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.

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...

Interaction of tea catechins with lipid bilayers was investigated with liposome systems, which enabled us to separate liposomes from the external medium by centrifugation. We found that epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure.

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