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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Obesity (Silver Spring). 2015 Jun 5;23(7):1401–1413. doi: 10.1002/oby.21115

CYP2J2 Overexpression Ameliorates Hyperlipidemia via Increased Fatty Acid Oxidation Mediated by the AMPK Pathway

Shasha Zhang 1, Guangzhi Chen 1, Ning Li 2, Meiyan Dai 1, Chen Chen 1, Peihua Wang 1, Huiru Tang 2, Samantha L Hoopes 3, Darryl C Zeldin 3, Dao Wen Wang 1, Xizhen Xu 1
PMCID: PMC4565055  NIHMSID: NIHMS720403  PMID: 26053032

Abstract

Objective

The study aims to investigate the effect of Cytochrome P450 2J2 (CYP2J2) overexpression on hyperlipidemia in mice and further to explore their effect on fatty acid oxidation in vivo and in vitro.

Methods

The effects and mechanisms of endothelial-specific CYP2J2 transgene (Tie2-CYP2J2-Tr) on lipid and fatty acids metabolism were investigated in high fat diet (HFD)-treated mice. HepG2, LO2 cells and HUVECs were exposed to 0.4 mM free fatty acid (FFA) for 24h and used as a model to investigate the roles of CYP2J2 overexpression and epoxyeicosatrienoic acids (EETs) on fatty acid β oxidation in vitro.

Results

Tie2-CYP2J2-Tr mice had significantly lower plasma and liver triglycerides, lower liver cholesterol and fatty acids, and the reduction in HFD-induced lipid accumulation. CYP2J2 overexpression resulted in activation of the hepatic and endothelial AMPKα, increased ACC phosphorylation, increased expression of CPT-1 and PPARα, which were all reduced by HFD treatment. In FFA-treated HepG2, LO2 and HUVECs, both CYP2J2 overexpression and EETs significantly decreased lipid accumulation and increased fatty acid oxidation via activating the AMPK and PPARα pathway.

Conclusions

Endothelial specific CYP2J2 overexpression alleviates HFD–induced hyperlipidemia in vivo. CYP2J2 ameliorates FFA-induced dyslipidemia via increased fatty acid oxidation mediated by the AMPK and PPARα pathway.

Keywords: Cytochrome P450 epoxygenase, Epoxyeicosatrienoic acids, Hyperlipidemia, AMP-activated protein kinase, Peroxisome proliferator activated receptor α

Introduction

Dyslipidemia can be defined as a disease in which total triglycerides and cholesterol are increased while there is a decrease in HDL. Dyslipidemia is a prevalent risk factor for the development of cardiovascular disease (CVD) and is associated with insulin resistance and obesity (1). Although hyperlipidemia can be prevented by diet control and exercise, people with a higher risk of developing hyperlipidemia require hypolipidemic therapy to help prevent CVD. Recent studies have demonstrated that AMPK reduces plasma triglyceride level and attenuates hepatic lipid accumulation in mice with diet-induced obesity (2,3). PPARα is another key regulator of fatty acid metabolism that has been used for the treatment of dyslipidemia for many years. Therefore, AMPK and PPARα may be potential therapeutic targets for treatment of dyslipidemia, obesity and hepatic disorders.

Cytochrome P450 2J2 (CYP2J2), a human epoxygenase, metabolizes arachidonic acid to four regioisomeric epoxyeicosatrienoic acid (5,6-, 8,9-, 11,12- and 14,15-EET). EETs are expressed in both hepatic and extrahepatic organs and have diverse biological activities (4). Early studies have shown that EETs can cause vasodilation of vascular beds by activating calcium-sensitive potassium channels (5,6). EETs are also anti-inflammatory in endothelial cells (7), and furthermore EETs stimulate endothelial cell growth and angiogenesis (8), and protect endothelial cells from apoptosis (9). Moreover, transgenic mice with expressing human CYP2J2 that was subcloned downstream of the murine Tie2 promoter to drive endothelial expression were generated and have increased endothelial-derived EETs, increased vasodilation and lower blood pressure following induction of hypertension (10).

