Effect Of CYP450-2J3 Gene On Fructose-Induced Insulin Resistance And Hypertension In Rats

Background and Objective: It has been known for some time that increase in dietary carbohydrate intake can raise blood pressure in experimental animals. The increased intake of either sucrose or glucose was shown to enhance the development of either spontaneous hypertension or salt hypertension in rats. Hwang et al.first reported that hypertension could be induced in normal rats by feeding a high-fructose diet. Fructose feeding was also founded to cause insulin resistance, hyperinsulinemia and hypertriglyceridemia in normal rats. In the studies of the effects of fructose loading, rats were often given a diet containing 35-72% fructose to eat or 10% or 15% fructose in water to drink. This study mainly investigates whether fructose could induce hypertension hyperinsulinemia and hypertriglyceridemia in normal rats.Methods: Thirty male Sprague-Dawely rats were fed standard chow one week in room temperature, and then rats were randomly divided into two groups: fructose-treated rats and control animals. Fructose-treated rats were given 15% fructose solution and standard chow; control rats were given tap water and standard chow through out the whole experimental period. The caudal arterial pressure was measured once a week. At the end of the third week, the blood was collected for measuring the levels of blood glucose and insulin; Twenty-four hour urine samples were collected in brown bottles containing dimethylamine and stored at -80oC. Serum glucose, cholesterol, and triglyceride were measured in duplicate or triplicate on an AEROSET Clinical Chemistry system. Insulin resistance was calculated using the homeostasis model assessment (HOMA) method.Results: Compared with control rats given normal water, hypertension was induced in rats by addition of 15% fructose to water after 3 weeks(135.5±0.6 and 119.4±0.7, P﹤0.001). Fructose-fed rats also had increased serum insulin and serum triglycerides(P﹤0.05).Conclusion: High fructose diet mediated the development of hypertension, hyperinsulinemia and hypertriglyceridemia in rats. Fructose-induced metabolic syndrome in rats is one of ideal animal models.Part Two Cloning of CYP2J3 Gene and Its Expression in 293 CellsBackground and Objective : Arachidonic acid cytochrome P450 ( CYP450 )epoxygenase can generate epoxyeicosatrienoic acids(EETs)from arachidonic acid(AA) which is an important endothelium-derived relaxing factor ( EDRF ), and was found after nitrogen monoxidum (NO) and prostacyclin (PGI2). EETs elicit the hyperpolarization of smooth muscle cell (SMC) through opening Ca2+-sensitive potassium channel (KCa2+).Now, EETs was called endothelium-derived hyperpolaring factor (EDHF). Cytochrome P450 epoxygenase converts arachidonic acid to four epoxyeicosatrienoic acid regiosomes: 5, 6-EET,8, 9-EET,11, 12-EET and 14, 15-EET. In the recent studies, EETs were found having several physiological functions, including regulating blood vessel, cell proliferation, hormone secretion, anti-inflammatory, and also signal transduction in the cells. Rattus norvegicus cytochrome P450-2J3 gene can code monooxygenase (57,969Da) which has the heme-containing terminal. Many CYP450 Isoenzymes can metabolize arachidonic acid to EETs. We successfully clone CYP2J3 gene by RT-PCR and OE-PCR, then subclone into the pCDNA3.1 (+) to form an eukaryotic vector. Western Blotting was used to detect the expression of pCDNA 3.1 (+)-CYP2J3 in 293 cells. Successful cloneing of CYP2J3 gene and construction of pCDNA3.1 (+)-CYP2J3 provide the possibility for the further study of the biological function and related disease.Methods: The total RNA was extracted from rat hepatic tissue. CYP2J3-A and CYP2J3-B was amplified by RT-PCR, Cloning of CYP2J3 ORF gene was amplified by OE-PCR. After the identification of sequence analysis, CYP2J3 gene was subclone into the pCDNA3.1(+) eukaryotic vector. 293 cells were transfected with liposome/plasmid pCDNA3.1(+)-CYP2J3. Western Blotting was used to detect the expression of pCDNA3.1 (+)-CYP2J3 in 293 cells.Results: CYP2J3 gene was obtained by RT-PCR and OE-PCR. The recombinant eukaryotic expression vector for CYP2J3 gene had been successfully constructed. The results of Western Blotting showed the transfected 293 cells could express CYP2J3 protein.Conclusion: Cloning of CYP2J3 gene and construction of pCDNA3.1 (+)-CYP2J3 offer the tool for the further study of the protein structure and biological function and the related disease.Part Three CYP2J3 Gene Reduces Hypertension and Hyperinsulinemia in Fructose-Induced Hyperrensive RatsBackground and Objective: Clinical studies suggest that hypertension was associated with insulin resistance and diabetes mellitus, we found a high-fructose diet can induce hypertension and insulin-resistance in Sprague-Dawley rats. Now, EETs was regard as one of endothelium-derived relaxing factor ( EDRF ), EETs hyperpolarize VSM cells by increasing the open-state probability of KCa channels, then EETs reduces hypertension through regulating vascular tone. Early evidence suggests that EETs can dilate vascular tone and reduce hypertension in rat. These results indicated that EETs affects vascular tone which was associated with blood pressure regulation. This study mainly investigate CYP2J3 gene reduces fructose-induced hypertension. At the same time, we also found that CYP2J3 gene can reduce the plasma insulin levels in fructose-induced rat models and improves the insulin sensitivity.Methods: Male Sprague-Dawely rats were fed with 15% fructose water to develop hypertension and hyperinsulinemia models. Fructose-induced hypertensive rats and control rats were injected intravenously with recombinant plasmid containing CYP2J3 gene. The caudal arterial pressure was measured once a week until the rats were sacrificed.The blood was collected for measuring the levels of blood glucose and insulin after that plasmid was injected and rats were sacrificed. Twenty-four hour urine samples were collected in brown bottles containing dimethylamine and stored at -80oC. Serum glucose, cholesterol, and thiglyceride were measured in duplicate or triplicate on an AEROSET Clinical Chemistry system. Insulin resistance was calculated using the homeostasis model assessment (HOMA)method.Results: Compared with control rats given normal water, hypertension was induced in rats by addition of 15% fructose to water after 3 weeks. Fructose-fed rats also had increased serum insulin and serum triglycerides. A single intravenous injection of pCDNA3.1-CYP2J3 resulted in a significant reduction in blood pressure beginning 7 days after injection and the maximal effect appeared at 2 weeks and also reduction in plasma insulin levels in fructose-induced hypertensive rats.Conclusion: High fructose diet mediated the development of hypertension and hyperinsulinemia in rats. CYP2J3 gene transfer may reduce dominantly blood pressure and high levels of plasma insulin in fructose-induced hypertensive rats with hyperinsulinemia, which raises the feasibility of applying CYP2J3 gene to treat hypertension and insulin resistance.