| Research Backgrounds and AimsArachidonic acid (AA) is the most abundant and the most widely distributed polyunsaturated fatty acids in human body in organisms and its metabolites has avverse and potent biological activities. AA metabolic network is an important component of the multiple metabolic pathways and of endogenous bioactive substances including inflammation signaling pathway and oxidative stress network in vivo. Phospholipids inside the cell membrane was lipolysised by phospholipase A2 to release the arachidonic acid into cytoplasm under a variety of physiological or pathological stimulations, free AA would be metabolized by three different pathways, including yclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP) monooxygenases to different biologically active eicosanoids. CYP monooxygenase pathway includesω/ω-1 hydroxylases and epoxygenases. AA was metabolized by CYP epoxygenases to 4 epoxyeicosatrienoic acid (EET) regioisomers, namely 5,6-EET,8,9-EET,11,12-EET and 14,15-EET, which were then rapidly hydrolyzed by soluble epoxide hydrolase (sEH), one of the major enzymes that metabolize EETs, to their corresponding weak biologically active dihydroxyeicosatrienoic acids (DHET). CYP epoxygenase-EETs system plays an important biological role the regulation of various cellular and physiologic processes in various organs and tissues in organisms including human being, especially in cardiovascular system. EETs can activate calcium-sensitive potassium channels in vascular smooth muscle to cause hyperpolarization of vascular smooth muscle cells, which leading to vessel vasodilation and blood pressure reduction; EETs could promote the proliferation and migration of endothelial cells and angiogenesis resulted in vascular remodeling by activating the MAPK and PI3K/AKT signaling pathways, partially upregulating the expression of eNOS in endothelial cell and NO release; Increased EETs also possess the effects of inhibiting apoptosis of endothelial cells, anti-oxidative stress, anti-platelet, and fibrinolysis; EETs can also downregulate the increased expression of cell adhesion molecules induced by cell cytokine, inhibite the adhesion of lymphocytes to surface of vascular wall, and the activation of nuclear transcription factors (NF-kappa B) and monocyte/macrophage and the its downstream signaling cascades, all these effects suggest an anti-inflammatory effect of EETs in a variety of pathophysiologies. In addtion, EETs are closely related to islet functions, by stimulating the secretion of insulin and glucagon from isolated rat pancreatic islets. Our laboratory had previous proved that CYP2J3 overexpression reversed the insulin resistance of the various organs in the diabetic rats and mice, suggesting that CYP2J may be a possible target for treatment of diabetes, but the underling mechanisms of this effect in insulin resistance of CYP2J are not yet clearly known.Diabetes mellitus (DM) is a chronic progressive metabolic syndrome characterized by glucose, lipid and protein metabolism disorder either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced, it's macrovascular and microvascular complications pose a serious threat to human health, of which about 70% death is due to the consolidation of cardiovascular and cerebrovascular complications, Based on above theory, the consensus of "Type 2 diabetes is coronary heart disease risk syndrome" has basically reached in medical community and diabetes has become a serious social problem.Diabetes mellitus is classified into four broad categories:type 1, type 2, gestational diabetes and "other specific types" of diabetes. Type 2 diabetes (T2DM) is the most common type making up about 90% of the DM and the incidence rate keeps increasing every year. T2DM is characterized by insulin resistance which may be combined with relatively reduced insulin secretion and (3 cells failure, resulted in many serious concurrent diseases in various organs such as cardiovascular diseases, stroke and renal dysfuction and even death. Mounting experimental evidence suggests that diabetes is closed related to hypertension, dyslipidemia, abnormal glucose metabolism, obesity and other risk factors. With further understanding of natures of T2DM and insulin resistance, researches found that oxidative stress and inflammatory response involved in the insulin