval.(5)Clinical-grade cyclosporine A(CsA)was diluted fresh in PBS.PBS or CsA (10 mg·kg-1·d-1 or 20 mg·kg-1·d-1)were injected intraperitoneally daily to C57Bl/6j mice for six weeks.(6)Tunicamycin was soluble in DMSO at 10 mg·ml-1 and diluted fresh in PBS.PBS or tunicamycin(1 mg·kg-1·d-1)were injected intraperitoneally daily to mice for two days.2.Cell culture:HepG2 cells were cultured according to the guidance from ATCC.Primary hepatocytes were isolated from mice using the two-step collagenase perfusion protocol.3.In vivo Imaging:For in vivo imaging,anesthetized mice were detected by the LB983 NightOWL II Operating Manual System and the luciferase reporter for the mouse Srebplc or Ppif(CypD)promoter was analyzed.4.Indirect calorimetry:The systemic metabolism of mice was measured with a LabMaster system(TSE Systems),and the data were collected.5.Mitochondrial swelling:Mitochondria were isolated from the livers.The mitochondrial swelling was triggered by the addition of calcium and immediately recorded on the Microplate Reader.6.Malondialdehyde levels:The mitochondrial malondialdehyde(MDA)levels were measured using a Lipid Peroxidation MDA Assay Kit according to the manufacturer's directions.7.Blood samples from the mice were obtained for analysis of glucose,lipids and ALT/AST using Mindrary autoanalyzer.8.Liver lipids(TG and TC)were measured using commercial kits.9.Transmission electron microscope analysis was used to determine hepatic mitochondria and lipid accumulation.10.H&E and oil O staining were adopted to detect lipid accumulation in mouse livers.11.In situ detection of mitochondrial reactive oxygen species:We performed in situ measurements of ROS in liver slices and HepG2 cells as directions.12.Real time-PCR was used to determine the mRNA expression of SREBP-1cXbp-1,etc.13.PCR array were adopted to test genes related to fatty liver disease.14.Western Blot analysis was used to determine the protein expression of CypD?p38 MAPK?IRE1??SREBP-1c,etc.15.Mouse body temperature was measured using a rectal digital thermometer probe.16.The body fat mass of mice was analyzed with LUNAR Prodigy X-Ray Tube Housing Assembly.17.Mito-stress test:The oxygen consumption ratios(OCRs)of the spare respiratory capacity and ATP production were analyzed according to parameters calculated by Mito Stress Test Report Generator.18.Double-immunofluorescence was adopted to determine the expression of CypD in hepatic mitochondria.The intracellular concentration of Ca2+ was determined by Fura 2-AM.Results:1.Hepatic steatosis is secondary to the increase of CypD expressionWe fed mice with a chow diet or a high fat diet containing 60 kcal%fat(HFD)for different time periods,and hepatic steatosis and CypD expression were examined at the end of the 2nd,4th,8th and 12th weeks,respectively.The luciferase activity and protein expression(p<0.01)of CypD were increased as early as the end of the 4 week of HFD feeding and gradually enhanced as time passed,by the in vivo imaging and western blot analysis.However,the hepatic TG deposition(p<0.01)and steatosis were observed until the end of the 8th week of HFD feeding and continued to increase.The results showed that increased CypD expression occurred earlier than the TG accumulation in the liver,suggesting a potential relationship between CypD and the etiology of steatosis.2.CypD positively correlates with hepatic mitochondrial stress and steatosisWe explored whether CypD serves as a mitochondrial target potentiating steatosis in Ppif-/-(CypD deficiency)mice fed with chow diet and HFD.On the other hand,to directly determine the role of CypD in hepatic TG accumulation,we overexpressed CypD in mouse liver by infecting AdPPIF(adenoviral overexpression of CypD)through the cauda vein.a Indirect calorimetryMetabolic cage results showed no clear changes of systemic metabolism between Ppif-/-(CypD deficiency)and Ppif+/+(littermate counterparts)mice on chow diet.However,higher oxygen expenditure(VO2)and energy expenditure in Ppif-/-mice with HFD indicated accelerated fatty acid oxidation.Compared to AdEGFP(empty vector adenovirus),AdPPIF(adenoviral overexpression of CypD)in the mice didn't change the VO2 and energy expenditure significantly,which demonstrated that the change of systemic metabolism may not be a direct effect of CypD knockout.b Serological index analysisThe serum TG(p<0.05)and fasting blood glucose levels were decreased in Ppif-/-mice with HFD,while the serum total cholesterol(TC)level was not significantly changed.On the other hand,no significant changes in serum TG and TC levels were observed between Ppif-/-and Ppif+/+ mice on chow diet.c Hepatic mitochondrial stress and steatosis analysisThe hepatic TG levels in Ppif-/-mice were lower than that in Ppif+/+ mice,while this difference was enlarged by HFD treatment(p<0.01).However,the hepatic TC level has no significant change.To confirm whether remission of steatosis in Ppif-/-mice was related to the promotion of mitochondrial function,we detected hepatic mitochondrial stress in HFD-induced mice.The mitochondria in HFD-treated Ppif+/+mice showed a greater swelling than corresponding chow group(p<0.05),but this difference was eliminated between Ppif-/-mice fed with chow diet