| Previous studies suggest that arachidonic acid (AA) is the precursor of various important biological active substances. AA is released from membrane phospholipids by phospholipase A2, which is activated by a variety of physiological stimulus. It is well established that oxidative metabolism of AA by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP) epoxygenases to biologically active eicosanoids plays a critical role in the regulation of various cellular and physiologic processes. Cytochrome P450 epoxygenases convert AA to 4 epoxyeicosatrienoic acid (EET) regioisomers, namely 5,6-EET,8,9-EET,11,12-EET and 14,15-EET. Soluble epoxide hydrolase (sEH) is one of the major enzymes that metabolize EETs. sEH rapidly hydrolyzes EETs to their corresponding dihydroxyeicosatrienoic acids (DHET), which, in general, are much less biologically active than EETs.Experimental evidence indicates that cytochrome P450 epoxygenases-derived EETs play a vital biological role in the homeostasis of cardiovascular systems. It is well established that EETs possess potent vasodilatory effects, which are mediated by smooth muscle cell hyperpolarization via the activation of calcium-sensitive potassium channels (BKCa2+), as well as the release of nitric oxide (NO) from L-arginine metabolized by endothelial nitric oxide synthase (eNOS). AA epoxygenase metabolites EETs promote the proliferation of endothelial cells and angiogenesis involving the activation of MAPK and PI3K/AKT signaling pathways, and to some extent, the eNOS pathway. EETs upregulate eNOS expression via activation of MAPK and protein kinase C, as well as PI3K/AKT signaling pathways. CYP epoxygenase overexpression, which is known to increase EETs biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-alpha. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/AKT signaling. CYP-derived EETs possess potent anti-inflammatory properties in the vasculature, and improved endothelial dysfunction. Recent study shows that a specific inhibitor of sEH, whose biological activity was inhibited, has a significant protective effect in the cardiovascular system, as well as in other systems. These effects include vasodilation, reduced blood pressure, attenuated renal vascular and glomerular injury induced by hypertension, protection against ischemic stroke and vascular disease, and protection against myocardium injury induced by ischemia and reperfusion, as well as attenuated cardiac hypertrophy.Diabetes (DM) is a chronic progressive disease; including type 1 DM, type 2 DM, gestational diabetes, and other special types of DM. More than 90%of patients suffer from type 2 DM among the diabetic patients. The incidence of type 2 diabetes increased year by year, of which weight gain/obesity is a prominent manifestation of these patients. The main pathophysiological mechanisms of type 2 diabetes are insulin resistance and pancreaticβcell failure. Type 2 diabetes leads to cardio-cerebral vascular system and kidneys and other serious complications, even death, and brings a heavy social and economic burden to the society and the families. Recent results show that CYP epoxygenase, EETs and DHETs play important roles in the pathogenesis of IR and fatty acidβoxidation, as well as DM. However, the effects of CYP2J3 overexpression on insulin resistance, peripheral tissue insulin sensitivity and isletβcell function as well as the progression of type 2 diabetes are not clear.In this study, we assume that CYP2J3 overexpression, as well as the subsequent increased EETs production has important protective effects in improving IR, enhancingβcell function and delaying the progression of DM. So, the effects of CYP2J3 gene therapy on DM and IR and the possible molecular mechanisms were investigated in db/db type 2 diabetic mice models. In addition, the effects of exogenous EETs on the NIT-1 cell proliferation and insulin secretion, as well as the anti-injury and possible molecular mechanisms were also explored in vitro study.In Vivo Study Effects and Mechanisms of Cytochrome P450 Epoxygenases 2J3 Overexpression on Insulin Resistance in db/db Type 2 Diabetic MiceResearch aims:The effects and possible molecular mechanisms of CYP2J3 overexpression on insulin resistane