resistance and endothelial dysfunction, inflammatory response has become the "common soil" of the IR, isletβ-cell dysfunction, metabolic syndrome and cardiovascular complications. Essentially, diabetes is a chronic subclinical inflammation response disease and studies have shown that T2DM is often accompanied by the increased levels of acute phase response markers in circulating blood, the activation of innate immune response and chronic inflammation, which mediated the pathogenesis of diabetes. Elevated levels of inflammatory cytokines make a direct damage to vascular endothelium and other types of cells, induce the thrombosis and embolism and aggravated dyslipidemia in patients, which accelerates the deterioration of diabetic vascular lesions and organ damages. Previous evidence suggested that elevated levels of inflammatory factors induce insulin resistance involving the activation 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 involving inhibition of insulin-phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, and all these lead to weakening in insulin signal transduction and consequently to insulin 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 of diabetes-related liver disease pathophysiological process, the activation of NF-κB into the nucleus in the liver is a crucial step in the insulin resistance. The clinical study confirmed that elevated levels of serum TNF-a and IL-1βare risk factors for diabetes and insulin resistance, therefore, anti-inflammatory therapy has become a new way for treatment of diabetes.In this study, we assume that CYP2J2 overexpression and subsequent elevated generation in EETs have an important role in inhibiting IR and relieving DM primarily by inhibiting of hepatic inflammatory signaling pathway. Therefore, in T2DM model db/db mouse, we studied the effects of CYP2J2 gene delievery on DM, and IR inflammation, as well as the possible molecular mechanisms in vivo. We also studied the effects of exogenous CYP2J2 overexpression on IR, inflammation and possible molecular mechanisms in HepG2 cells, in order to understand the possible role of CYP2J2-EETs clearly in improving insulin resistance, peripheral tissue insulin sensitivity and reducing the liver inflammation, as well as further clarifing whether the CYP2J2 or EETs can attenuate the progress of type 2 diabetes.In Vivo Study:Effects of CYP Epoxygenases 2J2 on Insulin Resistance and inflammation in db/db Diabetic MiceResearch aims:The effects of CYP2J2 overexpression on insulin resistane and inflammation in db/db type 2 diabetic mice were investigated.Research methods:1.CYP2J2 gene was introduced into the recombinant adeno-associated virus-mediated vector (rAAV), all plasmids were prepared by alkaline lysis method and purified by cesium chloride-ethidium bromide gradient equilibrium centrifugation.293 cells were co-transfected with plasmid pXXUF1-2J2 or rAAV-GFP, packaging plasmid pXX8 and phelper by calcium phosphate precipitation, and the virus titer was detected by Real Time PCR after purification. 2. Sixty male db/db mice and 60 C57B/6 mice (8 weeks old) were randomly divided into 6 groups after one week adaptive feeding, respectively. The mice in C57BL/6 group were invided into normal saline group, C57BL/6+rAAV-GFP group, C57BL/6+rAAV-CYP2J2 group, C57BL/6+GW9662 group, C57BL/6+rAAV-GFP+ GW9662 group, C57BL/6+rAAV-CYP2J2+GW9662 group, db/db mice were also invided into normal saline group, db/db+rAAV-GFP group, db/db+rAAV-CYP2J2 group, db/db+GW9662 group, db/db+rAAV-GFP+GW9662 group, db/db+rAAV-CYP2J2+ GW9662 group, and all the saline group were injected with equal volume of saline saline, all the rAAV-GFP groups or rAAV-CYP2J2 groups were injected with corresponding rAAV-GFP or rAAV-CYP2J21×1011p.f.u, separately. Eight weeks later all the GW9662 groups were gavaged with GW9662 with the dose of lmg/kg/day for the last 4 weeks.3. During the experiments, the body weights of experimental animals were weighed and 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 as well Hyperinsulinemic-euglycemic clamp tests were performed; 24-hour urine samples were collected; Animals were sacrificed after the experiment, and blood samples, heart, aorta, pancreas, liver, skeletal muscle, adipose tissue and kidney were collected and stored in liquid nitrogen, and then transfered