The Arachidonic Acid Cytochrome P450 Epoxygenase Gene Attenuates Rats' Insulin Resistance And Hypertension Induced By Fructose Via Nitric Oxide

Background and objective Type2 diabetes mellitus (T2DM) is an insulin resistant state associated with atherosclerosis, hypertension, dyslipidemia, and abnormal vasomotor responses to insulin. This phenomenon can be copied in animal by high fructose diet. It was demonstrated that Arachidonic acid (AA) cytochrome P450 (CYP) epoxygenase metabolites, epoxyeicosatrienoic acids (EETs), can dilate not only artery via hyperpolarizing artery smooth muscle cells, but also have non-vascular dilatory effects, including anti-inflammation, upregulate endothelial nitric oxide synthase (eNOS), protect endothelial cells from apoptosis induced by tumor necrosis factor alpha. Some of the effects was induced by PI3K and MAPK signaling pathway, which play significant role in the insulin signaling pathway. Nitric oxide is one of the most important message molecular, insulin mediated skeletal muscle vasodilation is nitric oxide dependent,NO enhanced the uptake of glucose in skeletal muscle, and attenuated the insulin resistance of peripheral organs. However, how are therapeutic effects of CYP2J3 on diabetes mellitus and insulin resistance? Whether Gene delivery of CYP2J3 synchronously ameliorates diabetes mellitus and insulin resistance while reduces hypertension? Whether the signaling pathway and the expression of eNOS will be changed? This study investigates therapeutic effects of CYP2J3 gene delivery on the hypertension as well as insulin resistance and the possible mechanism of high fructose-induced hypertension in fructose-induced hypertensive rats.Methods Male Sprague-Dawley rats (n=21) fed on high-fructose (10%) water to develop mild hypertensive models within 4 weeks. Then the rats were assigned to three groups randomly; 10%Fructose drink only (F) n=7, 10%fructose and pcDNA-2J3 (F+2J3) n=7, 10%fructose, pcDNA-2J3 and L-NAME was added (F+2J3+L) n=7. The caudal arterial pressure was measured once a week for 7 weeks until the animals were sacrificed. Blood samples were collected between 8:30am and 10:30am after an overnight fast. Serum was prepared and stored at -20°C. Twenty-four hour urine samples were collected from 8:00am to 8:00am the following day in brown bottles containing dimethylamine. Serum glucose, cholesterol, and triglyceride were measured in duplicate or triplicate on an AEROSET Clinical Chemistry System (Abbott Laboratories, Abbott Park, IL). Serum insulin was measured using a magnetic sold phase enzyme immunoassay kit from BioChem ImmunoSystems (Rome, Italy). Insulin resistance was calculated using the homeostasis model assessment (HOMA) method. Urinary osmolarity was calculated on a model single-sample 030 osmometer (Gonotec osmomat, German). Vascular ET-1 and ETA-R were measured by RT-PCR. The expression of rats'CYP2J3, human eNOS, (p-)MAPK and (p-)AMPK in the liver was explored by Western blot.Results Compared with control rats given normal water, hypertension was induced in rats by addition of 10% fructose to drinking water after 4 weeks. A single intravenous injection of pcDNA-2J3 resulted in a significant reduction in blood pressure in the first week (115.2±6.1 to 112.4±2.6 mmHg) after injection and the maximal effect appeared at the second week (108.16±3.6 mmHg) after injection and the effect lasted for at least 3 weeks. And it is very surprised that two weeks after injection of CYP2J3 gene, serum insulin levels normalized, urine osmolarity increased, although all animals were kept in feeding with 10% fructose water. In contrast, the F+2J3+L-NAME group increased the blood pressure to 135.7±2.3 mmHg in the first week after L-NAME, in the second week, the blood pressure increased to 146.3±5.2 mmHg (p<0.01 vs F and F+2J3). RT-PCR showed that fructose-fed rats with CYP2J3 gene delivery had lower ET-1, ETA-R gene expression than the fructose only. The expression of CYP2J3 was detected in rat liver by Western blot in pcDNA-2J3 treated rats. The expression of eNOS, phosphorylated MAPK and AMPK were upregulated by CYP2J3, and reduced in the group of F+2J3+L, the amount of total MAPK and AMPK were the same in three groups.Conclusion These findings suggest that elevated vascular expression of ET-1, ETA-R genes and downregulation of the protein eNOS, p-AMPK and p-MAPK may mediate the development of hypertension and hyperinsulinemia in rats fed a fructose-rich diet. CYP2J3 gene transfer may reduce dominantly blood pressure and high levels of plasma insulin in fructose-induced hypertensive rats with hyperinsulinemia through the reduction of vascular expression of ET-1, ETA-R receptor, and upregulation of the protein eNOS, p-AMPK and p-MAPK, which raises the feasibility of applying CYP2J3 gene to treat human insulin resistance with hypertension. We also illustrate that all the effects above were NO dependent .