Recent studies have revealed that vascular cells can produce reactive oxygen species (ROS) through NAD(P)H oxidase, which may be involved in vascular injury. However, the pathological role of vascular NAD(P)H oxidase in diabetes or in the insulin-resistant state remains unknown. In this study, we examined the effect of high glucose level and free fatty acid (FFA) (palmitate) on ROS production in cultured aortic smooth muscle cells (SMCs) and endothelial cells (ECs) using electron spin resonance spectroscopy. Exposure of cultured SMCs or ECs to a high glucose level (400 mg/dl) for 72 h significantly increased the free radical production compared with low glucose level exposure (100 mg/dl). Treatment of the cells for 3 h with phorbol myristic acid (PMA), a protein kinase C (PKC) activator, also increased free radical production. This increase was restored to the control value by diphenylene iodonium, a NAD(P)H oxidase inhibitor, suggesting ROS production through PKC-dependent activation of NAD(P)H oxidase. The increase in free radical production by high glucose level exposure was completely restored by both diphenylene iodonium and GF109203X, a PKC-specific inhibitor. Exposure to palmitate (200 µmol/l) also increased free radical production, which was concomitant with increases in diacylglycerol level and PKC activity. Again, this increase was restored to the control value by both diphenylene iodonium and GF109203X. The present results suggest that both high glucose level and palmitate may stimulate ROS production through PKC-dependent activation of NAD(P)H oxidase in both vascular SMCs and ECs. This finding may be involved in the excessive acceleration of atherosclerosis in patients with diabetes and insulin resistance syndrome.

Abstract. Hyperglycemia seems to be an important causative factor in the development of micro-and macrovascular complications in patients with diabetes. Several hypotheses have been proposed to explain the adverse effects of hyperglycemia on vascular cells. Both protein kinase C (PKC) activation and oxidative stress theories have increasingly received attention in recent years. This article shows a PKC-dependent increase in oxidative stress in diabetic vascular tissues. High glucose level stimulated reactive oxygen species (ROS) production via a PKC-dependent activation of NAD(P)H oxidase in cultured aortic endothelial cells, smooth muscle cells, and renal mesangial cells. In addition, expression of NAD(P)H oxidase components were shown to be upregulated in vascular tissues and kidney from animal models of diabetes. Furthermore, several agents that were expected to block the mechanism of a PKCdependent activation of NAD(P)H oxidase clearly inhibited the increased oxidative stress in diabetic animals, as assessed by in vivo electron spin resonance method. Taken together, these findings strongly suggest that the PKC-dependent activation of NAD(P)H oxidase may be an essential mechanism responsible for increased oxidative stress in diabetes.Hyperglycemia seems to be an important causative factor in the development of micro-and macrovascular complications in patients with diabetes (1,2). Various pathophysiological and biochemical mechanisms have been proposed to explain the adverse effects of hyperglycemia on vascular cells (3-6). Among various possible mechanisms, it is widely accepted that high glucose level and a diabetic state induce the persistent activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway in micro-and macrovascular tissues of diabetic animals and of patients with diabetes (7-12). Because PKC is a critical intracellular signaling molecule that can regulate many vascular functions, it is to be expected that activation of PKC may cause alteration in various vascular functions in diabetes. However, accumulating evidence has shown that oxidative stress also may play a role in the development of diabetic vascular complications. A number of in vitro and in vivo studies suggest that the production of reactive oxygen species (ROS) is increased in diabetes (13-16). It has been postulated that ROS production in diabetes may be enhanced by hyperglycemia through various mechanisms such as enhanced formation of glycation products (17), altered polyol pathway activity (18), and increased superoxide release from mitochondria (19). In contrast, attention is increasingly focused on NAD(P)H oxidase as the most important source of ROS production in blood vessels (20 -23). Recent reports have implicated that this oxidase may be involved in the pathophysiology of various vascular diseases, including hypercholesterolemia (24), atherosclerosis (25-27), and hypertension (28). In this review, we show that a PKC-dependent activation of NAD(P)H oxidase may be an essential mechanism responsible for increased ROS ...