to-80℃refrigerator for further tests, and some of the above sample tissues were fixed in 4% formaldehyde solution, embedded in paraffin after dehydration for reservation;4. Oral glucose tolerance test and insulin tolerance test were used to detect the effect of CYP2J2 gene overexpression in insulin resistance in db/db type 2 diabetic mice.5. Hyperinsulinemic-euglycemic clamp test was performed to measure the effect of CYP2J2 gene overexpression in insulin sensitivity and whole body glucose metabolism of db/db mice.6. ELISA method was used to detect the content of the serum insulin and urinary 14,15-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-a and CRP levels in serum; Real-time quantitative PCR was used to detect mRNA expression of inflammatory cytokines IL-1β, IL-6, TNF-a, 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-κB in db/db mice liver.Results:1. Western blot results showed that after three months of rAAV-2J2 gene injection into mice, the expression of CYP2J2 protein in the livers of db/db mice was significantly higher than the mice injected with rAAV-GFP. ELISA results showed that urine 14,15-DHET content of C57BL/6+rAAV-2J2 group is (35.52±4.26) ng/ml, significantly higher than that (6.53±1.89) ng/ml of C57BL/6+rAAV-GFP group and that (8.32±2.41) ng/ml of C57BL/6 saline mice (P<0.05), while 14,15-DHET content of db/db+rAAV-2J2 group 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). After GW9662 intervention, urine 14,15-DHET content didn't change,14,15-DHET content of db/db+rAAV-2J2+GW9662 group is (34.22±3.56) ng/ml, significantly higher than that (5.84±1.38) ng/ml of db/db+rAAV-GFP+GW9662 group and that (7.14±2.12) ng/ml of db/db+GW9662 mice (P<0.05), there is no obvious difference between db/db mice and db/db+GW9662 group. These results confirm the CYP2J2 can obtain lasting and stable expression in the mouse liver as long as three months, and significantly increased the level of EETs in vivo, while GW9662 does not affect the metabolism of CYP2J2.2. The results of glucose and lipid metabolism showed that, there was no significant difference in each group of control C57BL/6 mice (P>0.05). However, db/db mice showed significant glucose and lipid metabolism disorders, that is serum glucose, insulin, triglycerides and cholesterol were significantly higher than C57BL/6 mice (P<0.05). Compared with recombinant adeno-associated virus-mediated rAAV-GFP, rAAV-CYP2J2 gene transfection significantly reduced serum glucose insulin, triglyceride and cholesterol levels 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 significant differences (P>0.05) between each group in C57BL/6 mice before and after the glucose load. The fasting serum glucose level of db/db mice before glucose load and the serum glucose level after glucose load were significantly increased compared with C57BL/6 mice (P<0.05) (Figure 1-5A); However, the transfection of rAAV-2J2 gene made the serum glucose concentration of db/db group at 0,30,60,120 min (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 significantly decreased 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+GW9662 group (P<0.05). These data indicated CYP2J2 gene transfection improved the sensitivity of insulin in vivo.4. Insulin tolerance test results showed that the fasting serum glucose of C57BL/6 mice after insulin load reduced quickly, the percentages of serum glucose levels accounted for the basic serum glucose levels at different time point in six groups of db/db mice after insulin load were much higher than that in C57BL/6 group (P<0.05); transfection of rAAV-2J2 gene made the percentages of serum glucose to the basic serum glucose levels at different time 15,30,60,120 min in db/db+of rAAV-2J2 group (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+GW9662 group at 60,120 min (68.41±3.18)%(75.85±2.62)%, (P<0.05).5. Hyperinsulinemic-eug1ycemic clamp tests showed that glucose infusion rate, whole body glucose uptake, liver-specific glucose uptake, whole body glucose flux of C57BL mice were much higher than that of db/db mice (P<0.05), hepatic glucose production was significantly less than that of db/db mice (P<0.05). CYP2J2 gene transfection significantly increased the glucose infusion rate, whole body glucose uptake, liver-specific glucose uptake and whole body glucose flux in db/db mice (P<0.05), reduced