Membrane-Based Mechanisms of Hepatic Insulin Resistance

肝胰岛素抵抗的膜机制

• 批准号: 8758031
• 负责人: Baran Ersoy
• 金额: $ 15.79万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8758031
• 关键词: Ablation; Activation Analysis; Advisory Committees; Affect; Applications Grants; Attenuated; Automobile Driving; Basic Science; Binding; Binding Proteins; Biological; Bone Marrow Transplantation; Boxing; Calcium Channel; Cell Culture System; Cell Culture Techniques; Cells; Cellular biology; Chemicals; Committee Members; Data; Defect; Development; Diabetes Mellitus; Diet; Disease; Education; Endocrinologist; Endoplasmic Reticulum; Environment; Enzyme-Linked Immunosorbent Assay; Exhibits; Extrahepatic; Fasting; Fatty acid glycerol esters; Genetic; Glucose Metabolism Disorders; Goals; Harvest; Hepatic; Hepatocyte; Hepatology; Hospitals; Immune response; Immunoblotting; In Vitro; Inflammation; Inflammatory; Insulin; Insulin Receptor; Insulin Resistance; Interleukin-6; Intervention; JNK-activating protein kinase; JUN gene; Laboratories; Lead; Lecithin; Link; Lipid Binding; Lipids; Lipopolysaccharides; Liver; Liver diseases; MAPK8 gene; Macrophage Activation; Mass Spectrum Analysis; Measures; Mediating; Medication Management; Medicine; Membrane; Mentored Research Scientist Development Award; Mentors; Mentorship; Metabolic; Mission; Molecular; Molecular Biology; Molecular Immunology; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Outcome Study; Pathway interactions; Peritoneal Macrophages; Phospholipids; Physiological; Play; Positioning Attribute; Prevention; Proteins; Proteomics; Public Health; RNA Splicing; Reagent; Regulation; Research; Research Personnel; Research Proposals; Resistance; Role; Scientist; Signal Transduction; Small Interfering RNA; Starvation; TNF gene; Techniques; Testing; Time; Tissues; Training; Transgenic Mice; Tumor Necrosis Factor-alpha; Tunicamycin; Woman; base; biological adaptation to stress; blood glucose regulation; career; common treatment; cytokine; endoplasmic reticulum stress; experience; feeding; glucose metabolism; glucose production; human IRS2 protein; improved; inhibitor/antagonist; instructor; insulin sensitivity; insulin signaling; lipid metabolism; loss of function; medical schools; mouse model; new therapeutic target; non-alcoholic fatty liver; novel; novel therapeutic intervention; phosphatidylcholine transfer protein; professor; protein complex; protein expression; public health relevance; research study; response; skills; small molecule; success

DESCRIPTION (provided by applicant): This is an application for a K01 Award for Dr. Baran A. Ersoy, an Instructor in Medicine at the Brigham and Women's Hospital (BWH) and Harvard Medical School (HMS). Dr. Ersoy is a young investigator in basic research on disorders of glucose and lipid metabolism. His application outlines a short-term plan to further his education by gaining experience in advanced molecular biology and immunology techniques, including mass spectrometry of proteomics and bone marrow transplantation. This plan will build upon his previous experience and provide him the time and training to establish new research skills, strategies and projects that will contribute to his success upon becoming an independent scientist. It is his long-term goal to become an establish independent investigator in an academic setting and study the mechanisms driving insulin resistance and promoting type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). To achieve these goals, Dr. Ersoy will be mentored by Dr. David E. Cohen, Professor of Medicine at HMS and Director of Hepatology at BWH. Dr. Ersoy has also assembled an advisory committee comprised of Dr. Steven P. Gygi, Professor of Cell Biology at HMS, who focuses on mass spectrometry of proteomics; Dr. Umut Ozcan, an endocrinologist with expertise in endoplasmic reticulum stress, inflammation and insulin signaling; and Dr. Morris F. White, an expert in insulin signaling who played a major role in the discovery insulin receptor substrate proteins. Dr. Ersoy's mentor and advisory committee members have established mentorship and training record, whose previous trainees hold independent investigator positions throughout the world. Dr. Ersoy will conduct his training at BWH and HMS, which will provide an intellectually rich research environment and has a long- standing commitment to training young scientists for independent academic research careers. Obesity-induced resistance to insulin action is the primary pathophysiological defect that predisposes to NAFLD and type 2 diabetes. Because current management options remain limited, identification of new regulatory mechanisms that govern the metabolic response to insulin should serve to identify novel opportunities for pharmacologic intervention. The objective of this research proposal is to elucidate a fundamental new mechanism for the regulation of glucose homeostasis. The rationale is that the identification of a novel mechanism that regulates hepatic insulin sensitivity could lead to the identification of new therapeutic targets for the treatment of NAFLD and type 2 diabetes. Guided by extensive preliminary data, the central hypothesis of this research plan is that phosphatidylcholine transfer protein (PC-TP) regulates glucose homeostasis by controlling the activity of insulin receptor substrate 2 (IRS2) both directly and also indirectly by modulating endoplasmic reticulum (ER) stress and inflammation. This will be tested in three specific aims: 1) Define the mechanisms by which PC-TP suppresses IRS2 activity; 2) Determine the role of ER stress in PC- TP-mediated regulation of insulin signaling; 3) Elucidate the role of immune response in PC-TP-mediated inhibition of insulin sensitivity. In Aim 1, a small molecule inhibitor of PC-TP (compound A1) and IRS2 deficient mice will be used to examine the role of IRS2 in the regulation of insulin signaling by PC-TP. The biological pathways in the control of IRS2 inhibition by PC-TP will be unveiled using mass spectroscopy of proteomics in Pctp+/+ and Pctp-/- mice as well as compound A1-treated hepatocytes harvested from these mice. In Aim 2, Pctp+/+ and Pctp-/- mice and hepatocytes harvested from these mice will be used to determine whether PC-TP modulates key ER stress pathways that regulate insulin signaling and IRS2 activity by immunoblot analyses. ER stress signaling will be activated by refeeding following 24 h starvation in mice, and by the chemical reagent tunicamycin in cell culture systems. Aim 3 will utilize peritoneal macrophages harvested from Pctp+/+ and Pctp-/- mice and bone marrow transplantation of Pctp-/- donor cells into Pctp+/+ recipient mice. Inflammation will be induced by high fat diet feeding in mice, and by lipopolysaccharide treatment in cell culture. Inflammatory signals will be measured by ELISA, immunoblot and qPCR analyses. It is anticipated that PC-TP will regulate insulin signaling by regulating IRS2 activity both directly and by modulating ER stress and inflammation. This is important because loss of IRS2 expression and increased ER stress and inflammation are associated with development of insulin resistance. These studies are expected to identify new therapeutic targets for the management of insulin resistance and related disorders. The data generated from this research proposal will form the basis for an R01 grant application at the end of the K01 award.

描述（由申请人提供）：这是 Baran A. Ersoy 博士的 K01 奖申请，他是布莱根妇女医院 (BWH) 和哈佛医学院 (HMS) 的医学讲师。 Ersoy 博士是一位年轻的研究员，致力于葡萄糖和脂质代谢紊乱的基础研究。他的申请概述了一个短期计划，通过获得先进分子生物学和免疫学技术（包括蛋白质组学质谱分析和骨髓移植）的经验来进一步深造。该计划将以他之前的经验为基础，为他提供时间和培训，以建立新的研究技能、策略和项目，这将有助于他成为一名独立科学家后取得成功。他的长期目标是成为学术界的一名独立研究者，研究驱动胰岛素抵抗和促进 2 型糖尿病和非酒精性脂肪肝 (NAFLD) 的机制。为了实现这些目标，Ersoy 博士将得到 HMS 医学教授兼 BWH 肝病科主任 David E. Cohen 博士的指导。 Ersoy 博士还组建了一个咨询委员会，由 HMS 细胞生物学教授 Steven P. Gygi 博士组成，他专注于蛋白质组学的质谱分析； Umut Ozcan 博士是一位内分泌学家，在内质网应激、炎症和胰岛素信号传导方面具有专业知识； Morris F. White 博士是胰岛素信号传导专家，在胰岛素受体底物蛋白的发现中发挥了重要作用。 Ersoy博士的导师和咨询委员会成员建立了指导和培训记录，其先前的学员在世界各地担任独立研究员职位。 Ersoy 博士将在 BWH 和 HMS 进行培训，这将提供一个智力丰富的研究环境，并长期致力于培养年轻科学家从事独立学术研究职业。肥胖引起的胰岛素抵抗是导致 NAFLD 和 2 型糖尿病的主要病理生理缺陷。由于当前的管理选择仍然有限，因此确定控制胰岛素代谢反应的新调节机制应有助于确定药物干预的新机会。本研究计划的目的是阐明调节葡萄糖稳态的基本新机制。其基本原理是，调节肝脏胰岛素敏感性的新机制的确定可能有助于确定治疗 NAFLD 和 2 型糖尿病的新治疗靶点。在大量初步数据的指导下，该研究计划的中心假设是磷脂酰胆碱转移蛋白（PC-TP）通过直接或间接通过调节内质网（ER）控制胰岛素受体底物2（IRS2）的活性来调节葡萄糖稳态压力和炎症。这将在三个具体目标中进行测试：1）定义 PC-TP 抑制 IRS2 活性的机制； 2）确定ER应激在PC-TP介导的胰岛素信号调节中的作用； 3）阐明免疫反应在PC-TP介导的胰岛素敏感性抑制中的作用。在目标 1 中，将使用 PC-TP（化合物 A1）的小分子抑制剂和 IRS2 缺陷小鼠来检查 IRS2 在 PC-TP 调节胰岛素信号传导中的作用。通过对 Pctp+/+ 和 Pctp-/- 小鼠以及从这些小鼠中收获的经化合物 A1 处理的肝细胞进行蛋白质组学质谱分析，将揭示 PC-TP 控制 IRS2 抑制的生物学途径。在目标 2 中，Pctp+/+ 和 Pctp-/- 小鼠以及从这些小鼠中收获的肝细胞将用于通过免疫印迹分析确定 PC-TP 是否调节关键的 ER 应激途径，从而调节胰岛素信号传导和 IRS2 活性。小鼠饥饿 24 小时后重新进食以及细胞培养系统中的化学试剂衣霉素将激活 ER 应激信号。目标 3 将利用从 Pctp+/+ 和 Pctp-/- 小鼠中收获的腹膜巨噬细胞，并将 Pctp-/- 供体细胞的骨髓移植到 Pctp+/+ 受体小鼠中。小鼠的高脂肪饮食喂养以及细胞培养物中的脂多糖处理会诱导炎症。将通过 ELISA、免疫印迹和 qPCR 分析来测量炎症信号。预计 PC-TP 将通过直接调节 IRS2 活性以及调节 ER 应激和炎症来调节胰岛素信号传导。这很重要，因为 IRS2 表达的丧失以及 ER 应激和炎症的增加与胰岛素抵抗的发展有关。这些研究有望确定治疗胰岛素抵抗和相关疾病的新治疗靶点。本研究提案生成的数据将构成 K01 奖项结束时 R01 资助申请的基础。