In our recent studies, CYP2J3 gene delivery or CYP2J2 overexpression were found to increase EETs generation, improve insulin sensitivity and ameliorate diabetic nephropathy in hypertensive rats and diabetic mice (11,12). Endothelial CYP2J2 overexpression attenuated adiposity and vascular dysfunction in mice fed with HFD (13). As we all know, liver tissue, the most important organ in regulating plasma lipoproteins and endogenous lipids, is highly vascularized, and, vice versa, most of the vasculature is in the liver tissue. Moreover, our previous study used endothelial-specific CYP2J2 overexpression (Tie2-CYP2J2-Tr) mice to investigate the protective effect of endothelial-specific CYP2J2 overexpression on NAFLD induced by HFD (14). Taken together, these data indicated that CYP2J2 overexpression played an important role in lipid metabolism. So, whether CYP2J2 overexpression has beneficial effects on hyperlipidemia remains unknown. In this study, we used Tie2-CYP2J2-Tr mice to investigate the effect of CYP2J2 on HFD-induced hyperlipidemia. Interestingly, CYP2J2 overexpression ameliorated hyperlipidemia through enhanced fatty acid β-oxidation mediated by AMPK and PPARα pathway activation.

Methods

Animals

All animal studies were approved by The Academy of Sciences of China and complied with standards stated in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Tie2-CYP2J2-Tr mice were generated on a pure C57BL/6 genetic background as previously described (10). Eight-week-old male Tie2-CYP2J-Tr mice and wild type (WT) controls were divided into normal chow and high fat diet (HFD) (fat, 60.0%) groups (4 groups, 10 mice each) for 16 weeks. At the end of the experiment, all the mice were fasted for 12 hours and anesthetized with pentobarbital (50 mg/kg body weight). Plasma was collected for measuring triglyceride (TG), cholesterol (TC) levels. Serum levels of liver enzymes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined on a clinical autoanalyser (Beckman coulter DX). Liver samples were collected, immediately snap-frozen in liquid nitrogen, and then stored at −80°C.

Cell culture and treatment

HepG2, LO2 and HUVECs, which were obtained from the ATCC, were infected with rAAV2-GFP and rAAV2-CYP2J2 (about 100 100 vector genomes/cell) that were packaged and purified by a single-step gravity-flow column purification method as previously described (8). After synchronization, the cells were incubated in modified DMEM with 1% (wt/vol) BSA alone or 0.4 mM free fatty acid (FFA) complexed to 1% (wt/vol) BSA for 24h. FFA/BSA complex solution was prepared as previously described (15). Compound C (20 µM) or GW6471 (1 µM) was added into medium 30 min prior to FFA treatment. For some experiments, the cells were cultured with 14,15-EET (1 µM) and 14,15-EEZE (10 µM) 30 min prior to treatment with FFA.

Determination of triglyceride (TG), cholesterol (TC) and β-hydroxybutyrate in plasma and liver tissue

The tissues and cell lysates were homogenized and total lipids were extracted using of a mixture of chloroform:methanol (2:1). TG, TC and β-hydroxybutyrate concentrations in plasma and liver were measured by the commercial kits (Zhongsheng, Beijing, China) (16).

GC-FID/MS Analysis of Fatty Acid Composition

Plasma fatty acids were methylated as previously described (17) with some modifications. For liver tissues, 10 mg of sample was homogenized with methanol (500 µl) using a Tissue Lyser at 20 Hz for 90 s. 100 µl of such homogenate mixture was transferred into a Pyrex tube to be methylated according to the above procedures. Methylated fatty acids were performed on a Shimadzu GC2010Plus GC-MS spectrometer (Shimadzu Scientific Instruments, USA) equipped with a flame ionization detector (FID) and a mass spectrometer with an electron impact (EI) ion source. Each fatty acid was quantified with the FID data from its signal integrals and internal standards.

Western blotting analysis

Western blots were performed as described previously (18). Briefly, equal amounts of protein were separated on a 10% SDS-PAGE and electrophoretically transferred onto PVDF membranes. The membranes were incubated with 5% non-fat dry milk in TBST for 2 hours, then incubated overnight at 4°C with the indicated primary antibodies. The proteins were visualized by enhanced chemiluminescence (ECL).

RNA isolation and quantitative real-time PCR

Total RNA was extracted from frozen livers using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was reversely transcribed using the TransScript First-Strand cDNA Synthesis kit and quantitative real-time PCR was performed on an ABI7900 PCR system (Applied Biosystems, Darmstadt, Germany) using the TransStart™ Eco Green qPCR Kit (Qiagen, Valencia, CA). The sequences of the sense and antisense primers used for amplification were as follows: CPT-1, 5’-AGATCAATCGGACCCTAGACAC-3’ and 5’-CAGCGAGTAGCGCATAGTCA-3’; PPARα, 5’-AACATCGAGTGTCGAATATGTGG-3’ and 5’-CCGAATAGTTCGCCGAAAGAA-3’; GAPDH, 5’-TGGCCTTCCGTGTTCCTAC-3’ and 5’-GAGTTGCTGTTGAAGTCGCA-3’.

