| Arachidonic acid （AA） is the most abundant and the most widely distributedpolyunsaturated fatty acids in human body in organisms and its metabolites has avverse andpotent biological activities. AA metabolic network is an important component of themultiple metabolic pathways and of endogenous bioactive substances includinginflammation signaling pathway and oxidative stress network in vivo. Phospholipids insidethe cell membrane was lipolysised by phospholipase A2to release the arachidonic acid intocytoplasm under a variety of physiological or pathological stimulations, free AA would bemetabolized by three different pathways, including yclooxygenases （COX）, lipoxygenases（LOX）, and cytochrome P450（CYP） monooxygenases to different biologically activeeicosanoids. CYP monooxygenase pathway includes ω/ω-1hydroxylases andepoxygenases. AA was metabolized by CYP epoxygenases to4epoxyeicosatrienoic acid（EET） regioisomers, namely5,6-EET,8,9-EET,11,12-EET and14,15-EET, which werethen rapidly hydrolyzed by soluble epoxide hydrolase （sEH）, one of the major enzymes thatmetabolize EETs, to their corresponding weak biologically active dihydroxyeicosatrienoicacids （DHET）. CYP epoxygenase-EETs system plays an important biological role the regulation ofvarious cellular and physiologic processes in various organs and tissues in organismsincluding human being, especially in cardiovascular system. EETs can activatecalcium-sensitive potassium channels in vascular smooth muscle to cause hyperpolarizationof vascular smooth muscle cells, which leading to vessel vasodilation and blood pressurereduction; EETs could promote the proliferation and migration of endothelial cells andangiogenesis resulted in vascular remodeling by activating the MAPK and PI3K/AKTsignaling pathways, partially upregulating the expression of eNOS in endothelial cell andNO release; Increased EETs also possess the effects of inhibiting apoptosis of endothelialcells，anti-oxidative stress, anti-platelet, and fibrinolysis; EETs can also downregulate theincreased expression of cell adhesion molecules induced by cell cytokine, inhibite theadhesion of lymphocytes to surface of vascular wall, and the activation of nucleartranscription factors （NF-kappa B） and monocyte/macrophage and the its downstreamsignaling cascades. all these effects suggest an anti-inflammatory effect of EETs in avariety of pathophysiologies. In addtion, EETs are closely related to islet functions, bystimulating the secretion of insulin and glucagon from isolated rat pancreatic islets. Ourlaboratory had previous proved that CYP2J3overexpression reversed the insulin resistanceof the various organs in the diabetic rats and mice, suggesting that CYP2J may be apossible target for treatment of diabetes, but the underling mechanisms of this effect ininsulin resistance of CYP2J are not yet clearly known.Diabetes mellitus （DM） is a chronic progressive metabolic syndrome characterized byglucose, lipid and protein metabolism disorder either because the body does not produceenough insulin, or because cells do not respond to the insulin that is produced, it’smacrovascular and microvascular complications pose a serious threat to human health, ofwhich about70%death is due to the consolidation of cardiovascular and cerebrovascularcomplications, Based on above theory, the consensus of “Type2diabetes is coronary heartdisease risk syndrome” has basically reached in medical community and diabetes has become a serious social problem.Diabetes mellitus is classified into four broad categories: type1, type2, gestationaldiabetes and “other specific types” of diabetes. Type2diabetes （T2DM） is the mostcommon type making up about90%of the DM and the incidence rate keeps increasingevery year. T2DM is characterized by insulin resistance which may be combined withrelatively reduced insulin secretion and β cells failure, resulted in many serious concurrentdiseases in various organs such as cardiovascular diseases, stroke