in db/db type 2 diabetic mice were investigated.Research methods:1. Screening of pcDNA-2J3 eukaryotic expression plasmid and identification by restriction enzymes digestion;2. Extraction of pcDNA3.1 and pcDNA-2J3 eukaryotic expression plasmid by alkaline lysis methods and purification by silica;3. Animals were randomly assigned to different treatment groups and subjected to a one-week-adaptation period. Animals were randomly divided into six groups, and each group has 10 mice. The mice in C57BL/6 group were injected with normal saline, C57BL/6 +pcDNA3.1 group were injected with pcDNA3.1, C57BL/6+pcDNA-2J3 (+) group were injected with pcDNA-2J3 (+), db/db group were injected with normal saline, db/db+ pcDNA3.1 group were injected with pcDNA3.1, db/db+pcDNA-2J3 group were injected with pcDNA-2J3. Before the beginning of the experiment, blood samples and 24-hour urine specimens during 8AM to 8AM the next day were collected. During the experiments, the body weights of experimental animals were weighed once a week until the end of the experiment. 4. After the fixation of experimental animals, the target gene pcDNA-2J3 and control gene pcDNA3.1 as well as the equal volume of saline were injected into the bodies of mice through tail veins, and the dose of plasmid injected was 5 mg/kg body weight.2 weeks after gene transfer,24-hour water intake and 24-hour urine samples were collected. Meanwhile, intraperitoneal injection of glucose tolerance test was carried out. Animals were sacrificed after the experiment. Blood samples, heart, aorta, pancreas, fat, liver, skeletal muscle and kidney were taken and placed in liquid nitrogen, and then transfered into the-80℃refrigerator, and some of the above tissues were fixed with formaldehyde solution, embedded in paraffin after dehydration at room temperature;5. Intraperitoneal injection of glucose tolerance was carried out to evaluate the effects of CYP2J3 overexpression on insulin resistance in db/db type 2 diabetic mice;6. ELISA was carried out to measure insulin levels in serum and 14,15-DHET levels in urine; Serum concentrations of glucose and lipid profile were also measured by the related method, and insulin resistance index HOMA-IR was calculated;7. Western blot was carried out to detect CYP2J3 expression in aorta, heart, liver and kidney of db/db type 2 diabetic mice; P-Y989-IRS-1, P-S307-IRS-1, PI3K, P-T308-AKT, P-S1177-eNOS, P-T495-eNOS and P-T172-AMPK expression were also detected in livers and skeletal muscles of db/db and C57BL/6 mice.Research results:1. Results showed that compared with the mice injected with pcDNA3.1, the CYP2J3 expression in aorta, heart, liver, and kidney of mice injected with pcDNA-2J3 increased significantly (P<0.05). Results of ELISA indicated that 14,15-DHET content (38.68±2.86) ng/mL of C57BL/6+pcDNA-2J3 (+) group in urine was significantly higher than that (6.32±1.12) ng/mL of C57BL/6+pcDNA3.1 group (P<0.05), and that 14,15-DHET content (34.69±3.2) ng/mL of db/db+pcDNA-2J3 group in urine was significantly higher than that (5.48±1.98) ng/mL of db/db+pcDNA3.1 group (P<0.05).2. A glucose tolerance test was performed in C57BL/6 and db/db mice 2 weeks after gene delivery. Fasting plasma glucose levels before glucose loading and plasma glucose levels after glucose loading were not altered by the various gene therapy treatments in C57BL/6 mice (P>0.05). Compared with levels in C57BL/6 mice, fasting plasma glucose levels before glucose loading and plasma glucose levels after glucose loading were significantly higher in all db/db mice groups. However, the glucose levels before and after glucose loading in db/db+CYP2J3(+) mice were significantly less than those in the db/db control and db/db+pcDNA groups (P<0.05). The results showed that 0,30,60,120 min the glucose levels (12.65±1.14) mmol/L, (34.28±3.33) mmol/L, (19.01±3.23) mmol/L, (17.07±1.47) mmol/L in db/db+pcDNA-2J3 group were lower than that (18.78±1.92) mmol/L, (43.47±2.5) mmol/L, (27.73±1.36) mmol/L, (23.49±1.44) mmol/L in db/db+ pcDNA3.1 group (P<0.05). A similar profile for fasting plasma insulin levels and for plasma insulin levels after glucose loading was observed, with db/db+CYP2J3(+) mice having levels lower than those of the other db/db groups