Statistical analysis

All data are expressed as the mean ± standard error (SEM). The data was analyzed by a Student’s t test for two groups and by one-way analysis of variance (ANOVA) for multiple groups. P<0.05 was considered statistically significant.

Results

CYP2J2 overexpression attenuates metabolic dysfunction in hyperlipidemic mice

As expected, HFD treatment resulted in significantly increased body weight, subcutaneous and visceral fat mass in WT mice; Tie2-CYP2J2-Tr mice treated with HFD had lower body weight, subcutaneous and visceral fat mass than HFD-fed WT mice as shown in Figure S1. However, there was no significant difference in body weight and fat mass between WT and Tie2-CYP2J2-Tr mice treated with normal chow (Figure S1). In addition, food intake was also measured; and interestingly, there was no significant difference in food intake between WT mice and Tie2-CYP2J2-Tr mice fed with HFD (Figure S1). These data indicated that endothelial specific CYP2J2 overexpression markedly prevented body weight gain in mice treated with high fat diet.

As expected, HFD resulted in significantly increased plasma and liver triglycerides and cholesterol in WT mice; however, Tie2-CYP2J2-Tr mice with HFD had lower plasma (Figure 1A) and liver (Figure 1B) triglycerides than HFD-fed WT mice, indicating that CYP2J2 overexpression significantly decreased plasma and liver triglyceride level in hyperlipidemic mice. HFD resulted in significantly increased plasma and liver cholesterol in WT mice (Figure 1C and D). Tie2-CYP2J2-Tr mice on HFD had lower liver cholesterol than HFD-fed WT mice (Figure 1D). There was a reduction tendency in plasma cholesterol in Tie2-CYP2J2-Tr mice on HFD, it did not reach the significant difference (Figure 1C). Both plasma and liver β-hydroxybutyrate was significantly decreased in HFD treated mice, but these effects were blocked by CYP2J2 overexpression, demonstrating that there is an actual increase in fatty acid oxidation (19) (Figure 1 E and F). Moreover, CYP2J2 overexpression also reversed ALT and AST abnormalities in mice caused by HFD treatment (Figure 1G and H). These results indicate that CYP2J2 overexpression attenuates metabolic dysfunction in hyperlipidemic mice.

Figure 1. CYP2J2 overexpression attenuates metabolic dysfunction in HFD induced mice.

Figure 1

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WT and Tie2-CYP2J2-Tr mice were fed normal chow or HFD for 16 weeks. (A, B) Triglyceride (TG) content in plasma and liver, (C, D) cholesterol levels and (E, F) β-hydroxybutyrate in plasma and liver from WT and Tie2-CYP2J2-Tr mice in both normal chow and HFD groups. (G, H) Serum ALT (G) and AST (H) levels in WT and Tie2-CYP2J2-Tr mice from both normal chow and HFD groups. Data are expressed as mean ± SEM (n=6 per group); *P<0.05 vs. WT mice in normal chow group; #P<0.05 vs. WT mice in HFD group.

CYP2J2 overexpression regulates fatty acid composition in both plasma and liver

GC-FID/MS results showed CYP2J2 overexpression induced changes in fatty acids composition in both plasma and liver samples in hyperlipidemic mice. In addition to C22:6n3, HFD significantly increased plasma total fatty acid (ToFA), unsaturated fatty acid (UFA), saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) level in WT mice. HFD induced the increase in many fatty acids levels in plasma including some PUFAs (C20:3n6, C20:4n6), MUFAs (C18:1n9), and SFAs (C18:0), whereas the decrease in some PUFAs (C18:2n6, C18:3n6, C18:3n3 and C20:5n3) and MUFAs (C16:1n7) (Figure 2). However, there was no significant difference in fatty acid composition (UFA, SFA, MUFA, PUFAs, MUFAs and SFAs) in plasma between WT and Tie2-CYP2J2-Tr mice in HFD group (Figure 2). For liver, HFD significantly increased ToFA, UFA, MUFA and SFA, as well as many fatty acids including some SFAs (C14:0, C16:0, C18:0, and C20:0), MUFAs (C16:1n7, C18:1n7, C18:1n9 and C20:1) and PUFAs (C20:2, C18:3n6, C20:3n6 and C20:4n6) level, whereas some PUFAs (C18:3n3, C20:5n3 and C22:6n3) levels were significantly decreased in HFD treated mice (Figure 3). However, HFD had no significant effect on PUFA and C18:2n6 fatty acid level. Interestingly, Tie2-CYP2J2-Tr mice in HFD group partly decreased liver fatty acid composition (ToFA, UFA, SFA, MUFA, SFAs, MUFAs and PUFAs) except for C18:0 fatty acid (Figure 3). GC-FID/MS results indicated that HFD caused widespread metabolic changes in fatty acid composition in plasma and liver. CYP2J2 overexpression markedly attenuated liver fatty acids composition induced by HFD, but it had no significant effect on plasma fatty acids composition.