and renal dysfuction andeven death. Mounting experimental evidence suggests that diabetes is closed related tohypertension, dyslipidemia, abnormal glucose metabolism, obesity and other risk factors.With further understanding of natures of T2DM and insulin resistance, researches foundthat oxidative stress and inflammatory response involved in the insulin resistance andendothelial dysfunction, inflammatory response has become the “common soil” of the IR,islet β-cell dysfunction, metabolic syndrome and cardiovascular complications. Essentially,diabetes is a chronic subclinical inflammation response disease and studies have shown thatT2DM is often accompanied by the increased levels of acute phase response markers incirculating blood, the activation of innate immune response and chronic inflammation,which mediated the pathogenesis of diabetes. Elevated levels of inflammatory cytokinesmake a direct damage to vascular endothelium and other types of cells, induce thethrombosis and embolism and aggravated dyslipidemia in patients, which accelerates thedeterioration of diabetic vascular lesions and organ damages. Previous evidence suggestedthat elevated levels of inflammatory factors induce insulin resistance involving theactivation of nuclear factor-kappa B （NF-κB）, protein kinase C （PKC）, inhibitor kinase（IKK）, mitogen-activated protein kinase （MAPK） and c-Jun N-terminal kinase（JNK）/stress-activated protein kinase （SAPK） signaling pathway, and also involvinginhibition of insulin-phosphatidylinositol3-kinase/protein kinase B （PI3K/Akt） signalingpathway, and all these lead to weakening in insulin signal transduction and consequently toinsulin resistance. Apart from being the main organ of glucose metabolism, the liver is a place of secretion or release of inflammatory factors, especially during the exacerbation ofdiabetes-related liver disease pathophysiological process, the activation of NF-κB into thenucleus in the liver is a crucial step in the insulin resistance. The clinical study confirmedthat elevated levels of serum TNF-α and IL-1β are risk factors for diabetes and insulinresistance, therefore, anti-inflammatory therapy has become a new way for treatment ofdiabetes.In this study, we assume that CYP2J2overexpression and subsequent elevatedgeneration in EETs have an important role in inhibiting IR and relieving DM primarily byinhibiting of hepatic inflammatory signaling pathway. Therefore, in T2DM model db/dbmouse, we studied the effects of CYP2J2gene delievery on DM, and IR inflammation, aswell as the possible molecular mechanisms in vivo. We also studied the effects ofexogenous CYP2J2overexpression on IR, inflammation and possible molecularmechanisms in HepG2cells, in order to understand the possible role of CYP2J2-EETsclearly in improving insulin resistance, peripheral tissue insulin sensitivity and reducing theliver inflammation, as well as further clarifing whether the CYP2J2or EETs can attenuatethe progress of type2diabetes.In Vivo Study: Effects of CYP Epoxygenases2J2on Insulin Resistance andinflammation in db/db Diabetic MiceResearch aims: The effects of CYP2J2overexpression on insulin resistane andinflammation in db/db type2diabetic mice were investigated.Research methods:1.CYP2J2gene was introduced into the recombinant adeno-associated virus-mediatedvector （rAAV），all plasmids were prepared by alkaline lysis method and purified by cesiumchloride-ethidium bromide gradient equilibrium centrifugation.293cells wereco-transfected with plasmid pXXUF1-2J2or rAAV-GFP, packaging plasmid pXX8andphelper by calcium phosphate precipitation，and the virus titer was detected by Real TimePCR after purification. 