but higher than those of all of the C57BL/6 groups at all time points assessed. The results showed that 0,30,60,120 min the plasma insulin levels (1.43±0.36) ng/mL, (1.86±0.1) ng/mL, (2.09±0.322) ng/mL, (1.84±0.28) ng/mL in db/db+pcDNA-2J3 group were significantly lower than that (2.5±0.27) ng/mL, (3.16±0.28) ng/mL, (3.40±0.287) ng/mL, (3.29±0.32) ng/mL in db/db+ pcDNA3.1 group (P<0.05). These data indicate that CYP2J3 gene delivery attenuated insulin resistance and has anti-diabetic effects in db/db mice.3. The levels of a variety of biochemical parameters, particularly parameters of glucose and lipid metabolism in three groups of C57BL/6 mice had no significant differences (P>0.05). The diabetic phenotype of db/db mice (injected with the empty pcDNA vector) was confirmed by significantly higher levels of serum glucose, serum insulin, insulin resistance (HOMA-IR), serum triglycerides, serum cholesterol and urine volume in these animals compared with those in C57BL/6 mice (all P<0.05). Injection of pcDNA-CYP2J3 significantly reduced insulin resistance index (HOMA-IR) and urine volume in db/db mice, whereas injection of the empty pcDNA vector did not. Although not all parameters of the diabetic phenotype in db/db mice were reversed by pcDNA-CYP2J3 treatment, these data provide further evidence that CYP2J3 can attenuate insulin resistance in this animal model.4. S307-IRS-1 phosphorylation was significantly increased and Y989-IRS-1 phosphorylation was significantly decreased in livers of db/db control and db/db+pcDNA mice, compared with C57BL/6 mice. Similar effects were observed in skeletal muscle. Administration of pcDNA-CYP2J3 to db/db mice prevented these changes in both tissues. The effects of pcDNA-CYP2J3 treatment on the phosphorylation status of eNOS were examined in liver and skeletal muscle. The level of phospho-T495-eNOS was higher, but phospho-S1177-eNOS was lower in livers of db/db mice, compared with C57BL/6 mice. CYP2J3 gene delivery prevented the downregulation of phospho-S1177-eNOS, but had no impact on phospho-T495-eNOS. Similar results were found in skeletal muscle. These results suggest that pcDNA-CYP2J3 treatment in db/db mice altered eNOS phosphorylation status. Compared with C57BL/6 mice, the PI3K, P-T172-AKT, and P-T172-AMPK expression levels in liver and skeletal muscle of db/db group and db/db+ pcDNA group were significantly lower (P<0.05). CYP2J3 overexpression in db/db+ pcDNA-2J3 group significantly prevented the decrease of PI3K, P-T172-AKT, and P-T172-AMPK expression levels in liver and skeletal muscle (P<0.05).In Vitro Study Effects and Mechanisms of Exogenous EETs on NIT-1 Cells Proliferation and Insulin SecretionResearch aims:The effects and mechanisms of exogenous EETs on NIT-1 cells proliferation and insulin secretion were investigated.Research Methods:1. NIT-1 cells were cultured in DMEM media containing 10%FBS, and were kept in an incubator with 37℃and 5%CO2. When grow to 90%confluence in 75 cm2 flasks, NIT-1 cells were passaged by 0.25%trypsin, and then were treated with synchronization. That is, when cell growth to 70%-80%confluence, NIT-1 cells were placed in reduced serum (0.5%FBS) media for 12 h after cell attachment, and then reduced serum media were changed to media without serum.2. After synchronization, SRB assays were carried out to measure the effects of exogenous EETs on NIT-1 cells activities;3. After synchronization, flow cytometry assays were carried out to determine the effects of exogenous EETs on NIT-1 cells cell cycle;4. After synchronization, MAPK, MEK, PI3K, and PPARy inhibitors Apigenin, PD98059, LY294002 and GW9662 and NIT-1 cells, as well as 14,15-EET were incubated together for 24 h, and then SRB assays were carried out to measure the effects of exogenous EETs on NIT-1 cells activities;5. After treated with EETs for 1 h, NIT-1 cells were collected, and proteins were extracted. Western blot was carried out to detect PI3K, P-T308-AKT, P-MAPK and PPARγexpression in NIT-1 cells.6. After synchronization, NIT-1 cells were randomly divided into Control group, Vehicle group, EETs 10 nmol/L group, EETs 50 nmol/L group and EETs 250 nmol/L groups, and then stimulated with different varieties and different concentrations