Molecular Pharmacology of Insulin Resistance in Burns

烧伤胰岛素抵抗的分子药理学

• 批准号: 6729136
• 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/6729136
• 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）烧伤的多代谢状态为 与蛋白质，脂肪和碳水化合物的不受控制的分解代谢以及 影响发病率和死亡率。相关的主要代谢异常是 抗胰岛素的抗性，胰岛素的作用，胰岛素的关键性合成代谢激素。在 信号传导级联反应由胰岛素激活，胰岛素受体（IR），胰岛素 受体底物（IRS），磷脂酰肌醇-3-磷酸激酶（PI 3-K） AKT/PKB对于能量代谢和葡萄糖稳态都是中心。 激活的AKT/PKB反过来抑制其下游分子，葡萄糖合酶 激酶-3（GSK-3），导致蛋白质和糖原合成增加。 所有这些信号分子的激活改变发生后发生 受伤，但是诱导这些变化的分子机制尚未 阐明。许多细胞因子在本地和系统地表达 烧伤损伤，导致诱导一氧化氮（INOS）的表达增加， 并释放高水平的一氧化氮（NO）。基于引人注目的和 令人信服的初步数据，我们假设Inos通过释放NO 超氧化物，在改变的燃烧胰岛素抵抗中起重要作用 通过IR，IRS，PI 3-K，AKT/PKB和GSK-3发出信号。 以下特定目的将在烧伤/虚假的损伤中检验上述假设 啮齿动物在体内，培养细胞和重建的体外系统中： 特定目标1将检验胰岛素需要iNOS的假设 反抗。特定目标2将检验夸张的假设 INOS的NO生产降低了IR和酪糖基的酪氨酸激酶活性 IRS的磷酸化。 JR和IRS灭活的分子机制 （S-硝基化与硝化）也将被鉴定。具体的目标3将 检验以下假设：iNOS夸大NO的产生有所改变 AKT/PKB的激酶活性，PI 3-K的进一步下游分子， 独立于IR和IRS。负责的分子机制 AK/IPKB的灭活（翻译后修改）也将是 确定。特定目标4将检验夸张产量的假设 INOS的NO属于GSK-3的活性，这是由于与两者相关的影响 降低了AKTJPKB活性和NO对GSK-3的直接影响。直接角色 GSK-3（独立于Aktipkb）的激活NO将进行测试 捐助者和清道夫。体内研究将包括使用烧伤和 损伤的大鼠，iNOS敲出（ - / - ）和野生型（+/+）小鼠。胰岛素 介导的信号变化以及在 具有和没有特定iNOS抑制剂的上述列举的信号分子 （1400W）将进行评估。功能变化，使用2-脱氧葡萄糖评估 肌肉和脂肪细胞的吸收将与信号变化相关。使用 来自iNOS的脂肪细胞和肌细胞细胞系以及原发性培养物 - / - 和iNOS +/+小鼠，iNOS/否的作用将在没有供体的情况下进行评估或 清道夫。 NO的作用将在体外重建系统中得到确认 包含活性信号分子。翻译后修改 （硝酸盐与S-硝基化）与1NOS/NO相关的 生化，分光光度法和免疫印迹技术。几行 有证据表明蛋白质S-亚硝基化 - 决定和酪氨酸 硝化/否定可以用作调节成分。否参与 根据这个新概念，将评估胰岛素抵抗。 因此，这些研究的直接短期目标是表征 诱导胰岛素抵抗的分子和生化机制，以便 从长远来看，可以逆转人类烧伤损伤的胰岛素抵抗。 因此，研究将共同​​提供对 胰岛素抵抗的发病机理，并提供有关新型治疗的信息 治疗燃烧以及其他压力或炎症引起的胰岛素的策略 反抗。