Inoguchi T, Li P, Umeda F, Yu H Y, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H
Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushyu University, Fukuoka, Japan.
Diabetes. 2000 Nov;49(11):1939-45. doi: 10.2337/diabetes.49.11.1939.
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 micromol/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.
最近的研究表明，血管细胞可通过NAD(P)H氧化酶产生活性氧（ROS），这可能与血管损伤有关。然而，血管NAD(P)H氧化酶在糖尿病或胰岛素抵抗状态下的病理作用仍不清楚。在本研究中，我们使用电子自旋共振光谱法检测了高糖水平和游离脂肪酸（FFA）（棕榈酸酯）对培养的主动脉平滑肌细胞（SMC）和内皮细胞（EC）中ROS产生的影响。与低葡萄糖水平暴露（100mg/dl）相比，将培养的SMC或EC暴露于高葡萄糖水平（400mg/dl）72小时可显著增加自由基产生。用蛋白激酶C（PKC）激活剂佛波醇肉豆蔻酸酯（PMA）处理细胞3小时也会增加自由基产生。用NAD(P)H氧化酶抑制剂二亚苯基碘鎓可将这种增加恢复到对照值，提示通过PKC依赖性激活NAD(P)H氧化酶产生活性氧。高葡萄糖水平暴露导致的自由基产生增加可被二亚苯基碘鎓和PKC特异性抑制剂GF109203X完全恢复。暴露于棕榈酸酯（200μmol/l）也会增加自由基产生，这与二酰甘油水平和PKC活性增加同时发生。同样，这种增加也可被二亚苯基碘鎓和GF109203X恢复到对照值。目前的结果表明，高糖水平和棕榈酸酯均可通过PKC依赖性激活血管SMC和EC中的NAD(P)H氧化酶来刺激ROS产生。这一发现可能与糖尿病和胰岛素抵抗综合征患者动脉粥样硬化的过度加速有关。