of EETs. EETs were added every other 6 hours to minimize reduction in levels of EETs due to autooxidation. After 24 h, the medium was removed, and cells were first equilibrated in Krebs-Ringer bicarbonate-HEPES (KRBH) buffer containing 1.67 mmol/L glucose for 1 h, and then buffer was washed off and KRBH containing 16.7 mmol/L glucose was added. The concentration of insulin in the buffer was then determined by ELISA kit after incubation with KRBH containing 16.7 mmol/L glucose for 2 h.7. After synchronization, MAPK, MEK, PI3K, and PPARy inhibitors Apigenin, PD98059, LY294002 and GW9662 and NIT-1 cells, as well as 14,15-EET were incubated together for 24 h, and then stimulated with 14,15-EET (100 nmol/L).14,15-EET was added every other 6 hours to minimize reduction in levels of EETs due to autooxidation. After 24 h, the medium was removed, and cells were first equilibrated in Krebs-Ringer bicarbonate-HEPES (KRBH) buffer containing 1.67 mmol/L glucose for 1 h, and then buffer was washed off and KRBH containing 16.7 mmol/L glucose was added. The concentration of insulin in the buffer was then determined by ELISA kit after incubation with KRBH containing 16.7 mmol/L glucose for 2 h.Research results:1.8,9-EET,11,12-EET and 14,15-EET significantly promoted NIT-1 cell proliferation in a time-and dose-dependent manner. MAPK inhibitor Apigenin, MEK inhibitor PD98059, PI3K inhibitor LY294002 and the PPARy inhibitor GW9662 significantly inhibited NIT-1 cell proliferation stimulated by.14,15-EET.2.8,9-EET,11,12-EET and 14,15-EET significantly promoted NIT-1 cell division, characterized by significantly increased cell ratio of S-phase cells and S+G2/M phase cells. The cell ratio of S+G2/M phase cells in the control group and the vehicle group were (37.3±0.7)%and (31.0±2.0)%, while in the group of 8,9-EET,11,12-EET, 14,15-EET, were (43.0±1.1)%, (45.6±2.9)%and (45.3±1.8)%respectively. The differences of S+G2/M phase cell ratio between group of three kinds of EETs and vehicle group were statistically significant (P<0.05).3. Results showed that 8,9-EET,11,12-EET and 14,15-EET significantly increased P-MAPK, PI3K, P-AKT expression, as well as PPARy expression in NIT-1 cells, indicating that EETs activated the MAPK, PI3K/AKT and PPARy signaling pathways.4. Results of ELISA showed that 8,9-EET,11,12-EET and 14,15-EET significantly increased NIT-1 cells insulin secretion in a dose-dependent manner after glucose loading independent on the activations of MAPK, PI3K/AKT and PPARy signaling transduction pathways.In Vitro Study Effects and Mechanisms of Exogenous EETs on NIT-1 Cells Apoptosis Induced by TNF-a Research aims:The effects and mechanisms of exogenous EETs on NIT-1 cells apoptosis induced by TNF-aResearch methods:1. NIT-1 cells were cultured in DMEM media containing 10%FBS, and were kept in an incubator with 37℃and 5%CO2. When grow to 90%confluence in 75 cm2 flasks, NIT-1 cells were passaged by 0.25%trypsin, and then were treated with synchronization. That is, when cell growth to 70%-80%confluence, NIT-1 cells were placed in reduced serum (0.5%FBS) media for 12 h after cell attachment, and then reduced serum media were changed to media without serum.2. After synchronization, flow cytometry assays were carried out to determine the effects of exogenous EETs on NIT-1 cells apoptosis induced by TNF-a;3. After synchronization, MAPK, MEK, PI3K, and PPARy inhibitors Apigenin, PD98059, LY294002 and GW9662 and NIT-1 cells, as well as 14,15-EET were incubated together for 24 h, and then flow cytometry assays were carried out to determine the effects of exogenous EETs on NIT-1 cells apoptosis induced by TNF-a;4. After synchronization, TNF-a was added into the medium for the induction of NIT-1 cells apoptosis. EETs and NIT-1 cells were incubated together for 24 h, and then cells were collected, and cell proteins were extracted. Western blot was carried out to detect Bcl-2, Bcl-XL, Bax, PI3K, P-T308-AKT and P-MAPK expression in NIT-1 cells;5. After synchronization, TNF-a was added into the medium for the induction of NTT-1 cells apoptosis. MAPK, MEK, PI3K, and PPARy inhibitors Apigenin, PD98059, LY294002 and GW9662 and NIT-1 cells, as well as 14,15-EET were incubated together for 24 h, and