Translational investigation of abnormal fat metabolism in mitochondrial disease

 描述(由申请人提供)： 儿童肥胖是一种日益普遍的公共健康危机，导致全球2型糖尿病负担不断增加。在最基本的层面上，肥胖和糖尿病代表着能量平衡的紊乱。在肥胖症中，能量储存超过了利用。在糖尿病患者中，可用的能量来源(葡萄糖)被不当使用。能量平衡是由细胞的线粒体感知和控制的。对能量平衡紊乱的关键线粒体通路的重点研究将提高我们对肥胖症内分泌并发症的理解，并可能导致新的治疗方法。为了更好地了解线粒体功能障碍和肥胖相关内分泌疾病的交集，一个特别相关的研究群体是患有原发(遗传)线粒体疾病的患者，他们即使没有明显的糖尿病或肥胖，也会出现类似的脂肪代谢紊乱，包括高甘油三酯血症和低高密度脂蛋白胆固醇。通过建议的K23指导性患者导向研究职业发展奖，我将调查原发性线粒体疾病脂代谢紊乱的机制基础和代谢后果。随着我作为一名内科科学家走向独立，拟议的研究将直接支持我的主要职业目标。这些目标是[1]促进我们对线粒体、能量平衡和代谢在儿科内分泌疾病中的作用的理解，并[2]应用生理学见解来开发合理、有针对性和有效的治疗干预措施，以改善内分泌和代谢性疾病患者的健康。这些目标将在K23颁奖期间通过追求3个首要的培训和职业发展目标来推进：首先，我将获得教书 以及对这一领域以患者为中心的研究的成功设计和实施至关重要的技术知识。在拟议奖项的三年时间里，我将学习在内容领域专家顾问的指导下进行有价值的体外和体内表型鉴定技术。具体地说，稳定同位素的体外利用和“胞核”细胞的操作，以及由稳定同位素研究补充的高胰岛素-正常血糖钳研究的体内表现，将是非常宝贵的技能。其次，我将在诊断、评估和管理原发线粒体疾病患者方面积累新的专业知识。这一群体对所有领域，特别是包括儿科内分泌学在内的更有见识的专科医生有着巨大的、尚未得到满足的临床需求。他们进一步提供了一个有重点的机会来研究线粒体功能障碍在常见内分泌疾病中的作用。这一目标将通过为线粒体疾病患者提供临床护理以及在体外和体内进行试验来实现 研究调查，以确定他们血脂异常的程度和基础。第三，我将过渡到作为一名调查员的独立。我将继续与我的指导委员会的专家成员合作，开展富有成效的多学科合作。我将获得必要的专业知识，通过完成拟议的研究、在全国会议上发表报告、在同行评议的期刊上发表文章，以及获得后续的R01资金来过渡到独立。执行这个拟议的项目将有助于实现这些目标。这些实验将检验我们的中心假设：[1]在初级呼吸链(RC)受损的背景下，细胞NAD+/NADH氧化还原平衡改变导致α-酮戊二酸(αKG)衍生的柠檬酸盐从头合成脂质增加，并降低脂肪酸氧化(FAO)能力；[2]由此导致的骨骼肌脂质过度积累导致骨骼肌胰岛素抵抗。为了验证这些假说，我们将采用体外和体内两种方法。我们将使用体外人类细胞系模型来测试RC抑制是否通过“反向”柠檬酸循环流动定量地增加从头开始的脂肪生成。一种补充的体内方法将寻找(I)有高甘油三酯血症但没有糖尿病的成人原发RC疾病患者新生脂肪生成增加、肌肉脂肪堆积和胰岛素敏感性降低的证据。我们将把这一组与(Ii)适当匹配的健康个体进行比较。为了评估“反向”柠檬酸循环通量是否也可能导致“典型”非糖尿病肥胖者的高甘油三酯血症，我们还将研究(Iii)适当匹配的肥胖者。未来，我们可以将这些研究的各个方面扩展到儿科人群。该项目利用了CHOP和宾夕法尼亚大学的许多独特资源。首先，我的主要导师马尼·J·福尔克博士有一大群表型良好的患者，他们都有明确的线粒体疾病基因诊断，这为开展拟议的研究提供了理想的受试者和组织来源。CHOP的线粒体和表观基因组医学中心(CMEM)进一步提供了这一领域的世界级基础设施。宾夕法尼亚大学糖尿病研究中心(DRC)拥有教师和核心专业知识，以确保成功完成详细的代谢表型研究，其中关键教师是主持人。我们在宾夕法尼亚大学高级磁共振成像和光谱中心(CAMRIS)的合作伙伴已经开发出代谢成像技术来评估和定位线粒体功能。这项工作将使我能够建立一个坚实的背景，在此基础上进行未来独立资助的关于线粒体、能量平衡和新陈代谢在儿童内分泌疾病中的作用的研究。

 DESCRIPTION (provided by applicant): Pediatric obesity is an increasingly prevalent public health crisis that contributes to the rising global burden of type 2 diabetes. Obesity and diabetes, at the most fundamental level, represent disorders of energy balance. In obesity, energy storage exceeds utilization. In diabetes, available energy sources (glucose) are improperly used. Energy balance is sensed and controlled by the cell's mitochondria. Focused study of key mitochondrial pathways that are disrupted in disorders of energy balance will improve our understanding of endocrine complications of obesity, and may lead to new treatment approaches. A particularly relevant group to study to better understand the intersection of mitochondrial dysfunction and obesity-related endocrine disorders are patients with primary (genetic) mitochondrial diseases, who develop similar profiles of disordered fat metabolism even in the absence of overt diabetes or obesity, including hypertriglyceridemia and low high density lipoprotein (HDL) cholesterol. Through the proposed K23 Mentored Patient-Oriented Research Career Development Award, I will investigate the mechanistic basis and metabolic consequences of disrupted lipid metabolism in primary mitochondrial disease. The proposed studies will directly support my main career goals as I progress towards independence as a physician-scientist. These goals are to [1] advance our understanding of the role of mitochondria, energy balance, and metabolism in pediatric endocrine disease, and to [2] apply physiologic insights to develop rational, targeted, and effective therapeutic interventions to improve the health of patients with endocrine and metabolic diseases. These goals will be advanced during the K23 award period through the pursuit of 3 overarching training and career development objectives: First, I will acquire didactic and technical knowledge critical for the successful design and execution of patient-oriented research in this