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...

This study has identified constitutive IL-1 beta expression by glomerular endothelium and tubular epithelial cells in normal rat kidney. In addition, the marked upregulation of IL-1 beta expression by intrinsic glomerular cells and tubules in rat anti-GBM disease suggests an important role for these cells in IL-1 dependent crescent formation and tubulointerstitial injury.

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.

Background We investigated the effects of metformin on neurological function and oxidative stress in patients with type 2 diabetes mellitus with acute stroke. Material/Methods We randomly assigned 80 acute stroke patients to 2 groups: the metformin combined group and the insulin group. Each group had 40 patients and all were treated with standard stroke treatment. The indexes of nervous functional score and oxidative stress were measured before and 2 weeks after treatment. The primary fetal rat hippocampal neurons were gradually matured after 7 days of culture, and divided into the control group (Con), the oxygen-glucose deprivation model group (Mod), and the metformin group (Met). In the Met group, 10 mmol/L metformin was added, and the Con group and the Mod group received equal volumes of cell culture fluid. Cell viability, cell apoptosis rate, and the expression of Bax, Bcl-2, AMPK, pAMPK and mTOR were detected; MDA content and SOD activity were also detected. Results Before treatment, there was no difference in the metrical indexes between the 2 groups. After treatment, the treatment group was better than the control group in neurological function scores and multiple oxidative stress-related indicators. The experimental results of primary fetal rat hippocampal neuronal cells suggest that this mechanism of improvement is closely related to the AMPK/mTOR signaling pathway. Conclusions Metformin can improve the neurological function and oxidative stress status of acute stroke patients with type 2 diabetes, and its mechanism may be related to the AMPK/mTOR signaling pathway and oxidative stress.