Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis
Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis
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DOI：
10.1152/ajpendo.00015.2008
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发表时间：
2008-08-01
影响因子：
5.1
通讯作者：
Kadiiska, Maria B.
中科院分区：
文献类型：
--
作者：
Stadler, Krisztian;Bonini, Marcelo G.;Kadiiska, Maria B.
糖尿病患者经常遇到酮症，其特征在于脂质的分解，随之而来的是酮体的积累。几项研究表明，反应性物质可能会诱导糖尿病组织损伤，但酮体在该过程中的作用尚未得到充分研究。在这项研究中，电子顺磁共振（EPR）光谱结合新的自旋捕获和免疫学技术已被用来研究在丙酮诱导的酮症小鼠模型体内自由基的形成。在肝脏提取物中检测到与碳中心脂质衍生自由基的α-（4-吡啶基-1-氧化物）-N-叔丁基硝酮自由基加合物一致的六线EPR光谱。为了研究这些自由基的可能的酶源，使用诱导型一氧化氮合酶（iNOS）和NADPH氧化酶敲除小鼠。自由基的产生在NADPH氧化酶敲除小鼠中没有变化，但在iNOS敲除小鼠中大大减少，表明iNOS在自由基产生中的作用。长期暴露于丙酮揭示了iNOS过度表达在肝脏中的蛋白质自由基的形成，这是通过共聚焦显微镜和一种新的免疫自旋捕获方法检测。免疫组织化学分析显示，增强的脂质过氧化和蛋白质氧化的结果，持续的自由基产生后，丙酮处理21天的控制和NADPH氧化酶敲除，但不是在iNOS敲除小鼠。两者合计，我们的数据表明，丙酮管理，酮病模型，可以导致蛋白质氧化和脂质过氧化，通过自由基依赖性的机制，主要由iNOS过表达驱动。
Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression.