This study was undertaken to examine the effects of CYP2J3 gene delivery on insulin resistance and diabetes in fructose-induced insulin-resistant rats and db/db diabetic mice. Results showed that a single intravenous injection of CYP2J3 in the eukaryotic expression plasmid pcDNA reduced blood pressure in fructose-fed rats and improved sensitivity to insulin in peripheral tissues and organs in both animal models. CYP2J3 overexpression significantly reduced blood pressure with upregulated eNOS expression and downregulated ET-1 and ETA expression. Furthermore, CYP2J3 overexpression significantly improved insulin resistance in fructose-induced insulin-resistant rats and db/db diabetic mice, at least in part through eNOS, IRS-1, and PI 3-kinase/AKT signaling pathways, as well as AMPK signaling pathways in liver, muscle, heart, and kidney. These data provide direct evidence that CYP2J3-derived EETs may alleviate insulin resistance through a variety of beneficial effects on critical intracellular signaling pathways.
Previous studies have demonstrated that rats treated with high-fructose drinking water develop systemic hypertension, hyperinsulinemia, and hypertriglyceridemia (24
). Although the pathophysiological mechanisms responsible for elevated blood pressure and hyperinsulinemia in fructose-treated animals are not completely understood, elevated sympathetic nervous system activity, impaired endothelium-dependent dilation, reduction of capillary permeability, elevated vascular expression of ET-1 and ETA receptor genes, decreased eNOS activity, and increased salt absorption by the intestine and kidney have all been implicated (12
). Polymorphisms in CYP2J2
(the human homolog of CYP2J3
) have been associated with essential hypertension (26
), and reduced renal CYP-derived eicosanoid synthesis has been reported in rats with high-fat diet–induced hypertension (27
). Furthermore, EETs have direct vasodilatory activity (28
). In the present study, CYP2J3
overexpression significantly elevated urinary levels of 14,15-DHET in rats and mice and attenuated fructose-induced changes in eNOS, ET-1, and ETA-R expression in rats. These effects may underlie the beneficial effects of CYP2J3
overexpression on blood pressure and hyperinsulinemia that we observed.
Fructose induces inflammatory changes in human aortic endothelial cells and vessel walls in rats (30
). Cytokines such as TNF-α induce insulin resistance in endothelial cells via a p38 mitogen–activated protein kinase–dependent pathway (32
). This inflammatory signaling is relevant to diabetes, as TNF receptor 1 blockade protects Wistar rats from diet-induced obesity and insulin resistance (33
). Physiological concentrations of EETs or overexpression of CYP2J2
decreases cytokine-induced endothelial cell adhesion molecule expression, indicating that EETs have anti-inflammatory properties independent of their membrane-hyperpolarizing effects (34
). Furthermore, increased NO release improves insulin resistance in fructose-treated rats (23
). Potassium depletion in rats exacerbates endothelial dysfunction and lowers the bioavailability of NO, which blocks insulin activity and causes insulin resistance (35
). We observed that CYP2J3
expression resulted in a significant reduction in urine volume and urine potassium in fructose-treated rats. This suggests a potential ameliorative effect of CYP2J3
treatment against hypertension-related end organ (kidney) damage and attenuation of insulin resistance.
The precise molecular mechanisms for attenuation of insulin resistance in CYP2J3
-injected, fructose-treated rats remain to be elucidated. Recent studies indicate that the ability of insulin to vasodilate skeletal muscle vasculature is mediated by endothelium-derived NO (36
). These actions may partially explain our observation of improved insulin sensitivity after CYP2J3
gene delivery. In addition, we observed effects on insulin signaling in tissues directly involved in insulin sensitivity (37
), including liver, muscle, heart, and kidney, as well as in an islet cell line. Our data show that insulin-dependent signaling was significantly inhibited in fructose-treated rats and db/db
mice, but dramatically reversed by CYP2J3
overexpression. These results indicate that CYP2J3
overexpression potentiates insulin receptor signaling in liver, muscle, heart, and kidney and thus improves insulin sensitivity.
We also evaluated the phosphorylation status of AMPK. Small molecule–mediated activation of AMPK improves insulin resistance in ob/ob
) and represents a promising approach for the treatment of type 2 diabetes and metabolic syndrome (42
). We found that CYP2J3 overexpression resulted in a significant increase in AMPK phosphorylation, which may contribute to the EET-mediated alleviation of diabetes and insulin resistance we observed. CYP2J3
overexpression induced a significant increase in urine cAMP and cGMP secretion, which may result from GPCR activation by EETs. Thus, EET-induced GPCR activation may play an important role in EET-mediated AMPK activation and insulin sensitization. Taken together, these data suggest that the improvement of insulin resistance after CYP2J3
gene delivery was due to, at least in part, increased activation of IR/IRS-1/PI 3-kinase/AKT and AMPK signaling pathways (43
), as well as the upregulation of eNOS.
In conclusion, we have demonstrated alleviation of insulin resistance and diabetes by CYP2J3 gene therapy in db/db diabetic mice and a model of fructose-induced insulin resistance in rats. These effects were associated with increased insulin sensitivity in peripheral tissues and organs via upregulation of systemic eNOS and activation of the IRS-1/PI 3-kinase/AKT and AMPK signaling pathways in muscle, liver, heart, kidney, and aorta. However, the more precise mechanisms need to be further investigated. The ability of CYP epoxygenase delivery to exert a broad spectrum of beneficial effects in these animal models warrants further investigation of this approach in the treatment of hypertension associated with insulin resistance and diabetes in humans.