Molecular Pharmacology of Insulin Resistance in Burns

烧伤胰岛素抵抗的分子药理学

• 批准号: 6625958
• 负责人: Jeevendra Martyn
• 金额: $ 38.01万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6625958
• 关键词: adipocytes; bioenergetics; biological signal transduction; burns; calmodulin dependent protein kinase; enzyme activity; enzyme inhibitors; gene targeting; genetically modified animals; glycogenesis; immunoprecipitation; insulin receptor; insulin sensitivity /resistance; laboratory mouse; laboratory rat; muscle cells; nitric oxide; nitric oxide synthase; phosphatidylinositol 3 kinase; phosphorylation; posttranslational modifications; protein metabolism; receptor sensitivity; spectrometry; tissue /cell culture; western blottings

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.

描述（由申请人提供） 烧伤的高代谢状态是 与蛋白质、脂肪和碳水化合物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 对胰岛素（关键的合成代谢激素）作用的抵抗。其中 由胰岛素、胰岛素受体 (IR)、胰岛素激活的信号级联 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稳态的核心。 激活的 Akt/PKB 进而抑制其下游分子葡萄糖合酶 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会发生改变 损伤，但引起这些变化的分子机制尚未阐明 阐明了。许多细胞因子在局部和系统地表达 烧伤，导致诱导型一氧化氮（iNOS）表达增加， 并释放高浓度的一氧化氮 (NO)。基于令人信服和 根据令人信服的初步数据，我们假设 iNOS 通过释放 NO 超氧化物，通过改变在烧伤的胰岛素抵抗中发挥重要作用 通过 IR、IRS、PI 3-K、Akt/PKB 和 GSK-3 发出信号。 以下具体目标将在烧伤/假伤中检验上述假设 啮齿动物体内、培养细胞和体外重建系统： 具体目标 1 将检验胰岛素需要 iNOS 的假设 反抗。具体目标 2 将检验夸大的假设 iNOS 产生 NO 会降低 IR 和酪氨酰的酪氨酸激酶活性 IRS 的磷酸化。 JR和IRS失活的分子机制 （S-亚硝基化与硝化）也将被识别。具体目标 3 将 检验以下假设：iNOS 过量产生 NO 会改变 Akt/PKB（PI 3-K 的更下游分子）的激酶活性， 独立于 IR 和 IRS。负责的分子机制 NO 也会导致 Ak/IPKB 失活（翻译后修饰） 确定。具体目标 4 将检验夸大产量的假设 iNOS 产生的 NO 会增加 GSK-3 的活性，这是由于与两者相关的影响 降低 AktJPKB 活性以及 NO 对 GSK-3 的直接影响。直接作用 NO 对 GSK-3 激活的影响（独立于 AktIPKB）将用 NO 进行测试 捐助者和拾荒者。体内研究将包括使用烧伤和 假损伤大鼠、iNOS 敲除 (-/-) 和野生型 (+/+) 小鼠。胰岛素 介导的信号传导变化以及翻译后修饰 上面列举的信号分子，有或没有特定的 iNOS 抑制剂 （1400W）将被评估。使用 2-脱氧葡萄糖评估功能变化 肌肉和脂肪细胞的摄取将与信号传导变化相关。使用 来自 iNOS -/- 和 iNOS 的脂肪细胞和肌细胞细胞系和原代培养物 +/+ 小鼠，将在有或没有 NO 供体的情况下评估 iNOS/NO 的作用或 拾荒者。 NO的作用将在体外重建系统中得到证实 含有活性信号分子。翻译后修饰 与 1NOS/NO 相关的（硝化与 S-亚硝基化）将通过以下方式进行研究 生物化学、分光光度和免疫印迹技术。几行 有证据表明蛋白质 S-亚硝基化/去亚硝基化和酪氨酸 硝化/脱硝可以作为调节成分。 NO的参与 将根据这一新概念评估胰岛素抵抗的情况。 因此，这些研究的近期目标是表征 诱导胰岛素抵抗的分子和生化机制 从长远来看，人类烧伤的胰岛素抵抗是可以逆转的。 因此，这些研究将共同​​提供对以下问题的重要见解： 胰岛素抵抗的发病机制并提供新治疗方法的信息 治疗烧伤和其他压力或炎症引起的胰岛素的策略 反抗。