area. During the three years of the proposed award, I will learn to perform valuable in vitro and in vivo phenotyping techniques under the guidance of content-area expert advisors. Specifically, in vitro utilization of stable isotopes and manipulation of "cybrid" cell lnes, and in vivo performance of hyperinsulinemic-euglycemic clamp studies complemented by stable isotope studies will be invaluable skills. Second, I will accrue new expertise in the diagnosis, evaluation, and management of patients with primary mitochondrial disease. This population has a large, unmet clinical need for better-informed subspecialists in all areas, particularly including Pediatric Endocrinology. They further provide a focused opportunity to study the role of the mitochondrial dysfunction in common endocrine disorders. This goal will be met by providing clinical care for mitochondrial disease patients and performing both in vitro and in vivo research investigations to characterize the extent and basis of their dyslipidemia. Third, I will transition to independence as an investigator. I will continue to work and develop productive multi-disciplinary collaborations with the expert members of my mentoring committee. I will gain necessary expertise to transition to independence through completing of the proposed studies, presenting at national meetings, publishing in peer-reviewed journals, and securing subsequent R01 funding.. Performing this proposed project will help accomplish these goals. These experiments will test our central hypotheses that: [1] altered cellular NAD+/NADH redox balance in the setting of primary respiratory chain (RC) impairment leads to increased de novo lipid synthesis from alpha-ketoglutarate (αKG)- derived citrate and decreased fatty acid oxidation (FAO) capacity and [2] the resulting excess accumulation of lipids in skeletal muscle causes skeletal muscle insulin resistance. To test these hypotheses, we will employ both in vitro and in vivo approaches. We will use in vitro human cell line models to test whether RC inhibition quantifiably increases de novo lipogenesis via "reversed" citric acid cycle flow. A complementary in vivo approach will look for evidence of increased de novo lipogenesis, muscle lipid accumulation, and decreased insulin sensitivity in (i) adults with primary RC disease, with hypertriglyceridemia but without DM. We will compare this group to (ii) appropriately matched healthy individuals. To assess whether "reversed" citric acid cycle flux might also contribute to hypertriglyceridemia in "typical" non-diabetic obese individuals, we will also study (iii) appropriately matched obese individuals. In future, we can extend aspects of these studies to the pediatric population. This project leverages many unique resources at CHOP and Penn. First, my primary mentor, Dr. Marni J. Falk, has a large and well-phenotyped cohort of patients with clear genetic diagnoses of mitochondrial disease that provides a ready source of ideal subjects, and tissues, in which to perform the proposed studies. CHOP's Center for Mitochondrial and Epigenomic Medicine (CMEM) further offers world-class infrastructure in this field. Penn's Diabetes Research Center (DRC) has faculty and core expertise already in place to assure successful completion of detailed metabolic phenotyping studies, where key faculty are comentors. Our collaborators at Penn's Center for Advanced Magnetic Resonance Imaging and Spectroscopy (CAMRIS) have developed metabolic imaging technologies to estimate and localize mitochondrial function. This work will allow me to establish a solid background from which to pursue future, independently-funded studies on the role of mitochondria, energy balance, and metabolism in pediatric endocrine disease