Molecular Pharmacology of Insulin Resistance in Burns

描述(由申请人提供)烧伤的高代谢状态为 与蛋白质、脂肪和碳水化合物的不受控制的分解代谢有关，以及 影响发病率和死亡率。相关的主要代谢异常是 抵抗胰岛素的影响，胰岛素是关键的合成代谢激素。在这些人中 胰岛素、胰岛素受体(IR)、胰岛素激活的信号转导 受体底物(IRS)、磷脂酰肌醇-3-磷酸激酶(PI 3-K) Akt/PKB是能量代谢和葡萄糖动态平衡的中心。 激活的Akt/PKB进而抑制其下游分子葡萄糖合成酶 激酶-3(GSK-3)，导致蛋白质和糖原合成增加。 烧伤后，所有这些信号分子的激活都发生了变化 损伤，但导致这些变化的分子机制尚未得到 已澄清。许多细胞因子在以下局部和系统中表达 烧伤，导致诱导型一氧化氮(INOS)表达增加， 和释放高水平的一氧化氮(NO)。基于令人信服的 令人信服的初步数据，我们假设iNOS，通过释放NO和 超氧化物歧化酶在烧伤胰岛素抵抗中的作用 通过IR、IRS、PI 3-K、Akt/PKB和GSK-3等途径进行信号传导。 以下具体目标将在烧伤/假损伤中检验上述假设 在活体、培养细胞和体外重组系统中的啮齿动物： 特定目标1将检验胰岛素需要iNOS这一假设 抵抗。具体目标2将检验这样一个假设，即夸张的 诱导型一氧化氮合酶产生NO降低胰岛素抵抗和酪氨酸氨基转移酶活性 IRS的磷酸化。JR和IRSS失活的分子机制 (S-亚硝化和硝化)也将被识别。具体目标3将 检验iNOS夸大产生NO会改变 PI3-K的下游分子Akt/PKB的激酶活性， 独立于IR和IRSS。导致这一现象的分子机制 NO对AK/IPKB的失活(翻译后修改)也将是 已确认身份。具体目标4将检验夸大产量的假设 诱导型一氧化氮合酶抑制GSK-3活性 AktJPKB活性降低及NO对GSK-3的直接影响。直接作用 NO对GSK-3(独立于AktIPKB)激活的影响将与NO一起测试 捐赠者和食腐动物。体内研究将包括使用烧伤和 假损伤大鼠，iNOS基因敲除(-/-)和野生型(+/+)小鼠。胰岛素 介导的信号变化，以及翻译后的修改 在使用和不使用特定iNOS抑制剂的情况下以上列举的信号分子 (1400W)将进行评估。功能改变，用2-脱氧葡萄糖评估 肌肉和脂肪细胞的摄取，将与信号变化相关。vbl.使用 INOS-/-和iNOS来源的脂肪细胞和心肌细胞系及原代培养 +/+小鼠，iNOS/NO的作用将在有或没有供体或 食腐动物。NO在体外重建系统中的作用将得到证实 含有活跃的信号分子。翻译后修饰语 (硝化与S-亚硝化)与1NOS/NO相关的研究将通过 生化、分光光度和免疫印迹技术。几行 有证据表明，蛋白质S-亚硝基-反硝化和酪氨酸 硝化/反硝化可以作为调节成分。NO的参与 将根据这一新概念对胰岛素抵抗进行评估。 因此，这些研究的近期短期目标是 诱导胰岛素抵抗的分子和生化机制，从而 从长远来看，人类烧伤的胰岛素抵抗是可以逆转的。 因此，这些研究将为我们提供对 胰岛素抵抗的发病机制和新的治疗方法 治疗烧伤和其他应激或炎症诱导的胰岛素的策略 抵抗。

DESCRIPTION (provided by applicant) The hypermetabolic state of burns is associated with uncontrolled catabolism of proteins, fat and carbohydrates, and affects morbidity and mortality. The associated major metabolic anomaly is resistance to the effects of insulin, the pivotal anabolic hormone. Among the signaling cascades activated by insulin, the insulin receptor (IR), insulin receptor substrates (IRSs), phosphatidylinositol-3-phosphate kinase (PI 3-K) and Akt/PKB are central for energy metabolism and glucose homeostasis. Activated Akt/PKB in turn inhibits its downstream molecule, glucose synthase kinase-3 (GSK-3), resulting in increased protein and glycogen synthesis. Altered activation of all these signaling molecules occurs following burn injury, but the molecular mechanisms inducing these changes have not been elucidated. Many cytokines are expressed locally and systematically following burn injury, leading to increased expression of inducible nitric oxide (iNOS), and release of high levels of nitric oxide (NO). Based on compelling and convincing preliminary data, we hypothesize that iNOS, via release of NO with superoxide, plays an important role in insulin resistance of burn by altered signaling via IR, IRSs, PI 3-K, Akt/PKB and GSK-3. The following Specific Aims will test the above hypothesis in burn/sham-injured rodents in vivo, in cultured cells and in reconstituted in vitro systems: Specific Aim 1 will test the hypothesis that iNOS is required for insulin resistance. Specific Aim 2 will test the hypothesis that the exaggerated production of NO by iNOS decreases tyrosine kinase activity of IR and tyrosyl phosphorylation of IRSs. The molecular mechanism of inactivation of JR and IRSs (S-nitrosylation vs. nitration) will also be identified. Specific Aim 3 will test the hypothesis that the exaggerated production of NO by iNOS alters the kinase activity of Akt/PKB, the further downstream molecule of PI 3-K, independent of IR and IRSs. The molecular mechanisms responsible for inactivation (post-translational modifications) of Ak/IPKB by NO will also be identified. Specific Aim 4 will test the hypothesis that exaggerated production of NO by iNOS increases activity of GSK-3, due to effects related to both decreased AktJPKB activity and direct effects of NO on GSK-3. The direct role of NO on activation of GSK-3 (independent of AktIPKB) will be tested with NO donors and scavengers. The in vivo studies will include the use of burn and sham-injured rats, and iNOS knock out (-/-) and wild type (+/+) mice. Insulin mediated signaling changes, and the post-translational modifications in the signaling molecules enumerated above with and without specific iNOS inhibitor (1400W) will be evaluated. Functional changes, evaluated using 2-deoxyglucose uptake in muscle and adipocyte, will be correlated to signaling changes. Using adipocyte and myocyte cell lines and primary cultures from iNOS -/- and iNOS +/+ mice, the role of iNOS/NO will be evaluated with and without NO donors or scavengers. The role of NO will be confirmed in in vitro reconstitution system containing active signaling molecules. The post-translational modifications (nitration vs. S-nitrosylation) associated with 1NOS/NO will be studied by biochemical, spectrophotometric and immunoblot techniques. Several lines of evidence suggest that protein S-nitrosylationl-denitrosylation and tyrosine nitration/denitration may serve as regulatory components. The involvement of NO in insulin resistance will be assessed in the light of this new concept. The immediate short-term goals of these studies are, therefore, to characterize the molecular and biochemical mechanisms inducing insulin resistance, so that in the long-term, insulin resistance of burn injury in humans can be reversed. The studies together will thus provide significant insights into the pathogenesis of insulin resistance and provide information on novel therapeutic strategies to treat burn, and other stress or inflammation-induced insulin resistance.