This study provides evidence that NAD(P)H oxidase subunits, NOX4 and p22phox, were increased in the kidney of diabetic rats. Thus, NAD(P)H-dependent overproduction of ROS could cause renal tissue damage in diabetes. This might contribute to the development of diabetic nephropathy.

Oxidative stress may contribute to the pathogenesis of diabetic nephropathy. However, the detailed molecular mechanism remains uncertain. Here, we report oxidative mitochondrial DNA (mtDNA) damage and accumulation of mtDNA with a 4,834-bp deletion in kidney of streptozotocin-induced diabetic rats. At 8 weeks after the onset of diabetes, levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), which is a marker of oxidative DNA damage, were significantly increased in mtDNA from kidney of diabetic rats but not in nuclear DNA, suggesting the predominant damage of mtDNA. Semiquantitative analysis using PCR showed that the frequency of 4,834-bp deleted mtDNA was markedly increased in kidney of diabetic rats at 8 weeks, but it did not change at 4 weeks. Intervention by insulin treatment starting at 8 weeks rapidly normalized an increase in renal 8-OHdG levels of diabetic rats, but it did not reverse an increase in the frequency of deleted mtDNA. Our study demonstrated for the first time that oxidative mtDNA damage and subsequent mtDNA deletion may be accumulated in kidney of diabetic rats. This may be involved in the pathogenesis of diabetic nephropathy. Diabetes 51:1588 -1595, 2002 D iabetic nephropathy is the major cause of morbidity and mortality in diabetic patients. It is more rapidly liable to functional deterioration compared with other types of chronic renal disease and finally progresses to renal failure requiring dialysis therapy. Several mechanisms have been proposed for the pathogenesis of diabetic vascular complications that include nephropathy, such as hyperfiltration (1), increased production of advanced glycation end products (AGEs) (2), activation of protein kinase C (3-5), enhanced polyol pathway (6,7), and enhanced oxidative stress (8 -10). A number of in vitro and in vivo studies suggest that oxidative stress is increased in diabetic patients and animal models of diabetes (8 -14). Although enhanced oxidative stress may contribute to the initiation and development of diabetic nephropathy, the detailed molecular mechanism remains uncertain.In general, oxidative stress, including reactive oxygen species (ROS), can damage cellular macromolecules. Among the oxidative damages, base modifications, such as oxidation of deoxyguanosine to 8-hydroxy-2Ј-deoxyguanosine (8-OHdG) and subsequent mutations of mitochondrial DNA (mtDNA), have received increasing attention in recent years. It is widely accepted that mtDNA is 10 -20 times more vulnerable to oxidative damage and subsequent mutations than nuclear DNA (15-17). More than 50 pathogenic mtDNA mutations associated with or responsible for specific human diseases have been reported. Congenital mtDNA mutations as well as oxidative stress-induced mtDNA mutations may be related to the pathophysiology of various diseases. It has been shown that oxidative stress-induced mtDNA mutations may be related to aging-related organ dysfunction (18 -23) and several degenerative diseases (24,25). We speculated that enhanced oxidative stress might induce mtDNA damage and subsequent mtDNA...

Hyperglycemia appears to be an important etiologic factor in the development of micro- and macrovascular complications in diabetic patients. However, its detailed molecular mechanism remains unclear. Among various possible mechanisms, it is widely accepted that high glucose level and a diabetic state induce protein kinase C (PKC) activation in vascular cells in cultured and vascular tissues of diabetic animals. Gap junctions are clusters of membrane channels that permit the intercellular exchange of ions and second messengers between adjacent cells. Gap junctional intercellular communication (GJIC) plays an important role in cardiovascular tissue homeostasis. Here we report that GJIC in cultured vascular cells such as endothelial cells and smooth muscle cells is inhibited by high glucose level. Furthermore, we show that it is mediated by PKC-dependent excessive phosphorylation of connexin-43 which is the main functional component of gap junction in vascular cells. In addition, we also show that in diabetic rats, PKC-dependent excessive phosphorylation of connexin-43 induces the impairment of ventricular conduction in the heart. These results suggest that PKC-dependent impairment of GJIC may lead to various disorders of cardiovascular homeostasis and contribute to cardiovascular dysfunctions associated with diabetes.