Figure 2. CYP2J2 overexpression regulates fatty acids changes in plasma of hyperlipidemic mice.

Figure 2

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WT and Tie2-CYP2J2-Tr mice were fed normal chow or HFD for 16 weeks. Fatty acids composition for plasma calculated from GC-FID/MS results. Data are mean ± SEM (n=6 per group). *P<0.05 vs. WT mice in normal chow group.

Figure 3. CYP2J2 overexpression regulates fatty acids changes in livers of hyperlipidemic mice.

Figure 3

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WT and Tie2-CYP2J2-Tr mice were fed normal chow or HFD for 16 weeks. Fatty acids composition for livers calculated from GC-FID/MS analysis. Data are mean ± SEM (n=6 per group). *P<0.05 vs. WT mice in normal chow group; #P<0.05 vs. WT mice in HFD group.

CYP2J2 overexpression regulates key enzymes involved in FFA metabolism in liver and blood vessel

We next detected the expression of genes involved in fatty acid β-oxidation in the liver. Quantitative real-time PCR analysis revealed that the expression of CPT-1 and PPARα was reduced in WT mice treated with HFD, while CYP2J2 overexpression abrogated this response (Figure 4A). Hepatic AMPKα phosphorylation was decreased (Figure 4B and C), and moreover phosphorylated ACC and CPT-1, the rate-limiting enzymes for fatty acid synthesis and β-oxidation in liver (20,21), were also reduced in response to HFD. However, CYP2J2 overexpression partly reversed these changes (Figure 4B and C). CYP2J2 overexpression also abolished HFD-induced reduction in hepatic PPARα expression (Figure 4B and C). Endothelial-specific CYP2J2 overexpression has the similar effect on vascular fatty acid oxidation related gene and proteins (Figure 4D–F). These data suggest that CYP2J2 overexpression inhibited the HFD-induced reduction in genes and enzymes involved in hepatic and vascular fatty acid β-oxidation.

Figure 4. CYP2J2 overexpression attenuates the HFD-induced reduction in FFA β-oxidation enzymes in livers and blood vessel in vivo.

Figure 4

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WT and Tie2-CYP2J2-Tr mice were fed normal chow or HFD. After 16 weeks, livers and blood vessel were used for RT-PCR and immunoblotting analysis. (A) CPT-1 and PPARα mRNA expression in liver. Western blots of p-AMPKα, p-ACC, CPT-1, and PPARα expression (B) followed by densitometry quantitation (C). (D) CPT-1 and PPARα mRNA expression in blood vessel. Western blots of vascular p-AMPKα, p-ACC, CPT-1, and PPARα expression (E) followed by densitometry quantitation (F). Data are mean ± SEM (n=6 per group). *P<0.05 vs. WT mice in normal chow group; #P<0.05 vs. WT mice in HFD group.

CYP2J2 overexpression or 14,15-EET treatment decreases intracellular triglyceride accumulation in FFA-treated cells

In order to investigate the effects of EETs on triglyceride production in vitro, two liver cell lines, HepG2 and LO2, and endothelial cell line, HUVEC cells, were treated with FFA after being transfected with rAAV-CYP2J2 or pre-incubated with 14,15-EET. Results revealed that FFA-treated cells had an increased accumulation of triglyceride compared to untreated cells (Figure 5 and Figure S1); however, CYP2J2 overexpression or 14,15-EET treatment suppressed intracellular triglyceride accumulation in FFA-treated cells. These effects were inhibited by antagonists of EETs (14,15--EEZE), AMPK (Compound C), and PPARα (GW6471) (Figure 5 and Figure S1). These results suggest that CYP2J2 and EETs decrease intracellular triglycerides via activating AMPKα and PPARα signaling pathways.