hepatic glucose production(P<0.05). GW9662 treatment partially inhibited the effects of CYP2J2 transfection (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 the results in the clamp test, that is, compared with C57B/6 mice, G6P and PEPCK mRNA expression levels increased in db/db mice liver tissue, while GCK mRNA expression level decreased (P<0.05). However, CYP2J2 overexpresson downregulated G6P and PEPCK mRNA 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 little effect 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-a and CRP concentrations in db/db groups were significantly higher than that of C57BL mice (P<0.05). inflammatory cytokines in db/db saline group and that of db/db with GFP group had little significant difference (P>0.05). rAAV-2J2 gene transfection into db/db mice significantly reduced four inflammatory factors levels compared with db/db saline group or db/db+rAAV-GFP group (P<0.05). Serum four inflammatory cytokines in db/db+rAAV-CYP2J2 mice increased after GW9662 intervention (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/6 mice had no significant difference (P>0.05). However, IL-1β, IL-6, TNF-alpha, CRP and PPAR gamma mRNA levels in livers of all the db/db mice significantly increased compared with C57BL/6 mice (P<0.05), rAAV-CYP2J2 gene transfection into db/db mice significantly lowered the mRNA levels of four inflammatory factors and PPAR gamma in the livers of db/db+rAAV-CYP2J2 mice compared with db/db saline group or db/db+rAAV-GFP group (P<0.05), while these factors mRNA levels were decreased by GW9662 intervention (P<0.05).Western blot analysis showed that although PPAR gamma and NF-κB protein expression level in the nucleus in liver tissues of C57BL/6 mice had little difference between each group (P>0.05), their protein levels obviously increased in all the db/db mice (P<0.05). After rAAV-CYP2J2 transfection, PPAR gamma and nuclear NF-κB protein expression levels in livers of db/db+ rAAV-CYP2J2 mice were significantly reduced (P<0.05), but they raised again after GW9662 intervention (P<0.05). these data suggested that inflammation response was extremely active in db/db type 2 diabetic mice, nsulin resistance, inflammatory pathways were activated via increased NF-κB transferring into the nucleus under insulin resistance, while rAAV-CYP2J2 overexpression significantly increase PPAR gamma expression but lowered nuclear NF-κB expression in livers of db/db mice, suggesting CYP2J2 overexpression inhibited NF-κB transferring into the nucleus, leading to reduction of inflammation in livers suffer from insulin resistance. rAAV-GFP gene transfection does not have an impact in the expression of PPAR gamma and nuclear NF-κB expression in db/db mice (P>0.05).8. Western blot analysis showed that P-S473-AKT expression in livers of db/db group and db/db+rAAV-GFP group was significantly lower than C57BL/6 mice (P<0.05), while the expression of P-ERK and P-JNK significantly increased CP<0.05); However, CYP2J2 overexpression in db/db+rAAV-CYP2J2 mice significantly increased the 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 P450 Epoxygenases 2J2 Overexpression on Insulin Resistance and inflammation in HepG2 cells insulin resistance modelResearch aims:The effects of CYP2J2 overexpression on insulin resistane and inflammation in HepG2 cells were investigated.Methods:1. HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37℃incubator with 5% CO2. When the cell growed to about 90% confluence,0.25% trypsin was used to digest and passage, when cells growed to 70% -80%confluence under microscope, discarded the medium, cells were cultured in DMEM medium containing 0.1% fetal bovine serum for starvation of 12 h to pursue subsequent treatments.2. After 24h Recombinant adeno-associated virus-mediated rAAV-CYP2J2 and rAAV-GFP gene transfection into HepG2 cells, GW9662 was added for 2h, and then insulin resistance was induced by palmitic acid(PA) for 24h.3. ELISA and real-time quantitative PCR methods analysis IL-1(3, IL-6, TNF-alpha and CRP levels in HepG2 cell supernatant;4. After intervention, total protein and nuclear protein were extracted, Western blot detected the protein expression of CYP2J2, PPAR gamma, AKT, P-JNK, P-ERK and NF-κB.5. After intervention,2-deoxy-[1-14C] glucose and insulin were added and glucose uptake rate of HepG2 was