2. Sixty male db/db mice and60C57B/6mice（8weeks old）were randomly dividedinto6groups after one week adaptive feeding, respectively. The mice in C57BL/6groupwere invided into normal saline group, C57BL/6+rAAV-GFP group,C57BL/6+rAAV-CYP2J2group, C57BL/6+GW9662group, C57BL/6+rAAV-GFP+GW9662group, C57BL/6+rAAV-CYP2J2+GW9662group, db/db mice were also invidedinto normal saline group, db/db+rAAV-GFP group, db/db+rAAV-CYP2J2group,db/db+GW9662group, db/db+rAAV-GFP+GW9662group, db/db+rAAV-CYP2J2+GW9662group, and all the saline group were injected with equal volume of saline saline,all the rAAV-GFP groups or rAAV-CYP2J2groups were injected with correspondingrAAV-GFP or rAAV-CYP2J21×1011p.f.u, separately. Eight weeks later all the GW9662groups were gavaged with GW9662with the dose of1mg/kg/day for the last4weeks.3. During the experiments, the body weights of experimental animals were weighedand blood sample were extracted once every two weeks until the end of the experiment.Twelve weeks after gene transfection, oral glucose tolerance and insulin tolerance tests aswell Hyperinsulinemic-euglycemic clamp tests were performed;24-hour urine sampleswere collected; Animals were sacrificed after the experiment, and blood samples, heart,aorta, pancreas, liver, skeletal muscle, adipose tissue and kidney were collected and storedin liquid nitrogen, and then transfered to-80℃refrigerator for further tests, and some ofthe above sample tissues were fixed in4%formaldehyde solution, embedded in paraffinafter dehydration for reservation;4. Oral glucose tolerance test and insulin tolerance test were used to detect the effectof CYP2J2gene overexpression in insulin resistance in db/db type2diabetic mice.5. Hyperinsulinemic-euglycemic clamp test was performed to measure the effect ofCYP2J2gene overexpression in insulin sensitivity and whole body glucose metabolism ofdb/db mice.6. ELISA method was used to detect the content of the serum insulin and urinary14,15-DHET; The glucose oxidase method was applicated to detect serum glucose concentrations; Appropriate kits were used to detect the concentration of serum lipid.7. ELISA was used to detect inflammatory cytokines IL-1β, IL-6, TNF-α and CRPlevels in serum; Real-time quantitative PCR was used to detect mRNA expression ofinflammatory cytokines IL-1β, IL-6, TNF-α, CRP and glucose metabolizing enzymes G6P,PEPCK, GCK, PPAR gamma in mouse liver tissue.8. Western blot was performed to detect the expression of CYP2J2, PPAR gamma,P-Y989-IRS-1, P-S307-IRS-1, PI3K, P-S473-AKT, P-ERK, P-C-JUN and nuclear NF-κBin db/db mice liver.Results:1. Western blot results showed that after three months of rAAV-2J2gene injectioninto mice, the expression of CYP2J2protein in the livers of db/db mice was significantlyhigher than the mice injected with rAAV-GFP. ELISA results showed that urine14,15-DHET content of C57BL/6+rAAV-2J2group is （35.52±4.26） ng/ml, significantlyhigher than that （6.53±1.89） ng/ml of C57BL/6+rAAV-GFP group and that （8.32±2.41）ng/ml of C57BL/6saline mice （P<0.05）, while14,15-DHET content of db/db+rAAV-2J2group is （35.52±4.26） ng/ml, significantly higher than that （5.88±1.24） ng/ml of db/db+rAAV-GFP group and that （6.73±1.45） ng/ml of db/db saline mice （P<0.05）. AfterGW9662intervention, urine14,15-DHET content didn’t change,14,15-DHET content ofdb/db+rAAV-2J2+GW9662group is （34.22±3.56） ng/ml, significantly higher than that（5.84±1.38） ng/ml of db/db+rAAV-GFP+GW9662group and that （7.14±2.12） ng/mlof db/db+GW9662mice （P<0.05）, there is no obvious difference between db/db mice anddb/db+GW9662group. These results confirm the CYP2J2can obtain lasting and stableexpression in the mouse liver as long as three months, and significantly increased the levelof EETs in vivo, while GW9662does not affect the metabolism of CYP2J2.2. The results of glucose and lipid metabolism showed that, there was no significantdifference in each group of control C57BL/6mice （P>0.05）. However, db/db mice showedsignificant glucose and lipid metabolism disorders, that is serum glucose, insulin, triglycerides and cholesterol were significantly higher than C57BL/6mice （P<0.05）.Compared with recombinant adeno-associated virus-mediated rAAV-GFP, rAAV-CYP2J2gene transfection significantly reduced serum glucose