and HFD diet,implying the indispensable role of CypD in HFD-induced swelling.Ppif-/-mice showed much less MitoSox staining compared to Ppif+/+ mice(p<0.01),indicating that the absence of CypD reduced HFD-induced mitochondrial ROS generation.In addition,compared to AdEGFP(empty vector adenovirus),AdPPIF(adenoviral overexpression of CypD)in the mice increased TG accumulation(p<0.05),mitochondrial swelling(p<0.05)and ROS generation.In conclusion,ablation of CypD prevented diet-induced hepatic mitochondrial stress and steatosis,while hepatic CypD overexpression increased mitochondrial stress and lipid accumulation,implying that CypD positively correlates with hepatic mitochondrial stress and steatosis.3.CypD mediates hepatic TG accumulation through up-regulating SREBP-lcWe performed RT2 PCR array to analyze the expression of fatty liver disease-focused genes involved in TG biosynthesis,fatty acid uptake and oxidation and the representative genes were validated by real-time quantitative PCR.It was demonstrated that the mRNA expression of SREBP-lc was significantly changed in Ppif-/-(CypD deficiency)mice fed a HFD for 8 weeks(p<0.05).The n-SREBP-1c expression was increased at the end of the 8th week of HFD treatment,while CypD deficiency eliminated HFD-induced SREBP-lc upregulation,implying the potential regulation of SREBP-lc by CypD.Further,the promoter activity and protein expression of SREBP-lc in AdPPIF(CypD overexpression adenovirus)-infected mice were increased compared with AdEGFP(empty vector adenovirus)group.To determine whether SREBP-lc was indispensable for CypD-induced TG accumulation,the Srebp1c-/-and Srebp1c+/+(littermate counterparts)mice were injected with AdPPIF and AdEGFP,respectively.Unlike the littermate Srebp1c+/+ mice,the lipid contents in Srebp1c-/-mice were not increased even in those treated with AdPPIF(p<0.01),which supported that CypD stimulated TG accumulation via SREBP-lc.4.IREla is involved in the process of CypD-stimulated SREBP-lc expressionBased on the above results,we wondered how CypD affected SREBP-lc.CypD-induced ROS accumulation could activate downstream signal pathway p38 mitogen activated protein kinase(MAPK)in synaptic damage.P38 MAPK is a connection point between mitochondria and endoplasmic reticulum.In our study,we found that the phosphorylation of p38 MAPK and IREla and ER dilatation were suppressed in HFD-induced Ppif-/-(CypD deficiency)mice.ER stress can trigger SREBP-lc expression and/or activation.Next,to determine the role of IREla in CypD-induced SREBP-1c expression,we established a liver-specific Ire1? knockout(Ire1?-LKO)mouse model.The expression of hepatic SREBP-lc and lipid accumulation in Ire1?-LKO mice were not increased even after treatment with AdPPIF compared to littermate flox/flox mice.These results suggested the indispensable role of IRE1? in CypD-induced SREBP-lc expression.5.CypD increases SREBP-lc expression through Ca2+/p38 MAPK/IRE1?signalingThe pLV-PPIF(CypD overexpression plasmid)was used to confirm the effects of CypD on TG accumulation in HepG2 cells and mouse primary hepatocytes,respectively.The results showed that CypD stimulated mPTP excessive opening and mitochondrial stress,subsequently causing ER stress via Ca2+/p38 MAPK activation and finally resulted in the increase of SREBP-1c mediated TG biosynthesis.SB203580(p38 MAPK inhibitor)or 4?8C(IREla inhibitor)significantly decreased the expression of SREBP-lc promoted by CypD overexpression in HepG2 cells.Similar to the results of HepG2 cells,in the primary hepatocytes,SB203580(p38 MAPK inhibitor)or liver-specific Irela knockout significantly decreased the expression of SREBP-1c and TG accumulation promoted by CypD overexpression plasmid.In conclusion,CypD increased hepatic TG accumulation through Ca2+/p38 MAPK/IREla/SREBP-lc signaling.6.Prevention and therapeutic effect of CypD inhibitor in hepatic steatosisConsidering the effect of hepatic CypD on hepatic TG accumulation,we applied clinical-grade CsA(Sandimmune),an inhibitor of CypD,to determine the preventive and therapeutic effects of CypD as a potential target on NAFLD.HFD-fed mice were given successive intraperitoneal injections of CsA at the two specific time points(4 weeks of HFD feeding or 30 weeks of HFD feeding),respectively.Following CsA usage,the mitochondrial dysfunction/stress was decreased and,astonishing,TG accumulation was blocked for early treatment,while fatty liver was ameliorated for relative later application.Considering this wide pharmacological effects of CsA,it is necessary to eliminate the possibility that decreased hepatic TG accumulation was derived from secondary effects.We used the siRNA of CypD in vitro and gained consistent results,suggesting that CypD is a specific target for prevention and therapy of NAFLD.Conclusions:1.Increased CypD expression occurred earlier than the TG accumulation in HFD-induced NAFLD mouse model.2.Hepatic CypD overexpression induced mitochondrial stress and lipid accumulation.3.CypD increased hepatic TG accumulation through Ca2+/p38 MAPK/IREla/SREBP-lc signaling.4.CypD might be a preventive and therapeutic target for hepatic TG accumulation and that CsA would be a new pharmacological approach for improving NAFLD.