Figure 5. CYP2J2 overexpression or 14,15-EET treatment decreases intracellular triglyceride accumulation in FFA-treated cells in vitro.

Figure 5

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HepG2 cells and HUVECs were incubated with 0.4 mmol/L FFA and/or transfected with CYP2J2 or treated with 14,15-EET for 24 h. Compound C, GW6471 or 14,15-EEZE were added to the medium 30 min prior to the FFA treatment. HepG2 cells (A, C) and HUVECs (B, D) were lysed and the intracellular TGs were quantified. The data are presented as mean ± SEM (n=6 per group). *P<0.05 vs. untreated control; #P<0.05 vs. FFA-treated cells; &P<0.05 vs. CYP2J2 or 14,15-EET and FFA-treated cells.

CYP2J2 overexpression or 14,15-EET treatment increases AMPK phosphorylation and enhances fatty acid β-oxidation in hepatic cells

To further investigate the involvement of CYP2J2 and 14,15-EET in enhanced FFA catabolism, we determined the protein levels of p-AMPK, p-ACC and CPT-1 in HepG2 (Figure 6A–C) and LO2 (Figure S2A–C) cells after rAAV-CYP2J2 transfection. CYP2J2 overexpression significantly increased p-AMPK, p-ACC and CPT-1 expression (Figure 6A–C; Figure S2A–C) which were reduced by FFA. These effects of CYP2J2 overexpression were partially blocked with Compound C (AMPK antagonist) pretreatment, suggesting that the triglyceride lowering effect of CYP2J2 overexpression is mediated by the AMPK pathway. Interestingly, CYP2J2 overexpression significantly increased PPARα expression (Figure 6C; Figure S2A and C) compared to cells incubated with only FFA. Compound C markedly blocked this effect, suggesting that CYP2J2 overexpression activated AMPK, and ultimately resulting in PPARα activation. Similar to the effects observed with CYP2J2 overexpression, pretreatment with 14,15-EET also activated the AMPK pathway and PPARα in both HepG2 (Figure 6D–F) and LO2 (Figure S2D–F) cells as evidenced by increased p-AMPK, p-ACC and CPT-1, PPARα expression, which were all attenuated by 14,15-EEZE treatment. These results demonstrated that the protective role of EETs in reducing triglyceride accumulation occurred through enhancement of mitochondrial β-oxidation mediated by AMPK and PPARα pathway.

Figure 6. CYP2J2 overexpression or 14,15-EET regulates key enzymes of mitochondrial β-oxidation in FFA-treated HepG2 cells.

Figure 6

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(A, B, C) p-AMPKα, AMPKα, p-ACC, ACC, CPT-1, PPARα and β-actin expression regulated by CYP2J2 in FFA-treated HepG2 cells were assessed by immunoblotting and densitometry analysis. (D, E, F) Fatty acid oxidation related proteins regulated by 14,15-EET were assessed by immunoblotting (D) and densitometry analysis (E, F). The data are presented as mean ± SEM (n=6 per group). *P<0.05 vs. untreated control; #P<0.05 vs. FFA-treated cells; &P<0.05 vs. CYP2J2 or 14,15-EET and FFA-treated cells.

AMPK activation induced by CYP2J2 or 14,15-EET enhances mitochondrial β-oxidation partially through PPARα in hepatic cells

In FFA-treated HepG2 and LO2 cells, CYP2J2 overexpression or 14,15-EET treatment significantly increased CPT-1 and PPARα expression (Figure 7A and B; Figure S3A and B), which could be partially blocked by GW6471.These results demonstrated that CYP2J2 overexpression or EETs regulates fatty acid β oxidation in vitro via activating PPARα pathway mediated by AMPK.

Figure 7. CYP2J2 overexpression or 14,15-EET treatment increases FFA β-oxidation via PPARα in FFA-treated HepG2 cells.

Figure 7

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HepG2 cells were incubated with 0.4mmol/L FFA only or with rAAV-CYP2J2 and 14,15-EET. GW6471 was added into medium 30 min prior to the FFA treatment. (A, B) Protein expression of CPT-1 and PPARα were assessed by immunoblotting and densitometry analysis. (C, D, E, F) Fatty acid oxidation related proteins increased by CYP2J2 expression were reduced by 14,15-EET inhibitor 14,15-EEZE. The data are presented as mean ± SEM (n=6 per group). *P <0.05 vs. untreated control; #P<0.05 vs. FFA-treated cells; &P<0.05 vs. 14,15-EET and FFA-treated cells.