calculated in scintillation counter.Results:1. Western blot results showed that the expression of CYP2J2 protein in HepG2 transfected with rAAV-2J2 gene was obviously higher than that in cells transfected with rAAV-GFP (P>0.05). CYP2J2 protein expression in HepG2 and HepG2 transfected with rAAV-GFP had no obvious difference (P<0.05).2. Inflammation-related indicators results: ELISA data showed that IL-1β, IL-6, TNF-αand CRP concentrations in the supernatant of PA-treated HepG2 were significantly higher than that of untreated HepG2 cells (P<0.05). There was had little significant difference between PA-treated HepG2 and PA+rAAV-GFP HepG2 (P>0.05). rAAV-2J2 gene transfection significantly reduced IL-1β, IL-6, TNF-αand CRP levels in supernatant of PA-treated HepG2 compared with PA-treated alone cells or PA+rAAV-GFP cells (P<0.05), while the four inflammatory cytokines levels in PA+rAAV-CYP2J2+GW9662 cells decreased compared with PA+ rAAV-CYP2J2 cells (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 PPARy mRNA levels in PA+rAAV-2J2 cells were obviously lower than PA-treated cells and PA+rAAV-GFP cells (P<0.05). GW9662 intervention weakened these influences of CYP2J2 on IL-1β, IL-6, TNF-alpha, CRP and PPARy mRNA expression (P<0.05).Western blot analysis showed that PPAR gamma protein expression and nuclear NF-κB protein expression level were significantly higher in PA-treated cells than untreated cells (P<0.05). However, PPAR gamma protein expression and nuclear NF-κB protein expression in PA+rAAV-2J2 cells were obviously lower than PA-treated cells and PA+rAAV-GFP cells (P<0.05). GW9662 intervention weakened these influences of CYP2J2 on 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 was significantly lower than untreated cells (P0<.05), while the expression of P-ERK and P-JNK significantly increased (P<0.05); However, CYP2J2 overexpression in PA-treated cells significantly increased the expression of P-S4732-AKT meanwhile decreased the expression of P-ERK and P-JNK compared with that of PA-treated alone cells and PA+rAAV-GFP cells (P<0.05). 4. DG uptake assay results showed that CYP2J2 overexpression significantly promoted glucose uptake rate of PA-treated HepG2 cells, meanwhile GFP had no effect, but GW9662 intervention weakened the effects of CYP2J2 overexpression on promotting glucose uptake rate of PA-treated HepG2 cells.Statistical AnalysisData are expressed as means±S.E.M. Statistical comparisons were performed by one-way analysis of variance (ANOVA). Values of P<0.05 were considered as significant.Conclusion1. Recombinant adeno-associated virus mediated CYP2J2 gene obtained stable and durable expression in C57BL/6 and db/db mice and metabolize arachidonic acid to EETs to play the biological role in vivo.2. CYP2J2 gene overexpression significantly improved insulin resistance in db/db type 2 diabetic mice, the possible molecular mechanism maybe related with the inhibition of hepatic inflammation by CYP2J2 overexpression, that is, CYP2J2 overexpression inhibited NF-κB transfer into the nucleus and activation of P-ERK, P-JNK pathways, inhibited expression and secretion of inflammatory cytokines, activated expression of PPAR gamma in livers of db/db mice.3. Exogenous CYP2J2 overexpression improved insulin resistance and inflammatory response in HepG2 cells suffered insulin resistance induced by PA, including inhibition of NF-κB transfer into nucleus and the P-ERK, P-JNK pathways activation, inhibition of inflammatory cytokines secretion and activation of PPAR gamma mRNA.4. CYP2J2-EETs ameliorated type 2 diabetes possiblely through improving insulin resistance, improving the insulin sensitivity of peripheral tissues, inhibiting the expression and release of inflammatory cytokines, inhibiting the activation of inflammation signaling pathways, delay the progress and may improve survival of insulin resistance and type 2 diabetes, 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 gene therapy would be a new drug target for diabetes. CYP2J2-EETs improved insulin resistance and type 2 diabetes pobably through inhibition of liver inflammation factors expression and inflammatory signaling pathways, and activation of PPARy expression in liver, which resulted in promotion in sensitivity of peripheral tissue to insulin. |
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