insulin, triglyceride and cholesterollevels in the db/db mice, compared with db/db+rAAV-GFP mice and db/db saline mice（P<0.05）, rAAV-GFP transfection had no significant effect on the four parameters of db/db mice （P>0.05）.3. OGTT results showed that the variation of serum glucose levels had no significantdifferences （P>0.05） between each group in C57BL/6mice before and after the glucoseload. The fasting serum glucose level of db/db mice before glucose load and the serumglucose level after glucose load were significantly increased compared with C57BL/6mice（P<0.05）（Figure1-5A）; However, the transfection of rAAV-2J2gene made the serumglucose concentration of db/db group at0,30,60,120min （16.35±3.03） mmol/L,（22.83±2.04） mmol/L,（18.74±1.38） mmol/L,（16.08±3.06） mmol/L separately was significantlydecreased compared with that （24.9±3.06） mmol/L,（33.6±1.24） mmol/L,（37.95±1.34）mmol/L,（27.26±2.79） mmol/L of db/db+rAAV-GFP group separately （P<0.05） and that（21.48±2.48） mmol/L,（28.14±1.66） mmol/L,（31.95±2.16） mmol/L,（21.57±1.6）mmol/L of db/db+rAAV-GFP+GW9662group （P<0.05）. These data indicated CYP2J2gene transfection improved the sensitivity of insulin in vivo..4. Insulin tolerance test results showed that the fasting serum glucose of C57BL/6mice after insulin load reduced quickly, the percentages of serum glucose levels accountedfor the basic serum glucose levels at different time point in six groups of db/db mice afterinsulin load were much higher than that in C57BL/6group （P<0.05）; transfection ofrAAV-2J2gene made the percentages of serum glucose to the basic serum glucose levels atdifferent time15,30,60,120min in db/db+of rAAV-2J2group （83.16±2.56）%（69.29±3.76）%（56.22±2.6）%,（61.56±6.56）%was significantly lower than that （90.34±2.08）%,（87.13±2.98）%（75.83±6.69）%（84.32±6.83）%in db/db+rAAV-GFP group and that of db/db+rAAV-2J2+GW9662group at60,120min （68.41±3.18）%（75.85±2.62）%,（P<0.05）.5. Hyperinsulinemic-euglycemic clamp tests showed that glucose infusion rate, wholebody glucose uptake, liver-specific glucose uptake, whole body glucose flux of C57BLmice were much higher than that of db/db mice （P <0.05）, hepatic glucose production wassignificantly less than that of db/db mice （P <0.05）. CYP2J2gene transfection significantlyincreased the glucose infusion rate, whole body glucose uptake, liver-specific glucoseuptake and whole body glucose flux in db/db mice （P<0.05）, reduced hepatic glucoseproduction（P<0.05）. GW9662treatment partially inhibited the effects of CYP2J2transfection （P<0.05）.6. Real-time quantitative PCR data showed the mRNA expression level of sugar-metabolizing enzymes G6P, PEPCK and GCK in the mice liver were consistent with theresults in the clamp test, that is, compared with C57B/6mice, G6P and PEPCK mRNAexpression levels increased in db/db mice liver tissue, while GCK mRNA expression leveldecreased （P<0.05）. However, CYP2J2overexpresson downregulated G6P and PEPCKmRNA levels, upregulated GCK mRNA level in the db/db mice liver tissue （P<0.05）,resulted in improved glucose metabolism in the liver of db/db mice. GFP gene have littleeffect on the mRNA levels of these enzymes in db/db mice （P>0.05）.7. Inflammation-related indicators results:ELISA data showed that serum IL-1β, IL-6, TNF-α and CRP concentrations in db/dbgroups were significantly higher than that of C57BL mice （P<0.05）. inflammatorycytokines in db/db saline group and that of db/db with GFP group had little significantdifference （P>0.05）. rAAV-2J2gene transfection into db/db mice significantly reducedfour inflammatory factors levels compared with db/db saline group or db/db+rAAV-GFPgroup （P<0.05）. Serum four inflammatory cytokines in db/db+rAAV-CYP2J2miceincreased after GW9662intervention （P<0.05）.Real-time quantitative PCR showed that four inflammatory factors IL-1β, IL-6, TNF-alpha, CRP and PPAR gamma mRNA levels in each group of C57BL/6mice had nosignificant difference （P>0.05）. However, IL-1β, IL-6, TNF-alpha, CRP and PPAR gammamRNA levels