Effects Of Fenofibrate On Relative Genes Of Myocardial Energy Metabolism In Mice

BackgroundPeroxisome proliferator-activated receptora (PPARa) is a ligand-inducible transcription factor that belongs to the nuclear-hormone-receptor family and function as transcription factors regulating the expression of genes involved in glucose and lipid metabolism. PPARs play essential roles in the regulation of cellular differentiation, development, and metabolism. Peroxisome proliferator-activated receptor gamma family genes contains PGC-la and PGC-1β that are closely related to mitochondria biogenesis, energy metabolism, fatty acid oxidation, glycometabolism and insulin secretion. PPAR aagonist fenofibrate is mainly used in the treatment of dyslipidaemia, and play a role in the therapy of high blood cholesterol or lipid II diabetes caused by metabolic disorders The mechanism is to accelerate its catabolism by inhibiting the production of VLDL and LDL, thereby reducing the very low density lipoprotein cholesterol and triglyceride content. In order to further understand its role in metabolism, we determined the expression level of genes involved in cardiac mitochondrial function following fenofibrate administration. At first, mice were treated with fenofibrate, then the RNA was extracted and genes involved in mitochondria function were detected, also analyzed the impact of fenofibrate in mice ventricular lipids. The above study was to investigate the role of fenofibrate on cardiac energy metabolism in mice.Fibrates can also enhance the body’s insulin sensitivity, and have an important role in alleviating diabetes and the resulting cardiovascular disease. We have found that in the research that fenofibrate receptor PPARa regulatory mechanism of glucose metabolism:when cells were treated with insulin, PPARa and the related protein GATA6both induced glucose consumption and the activities of citrate synthase, but high concentration of insulin induced cells apoptosis. To further investigate the relationship about fenofibrate, PPARa, GATA6, insulin and energy metabolism, we investigate the in vitro effect of insulin on cell proliferation, cell apoptosis and cell cycle distribution of P19mouse embryonal carcinoma cell-line and stable-transfected ppara or gata6cells. All these experiments explored the mechanism of different concentrations of insulin in the action of P19CL6cells and to study the relationship between insulin and cellular metabolism.PurposeIn this study, we determined the mRNA level of genes involved in cardiac mitochondrial function following7or14days of fenofibrate administration, such as NRF-l-L, NRF-l-S, Tom40, Lias, MCAD and PGC-la. On the other hand, we performed ventricular triglyceride analysis following7or14days of fenofibrate administration. Our analyses identified the relationship between PPARa ligand fenofibrate and cardiac energy metabolism in mice. In addition, we also explored the role of insulin that related to fenofibrate in P19CL6cells. Aims to explore the relationship between the mechanism of insulin and cell metabolism.Methods1. Eight-week-old mice were randomly divided into three groups:Group1, vehicle control; Group2, fenofibrate treatment for7days; and Group3, fenofibrate treatment for14days. Each group consisted of6-8mice. Fenofibrate was administered at a dose of1OOmg/kg/day. The vehicle control was orally administred in the0.5%sodium carboxymethylcellulose suspension for14days. RNA from ventricles was extracted with Trizol, according to the manufacture’s protocol. The cDNAs were synthesized from total RNA using the Omniscript RT kit. Real-time PCR was performed using MX3000P real-time PCR machine. Lipids were extracted from the ventricular tissue using a modified Bligh&Dyer technique. The triglyceride was quantified using a triglyceride quantification kit.2. Stable P19CL6cell lines overexpression ppara or gata6were generated using lipofectamine2000. The neo expression vector carrying the ppara or gata6coding sequence was transfected into P19CL6cells using lipofectamine2000according to the manufacturer’s protocol. Two days after transfection,400μg/mL of G418was added to the growth medium for selection of stable ppara or gata6-expressing cells. Drug-resistant cells began to form small colonies2weeks after G418addition.3. P19CL6cells and the stable ppara or gata6-transfected P19CL6cells were cultivated in a-MEM medium with10%FBS overnight. Then the cells were treated with insulin at different concentration for24h. MTT assay was used to test the proliferation of cells. The cell cycle distribution was detected by flow cytometry. DAPI staining was used to investigated the morphological changes.Results1. Transcript levels for NRF-l-L and NRF-l-S were downregulated at baseline in ventricles following treatment with fenofibrate for7days. However, expression levels of NRF-l-L and NRF-l-S were decreased by16%and increased by15%, respectively, following treatment with fenofibrate for14days.2. The mRNA level of Lias showed no significant changes following treatment with fenofibrate for7and14days. However, a7-day treatment with fenofibrate also caused no significant changes in Tom40mRNA level. After14days of treatment, Tom40expression level was increased by15%compared with the control.3. Both cytochrome b and MCAD mRNA levels were steadily increased in response to fenofibrate from7to14days. Following14days of treatment, expression of the two genes was significantly increased by31%and32%, respectively.4. The expression of PGC-la gene showed insignificant changes at baseline following the7-day administration of fenofibrate, but upon administration for14days, the PGC-la mRNA level was significantly reduced by31%compared with control.5. In the ventricle of mice treated with fenofibrate for14days, myocardial lipids content were significantly increased.6. MTT assay indicated that compared with the control group, insulin had no significant effect on P19CL6cell in low and middle concentration. On the contrast, P19CL6cell were significantly inhibited by insulin in high concertration(P<0.05). There were no significances compared the effect of control, low and middle concentration of insulin on stable P19CL6cell lines overexpression PPARa, while insulin of high concentration took significant inhibit effect on stable P19CL6cell lines overexpression PPARa(P<0.05). The proliferation of stable P19CL6cell lines overexpression GATA6were stimulated in insulin of low concentration(P<0.05), while after treated with high concentration, a significant inhibition of cell growth coule be observed(P<0.05).7. DAPI test showed that compared with control cells, the morphological features of P19CL6cell and stable P19CL6cell lines overexpression PPARa or GATA6apoptosis were the presence of condensation of the nucleus, nuclear fragmentation and the appearance of apoptosis body, which were treated with insulin of high concentration.8. Flow cytometry showed that the proportion of S phase increased when treated with insulin of low, middle and high concentration in P19CL6cell and stable P19CL6cell lines overexpression PPARa or GATA6.Conclusions1. The PGC-la mRNA level was significantly reduced following treatment with fenofibrate for14days, at the same time fenofibrate administration results in myocardial lipid accumulation.2. The low concentration insulin could significantly increased the growth of P19CL6cell and stable P19CL6cell lines overexpression PPARa or GATA6, but the high concentration insulin could significantly inhibited the growth of P19CL6cell and stable P19CL6cell lines overexpression PPARa or GATA6.