Additionally, to confirm whether the effects of CYP2J2 was EET-dependent, 14,15-EEZE was used in FFA-induced dyslipidemia in vitro, and we found that CYP2J2 overexpression activated the AMPK pathway in hepatic cells as evidenced by increased p-AMPK, p-ACC and CPT-1 expression, which were partially reversed by 14,15-EEZE treatment. The effect of CYP2J2 on PPARα expression was also partially blocked by 14,15-EEZE (Figure 7C–F; Figure S3C and D), suggesting that the hypolipidemic effect of CYP2J2 overexpression is EET-dependent.

CYP2J2 overexpression or 14,15-EET treatment enhances FFA β-oxidation via activation AMPK and PPARα in HUVECs

In this study, Tie2-CYP2J2-Tr mice were used, so the effects of CYP2J2 or 14,15-EET on endothelial cells FFA β oxidation were explored. The effects of CYP2J2 overexpression or 14,15-EET on p-AMPK, p-ACC and CPT-1 were partially blocked with Compound C or 14,15-EEZE. Moreover, compared to cells incubated with only FFA, the expression of PPARα were also increased by CYP2J2 or 14,15-EET treatment, which were markedly blocked by Compound C or 14,15-EEZE addition (Figure 8A–C). The effects of CYP2J2 overexpression or 14,15-EET on CPT-1 and PPARα expression could be partially blocked by pretreatment with GW6471 (Figure 8D). The effects of CYP2J2 on fatty acid β oxidation related proteins were also partially blocked by 14,15-EEZE (Figure 8E–G), confirming that the effects of CYP2J2 is EET-dependent. These results demonstrated that the protective role of EETs on HUVECs fatty acid β-oxidation was partially mediated by the AMPK/PPARα pathway activation.

Figure 8. CYP2J2 overexpression or 14,15-EET regulates key enzymes of FFA β-oxidation by activating AMPK pathway in FFA-treated HUVECs.

Figure 8

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HUVECs were incubated with 0.4mmol/L FFA only or with rAAV-CYP2J2 and 14,15-EET. Compound C, 14,15-EEZE and GW6471 were added into medium 30 min prior to the FFA treatment. (A, B, C, D) Western blot and densitometry analysis were done for fatty acid oxidation related proteins. (E, F, G) Fatty acid oxidation related proteins increased by CYP2J2 expression were reduced by 14,15-EEZE. The data are presented as mean ± SEM (n = 6 per group). *P <0.05 vs. untreated control; #P<0.05 vs. FFA-treated cells; &P<0.05 vs CYP2J2 or 14,15-EET and FFA-treated cells.

Discussion

This study was undertaken to determine the involvement of endothelial CYP2J2 overexpression in regulating lipid metabolism in response to HFD treatment using Tie2-CYP2J2-Tr mice in vivo. We also examined the effects and mechanisms of CYP2J2 overexpression or EET treatment on FFA-induced dyslipidemia in HepG2, LO2 and HUVECs. Results showed that WT mice fed with HFD exhibited increased plasma and liver triglycerides and cholesterol, liver fatty acids, as well as increased serum ALT and AST; however, in addition to plasma cholesterol, these increases were not observed in the Tie2-CYP2J2-Tr mice fed with HFD. In vitro, CYP2J2 overexpression or 14,15-EET treatment significantly improved lipid metabolism via enhanced fatty acid β-oxidation in hepatocytes and HUVECs with FFA treatment.

Previous studies have demonstrated that mice treated with long-term HFD develop hypertriglyceridemia, hepatic steatosis, systemic hypertension and hyperinsulinemia (22). However, the pathophysiological mechanisms responsible for elevated plasma triglycerides and hepatic steatosis in HFD-induced animals are not completely understood. FFA also plays an important role in regulating plasma lipid levels, especially during triglyceride metabolism (23). Promoting FFA catabolism could ameliorate HFD-induced hyperlipidemia and lipid accumulation in the liver demonstrating the critical role of lipid metabolism in hyperlipidemia and fatty liver (19). In this study, we fed mice with HFD to induce hyperlipidemia in vivo and demonstrated that HFD induced higher plasma triglycerides and cholesterol, increased lipid accumulation in liver as compared to those on a normal chow. In vitro, we used FFA to stimulate lipid accumulation in hepatic and endothelial cell lines, which were typically associated with increased plasma triglycerides in both animal and human experiments. FFA significantly increased intracellular triglyceride content indicating that using FFA-induced hepatic and endothelial cells to study the effect of CYP2J2 or EETs on lipids metabolism is feasible.