then cells were collected, and cell proteins were extracted. Western blot was carried out to detect Bcl-2, Bcl-XL, and Bax expression in NIT-1 cells.Research results:1.8,9-EET,11,12-EET and 14,15-EET significantly inhibited NIT-1 cells apoptosis induced by TNF-a. The results showed that EETs significantly reduced the ratio of apoptotic cells.The ratios of apoptotic cells in the group of Control,PBS,DMSO and PBS +DMSO were(18.33±1.45)％,(17.33±1.42)％,(29.3±1.72)％and(29.5±2.1)％respectively.The ratios of apoptotic cells in the group of TNF-αand TNF-α+DMSO were (62.1±4.4)％and(77.0±3.1)％respectively,while in the group of TNF-α+8,9-EET, TNF-α+11,12-EET,and TNF-α+14,15-EET were(37.7±1.1)％,(50.7±4.8)％and(35.2±3.3)％respectively.The differences of the ratio of apoptotic cells between group of TNF-α+8,9-EET,TNF-α+11,12-EET and TNF-α+14,15-EET and TNF-αgroup,as well as TNF-α+DMSO group were statistically significant(P<0.05).2.MAPK inhibitor Apigenin,MEK inhibitor PD98059 and P13K inhibitor LY294002 significantly inhibited the decrease of NIT-1 cell apoptosis induced by 14,15-EET.The ratios of apoptotic cells in the DMSO group were (32.0±2.3)％,and in the group of TNF-αand TNF-α+DMSO were(68.7±4.1)％and(78.3±2.6)％respectively.The ratios of apoptotic cells in the group of TNF-α+14,15-EET+Apigenin,TNF-α+14,15-EET+PD98059,TNF-α+14,15-EET+LY294002 and TNF-α+14,15.EET+ GW9662 were(65.1±2.5)％,(63.7±3.0)％,(64.0±3.5)％and(51.3±2.2)％.The differences of ratios of apoptotic cells between TNF-α+14,15.EET+Apigenin groups, TNF-α+14,15-EET+PD98059 group,as well as TNF-α+14,15-EET+LY294002 group and TNF-α+14,15-EET group were statistically significant(P<0.05).In contrast,the difierences of ratios of apoptotic cells between TNF-α+14,15-EET+GW9662 group and TNF-α+14,15-EET group were not statistically significant(P>0.05).3.TNF-αsignificantly decreased Bcl-2,Bcl-XL,P-MAPK,P13K and P-AKT expression levels(P<0.05),and significantly increased Bax expression levels in NIT-1 cells (P<0.05).8,9-EET,11,12-EET and 14,15-EET significantly inhibited the decrease of Bcl-2, Bcl-XL,P-MAPK,PI3K and P-AKT expression levels(P<0.05),and the increase of Bax expression levels induced by TNF-αin NIT-1 cells(P<0.05).4.The results showed that MAPK,MEK and P13K Inhibitors Apigenin,PD98059 and LY294002 significantly inhibited the increase of Bcl-2 and Bcl-XL expression levels (P<0.05), and the decrease of Bax expression levels induced by 14,15-EET in NIT-1 cells (P<0.05), while PPARγinhibitor GW9662 had no significant effects on the increase of Bcl-2 and Bcl-XL expression levels, and the decrease of Bax expression levels induced by 14,15-EET in NIT-1 cells (P>0.05).Statistical AnalysisContinuous data were expressed as means±S.E.M. Comparisons between groups were performed by a one-way analysis of variance. Two-way analysis of variance was used to examine differences in response to treatments and between groups, with post hoc analyses performed using the Student-Newman-Keuls method. Statistical significance was defined as P< 0.05.Conclusions1. CYP2J3 was successfully expressed in various tissues and organs of C57BL/6 and db/db type 2 diabetic mice, and was functionally active in the metabolism of arachidonic acid into EETs.2. CYP2J3 overexpression improved insulin resistance in db/db type 2 diabetic mice, and its possible molecular mechanisms are that CYP2J3 activated IRS-1/PI3K/AKT/eNOS and AMPK signaling pathways, indicating that EETs is an endogenous insulin-sensitizing agent.3. Exogenous EETs promoted NIT-1 cells proliferation, and protected NIT-1 cells from apoptosis induced by TNF-a via activation of MAPK and PI3K/AKT signaling pathways.4. Exogenous EETs promoted NIT-1 cell insulin secretion after glucose loading.5. CYP2J3 and EETs system slowed the progression of type 2 diabetes via improving insulin resistance and improving peripheral tissue sensitivity to insulin, as well as enhancing pancreatic (3 cells function, indicating that EETs possess potent anti-diabetic properties. 6. The findings of this study provide a new research direction, ideas and theoretical basis for future studies of prevention and treatment of insulin resistance and diabetes, as well as a new strategy to find new drug targets.
|
PDF Full Text Request