Evidence for contribution of vascular NAD(P)H oxidase to increased oxidative stress in animal models of diabetes and obesity

Abstract

It is well established that oxidative stress is enhanced in diabetes. However, the major in vivo source of oxidative stress is not clear. Here we show that vascular NAD(P)H oxidase may be a major source of oxidative stress in diabetic and obese models. In vivo electron spin resonance (ESR)/spin probe was used to evaluate systemic oxidative stress in vivo. The signal decay rate of the spin probe (spin clearance rate; SpCR) significantly increased in streptozotocin-induced diabetic rats 2 weeks after the onset of diabetes. This increase was completely normalized by treatment with the antioxidants α-tocopherol (40 mg/kg) and superoxide dismutase (5000 units/kg), and was significantly inhibited by treatment with a PKC-specific inhibitor, CGP41251 (50 mg/kg), and a NAD(P)H oxidase inhibitor, apocynin (5 mg/kg). Both obese ob/ob mice (10 weeks old) with mild hyperglycemia and Zucker fatty rats (11 weeks old) with normoglycemia exhibited significantly increased SpCR as compared with controls. Again, this increase was inhibited by treatment with both CGP41251 and apocynin. Oral administration of insulin sensitizer, pioglitazone (10 mg/kg), for 7 days also completely normalized SpCR values. These results suggest that vascular NAD(P)H oxidase may be a major source of increased oxidative stress in diabetes and obesity.

Introduction

Enhanced oxidative stress may be an important contributor to the pathogenesis of vascular diseases such as atherosclerosis [1], [2], [3], [4], hypertension [5], and diabetic angiopathy [6], [7], [8], [9]. Free radicals including reactive oxygen species (ROS) have been implicated in various aspects of vascular injury including lipoprotein oxidation [2], [3], [4], smooth muscle cell hypertrophy [10], and endothelial cell dysfunction [11], [12]. It is well established that oxidative stress is enhanced in diabetic patients and in animal models of diabetes. Various underlying mechanisms have been postulated, such as increased formation of advanced glycosylation end products [8], enhanced polyol pathway [7], increased superoxide release from mitochondria [13], increased xanthine oxidase activity [14], and activation of NAD(P)H oxidase [15]. However, the major source of increased oxidative stress in vivo remains uncertain.

Obesity, as well as diabetes, is increasingly recognized as a high risk factor for atherosclerosis [16], [17]. Accumulating evidence has suggested that obesity or insulin resistance is associated with endothelial dysfunction, including impaired endothelium-dependent vasodilation [18], [19], which could be explained by decreased nitric oxide (NO) levels or increased oxidative stress. A recent report showed that 8-epi-prostaglandin F2α, formed by peroxidation of lipid-esterified arachidonic acid, an oxidative stress marker, increased in obese Zucker fatty rats [20], suggesting that oxidative stress may increase in obesity or insulin-resistant states. The underlying mechanism remains to be elucidated.

Oxidative stress is derived mainly from vascular tissues. Among various potential sources, vascular NAD(P)H oxidase has received increasing attention as the most important source of ROS production in vascular tissues [21], [22], [23]. We previously reported that high glucose levels and free fatty acids stimulate superoxide production via protein kinase C (PKC)-dependent activation of vascular NAD(P)H oxidase in cultured aortic endothelial cells and smooth muscle cells. This evidence may, at least in part, account for the increased oxidative stress in diabetes and insulin-resistant states. On the basis of this hypothesis, we evaluate whether vascular NAD(P)H oxidase contributes to increased oxidative stress in streptozotocin-induced diabetic rats, obese animal models of ob/ob mice, and Zucker fatty rats. We used in vivo electron spin resonance (ESR) measurement, which has been reported to be useful for evaluating oxidative stress in vivo [24], [25], [26], [27], [28], [29], [30].