Numerous studies have shown that CYP2J2 and EETs have diverse biological effects in metabolic disorders including type 2 diabetes, obesity and dyslipidemia. For example, EETs directly stimulated isolated pancreatic islet cells to release insulin (24). CYP2J3 overexpression ameliorated diabetic symptoms and reduced insulin resistance in fructose-induced diabetic rats (25). Although EETs agonists attenuated fatty acid synthase expression and lipid accumulation (26), the underlying effects of CYP2J2 on hyperlipidemia is not completely understood. In the present study, CYP2J2 overexpression significantly attenuated HFD-induced changes in plasma and liver triglyceride levels and liver cholesterol level in mice. CYP2J2 overexpression or 14,15-EET also attenuated lipid accumulation in FFA-induced HepG2, LO2 and HUVECs respectively.

Systemic biology approaches, such as transcriptomics, proteomics and metabolomics methods, played important roles in finding the changes in metabolic disorders. Previous studies have reported that HFD significantly led to transcript and proteomic alterations of many genes including up-regulation of genes involving glycogen synthesis, lipogenesis and down-regulation of genes regulating lipid catabolism (27,28). In this study, we demonstrated that HFD not only reduced the hepatic mRNA expression of CPT-1 and PPARα in WT mice, but also decreased AMPKα and ACC phosphorylation level, as well as CPT-1 and PPARα expression. Interestingly, CYP2J2 overexpression significantly attenuated these changes. In addition, we primarily observed that HFD treatment led to higher levels of many fatty acids in plasma and liver in mice. CYP2J2 overexpression markedly reversed HFD-induced fatty acids changes in liver. Fatty acid played an important role in regulating triglyceride metabolism. Thus, the reduction of hepatic fatty acids in Tie2-CYP2J2-Tr mice may be involved in the hypolipidemic effect of CYP2J2. However, CYP2J2 had no significant effect on fatty acid composition in plasma. Plasma TG must first be hydrolyzed into FFA, and then is taken up and utilized by hepatic or exhepatic tissues (19). In other words, the level of plasma fatty acid may be regulated by many factors, such as the lipolysis of plasma TG, the transport of FFA, the uptake or utilization of hepatic or exhepatic tissues. These may be the reasons why CYP2J2 could decrease hepatic TG level and fatty acid composition, which were a major contributor to reduced plasma lipid, but still had no impact on plasma fatty acid composition.

AMPK is a heterotrimeric protein composed of catalytic α, regulatory β, and γ subunits (29). AMPK, an upstream regulator of ACC and CPT-1, was a critical enzyme involved in lipid metabolism and FFA oxidation (30). Increased AMPK phosphorylation reduced ACC activity resulting in a subsequent decrease in malonyl-CoA content and increased CPT-1 expression, ultimately resulting in increased β-oxidation (31). Moreover, CYP2J3 gene delivery or CYP2J2 overexpression increased adiponectin levels, reduced blood pressure, alleviated insulin resistance and diabetic cardiomyopathy via activating AMPKα in hypertensive rats and diabetic mice (12,25,32,33). EETs agonist and CYP2J2 overexpression increased adiponectin levels and restored the decreased adipocyte AMPK activation, in animals fed with a high-fat diet (13,34). These data indicated that AMPK activation by EETs mediated adiponectin upregulation is involved in improved insulin resistance and adiposity. In this study, our data indicated that endothelial CYP2J2 overexpression markedly improved lipid metabolism through enhancement of liver fatty acid β-oxidation mediated by AMPK activation and subsequent phosphorylation of ACC and increased CPT-1 expression as well as increased both plasma and liver β-hydroxybutyrate level. In addition, fatty acid β oxidation by peripheral tissues (i.e. skeletal muscle, brown adipose tissue) may actually underlie the plasma lipid differences, since CYP2J2 is also overexpressed with Tie2- promoter in these vascular beds. Likewise, circulating EETs may also play a signaling role in these very metabolically active tissues. It is speculated that fatty acid β oxidation in muscle, brown adipose tissue et al may also be involved in the beneficial effects of CYP2J2 overexpression on plasma lipid level in mice treated with high fat diet.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor subfamily that play an important role in regulating lipid and glucose metabolism (35). PPARα was the first isoform to be identified and is expressed at high levels in tissues with increased rates of fatty acid oxidation including the liver, kidney, heart, and skeletal muscle (36). Targeted disruption of the PPARα gene in mice revealed that PPARα plays an important role in mitochondrial fatty acid β-oxidation (37). However, the relationship between AMPK and PPARα in the regulation of fatty acid oxidation is not completely understood. In the present study, we demonstrated that CYP2J2 overexpression and 14,15-EET treatment activated PPARα both in vivo and in vitro and these effects were blocked by Compound C and 14,15-EEZE treatment. These inhibitors also altered CYP2J2 overexpression and EET induced changes in expression of enzymes involved in fatty acid β-oxidation. These results demonstrated that CYP2J2 overexpression or EETs increased FFA oxidation via activating PPARα mediated by the AMPK pathway.