in livers of all the db/db mice significantly increased compared withC57BL/6mice （P<0.05）, rAAV-CYP2J2gene transfection into db/db mice significantlylowered the mRNA levels of four inflammatory factors and PPAR gamma in the livers ofdb/db+rAAV-CYP2J2mice compared with db/db saline group or db/db+rAAV-GFP group（P<0.05）, while these factors mRNA levels were decreased by GW9662intervention（P<0.05）.Western blot analysis showed that although PPAR gamma and NF-κB proteinexpression level in the nucleus in liver tissues of C57BL/6mice had little differencebetween each group （P>0.05）, their protein levels obviously increased in all the db/db mice（P<0.05）. After rAAV-CYP2J2transfection, PPAR gamma and nuclear NF-κB proteinexpression levels in livers of db/db+rAAV-CYP2J2mice were significantly reduced（P<0.05）, but they raised again after GW9662intervention （P<0.05）. these data suggestedthat inflammation response was extremely active in db/db type2diabetic mice, nsulinresistance, inflammatory pathways were activated via increased NF-κB transferring into thenucleus under insulin resistance, while rAAV-CYP2J2overexpression significantlyincrease PPAR gamma expression but lowered nuclear NF-κB expression in livers of db/dbmice, suggesting CYP2J2overexpression inhibited NF-κB transferring into the nucleus,leading to reduction of inflammation in livers suffer from insulin resistance. rAAV-GFPgene transfection does not have an impact in the expression of PPAR gamma and nuclearNF-κB expression in db/db mice （P>0.05）.8. Western blot analysis showed that P-S473-AKT expression in livers of db/dbgroup and db/db+rAAV-GFP group was significantly lower than C57BL/6mice（P<0.05）, while the expression of P-ERK and P-JNK significantly increased （P<0.05）;However, CYP2J2overexpression in db/db+rAAV-CYP2J2mice significantly increasedthe expression of P-S4732-AKT meanwhile decreased the expression of P-ERK and P-JNK in livers compare with that in db/db+rAAV-GFP mice and db/db saline mice（P<0.05）.In Vitro Study Effects of Cytochrome P450Epoxygenases2J2Overexpression onInsulin Resistance and inflammation in HepG2cells insulin resistance modelResearch aims: The effects of CYP2J2overexpression on insulin resistane andinflammation in HepG2cells were investigated.Methods:1. HepG2cells were cultured in DMEM medium containing10%fetal bovine serumat37°C incubator with5%CO2. When the cell growed to about90%confluence,0.25%trypsin was used to digest and passage, when cells growed to70%-80%confluence undermicroscope, discarded the medium, cells were cultured in DMEM medium containing0.1%fetal bovine serum for starvation of12h to pursue subsequent treatments.2. After24h Recombinant adeno-associated virus-mediated rAAV-CYP2J2andrAAV-GFP gene transfection into HepG2cells，GW9662was added for2h, and theninsulin resistance was induced by palmitic acid（PA） for24h.3. ELISA and real-time quantitative PCR methods analysis IL-1β, IL-6, TNF-alphaand CRP levels in HepG2cell supernatant;4. After intervention, total protein and nuclear protein were extracted, Western blotdetected the protein expression of CYP2J2, PPAR gamma, AKT, P-JNK, P-ERK andNF-κB.5. After intervention,2-deoxy-[1-14C] glucose and insulin were added and glucoseuptake rate of HepG2was calculated in scintillation counter.Results:1. Western blot results showed that the expression of CYP2J2protein in HepG2transfected with rAAV-2J2gene was obviously higher than that in cells transfected withrAAV-GFP （P>0.05）. CYP2J2protein expression in HepG2and HepG2transfected withrAAV-GFP had no obvious difference （P<0.05）.2. Inflammation-related indicators results: ELISA data showed that IL-1β, IL-6, TNF-α and CRP concentrations in thesupernatant of PA-treated HepG2were significantly higher than that of untreated HepG2cells （P<0.05）. There was had little significant difference between PA-treated HepG2andPA+rAAV-GFP HepG2（P>0.05）. rAAV-2J2gene transfection significantly reduced IL-1β,IL-6, TNF-α and CRP