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）烧伤的高代谢状态是 与蛋白质、脂肪和碳水化合物的不受控制的代谢有关， 影响发病率和死亡率。相关的主要代谢异常是 抵抗胰岛素的作用，胰岛素是关键的合成代谢激素。中 由胰岛素、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） 和Akt/PKB是能量代谢和葡萄糖稳态的中心。 激活的Akt/PKB反过来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。 所有这些信号分子的激活都在烧伤后发生改变 损伤，但诱导这些变化的分子机制尚未被阐明 阐明。许多细胞因子表达的局部和系统性以下 烧伤，导致诱导型一氧化氮（iNOS）表达增加， 并释放高水平的一氧化氮（NO）。基于令人信服的和 令人信服的初步数据，我们假设，iNOS，通过释放NO， 超氧化物歧化酶在烧伤胰岛素抵抗中起重要作用， 通过IR、IRS、PI 3-K、Akt/PKB和GSK-3进行信号传导。 以下具体目的将在烧伤/假损伤中检验上述假设 啮齿类动物体内、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 阻力具体目标2将测试假设，夸大 诱导型一氧化氮合酶产生的一氧化氮降低了IR和酪氨酰酪氨酸激酶的活性， IRS的磷酸化。JR和IRS失活的分子机制 （S-亚硝基化与硝化）也将被确定。第3章将 检验iNOS过度产生NO改变了 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 通过NO使Ak/IPkB失活（翻译后修饰）也将被 鉴定具体目标4将检验夸大生产的假设， NO通过iNOS增加GSK-3的活性，由于与两者相关的作用， 降低AktJPKB活性和NO对GSK-3的直接作用。直接作用 NO对GSK-3活化的影响（不依赖于AktIPKB）将用NO 捐赠者和拾荒者体内研究将包括使用烧伤和 假损伤大鼠，以及iNOS敲除（-/-）和野生型（+/+）小鼠。胰岛素 介导的信号传导变化，和翻译后修饰， 在有和没有特异性iNOS抑制剂的情况下，上面列举的信号传导分子 （1400 W）将进行评估。功能变化，使用2-脱氧葡萄糖评价 在肌肉和脂肪细胞中的摄取将与信号传导变化相关。使用 脂肪细胞和肌细胞细胞系以及来自iNOS -/-和iNOS的原代培养物 +/+小鼠中，将在有和没有NO供体的情况下评估iNOS/NO的作用， 食腐动物NO的作用将在体外重组体系中得到证实 含有活性信号分子。的翻译后修饰 （硝化与S-亚硝基化）与1 NOS/NO相关的研究将通过 生物化学、分光光度计和免疫印迹技术。几行 有证据表明，蛋白质S-亚硝基化、脱亚硝基化和酪氨酸 硝化/反硝化可以用作调节组分。参与NO 将根据这一新概念对胰岛素抵抗进行评估。 因此，这些研究的直接短期目标是描述 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，这些研究将为以下方面提供重要的见解： 胰岛素抵抗的发病机制，并提供新的治疗信息 治疗烧伤和其他应激或炎症诱导的胰岛素的策略 阻力