In conclusion, we have determined the effects of CYP2J2 overexpression on hyperlipidemia and the underlying mechanism involved in this process. Endothelial specific CYP2J2 overexpression decreased plasma and liver triglycerides, liver fatty acids composition in hyperlipidemic mice via enhanced FFA β-oxidation. These effects were at least in part mediated by AMPK and PPARα pathway activation. Therefore, CYP2J2 overexpression induced beneficial effects including ameliorating hyperlipidemia and hepatic lipid accumulation in high fat diet treated mice that may ultimately help to improve metabolic syndromes.

What is already known about this subject?

  1. Hyperlipidemia is a prevalent risk factor for the cardiovascular disease and is associated with obesity and insulin resistance.

  2. Hyperlipidemia could be ameliorated by decreased lipid synthesis and/or increased lipid metabolism.

  3. CYP2J2-derived EETs have diverse biological effects on the cardiovascular system and metabolic disorders such as diabetes.

What does this study add?

  1. This study demonstrates that CYP2J2 overexpression attenuates hyperlipidemia in HFD-induced mice.

  2. CYP2J2 ameliorates hyperlipidemia characterized by decreased fatty acids level in livers but not in plasma in HFD-induced mice.

  3. CYP2J2 ameliorates hyperlipidemia by increased fatty acid oxidation via activating the AMPK pathway, which is partially mediated by PPARα.

Acknowledgments

Funding

This work was supported by the 973 program (Grant No. 2012CB517801), National Nature Science Foundation Committee of China (Grant Nos. 81400369 and 81471021).

Abbreviations

Tie2-CYP2J2-Tr

transgenic mice with endothelial-specific CYP2J2 overexpression

CYP2J2

cytochrome P450 2J2

WT

wild type

TG

triglyceride

ALT

alanine aminotransferase

AST

aspartate aminotransferase

CVD

cardiovascular disease

14, 15-DHET

14, 15-dihydroxyeicosatrienoic acid

14, 15-EEZE

14, 15-epoxyeicosa-5(Z)-enoic acid

AMPK

AMP-activated protein kinase

FFA

free fatty acid

ACC

acetyl CoA carboxlyase

CPT-1

carnitine palmitoyl transferase-1

PPARα

peroxisome proliferator activated receptor α

Compound C

6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine

H&E

hematoxylin and eosin

HDL

high-density lipoprotein

DMEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

EETs

epoxyeicosatrienoic acids

AA

arachidonic acid

ToFA

total fatty acid

UFA(s)

unsaturated fatty acid(s)

PUFA(s)

polyunsaturated fatty acid(s)

MUFA(s)

monounsaturated fatty acid(s)

SFA(s)

saturated fatty acid(s)

GC-FID/MS

gas chromatography-flame ionization detector/mass spectrometer

rAAV

recombinant adeno-associated viral vector

Footnotes

Disclosure

The authors have no competing interests.

Authors contributions

Shasha Zhang conceived and carried out experiments, collected data, analyzed data. Guangzhi Chen, Meiyan Dai, Ning Li and Huiru Tang carried out experiments, collected data; Chen Chen, Peihua Wang, Samantha L. Hoopes and Darryl C. Zeldin conceived the experiments and reviewed the manuscript. Dao Wen Wang and Xizhen Xu conceived the experiments, designed the experiments, reviewed and edited the manuscript. All authors were involved in writing the paper and had final approval of the submitted and published versions.

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