levels in supernatant of PA-treated HepG2compared withPA-treated alone cells or PA+rAAV-GFP cells （P<0.05）, while the four inflammatorycytokines levels in PA+rAAV-CYP2J2+GW9662cells decreased compared with PA+rAAV-CYP2J2cells （P<0.05）.Real-time quantitative PCR showed that IL-1β, IL-6, TNF-α, CRP, and PPARγmRNA levels were significantly higher in PA-treated cells than untreated cells （P<0.05）.However, IL-1β, IL-6, TNF-α, CRP, and PPARγ mRNA levels in PA+rAAV-2J2cellswere obviously lower than PA-treated cells and PA+rAAV-GFP cells （P<0.05）. GW9662intervention weakened these influences of CYP2J2on IL-1β, IL-6, TNF-alpha, CRP andPPARγ mRNA expression （P<0.05）.Western blot analysis showed that PPAR gamma protein expression and nuclearNF-κB protein expression level were significantly higher in PA-treated cells than untreatedcells （P<0.05）. However, PPAR gamma protein expression and nuclear NF-κB proteinexpression in PA+rAAV-2J2cells were obviously lower than PA-treated cells andPA+rAAV-GFP cells （P<0.05）. GW9662intervention weakened these influences ofCYP2J2on PPAR gamma protein expression and nuclear NF-κB protein expression（P<0.05）.3. Western blot analysis showed that P-S473-AKT expression in PA-treated cells wassignificantly lower than untreated cells （P<0.05）, while the expression of P-ERK andP-JNK significantly increased （P<0.05）; However, CYP2J2overexpression in PA-treatedcells significantly increased the expression of P-S4732-AKT meanwhile decreased theexpression of P-ERK and P-JNK compared with that of PA-treated alone cells andPA+rAAV-GFP cells （P<0.05）. 4. DG uptake assay results showed that CYP2J2overexpression significantlypromoted glucose uptake rate of PA-treated HepG2cells, meanwhile GFP had no effect,but GW9662intervention weakened the effects of CYP2J2overexpression on promottingglucose uptake rate of PA-treated HepG2cells.Statistical AnalysisData are expressed as means±S.E.M. Statistical comparisons were performed byone-way analysis of variance （ANOVA）. Values of P<0.05were considered as signifcant.Conclusion1. Recombinant adeno-associated virus mediated CYP2J2gene obtained stable anddurable expression in C57BL/6and db/db mice and metabolize arachidonic acid to EETs toplay the biological role in vivo.2. CYP2J2gene overexpression significantly improved insulin resistance in db/dbtype2diabetic mice, the possible molecular mechanism maybe related with the inhibitionof hepatic inflammation by CYP2J2overexpression, that is, CYP2J2overexpressioninhibited NF-κB transfer into the nucleus and activation of P-ERK, P-JNK pathways,inhibited expression and secretion of inflammatory cytokines, activated expression ofPPAR gamma in livers of db/db mice.3. Exogenous CYP2J2overexpression improved insulin resistance and inflammatoryresponse in HepG2cells suffered insulin resistance induced by PA, including inhibition ofNF-κB transfer into nucleus and the P-ERK, P-JNK pathways activation, inhibition ofinflammatory cytokines secretion and activation of PPAR gamma mRNA.4. CYP2J2-EETs ameliorated type2diabetes possiblely through improving insulinresistance, improving the insulin sensitivity of peripheral tissues, inhibiting the expressionand release of inflammatory cytokines, inhibiting the activation of inflammation signalingpathways, delay the progress and may improve survival of insulin resistance and type2diabetes, ultimately play the therapy role in T2DM.5. The results of this study provide a new research direction and a new strategy for prevention and treatment of insulin resistance and diabetes in the future, we expected genetherapy would be a new drug target for diabetes.CYP2J2-EETs improved insulin resistance and type2diabetes pobably through inhibitionof liver inflammation factors expression and inflammatory signaling pathways，andactivation of PPARγ expression in liver， which resulted in promotion in sensitivity ofperipheral tissue to insulin. |
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